WO2025054626A2 - Masked 2-aminoisobutyric acid and methods of use thereof - Google Patents

Masked 2-aminoisobutyric acid and methods of use thereof Download PDF

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WO2025054626A2
WO2025054626A2 PCT/US2024/045886 US2024045886W WO2025054626A2 WO 2025054626 A2 WO2025054626 A2 WO 2025054626A2 US 2024045886 W US2024045886 W US 2024045886W WO 2025054626 A2 WO2025054626 A2 WO 2025054626A2
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substituted
amino acid
alkylene
masked
aib
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PCT/US2024/045886
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French (fr)
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WO2025054626A3 (en
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Virginia Cornish
Kiavash GARAKANI
Krishna Kumar
Scott Snyder
Vladislav KHAMRAEV
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The Trustees Of Columbia University In The City Of New York
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C323/39—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton at least one of the nitrogen atoms being part of any of the groups, X being a hetero atom, Y being any atom
    • C07C323/43—Y being a hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/57—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C323/58—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C391/00—Compounds containing selenium
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • C07K5/06147—Dipeptides with the first amino acid being heterocyclic and His-amino acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y601/00—Ligases forming carbon-oxygen bonds (6.1)
    • C12Y601/01—Ligases forming aminoacyl-tRNA and related compounds (6.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae

Definitions

  • UAAs can be used to increase biological activity or improve the stability of desired proteins and peptides.
  • UAAs when applied to therapeutic proteins and peptides, UAAs are known to increase the potency and stability of the therapeutic proteins and peptides.
  • UAAs have been used in anti-obesity drugs, such as Glucagon-like Peptide- 1 (GLP-1) Receptor Agonists and dual GLP-1/GIP receptor agonists such as semaglutide, and tirzepatide.
  • GLP-1 Glucagon-like Peptide- 1
  • GLP-1 GLP-1 Receptor Agonists
  • dual GLP-1/GIP receptor agonists such as semaglutide, and tirzepatide.
  • these receptor agonists comprise the UAA 2-aminoisobutyric acid (Aib), which has the following structure: and differs from alanine by only the substitution of one hydrogen for one methyl group.
  • Incorporation of Aib into semaglutide can, for example, protects semaglutide from enzyme degradation (Mahapatra et al., Rev Endocr Metab Disord.2022:521-539). Semaglutide is typically produced using chemical synthesis (Yu et al., Adv. Drug Del. Rev. 2018:113-130) or hybrid recombinant and chemical synthesis. However, these chemical synthesis routes can have low yields or undesired byproducts, and can be very costly. Recombinant synthesis techniques, on the other hand, are more environmentally friendly and have lower production costs. In addition, methods which can synthesize GLP-1 receptor agonists via recombinant synthesis can increase the efficiency at which the products are created.
  • the present disclosure provides methods for the generation and purification of proteins and peptides comprising at least one or more than one 2-aminoisobutyric acid (Aib) and/or at least one masked form of Aib.
  • the present disclosure further provides compositions and cell media compositions comprising one or more masked forms of Aib and/or one or more recombinant cells for use in the disclosed methods.
  • the present disclosure provides masked forms of Aib or analogs thereof.
  • the masked form of Aib or analog thereof has the structure of any one of Formulas I-XVIII.
  • the masked form of Aib or analog thereof has the structure of Formula IX.
  • the masked form of Aib or analog thereof has the structure of Formula X.
  • the masked form of Aib or analog thereof has the structure of Formula XI.
  • the masked form of Aib or analog thereof has the structure of Formula XII.
  • the masked form of Aib or analog thereof has the structure of Formula XIII.
  • the masked form of Aib or analog thereof has the structure of Formula XIV. In certain embodiments, the masked form of Aib or analog thereof has the structure of Formula XV. In certain embodiments, the masked form of Aib or analog thereof has the structure of Formula XVI. In certain embodiments, the masked form of Aib or analog thereof has the structure of Formula XVII. In certain embodiments, the masked form of Aib or analog thereof has the structure of Formula XVIII.
  • the present disclosure further provides compositions comprising one or more masked forms of Aib or analogs thereof.
  • a composition of the present disclosure includes a cell culture medium and at least one masked form of Aib or analogs thereof, e.g., a masked form Aib or analog thereof that has the structure of any one of Formulas I-XVIII.
  • a composition of the present disclosure includes a buffer and at least one masked form of Aib or analogs thereof, e.g., a masked form Aib or analog thereof that has the structure of any one of Formulas I-XVIII.
  • the present disclosure provides methods for generating polypeptides, e.g., proteins, and peptides that include at least one or more than one Aib and/or at least one masked Active 121670349.1 2 Attorney Ref. No.070050.6868 form of Aib.
  • a method of the present disclosure includes providing a recombinant cell comprising a polynucleotide that includes at least one selector codon and encodes a protein or peptide.
  • the recombinant cell further comprises at least one or more polynucleotides encoding an orthogonal tRNA (O-tRNA) (e.g., that functions in the recombinant cell and recognizes the selector codon contained within the polynucleotide that encodes the protein or peptide) and encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) (e.g., where the O-RS aminoacylates the O-tRNA with a masked form of Aib).
  • O-tRNA orthogonal tRNA
  • O-RS orthogonal aminoacyl-tRNA synthetase
  • the method further includes culturing the recombinant cell in a medium and expressing the protein or peptide that includes at least one masked form of Aib.
  • a method of producing a peptide or protein comprising one or more 2- aminoisobutyric acid (Aib) amino acid residues in a recombinant cell includes (a) culturing the recombinant cell in a cell culture media, wherein the recombinant cell comprises (i) a polynucleotide encoding the peptide or protein and comprising at least one selector codon (e.g., where the recombinant cell expresses: an orthogonal tRNA (O-tRNA) that functions in the recombinant cell and recognizes the selector codon; and an orthogonal aminoacyl-tRNA synthetase (O-RS), wherein the O-RS preferentially aminoacylates the O-tRNA with a masked Aib amino acid or analog thereof); (b) providing the masked Aib amino acid or analog thereof to the cell culture media; and (c) incorporating the masked Aib amino acid into one
  • the medium for culturing the recombinant cells comprises the masked form of Aib.
  • the masked form of Aib present in the cell culture medium has the structure of any one of Formulas I-XVIII.
  • expressing the protein or peptide includes incorporating the masked form of Aib into one or more specified positions in the peptide or protein during translation of the polypeptide with the at least one selector codon.
  • the protein or peptide that includes the at least one masked form of Aib is secreted from the recombinant cell.
  • the protein or peptide that includes the at least one masked form of Aib is not secreted from the recombinant cell.
  • the method further includes purifying the protein or peptide comprising the at least Active 121670349.1 3 Attorney Ref. No.070050.6868 one masked form of Aib from the medium, supernatant and/or recombinant cell extract, e.g., using chromatography as described herein.
  • the present disclosure further provides methods for purifying a protein or peptide comprising at least one masked form of Aib.
  • a purification method of the present disclosure includes contacting a chromatography medium (e.g., within a chromatography column) or a solid support with a solution containing the protein or peptide comprising the at least one masked Aib.
  • the solution is a culture medium, supernatant and/or extract from recombinant cells.
  • the chromatography medium specifically binds to (e.g., non-covalently binds to or covalently binds to) a moiety (e.g., an azide group) contained within the masking group of the masked form of Aib present in the protein or peptide.
  • Non-limiting examples of reactions for binding the masking group to the chromatography medium or solid support are provided herein.
  • a method of the present disclosure can further include removing the masking group from the masked Aib present in the protein or peptide (e.g., purified protein or peptide), e.g., by performing a chemical reaction.
  • Non-limiting examples of reactions for removing the masking group from the masked Aib present in the protein or peptide and catalysts for performing such reactions are provided herein.
  • the chemical reaction is a desulfurization reaction.
  • the chemical reaction is a deselenization reaction.
  • the catalyst used for the chemical reaction is a metal reagent.
  • the catalyst used for the chemical reaction is Pd/Al 2 O 3 . In certain embodiments, the catalyst used for the chemical reaction is Pd/Carbon. In certain embodiments, the catalyst used for the chemical reaction is Raney nickel. In certain embodiments, the catalyst used for the chemical reaction is Pd/BaSO4. In certain embodiments, the catalyst used for the chemical reaction is PdO. In certain embodiments, the catalyst used for the chemical reaction is a Ni(0) catalyst. In certain embodiments, the catalyst used for the chemical reaction is Nickel boride. In certain embodiments, the catalyst used for the chemical reaction is Tin hydride. In certain embodiments, the chemical reaction is performed using a Raney nickel – sodium hypophosphite – acetate buffer system.
  • the present disclosure further provides recombinant cells and compositions thereof for use in the disclosed methods.
  • the recombinant cell comprises a polynucleotide that includes at least one selector codon and encodes a protein or peptide.
  • the recombinant cell further comprises at least one or more polynucleotides encoding an orthogonal tRNA (O-tRNA) (e.g., that functions in the recombinant cell and recognizes the selector codon of the polynucleotide encoding the protein or peptide) and encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) (e.g., where the O-RS aminoacylates the O-tRNA with a masked Aib amino acid).
  • OF-tRNA orthogonal tRNA
  • O-RS orthogonal aminoacyl-tRNA synthetase
  • the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl-tRNA synthetase derived from a species of the Methanococcus genus.
  • the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanococcus jannaschii tyrosyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanomethylophilus alvus tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanomethylophilus alvus tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanococcus jannaschii tyrosyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous Active 121670349.1 5 Attorney Ref. No.070050.6868 to the Saccharomyces cerevisiae glutaminyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O- RS comprises an amino acid sequence that is greater than 95% homologous to the Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the sequence of any one of SEQ ID NOs: 4-6 and 12-14.
  • the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 6.
  • the recombinant cell is a genetically modified bacterial cell. In certain embodiments, the recombinant cell is a genetically modified Escherichia coli cell. In certain embodiments, the recombinant cell is a genetically modified fungal cell. In certain embodiments, the recombinant cell is a genetically modified Saccharomyces cerevisiae cell. In certain embodiments, the recombinant cell is a genetically modified mammalian cell.
  • the present disclosure further provides kits and systems for use in the disclosed methods. In certain embodiments, a kit and/or system of the present disclosure can include one more recombinant cell compositions and/or one or more masked forms of Aib or analogs thereof (or compositions thereof).
  • a kit and/or system of the present disclosure can further include a chromatography medium for purifying a protein or peptide comprising at least one masked form of Aib. Active 121670349.1 6 Attorney Ref. No.070050.6868
  • the peptide or protein generated and/or purified by the methods of the present disclosure is a therapeutic protein or peptide, e.g., a therapeutic protein or peptide for treating diabetes (e.g., type 2 diabetes), for treating obesity, for weight loss and/or for reducing the risk of cardiovascular events (e.g., heart attack and/or stroke).
  • the therapeutic protein or peptide can be a glucagon-like peptide-1 (GLP-1) agonist, a GLP-1 analog, GLP-1 receptor agonist, a glucose-dependent insulinotropic polypeptide (GIP) agonist, a GIP receptor agonist or a dual GLP-1/GIP receptor agonist.
  • GLP-1 receptor agonist can be semaglutide.
  • the protein or peptide is a GLP-1 agonist.
  • the protein or peptide is a GLP-1 analog.
  • the protein or peptide is a GLP-1 receptor agonist.
  • the protein or peptide is a dual GLP-1/GIP receptor agonist.
  • the peptide is selected from the group consisting of semaglutide, tirzepatide, retatrutide, VK2735 and a combination thereof.
  • the peptide is semaglutide.
  • the peptide is tirzepatide.
  • the peptide is retatrutide.
  • the peptide is VK2735.
  • a composition of the present disclosure can include a cell culture medium and one or more recombinant cells.
  • a composition of the present disclosure can include lyophilized recombinant cells.
  • FIG. 2 shows the amino acid structure of tirzepatide.
  • FIG. 3 shows the amino acid structure of retatrutide.
  • FIG.4A shows the amber suppression in the presence of no amino acids or in the presence of unnatural amino acids with Methanomethylophilus alvus PylRS/tRNA pyl .
  • FIG.4B shows the amber suppression in the presence of no amino acids or in the presence of unnatural amino acids and masked amino acids with Methanosarcina mazei PylRS/tRNA pyl .
  • FIG. 4C shows the chemical structures of BocK and CbzK.
  • FIG. 5 shows the green fluorescent protein (GFP) amber suppression with the synthetases M. mazei WT and M.
  • GFP green fluorescent protein
  • FIG.6A compares the GFP fluorescence in the presence of no amino acids or (R)-2-amino- 3-((2-(((benzyloxy)carbonyl)amino)ethyl)thio)-2-methylpropanoic acid (Compound 2).
  • FIG. 6B compares the GFP fluorescence under increasing concentration conditions that include no amino acids or (R)-2-amino-3-((2-(((benzyloxy)carbonyl)amino)ethyl)thio)-2- methylpropanoic acid (Compound 2).
  • FIG. 7 provides an exemplary schematic of a method of the present disclosure for generating a peptide or protein comprising one or more Aib amino acids.
  • the presently disclosed subject matter for the synthesis and further purification of proteins and peptides which comprise at least one 2-aminoisobutyric acid (Aib) amino acid (or derivative thereof) and/or at least one masked form of Aib (or derivative thereof) in cells.
  • the presently disclosed subject matter further provides compositions comprising the proteins and peptides comprising Aib and/or masked Aib and kits and systems and methods of using the proteins and peptides comprising Aib and/or masked Aib.
  • incorporation of Aib into peptide and protein therapeutics can affect the stability and/or efficacy of the therapeutic.
  • the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
  • the terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms or words that do not preclude additional acts or structures.
  • the present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
  • expression refers to transcription and translation occurring within a cell, e.g., a recombinant cell, e.g., a recombinant E. coli cell.
  • the level of expression of a gene and/or nucleic acid in a cell can be determined on the basis of either the amount of corresponding mRNA that is present in the cell or the amount of the protein encoded Active 121670349.1 9 Attorney Ref. No.070050.6868 by the gene and/or nucleic acid that is produced by the cell.
  • mRNA transcribed from a gene and/or nucleic acid is desirably quantitated by northern hybridization.
  • Protein encoded by a gene and/or nucleic acid can be quantitated either by assaying for the biological activity of the protein or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay using antibodies that are capable of reacting with the protein.
  • assays for the biological activity of the protein or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay using antibodies that are capable of reacting with the protein.
  • polypeptide refers generally to peptides and proteins having about three or more amino acids.
  • the polypeptide can be endogenous to the cell, or preferably, can be exogenous, meaning that it is heterologous, i.e., foreign, to the cell being utilized.
  • protein refers to a sequence of amino acids for which the chain length is sufficient to produce the higher levels of tertiary and/or quaternary structure. This is to distinguish from “peptides” that typically do not have such structure.
  • the protein herein will have a molecular weight of at least about 4-100 kD, e.g., closer to about 15 kD.
  • a protein can include at least about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400 or about 500 amino acids.
  • amino acid amino acid monomer
  • amino acid residue refer to organic compounds composed of amine and carboxylic acid functional groups, along with a side-chain specific to each amino acid.
  • alpha- or ⁇ -amino acid refers to organic compounds in which the amine (-NH2) is separated from the carboxylic acid (-COOH) by a methylene group (-CH2), and a side-chain specific to each amino acid connected to this methylene group (-CH 2 ) which is alpha to the carboxylic acid (-COOH).
  • Different amino acids have different side chains and have distinctive characteristics, such as charge, polarity, aromaticity, reduction potential, hydrophobicity and pKa.
  • Amino acids can be covalently linked to form a polymer through peptide bonds by reactions between the carboxylic acid group of the first amino acid and the amine group of the second amino acid.
  • Amino acid in the sense of the disclosure refers to any of the twenty plus naturally occurring amino acids, non-natural amino acids, and includes both D and L optical isomers.
  • the term “nucleic acid,” “nucleic acid molecule” or “polynucleotide” as used herein refers Active 121670349.1 10 Attorney Ref. No.070050.6868 to any compound and/or substance that comprises a polymer of nucleotides.
  • Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group.
  • a purine- or pyrimidine base i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)
  • a sugar i.e., deoxyribose or ribose
  • phosphate group i.e., a sugar
  • the nucleic acid molecule is described by the sequence of bases, whereby the bases represent the primary structure (linear structure) of a nucleic acid molecule.
  • the sequence of bases is typically represented from 5’ to 3’.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including, e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules.
  • DNA deoxyribonucleic acid
  • cDNA complementary DNA
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • the nucleic acid molecule can be linear or circular.
  • nucleic acid molecule includes both, sense and antisense strands, as well as single stranded and double stranded forms.
  • the herein described nucleic acid molecule can contain naturally occurring or non-naturally occurring nucleotides.
  • nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of a nucleic acid of the disclosure in vitro and/or in vivo, e.g., in a bacterial cell.
  • a nucleic acid of the present disclosure can encode a therapeutic.
  • a nucleic acid of the present disclosure can encode an aminoacyl-tRNA synthetase.
  • DNA e.g., cDNA
  • RNA e.g., mRNA vectors can be unmodified or modified.
  • mRNA can be chemically modified to enhance the stability of the RNA vector and/or expression of the encoded molecule.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • recombinant cell refers to cells which have some genetic modification from the original parent cells from which they are derived. Such cells can also be referred to as “genetically-engineered cells.” Such genetic modification can be the result of an introduction of a heterologous gene (or nucleic acid) for expression of an aminoacyl transferase, e.g., an aminoacyl-tRNA synthetase.
  • recombinant protein refers generally to peptides and proteins. Such recombinant proteins are “heterologous,” i.e., foreign to the cell being utilized, such as a GLP-1 receptor agonist produced by a cell, e.g., a bacterial cell. Active 121670349.1 11 Attorney Ref. No.070050.6868 As used herein, “sequence identity” or “identity” in the context of two polynucleotide or polypeptide sequences makes reference to the nucleotide bases or amino acid residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • percentage of sequence identity or “percentage of identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window can include additions or deletions (gaps) as compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
  • determination of percent identity between any two sequences can be accomplished using certain well-known mathematical algorithms. Non- limiting examples of such mathematical algorithms are the algorithm of Myers and Miller, the local homology algorithm of Smith et al.; the homology alignment algorithm of Needleman and Wunsch; the search-for-similarity-method of Pearson and Lipman; the algorithm of Karlin and Altschul, modified as in Karlin and Altschul.
  • operative connection with regard to regulatory sequences of a gene indicate an arrangement of elements in a combination enabling production of an appropriate effect.
  • an operative connection indicates a configuration of the genes with respect to the regulatory sequence allowing the regulatory sequences to directly or indirectly increase or decrease Active 121670349.1 12 Attorney Ref. No.070050.6868 transcription or translation of the genes.
  • regulatory sequences directly increasing transcription of the operatively linked gene comprise promoters typically located on a same strand and upstream on a DNA sequence (towards the 5’ region of the sense strand), adjacent to the transcription start site of the genes whose transcription they initiate.
  • regulatory sequences directly increasing transcription of the operatively linked gene or gene cluster comprise enhancers that can be located more distally from the transcription start site compared to promoters, and either upstream or downstream from the regulated genes, as understood by those skilled in the art. Enhancers are typically short (50-1500 bp) regions of DNA that can be bound by transcriptional activators to increase transcription of a particular gene.
  • enhancers can be located up to 1 Mbp away from the gene, upstream or downstream from the start site.
  • secretable means able to be secreted, wherein secretion in the present disclosure generally refers to transport or translocation from the interior of a cell, e.g., within the cytoplasm or cytosol of a cell, to its exterior, e.g., outside the plasma membrane of the cell. Secretion can include several procedures, including various cellular processing procedures such as enzymatic processing of the peptide. In certain embodiments, secretion of a protein, e.g., a protein disclosed herein, can be continuous or induced.
  • detect indicates the determination of the existence and/or presence of a target in a limited portion of space, including but not limited to a sample, a reaction mixture, a molecular complex and a substrate.
  • the “detect” or “detection” as used herein can comprise determination of chemical and/or biological properties of the target, including but not limited to ability to interact, and in particular bind, other compounds, ability to activate another compound and additional properties identifiable by a skilled person upon reading of the present disclosure.
  • the detection can be quantitative or qualitative.
  • a detection is “quantitative” when it refers, relates to, or involves the measurement of quantity or amount of the target or signal (also referred as quantitation), which includes but is not limited to any analysis designed to determine the amounts or proportions of the target or signal.
  • a detection is “qualitative” when it refers, relates to, or involves identification of a quality or kind of the target or signal in terms of relative abundance to another target or signal, which is not quantified.
  • the term “derived” or “derive” is used herein to mean to obtain from a specified source.
  • No.070050.6868 the smallest fundamental unit of a chemical compound that can take part in a chemical reaction.
  • derivative or “analog” as used herein with reference to a protein or amino acid refers to a modified form of the protein or amino acid. Non-limiting examples of such derivatives include mutated forms of the protein.
  • “Pharmaceutically acceptable carrier,” as used herein, refers to a pharmaceutically acceptable material, composition or vehicle that is involved in carrying or transporting a compound or composition of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body.
  • the carrier can be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof.
  • each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it can come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.
  • subject refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be recipient of a particular treatment.
  • the term “modified” when referencing an organism, e.g., a cell refers to an organism that does not exist in nature.
  • a “therapeutically effective amount” or a “therapeutically effective level” refers to an amount of a protein or peptide produced by the methods of the present disclosure that prevents, decreases, alleviates or eliminates one or more symptoms of a condition, e.g., obesity.
  • “orthogonal” refers either to a tRNA molecule and/or to an aminoacyl synthetase molecule which reacts with reduced efficiency with the endogenous components of a translation system, either in vivo or in vitro.
  • an orthogonal tRNA in a translation system of interest e.g., in the translational system of E.
  • an orthogonal aminoacyl synthetase aminoacylates any endogenous tRNA in the translation system of interest, e.g., in the translational system of E. coli, with reduced or even zero efficiency as Active 121670349.1 14 Attorney Ref. No.070050.6868 compared to aminoacylation of the endogenous tRNA by an endogenous aminoacyl synthetase.
  • RS refers to an aminoacyl-tRNA synthetase.
  • O-RS refers to an orthogonal aminoacyl-tRNA synthetase.
  • O-tRNA refers to an orthogonal tRNA.
  • Sector codon refers to a codon (i.e., a series of 3 or more nucleic acids) recognized by an O-tRNA in the translation process and not recognized by an endogenous tRNA.
  • the O-tRNA anticodon loop recognizes the selector codon on an mRNA so that the amino acid it carries, e.g., an unnatural amino acid, is incorporated at the site in the peptide encoded by the selector codon.
  • a “suppressor tRNA” is a tRNA that alters the reading of a messenger RNA (mRNA) in a given translation system, e.g., by recognizing a stop codon or other nonsense codon and supplying an amino acid, thereby allowing the translation of codons located 3’ of the stop or nonsense codon.
  • “Unnatural amino acid” means any amino acid, amino acid derivative, amino acid analog, ⁇ -hydroxy acid, or other molecule other than a natural amino acid which can be incorporated into a polypeptide chain with an O-tRNA/O-RS pair and which allows extension of the polypeptide chain.
  • the term “non-proteogenic amino acid” refers to an unnatural amino acid.
  • the term “isolate” can refer to a therapeutic that has been separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting).
  • isolated therapeutics e.g., isolated protein or peptide therapeutics
  • isolated therapeutics can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of the other components with which they were initially associated.
  • isolated therapeutics e.g., isolated protein or peptide therapeutics
  • a therapeutic e.g., a protein or peptide therapeutic, is “pure” if it is substantially free of other components.
  • purify can refer to a protein or peptide therapeutic or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (e.g., whether in nature or in an experimental setting), or during any time after its initial production.
  • the one or more protein or peptide therapeutics present in the composition can be independently purified from other proteins or peptides present in the material or environment containing the protein or peptide therapeutic comprising the Aib amino acid.
  • first and second are used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another element. Accordingly, an element discussed below could be termed a second element, and similarly, a second element may be termed a first element without departing from the teachings of the present dsclosure.
  • alkyl refers to a saturated branched or unbranched carbon chain.
  • alkyl refers to a carbon chain with 1 to 12 carbon atoms (C 1 -C 12 alkyl), 1 to 8 carbon atoms (C 1 -C 8 alkyl), 1 to 6 carbon atoms (C 1 -C 6 alkyl), or 1 to 4 carbon atoms (C 1 -C 4 alkyl).
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, 1-methylbutyl, 2- methylbutyl, 3-methylbutyl, neopentyl, 3,3-dimethylpropyl, hexyl, 2-methylpentyl, and the like.
  • the alkyl group can be optionally substituted.
  • alkylene refers to an alkyl group as defined herein having two free radicals which can bond with another group.
  • alkaryl refers to an alkyl group substituted with an aryl radical.
  • alkenyl refers to a branched or unbranched carbon chain having at least one carbon-carbon double bond. In certain embodiments, alkenyl refers to a carbon chain with at least one carbon-carbon double-bond having 1 to 12 carbon atoms (C1-C12 alkenyl), 1 to 8 carbon atoms (C1-C8 alkenyl), 1 to 6 carbon atoms (C1-C6 alkenyl), or 1 to 4 carbon atoms (C1-C4 alkenyl).
  • the alkenyl group can be optionally substituted.
  • alkynyl refers to a carbon chain with at least one carbon-carbon triple-bond having 1 to 12 carbon atoms (C 1 -C 12 alkynyl), 1 to 8 carbon atoms (C 1 -C 8 alkynyl), 1 to 6 carbon atoms (C 1 -C 6 alkynyl), or 1 to 4 carbon atoms (C1-C4 alkynyl).
  • the alkynyl group can be optionally substituted.
  • the alkynyl group can be unbranched or branched. Representative examples include ethynyl, propynyl, butyn-1-yl, and butyn-2-yl.
  • alkoxy refers to –O–alkyl, wherein alkyl is as defined herein.
  • alkoxy refers to a carbon chain with –O–alkyl having 1 to 12 carbon atoms (C 1 -C 12 alkoxy), 1 to 8 carbon atoms (C 1 -C 8 alkoxy), 1 to 6 carbon atoms (C 1 -C 6 alkoxy), or 1 to 4 carbon atoms (C 1 -C 4 alkoxy).
  • alkoxy examples include, but are not limited to, methoxy (C1 alkoxy), ethoxy (C2 alkoxy), propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, cyclopropyloxy-, cyclohexyloxy- and the like.
  • the alkoxy group can be optionally substituted.
  • arylalkoxy refers to an alkoxy group substituted with an aryl group. In certain embodiments, the arylalkoxy group can be optionally substituted.
  • cycloalkyl or “carbocyclic ring” refers to nonaromatic rings that are either partially or fully hydrogenated and may exist as a single ring of 3 to 8 members or bicyclic ring of 9 to 14 members. Unless specified otherwise, the carbocyclic ring is generally a 3- to 8-membered ring (C3-C8 cycloalkyl) or 3- to 6-membered ring (C3-C6 cycloalkyl).
  • a cycloalkyl groups includes cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclpentenyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, norbornyl, norbornenyl, and the like.
  • cycloalkylene refers to cycloalkyl as defined herein having two free radicals which can bond with another group.
  • aryl refers to a monocyclic, bicyclic, or tricyclic aromatic ring. An aryl group contains 6 to 10 carbon atoms.
  • aryl group examples include phenyl (C6H6, Ph, or Ar) or naphthyl.
  • arylene refers to aryl as defined herein having a free radical which can bond with another group.
  • aralkyl refers to an aryl group substituted with an alkyl radical. Active 121670349.1 17 Attorney Ref. No.070050.6868 As used herein, a “heterocycle” or “heterocycloalkyl” refers to a monocyclic, bicyclic, or tricyclic aromatic ring system having 1 to 8 heteroatoms selected from N, O, or S.
  • a heterocycloalkyl is a 3-14 membered monocyclic, bicyclic or tricyclic ring system preferably containing 1 to 3 heteroatoms each independently selected from O, N, or S.
  • examples of hereocycloalkyl groups include oxiranyl, piperidinyl, tetrahydrofuranyl, 1,4-dioxanyl, azetidinyl, or tetrahydrothiophenyl.
  • heterocycloalkylene refers to heterocycloalkyl as defined herein having two free radicals available for bonding.
  • heteroaryl refers to a monocyclic, bicyclic, or tricyclic aromatic ring system having 1 to 8 heteroatoms selected from N, O, or S.
  • a heteroaryl is a 5-14 membered aromatic ring system preferably containing 1 to 4 heteroatoms independently selected from O, N, or S.
  • heteroaryl group examples include 2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrssolyl, 2-, 4-, or 5-imidazolyl, 3-, 4-, or 5- pyrazolyl, 2-, 4-, or 5-thiazolyl, 3-, 4-, or 5-isothiazolyl, 2-, 4-, or 5-oxazolyl, 3-, 4-, or 5-isoxazolyl, 3- or 5-1,2,4-triazolyl, 4- or 5-1,2, 3-triazolyl, tetrazolyl, 2-, 3-, or 4-pyridyl, 3- or 4-pyridazinyl, 3-, 4-, or 5-pyrazinyl, 2- pyrazinyl, and 2-, 4-, or 5-pyrimidinyl.
  • heteroarylene refers to heteroaryl as defined herein having two free radicals available for bonding.
  • halogen or “halo” refers to fluoro, chloro, bromo, or iodo.
  • the term “masking group” refers to a group that is introduced temporarily at a desired position of a molecule to “mask” the position so that a reaction does not occur at further synthetic steps. The masking group can be removed via a “demasking” reaction. Any suitable masking group can be used.
  • masking groups include, but are not limited, to thioethers (R-S-R) or selenoethers (R-Se-R), also called selenides.
  • the masking group is a thioether.
  • the masking group is a selenoether.
  • the term “protecting group” or “protective group” refers to a group that is introduced temporarily at a desired position of a molecule to “protect” the position so that a reaction does not occur at further synthetic steps. The protecting group can be removed via a “deprotecting” reaction. Any suitable protecting group can be used.
  • protecting groups to protect amines include, but are not limited to, t-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), 9-fluorenylmethoxycarbonyl (FMoc), or allyloxycarbonyl (Alloc).
  • protecting groups to protect carboxylic acids include, but are not limited to, tert- butyl (tBu), trityl (Trt), 2,4-dimethoxybenzyl (Dmb), 9-fluoromethyl (Fm), or benzyl (Bn). Active 121670349.1 18 Attorney Ref.
  • Examples of protecting groups to protect hydroxyl groups include, but are not limited to, tert- butyldimethylsilyl (TBDMS), allyl (Al), acetate (Ac) or o-nitrobenzyl (ONB).
  • Common ester protecting groups include, but are not limited to, acetate (Ac), pivalate (PV), and benzoate (Bz).
  • a masked form of Aib and/or analog thereof can be incorporated into a therapeutic peptide of the present disclosure.
  • a masked form of Aib and/or analog thereof can be used in a method of the present disclosure for preparing a therapeutic peptide that incorporates one or more Aib amino acids and/or the one or more masked forms of Aib.
  • the present disclosure provides an Aib amino acid or analog thereof that includes a masking group.
  • the masking group is selected from a thioether or a selenoether.
  • the masking group is a thioether.
  • the masking group is a selenoether.
  • the present disclosure provides an Aib amino acid coupled to a thioether masking group or a selenoether masking group.
  • the present disclosure provides an Aib amino acid coupled to a thioether masking group.
  • the present disclosure provides an Aib amino acid coupled to a selenoether masking group.
  • the present disclosure provides a masked Aib amino acid or analog thereof that has the structure of Formula I: wherein: the combination of X and R 3 are optional if the combination of Y and R 4 are present, but are otherwise required; Active 121670349.1 19 Attorney Ref.
  • R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide
  • R 2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide
  • each of X and Y when present is independently S or Se
  • each of R3 and R4 when present is independently an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”) 2
  • the present disclosure provides a masked Aib amino acid or analog thereof that has the structure of Formula II: wherein: the combination of X, A, and R3 are optional if the combination of Y, B, and R4 are present, but are otherwise required; the combination of Y, B, and R 4 are optional if the combination of X, A and R 3 are present, but are otherwise required;
  • R 1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide;
  • R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide;
  • each of X and Y when present is independently S or Se;
  • each of A and B are optional, and when present are independently an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyal
  • each of R3 and R4 when present is independently an azide, tetrazine, substituted tetrazine, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, trans-cyclooctene, substituted trans-cyclooctene, cyclooctyne, or substituted cyclooctyne; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the present disclosure provides a masked Aib amino acid or analog thereof that has the structure of Formula III: wherein: the combination of Y and B are optional; R 1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; X is S or Se; Y when present is S or Se; each of A and B are optional, and when present are independently an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted al
  • Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof;
  • L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, alkenylene, substituted alkenylene, alkynylene
  • the present disclosure provides a masked Aib amino acid or analog thereof that has the structure of Formula IV: Active 121670349.1 24 Attorney Ref. No.070050.6868 wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; each of X and Y is independently S or Se; R 3 is an H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alky
  • the present disclosure provides a masked Aib amino acid or analog thereof that has the structure of Formula V: wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; each of X and Y is independently S or Se; A is optional, and when present is an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)- ON(R”)2, -(alkylene or substituted
  • each R′′ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R′′ group is present, two R′′ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene,
  • the present disclosure provides a masked Aib amino acid or analog thereof that has the structure of Formula VI: Active 121670349.1 27 Attorney Ref. No.070050.6868 wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; X is S or Se; R 3 is an H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R
  • No.070050.6868 (alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, — C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O-(alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, — C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, — C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene
  • the present disclosure provides a masked Aib amino acid or analog thereof that has the structure of Formula VII: wherein: R 1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; X is S or Se; A is optional, and when present is an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)- Active 121670349.1 29 Attorney Ref.
  • each R′′ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R′′ group is present, two R′′ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene
  • the present disclosure provides a masked Aib amino acid or analog thereof that has the structure of Formula VIII: wherein: R 1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; and X is S or Se; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the present disclosure provides a masked Aib amino acid or amino acid analog of Formula III, wherein R1 is H, R2 is OH, X is S, and R3 is alkylalkoxy or substituted alkylalkoxy. In certain embodiments, the present disclosure provides a masked Aib amino acid or amino acid analog of Formula III, wherein R 1 is H, R 2 is OH, X is S, and R 3 is substituted alkylalkoxy.
  • the masked Aib amino acid or amino acid analog of Formula III is (R)-2-amino-3-((2-(((benzyloxy)carbonyl)amino)ethyl)thio)-2-methylpropanoic acid, Compound A, with the structure of Formula IX: F ormula IX or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • Active 121670349.1 31 Attorney Ref.
  • the present disclosure provides a masked Aib amino acid or amino acid analog of Formula III, wherein R1 is H, R2 is OH, X is S, and R3 is alkylalkoxy.
  • the masked Aib amino acid or amino acid analog of Formula III is (R)-2-amino-3-((2-((tert-butoxycarbonyl)amino)ethyl)thio)-2-methylpropanoic acid, Compound B, with the structure of Formula X: or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the present disclosure provides a masked Aib amino acid or amino acid analog of Formula VIII, wherein R1 is H, R2 is OH, and X is S.
  • the masked Aib amino acid or amino acid analog of Formula VIII is (R)-2-amino-3-((2-((((2-azidobenzyl)oxy)carbonyl)amino)ethyl)thio)-2-methylpropanoic acid, and has the structure of Formula XI: or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the present disclosure provides a masked Aib amino acid or amino acid analog of Formula III, wherein R 1 is H, R 2 is OH, X is Se, and R 3 is alkylalkoxy or substituted alkylalkoxy. In certain embodiments, the present disclosure provides a masked Aib amino acid or amino acid analog of Formula III, wherein R1 is H, R2 is OH, X is Se, and R3 is alkylalkoxy.
  • the masked Aib amino acid or amino acid analog of Formula III is (R)-2-amino-3-((2-(((benzyloxy)carbonyl)amino)ethyl)selanyl)-2-methylpropanoic acid, Compound D, with the structure of Formula XII: Active 121670349.1 32 Attorney Ref. No.070050.6868 or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the present disclosure provides a masked Aib amino acid or amino acid analog of Formula III, wherein R1 is H, R2 is OH, X is Se, and R3 is substituted alkylalkoxy.
  • the masked Aib amino acid or amino acid analog of Formula III is (R)-2-amino-3-((2-((tert-butoxycarbonyl)amino)ethyl)selanyl)-2-methylpropanoic acid, Compound E, with the structure of Formula XIII: Formula XIII or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the present disclosure provides a masked Aib amino acid or amino acid analog of Formula VIII, wherein R1 is H, R2 is OH, and X is Se.
  • the masked Aib amino acid or amino acid analog of Formula VIII is (R)-2-amino-3-((2-((((2-azidobenzyl)oxy)carbonyl)amino)ethyl)selanyl)-2-methylpropanoic acid, Compound F, and has the structure of Formula XIV: or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the present disclosure provides a masked Aib amino acid or amino acid analog of Formula III, wherein R 1 is H, R 2 is OH, X is S, and R 3 is alkenyl.
  • the masked Aib amino acid or amino acid analog of Formula III is (R)-3-(allylthio)-2-amino-2-methylpropanoic acid, Compound G, and has the structure of Formula XV: or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the present disclosure provides a masked Aib amino acid or amino acid analog of Formula III, wherein R1 is H, R2 is OH, X is Se, and R3 is alkenyl.
  • the masked Aib amino acid or amino acid analog of Formula III is (R)-3-(allylselanyl)-2-amino-2-methylpropanoic acid, Compound H, and has the structure of Formula XVI: or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the present disclosure provides a masked Aib amino acid or amino acid analog of Formula III, wherein R1 is H, R2 is OH, X is S, and R3 is alkynyl.
  • the masked Aib amino acid or amino acid analog of Formula III is (R)-2-amino-2-methyl-3-(prop-2-yn-1-ylthio)propanoic acid, Compound I, and has the structure of Formula XVII: or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the present disclosure provides a masked Aib amino acid or amino acid analog of Formula III, wherein R 1 is H, R 2 is OH, X is Se, and R 3 is alkynyl.
  • Active 121670349.1 34 Attorney Ref. No.070050.6868
  • the masked Aib amino acid or amino acid analog of Formula III is (R)-2-amino-2-methyl-3-(prop-2-yn-1-ylselanyl)propanoic acid, Compound J, and has the structure of Formula XVIII: or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the masked Aib amino acid or analog thereof of the present disclosure has the structure of any one of Formulas I-XVIII or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the masked Aib amino acid or analog thereof has the structure of Formula I or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the masked Aib amino acid or analog thereof has the structure of Formula II or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formula III or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the masked Aib amino acid or analog thereof has the structure of Formula IV or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the masked Aib amino acid or analog thereof has the structure of Formula V or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the masked Aib amino acid or analog thereof has the structure of Formula VI or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the masked Aib amino acid or analog thereof has the structure of Formula VII.
  • the masked Aib amino acid or analog thereof has the structure of Formula VIII or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the masked Aib amino acid or analog thereof has the structure of Formula IX.
  • the masked Aib Active 121670349.1 35 Attorney Ref. No.070050.6868 amino acid or analog thereof has the structure of Formula X or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the masked Aib amino acid or analog thereof has the structure of Formula XI or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the masked Aib amino acid or analog thereof has the structure of Formula XII or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the masked Aib amino acid or analog thereof has the structure of Formula XIII or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the masked Aib amino acid or analog thereof has the structure of Formula XIV or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formula XV or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the masked Aib amino acid or analog thereof has the structure of Formula XVI or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formula XVII or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the masked Aib amino acid or analog thereof has the structure of Formula XVIII or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the masked Aib amino acid or analog thereof has the structure of Formulas IX-XVIII or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the masked Aib amino acid or analog thereof has the structure of Formulas IX, X, XI, XII, XIII, XIV, XV, XVI, XVII or XVIII or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the masked Aib amino acid or analog thereof has the structure of Formulas IX, X or XI or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. Active 121670349.1 36 Attorney Ref.
  • the masked Aib amino acid or analog of Formulas I-XVIII is an (R) or (S) enantiomer.
  • the masked Aib amino acid or analog of Formulas I-XIII is an (R) enantiomer.
  • the masked Aib amino acid or analog of Formulas I-XIII is an (S) enantiomer.
  • the present disclosure further provides compositions comprising one or more masked Aib amino acid or an analog thereof described herein.
  • a composition of the present disclosure includes a masked Aib amino acid analog thereof that has a structure selected from the group consisting of Formulas I-XVIII.
  • a composition of the present disclosure includes comprises one compound, two compounds, three compounds, four compounds, five compounds, six compounds, seven compounds, eight compounds, nine compounds, or ten compounds selected from the group consisting of Formulas I-XVIII.
  • a composition of the present disclosure includes a masked Aib amino acid analog thereof that has the structure of Formula I.
  • a composition of the present disclosure includes a masked Aib amino acid analog thereof that has the structure of Formula II.
  • a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula III.
  • a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula IV.
  • a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula V. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula VI. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula VII. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula VIII. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula IX.
  • a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula X. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula XI. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula XII. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the Active 121670349.1 37 Attorney Ref. No.070050.6868 structure of Formula XIII. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula XIV.
  • a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula XV. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula XVI. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula XVII. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula XVIII. The present disclosure further provides a cell culture medium comprising one or more of the masked Aib amino acids or analogs thereof disclosed herein.
  • a cell culture medium comprising two or more, three or more, five or more, six or more, seven or more, eight or more, nine or more or ten or more masked Aib amino acids or analogs thereof of the present disclosure.
  • a culture medium of the present disclosure includes at least one masked Aib or amino acid analog thereof described herein.
  • a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having a structure of any one of Formulas I-XVIII.
  • a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having a structure of any one of Formulas I-XVIII.
  • a cell culture medium of the present disclosure comprises one masked Aib amino acid, two masked Aib amino acids, three masked Aib amino acids, four masked Aib amino acids, five masked Aib amino acids, six masked Aib amino acids, seven masked Aib amino acids, eight masked Aib amino acids, nine masked Aib amino acids or ten masked Aib amino acids selected from the group consisting of Formulas I-XVIII.
  • a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula I.
  • a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula II. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula III. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula IV. In certain embodiments, a Active 121670349.1 38 Attorney Ref. No.070050.6868 culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula V. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula VI.
  • a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula VII. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula VIII. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula IX. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula X. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula XI.
  • a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula XII. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula XIII. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula XIV. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula XV. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula XVI.
  • a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula XVII. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula XVIII.
  • the present disclosure further provides a composition comprising a buffer and a masked Aib amino acid or analog thereof described herein. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having a structure of any one of Formulas I-XVIII. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula I.
  • a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula II.
  • a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula III.
  • a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula IV.
  • a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula V.
  • a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula VI. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula VII. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula VIII. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula IX. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula X.
  • a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula XI. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula XII. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula XIII. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula XIV.
  • a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula XV. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula XVI. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula XVII. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula XVIII.
  • the masked Aib amino acids disclosed herein can be synthesized using synthesis strategies known in the art.
  • the masked Aib amino acids disclosed herein can be generated using the synthesis schemes provided in Example 1.
  • a masked Aib amino acid disclosed herein can be synthesized using scheme 1, scheme 2, scheme 3 or scheme 4 shown in Example 1.
  • a masked Aib amino acid disclosed herein can be synthesized using scheme 1 shown in Example 1.
  • a masked Aib amino acid disclosed herein can be synthesized using scheme 2 shown in Example 1.
  • a masked Aib amino acid disclosed herein can be synthesized using scheme 3 shown in Example 1.
  • a masked Aib amino acid disclosed herein can be synthesized using scheme 4 shown in Example 1.
  • compound A i.e., Formula IX
  • compound B i.e., Formula X
  • scheme 2 shown in Example 1.
  • Therapeutics The present disclosure provides methods for producing a therapeutic including one or more of the Aib amino acids and/or masked Aib amino acids disclosed herein.
  • the present disclosure provides methods for producing a peptide or protein therapeutic including one or more of the Aib amino acids and/or masked Aib amino acids disclosed herein in recombinant cells.
  • the peptide or protein therapeutic includes one or more Aib amino acids and/or masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic includes two or more Aib amino acids and/or masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic includes three or more Aib amino acids and/or masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic includes four or more Aib amino acids and/or masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic includes five or more Aib amino acids and/or masked Aib amino acids. In certain embodiments, the therapeutic can be a peptide or a derivative thereof or a functional fragment thereof.
  • the peptide therapeutic can include at least about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90 or about 100 amino acids. In certain embodiments, the peptide therapeutic can include from about 10 to about to about 50 amino acids. In certain embodiments, the peptide therapeutic can include from about 15 to about to about 50 amino acids. In certain embodiments, the peptide therapeutic can Active 121670349.1 41 Attorney Ref. No.070050.6868 include from about 20 to about to about 50 amino acids. In certain embodiments, the peptide therapeutic can include from about 25 to about to about 50 amino acids. In certain embodiments, the peptide therapeutic can include from about 25 to about to about 45 amino acids.
  • the peptide therapeutic can include from about 30 to about to about 45 amino acids. In certain embodiments, the peptide therapeutic can include from about 30 to about to about 40 amino acids. In certain embodiments, the peptide, e.g., peptide therapeutic can have a molecular weight of less than about 5 kD. In certain embodiments, the therapeutic can be a protein or a derivative thereof or a functional fragment thereof.
  • the proteins disclosed herein refer to a sequence of amino acids for which the chain length is sufficient to produce the higher levels of tertiary and/or quaternary structure. This is to distinguish from “peptides” that typically do not have such structure.
  • the protein e.g., protein therapeutic can have a molecular weight of at least about 5-100 kD, e.g., closer to about 15 kD.
  • the protein therapeutic can include at least about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500 amino acids, about 1,000 amino acids, about 1,500 amino acids, about 2,000 amino acids, about 2,500 amino acids, about 3,000 amino acids, about 35,000 amino acids or about 40,000 amino acids.
  • Non-limiting examples of protein therapeutics include all proteins, and, in general proteins that contain one or more disulfide bonds, including multi-chain polypeptides comprising one or more inter- and/or intrachain disulfide bonds.
  • the peptide or protein therapeutic can include other post- translation modifications including, but not limited to, glycosylation and lipidation. See, e.g., Prabakaran et al., WIREs Syst Biol Med (2012), which is incorporated herein by reference in its entirety.
  • the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for treating diabetes, for treating obesity, for weight loss and/or for reducing the risk of cardiovascular events.
  • the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for treating diabetes.
  • the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for treating type 2 diabetes. Active 121670349.1 42 Attorney Ref. No.070050.6868 In certain embodiments, the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for treating obesity. In certain embodiments, the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for reducing the risk of cardiovascular events. In certain embodiments, the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for reducing the risk of heart attack.
  • the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for reducing the risk of stroke.
  • the therapeutic protein or peptide can be a glucagon-like peptide- 1 (GLP-1) agonist, a GLP-1 analog, a GLP-1 receptor agonist, a glucose-dependent insulinotropic polypeptide (GIP) agonist, a GIP receptor agonist or a dual GLP-1/GIP receptor agonist.
  • GLP-1 analog a GLP-1 analog
  • the therapeutic protein or peptide is a GLP-1 agonist.
  • the therapeutic protein or peptide is a GLP-1 receptor agonist.
  • the therapeutic protein or peptide is a GIP agonist. In certain embodiments, the therapeutic protein or peptide is a GIP receptor agonist. In certain embodiments, the therapeutic protein or peptide is a dual GLP-1/GIP receptor agonist. In certain embodiments, the therapeutic protein or peptide is semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the GLP-1 receptor agonist can be semaglutide.
  • Semaglutide shares a 94% structural homology with native human GLP-1, and is distinguished by three modifications: 1) amino-acid substitutions at position 8 (alanine to ⁇ -aminoisobutyric acid or Aib) and position 34 (lysine to arginine), and acylation of the lysine in position 26 with a spacer consisting of two 8-amino-3,6-dioxaoctanoic acid (ADO) moieties, a glutamic acid moiety, and a C-18 fatty di-acid side chain (as shown in FIG.1) (see Lau et al. Journal of Medicinal Chemistry 58(18): 7370–7380 (2015)).
  • ADO 8-amino-3,6-dioxaoctanoic acid
  • the peptide therapeutic can be tirzepatide. In certain embodiments, tirzepatide has the amino acid structure shown in FIG.2. In certain embodiments, the peptide therapeutic can be retatrutide. In certain embodiments, retatrutide has the amino acid structure shown in FIG.3. Active 121670349.1 43 Attorney Ref. No.070050.6868 In certain embodiments, the peptide therapeutic can be VK2735 (Viking Therapeutics). In certain embodiments, retatrutide has the amino acid structure shown in Table 1.
  • the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous to a sequence comprising the sequence of any one of the above-noted peptides or proteins.
  • the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous to a sequence comprising the sequence of any one of the therapeutics disclosed herein.
  • the therapeutic comprises an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% or 100% homologous to a sequence provided in Table 1.
  • the therapeutic comprises an amino acid sequence that is at least about 97% homologous to a sequence provided in Table 1.
  • the therapeutic comprises an amino acid sequence that is at least about 98% homologous to a sequence provided in Table 1.
  • the therapeutic comprises an amino acid sequence that is at least about 99% homologous to a sequence provided in Table 1. In certain embodiments, the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence provided in Table 1. In certain embodiments, the therapeutic comprises an amino acid sequence provided in Table 1.
  • VK2735 comprises the amino acid structure of Tirzepatide, represented in FIG.3 and SEQ ID NO: 3.
  • the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous, e.g., about 97% to about 100% homologous, to a sequence comprising the sequence of any one of SEQ ID NOs: 1-3 and 9-11.
  • the therapeutic comprises an amino acid sequence that is at least about 97% homologous to a sequence of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the therapeutic comprises an amino acid sequence that is at least about 98% homologous to a sequence of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the therapeutic comprises an amino acid sequence that is at least about 99% homologous to a sequence of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the therapeutic comprises an amino acid sequence of SEQ ID NOs: 1-3 and 9-11.
  • the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous, e.g., about 97% to about 100% homologous, to a sequence comprising the sequence of SEQ ID NO: 1.
  • the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NO: 1. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous, e.g., about 97% to about 100% homologous, to a sequence comprising the sequence of SEQ ID NO: 2.
  • the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NO: 2. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous, e.g., about 97% to about 100% homologous, to a sequence comprising the sequence of SEQ ID NO: 3.
  • the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NO: 3. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at Active 121670349.1 46 Attorney Ref.
  • No.070050.6868 least about 97%, at least about 98%, at least about 99% or 100% homologous, e.g., about 97% to about 100% homologous, to a sequence comprising the sequence of SEQ ID NO: 9.
  • the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NO: 9.
  • the therapeutic comprises the amino acid sequence of SEQ ID NO: 9, where one or more amino acids of the therapeutic have been substituted by an Aib amino acid.
  • the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous, e.g., about 97% to about 100% homologous, to a sequence comprising the sequence of SEQ ID NO: 10.
  • the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NO: 10.
  • the therapeutic comprises the amino acid sequence of SEQ ID NO: 10.
  • the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous, e.g., about 97% to about 100% homologous, to a sequence comprising the sequence of SEQ ID NO: 11.
  • the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NO: 11. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 11.
  • IV. Recombinant Cells The present disclosure further provides recombinant cells and compositions thereof for use in the disclosed methods. For example, but not by way of limitation, the present disclosure provides recombinant cells and compositions thereof for synthesizing a peptide or protein therapeutic comprising one or more masked one or more masked Aib amino acids (or derivatives thereof). Active 121670349.1 47 Attorney Ref.
  • the present disclosure provides recombinant cells and compositions thereof for synthesizing a glucagon-like peptide-1 (GLP-1) agonist, a GLP-1 analog, a GLP-1 receptor agonist, a glucose-dependent insulinotropic polypeptide (GIP) agonist, a GIP receptor agonist or a dual GLP-1/GIP receptor agonist that includes one or more one or more masked Aib amino acids (or derivatives thereof).
  • GLP-1 glucagon-like peptide-1
  • GIP glucose-dependent insulinotropic polypeptide
  • the present disclosure provides recombinant cells and compositions thereof for synthesizing a GLP-1 analog.
  • the present disclosure provides recombinant cells and compositions thereof for synthesizing a GLP-1 receptor agonist.
  • the present disclosure provides recombinant cells and compositions thereof for synthesizing semaglutide. In certain embodiments, the present disclosure provides recombinant cells and compositions thereof for synthesizing tirzepatide. In certain embodiments, the present disclosure provides recombinant cells and compositions thereof for synthesizing retatrutide. In certain embodiments, the present disclosure provides recombinant cells and compositions thereof for synthesizing VK2735 (Viking Therapeutics).
  • the recombinant cells for use in expressing a protein or protein therapeutic that comprises one or more masked Aib amino acids (or derivatives thereof) can be a mammalian cell, a plant cell, a bacterial cell or a fungal cell.
  • the cell can be a mammalian cell, e.g., a genetically engineered mammalian cell.
  • the cell can be a plant cell, e.g., a genetically engineered plant cell.
  • the cell can be a bacterial cell, e.g., a genetically engineered bacterial cell.
  • the cell can be a fungal cell, e.g., a genetically engineered fungal cell.
  • the recombinant cell of the present disclosure is a bacterial cell.
  • bacteria include Caulobacter crescentus, Rodhobacter sphaeroides, Pseudoalteromonas haloplanktis, Shewanella sp.
  • strain Ac10 Pseudomonas fluorescens, Pseudomonas aeruginosa, Halomonas elongata, Chromohalobacter salexigens, Streptomyces lividans, Streptomyces griseus, Nocardia lactamdurans, Mycobacterium smegmatis, Corynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacterium lactofermentum, Bacillus subtilis, Bacillus brevis, Bacillus megaterium, Bacillus licheniformis, Bacillus Active 121670349.1 48 Attorney Ref.
  • the bacteria cell is Escherichia coli.
  • the cell can be a fungal cell, e.g., a genetically engineered fungal cell.
  • the genetically engineered fungal cell of the present disclosure is a species of phylum Ascomycota.
  • the species of the phylum Ascomycota is selected from Saccharomyces cerevisiae, Saccharomyces castellii, Saccharomyces var boulardii, Vanderwaltozyma polyspora, Torulaspora delbrueckii, Saccharomyces kluyveri, Kluyveromyces lactis, Zygosaccharomyces rouxii, Zygosaccharomyces bailii, Candida glabrata, Ashbya gossypii, Scheffersomyces stipites, Komagataella (Pichia) pastoris, Candida (Pichia) guilliermondii, Candida parapsilosis, Candida auris, Yarrowia lipolytica, Candida (Clavispora) lusitaniae, Candida albicans, Candida tropicalis, Candida tenuis, Lodderomyces elongisporous, Geotrichum candidum, Baudoin
  • the genetically-engineered cell of the present disclosure is a species of the Saccharomyces genus.
  • the recombinant cell of the present disclosure is a species of the Saccharomyces genus.
  • the species from the Saccharomyces genus is selected from the group consisting of Saccharomyces bayanus, Saccharomyces boulardii, Saccharomyces castellii, Saccharomyces cerevisiae, Saccharomyces dairenensis and Saccharomyces mikatae.
  • the genetically-engineered cell of the present disclosure is Saccharomyces cerevisiae.
  • the genetically-engineered cell of the present disclosure is Saccharomyces boulardii.
  • the recombinant cell of the present disclosure is a species from the Pichia genus.
  • species from the Pichia genus is selected from the group consisting of Pichia acacia, Pichia alni, Pichia americana, Pichia amethionina, Pichia amylophila, Pichia angophorae, Pichia angusta, Pichia anomala, Pichia antillensis, Pichia Active 121670349.1 49 Attorney Ref.
  • the genetically- engineered cell of the present disclosure is Pichia pastoris.
  • the recombinant cell of the present disclosure is a species of the Kluyveromyces genus.
  • the genetically-engineered cell of the present disclosure is Kluyveromyces lactis.
  • the recombinant cell of the present disclosure is a mammalian cell.
  • Non-limiting examples of mammalian cells include monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol.
  • TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci.
  • the recombinant cell of the present disclosure is not a mammalian Active 121670349.1 50 Attorney Ref. No.070050.6868 cell.
  • the recombinant cells express and/or secrete a protein or peptide therapeutic that includes an Aib amino acid (or derivative thereof) or a masked Aib amino acid (or derivative thereof) at high levels.
  • the recombinant cells express and/or secrete a protein or peptide therapeutic that includes an Aib amino acid (or derivative thereof) or a masked Aib amino acid (or derivative thereof) at levels greater than about 10 mg/L, e.g., greater than about 50 mg/L, greater than about 100 mg/L, greater than about 250 mg/L, greater than about 500 mg/L, greater than about 750 mg/L, greater than about 1 g/L, greater than about 10 g/L, greater than about 25 g/L, greater than about 50 g/L or greater than about 100 g/L.
  • a protein or peptide therapeutic that includes an Aib amino acid (or derivative thereof) or a masked Aib amino acid (or derivative thereof) at levels greater than about 10 mg/L, e.g., greater than about 50 mg/L, greater than about 100 mg/L, greater than about 250 mg/L, greater than about 500 mg/L, greater than about 750 mg/L, greater than about 1 g
  • the recombinant cells express and/or secrete a protein or peptide therapeutic that includes an Aib amino acid (or derivative thereof) or a masked Aib amino acid (or derivative thereof) at levels from about 10 mg/L to about 100 g/L.
  • the recombinant cells that have been genetically engineered to express one or more protein and/or peptide therapeutics.
  • the recombinant cells of the present disclosure have been genetically engineered to express two or more, three or more, five or more, six or more, seven or more, eight or more, nine or more or ten or more protein and/or peptide therapeutics.
  • a cell can be genetically engineered to express a protein or protein therapeutic that comprises one or more masked Aib amino acids (or derivatives thereof).
  • the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics disclosed in Table 1.
  • the recombinant cells of the present disclosure have been genetically engineered to express semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics).
  • the recombinant cells of the present disclosure have been genetically engineered to express semaglutide.
  • the recombinant cells of the present disclosure have been genetically engineered to express tirzepatide.
  • the recombinant cells of the present disclosure have been genetically engineered to express retatrutide. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express VK2735 (Viking Therapeutics). In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of Active 121670349.1 51 Attorney Ref. No.070050.6868 any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 1.
  • the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 9 (e.g., where one or more amino acids of the therapeutic have been substituted by an Aib amino acid).
  • the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the recombinant cell comprises a polynucleotide that encodes a protein or peptide therapeutic and includes at least one selector codon. For example, but not by way of limitation, a cell can be genetically engineered to express a protein or protein therapeutic that comprises one or more masked Aib amino acids (or derivatives thereof).
  • the masked amino acid or analog thereof will occur during translation of the nucleic acid that comprises at least one selector codon and encodes a protein.
  • the masked Aib amino acid is incorporated into the one or more specified positions in the peptide or protein during translation of the nucleic acid that comprises at least one selector codon and encodes a protein.
  • the selector codon is a stop codon.
  • the selector codon is selected from the group consisting of UAG, UAA and UGA.
  • the selector codon is UAG.
  • the selector codon is UGA.
  • the selector codon is UAA.
  • the selector codon is a 4-base codon.
  • 4-base codons include AGGA, AGUA, AGGU, CGGU, CCCU, CUCU, CUAU, CUAG and GGGU (see, also Shandell et al., Biochemistry 60(46): 3455–3469 (2021), the contents of which are incorporated herein by reference in its entirety).
  • the selector codon is Active 121670349.1 52 Attorney Ref. No.070050.6868 AGGU.
  • the selector codon is CGGU.
  • the selector codon is CCCU.
  • the selector codon is CUCU.
  • the selector codon is CGGU. In certain embodiments, the selector codon is CUAU. In certain embodiments, the selector codon is CGGU. In certain embodiments, the selector codon is GGGU. In certain embodiments, the selector codon is selected from the group consisting of UUU, UUA, UUG, UCA, CUG, AAA, UCG, UAU, GCA, UCU, CUG, AUG, UUA, AGA, AGG, CGU, CGC, CGA and CGG. Additional non-limiting of examples of codons that can be used as selector codons in the methods of the present disclosure can be found in Fredens et al.
  • the recombinant cells that have been genetically engineered to further express one or more aminoacyl-tRNA synthetases For example, but not by way of limitation, the recombinant cells that have been genetically engineered to further express one or more orthogonal aminoacyl-tRNA synthetase (O-RS).
  • the recombinant cell further comprises at least one or more polynucleotides encoding an orthogonal tRNA (O- tRNA).
  • the polynucleotide encoding the orthogonal tRNA (O-tRNA) and the polynucleotide encoding the orthogonal aminoacyl-tRNA synthetase (O-RS) are included in a single nucleic acid.
  • the polynucleotide encoding the orthogonal tRNA (O-tRNA) is included in a first nucleic acid and the polynucleotide encoding the orthogonal aminoacyl-tRNA synthetase (O-RS) is included in a second nucleic acid.
  • the O-tRNA functions in the recombinant cell and recognizes the selector codon of the polynucleotide encoding the protein or peptide therapeutic and the O-RS aminoacylates the O- tRNA with a masked Aib amino acid described herein.
  • the polynucleotides e.g., the polynucleotide encoding the orthogonal tRNA (O-tRNA) and/or the polynucleotide encoding the orthogonal aminoacyl-tRNA synthetase (O-RS) are integrated into the chromosome, e.g., for stability.
  • the O-RS can be homologous to a synthetase known in the art.
  • a synthetase useful in the present disclosure includes one or more, two or more, three or more or all four of the following features: lacks an editing domain, low selectivity for anticodon, has only minor interactions with amine and can accommodate diverse side chains.
  • Active 121670349.1 53 Attorney Ref. No.070050.6868 In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95%, or 99% homologous to a synthetase known in the art.
  • the synthetase is selected from pyrrolysyl-tRNA synthetase, a tyrosyl-tRNA synthetase, a glutaminyl-tRNA synthetase and a leucyl-tRNA synthetase.
  • the synthetase is an Escherichia coli synthetase or a Saccharomyces cerevisiae synthetase.
  • the pyrrolysyl-tRNA synthetase is derived from the Methanococcus genus, e.g., Methanosarcina mazei, Methanosarcina barkeri or Methanococcus jannaschii.
  • the Escherichia coli synthetase is Escherichia coli glutaminyl-tRNA synthetase or a tyrosyl-tRNA synthetase or a leucyl-tRNA synthetase.
  • the Saccharomyces cerevisiae synthetase is a Saccharomyces cerevisiae glutaminyl-tRNA synthetase. Additional non-limiting examples of tRNA synthetases (and O-RS/O-tRNA pairs for use in the present disclosure) are provided in S chandelierl et al., Biochemistry 60(46): 3455–3469 (2021) and Dumas et al., Chem Sci 6(1):50-69 (2015), the contents of each of which are incorporated herein by reference in their entireties.
  • the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl-tRNA synthetase, a tyrosyl-tRNA synthetase, a leucyl-tRNA synthetase or a glutaminyl-tRNA synthetase (e.g., an Escherichia coli glutaminyl-tRNA synthetase or a Saccharomyces cerevisiae glutaminyl-tRNA synthetase).
  • a pyrrolysyl-tRNA synthetase e.g., a tyrosyl-tRNA synthetase, a leucyl-tRNA synthetase or a glutaminyl-tRNA synthetase
  • a glutaminyl-tRNA synthetase e.
  • the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a pyrrolysyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a pyrrolysyl-tRNA synthetase. Active 121670349.1 54 Attorney Ref.
  • the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a pyrrolysyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a pyrrolysyl-tRNA synthetase derived from the Methanococcus genus.
  • the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a pyrrolysyl- tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a pyrrolysyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a pyrrolysyl-tRNA synthetase derived from the Methanococcus genus.
  • the O-RS comprises the amino acid sequence of a pyrrolysyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of a Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the Methanosarcina mazei pyrrolysyl- tRNA synthetase comprises the amino acid sequence set forth in SEQ ID NO: 5, 6 or 12.
  • the Methanosarcina mazei pyrrolysyl-tRNA synthetase comprises the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the Methanosarcina mazei pyrrolysyl-tRNA synthetase comprises the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the Methanosarcina mazei pyrrolysyl-tRNA synthetase comprises the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a Methanosarcina mazei pyrrolysyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Methanosarcina mazei Active 121670349.1 55 Attorney Ref. No.070050.6868 pyrrolysyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanosarcina barkeri pyrrolysyl-tRNA synthetase.
  • the Methanosarcina barkeri pyrrolysyl-tRNA synthetase comprises the amino acid sequence set forth in SEQ ID NO: 13 or 14.
  • the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a Methanosarcina barkeri pyrrolysyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Methanosarcina barkeri pyrrolysyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a Methanosarcina barkeri pyrrolysyl-tRNA synthetase.
  • the O-RS comprises the amino acid sequence of a Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanococcus jannaschii pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a Methanococcus jannaschii pyrrolysyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Methanococcus jannaschii pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Methanococcus jannaschii pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Methanococcus jannaschii pyrrolysyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater Active 121670349.1 56 Attorney Ref. No.070050.6868 than about 99% homologous to the sequence of a Methanococcus jannaschii pyrrolysyl-tRNA synthetase.
  • the O-RS comprises the amino acid sequence of a Methanococcus jannaschii pyrrolysyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase.
  • the Methanomethylophilus alvus pyrrolysyl-tRNA synthetase comprises the amino acid sequence set forth in SEQ ID NO: 4.
  • the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase.
  • the O-RS comprises the amino acid sequence of a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of glutaminyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a glutaminyl-tRNA synthetase. Active 121670349.1 57 Attorney Ref.
  • the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to an Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of an Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of an Escherichia coli glutaminyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of an Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of an Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of an Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of an Escherichia coli glutaminyl- tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Saccharomyces cerevisiae glutaminyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a Saccharomyces cerevisiae glutaminyl-tRNA synthetase.
  • the O-RS comprises the amino acid sequence of a Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% Active 121670349.1 58 Attorney Ref. No.070050.6868 homologous to the sequence of a tyrosyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a tyrosyl-tRNA synthetase.
  • the O-RS comprises the amino acid sequence of a tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a Methanococcus jannaschii tyrosyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Methanococcus jannaschii tyrosyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to an E. coli tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of an E.
  • the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of an E. coli tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of an E. coli tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence Active 121670349.1 59 Attorney Ref. No.070050.6868 that is greater than about 95% homologous to the sequence of an E. coli tyrosyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of an E. coli tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of an E. coli tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Saccharomyces cerevisiae synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a Saccharomyces cerevisiae synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Saccharomyces cerevisiae synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Saccharomyces cerevisiae. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Saccharomyces cerevisiae. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a Saccharomyces cerevisiae.
  • the O-RS comprises the amino acid sequence of a Saccharomyces cerevisiae. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a leucyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a leucyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to an Escherichia coli leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about Active 121670349.1 60 Attorney Ref. No.070050.6868 70% homologous to the sequence of an Escherichia coli leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of an Escherichia coli leucyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of an Escherichia coli leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of an Escherichia coli leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of an Escherichia coli leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of an Escherichia coli leucyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a sequence provided in Table 2. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to a sequence provided in Table 2. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to a sequence provided in Table 2. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to a sequence provided in Table 2. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to a sequence provided in Table 2.
  • the O-RS comprises an amino acid sequence that is greater than about 99% homologous to a sequence provided in Table 2. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 4.
  • the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than Active 121670349.1 61 Attorney Ref. No.070050.6868 about 99% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises the amino acid sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 5.
  • the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises the amino acid sequence of SEQ ID NO: 5.
  • the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 6.
  • the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the O-RS comprises the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 12. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 12. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 12.
  • the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 12. In certain embodiments, the O-RS comprises Active 121670349.1 62 Attorney Ref. No.070050.6868 an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 12. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 12. In certain embodiments, the O-RS comprises the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 13.
  • the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 13. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 13. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 13. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 13. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 13. In certain embodiments, the O-RS comprises the amino acid sequence of SEQ ID NO: 13.
  • the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 14.
  • the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 14. In certain embodiments, the O-RS comprises the amino acid sequence of SEQ ID NO: 14. Table 2 Active 121670349.1 63 Attorney Ref. No.070050.6868 Active 121670349.1 64 Attorney Ref. No.070050.6868 In certain embodiments, the recombinant cells that have been genetically engineered to express one or more O-tRNAs. In certain embodiments, the O-tRNA can be homologous to a tRNA known in the art.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 70%, 80%, 90%, 95%, or 99% homologous to a tRNA known in the art.
  • the tRNA is selected from a tRNA Tyr , a tRNA Leu , a tRNA Gln and a tRNA Pyl .
  • the tRNA is an Escherichia coli tRNA or a Saccharomyces cerevisiae tRNA.
  • the tRNA is derived from the Methanococcus genus, e.g., Methanosarcina mazei, Methanosarcina barkeri or Methanococcus jannaschii.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a sequence provided in Table 3.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 70% homologous to a sequence provided in Table 3.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 80% homologous to a sequence provided in Table 3.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 90% homologous to a sequence provided in Table 3. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to a sequence provided in Table 3. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 99% homologous to a sequence provided in Table 3. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a sequence of any one of SEQ ID NOs: 7-8 Active 121670349.1 65 Attorney Ref. No.070050.6868 and 15.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 70% homologous to a sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 80% homologous to a sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 90% homologous to a sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to a sequence of any one of SEQ ID NOs: 7-8 and 15.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 99% homologous to a sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 7.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, the O-tRNA comprises a nucleotide sequence of SEQ ID NO: 7.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 8.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is Active 121670349.1 66 Attorney Ref. No.070050.6868 greater than about 99% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, the O-tRNA comprises a nucleotide sequence of SEQ ID NO: 8. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 15.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 15.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence of SEQ ID NO: 15.
  • a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanosarcina mazei pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanosarcina mazei tRNA.
  • a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 95% homologous to a Methanosarcina mazei pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 95% homologous to a Methanosarcina mazei tRNA.
  • No.070050.6868 disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 99% homologous to a Methanosarcina mazei pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 99% homologous to a Methanosarcina mazei tRNA.
  • the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic, e.g., a peptide or protein therapeutic disclosed in Table 1.
  • the recombinant cell further comprises a polynucleotide encoding semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics) that comprises a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding semaglutide that comprises a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding tirzepatide that comprises a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding retatrutide that comprises a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding VK2735 that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising an amino acid of any one of SEQ ID NOs: 1-3 and 9- 11 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 1 and a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 2 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 3 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 9 and a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 10 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 11 and a selector codon.
  • a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 5 or 6 and (b) a Active 121670349.1 68 Attorney Ref. No.070050.6868 polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 7.
  • a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 5 or 6 and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 7.
  • a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 5 or 6 and (b) a polynucleotide encoding an O- tRNA comprising a nucleotide sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 7.
  • the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic, e.g., a peptide or protein therapeutic disclosed in Table 1 and a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics).
  • the recombinant cell further comprises a polynucleotide encoding semaglutide that includes a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding tirzepatide that includes a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding retatrutide that includes a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding VK2735 that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising an amino acid of any one of SEQ ID NOs: 1-3 and 9-11 and comprises a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 1 and a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 2 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 3 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 9 and a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding a Active 121670349.1 69 Attorney Ref. No.070050.6868 peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 10 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 11 and a selector codon.
  • a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanomethylophilus alvus tRNA.
  • a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 95% homologous to a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 95% homologous to a Methanomethylophilus alvus tRNA.
  • a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 99% homologous to a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 99% homologous to a Methanomethylophilus alvus tRNA.
  • the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic, e.g., a peptide or protein therapeutic disclosed in Table 1 and a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics).
  • the recombinant cell further comprises a polynucleotide encoding semaglutide that includes a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding tirzepatide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding retatrutide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding VK2735 that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising an amino acid of any one of SEQ ID NOs: 1-3 and 9-11 and comprises a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 1 and a selector codon.
  • the Active 121670349.1 70 Attorney Ref. No.070050.6868 recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 2 and a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 3 and a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 9 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 10 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 11 and a selector codon.
  • a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 4 and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 8.
  • a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 4 and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 8.
  • a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 4 and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 8.
  • a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanosarcina barkeri pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanosarcina barkeri tRNA.
  • a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 95% homologous to a Methanosarcina barkeri pyrrolysyl-tRNA synthetase and (b) a Active 121670349.1 71 Attorney Ref. No.070050.6868 polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 95% homologous to a Methanosarcina barkeri tRNA.
  • a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 99% homologous to a Methanosarcina barkeri pyrrolysyl- tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 99% homologous to a Methanosarcina barkeri tRNA.
  • the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic, e.g., a peptide or protein therapeutic disclosed in Table 1 and a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics).
  • the recombinant cell further comprises a polynucleotide encoding semaglutide that includes a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding tirzepatide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding retatrutide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising an amino acid of any one of SEQ ID NOs: 1-3 and 9-11 and comprises a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 1 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 2 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 3 and a selector codon.
  • the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 9 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 10 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 11 and a selector codon.
  • one or more polynucleotides (or nucleic acids comprising one or more polynucleotides) of the present disclosure can be introduced into cells, e.g., bacteria cells, Active 121670349.1 72 Attorney Ref. No.070050.6868 using vectors, such as plasmid vectors and cell transformation techniques such as electroporation, heat shock and others known to those skilled in the art and described herein.
  • cells e.g., bacteria cells, Active 121670349.1 72 Attorney Ref. No.070050.6868 using vectors, such as plasmid vectors and cell transformation techniques such as electroporation, heat shock and others known to those skilled in the art and described herein.
  • one or more polynucleotides encoding an O-RS (or nucleic acids comprising one or more polynucleotides) of the present disclosure can be introduced into cells, e.g., bacteria cells, using vectors, such as plasmid vectors and cell transformation techniques such as electroporation, heat shock and others known to those skilled in the art and described herein.
  • cells e.g., bacteria cells
  • vectors such as plasmid vectors and cell transformation techniques such as electroporation, heat shock and others known to those skilled in the art and described herein.
  • one or more polynucleotides encoding an O-tRNA (or nucleic acids comprising one or more polynucleotides) of the present disclosure can be introduced into cells, e.g., bacteria cells, using vectors, such as plasmid vectors and cell transformation techniques such as electroporation, heat shock and others known to those skilled in the art and described herein.
  • cells e.g., bacteria cells
  • vectors such as plasmid vectors and cell transformation techniques such as electroporation, heat shock and others known to those skilled in the art and described herein.
  • one or more polynucleotides encoding a peptide or protein therapeutic (or nucleic acids comprising one or more polynucleotides) of the present disclosure can be introduced into cells, e.g., bacteria cells, using vectors, such as plasmid vectors and cell transformation techniques such as electroporation, heat shock and others known to those skilled in the art and described herein.
  • the genetic molecular components are introduced into the cell to persist as a plasmid or integrate into the genome.
  • the nucleic acid can be incorporated into the genome of the genetically-engineered cell.
  • the cells can be engineered to chromosomally integrate a polynucleotide of one or more genetic molecular components described herein, using methods identifiable to skilled persons upon reading the present disclosure.
  • one or more polynucleotides (or nucleic acids comprising one or more polynucleotides) can be inserted into the genome of a genetically engineered cell using a CRISPR/Cas9 system.
  • a polynucleotide encoding one or more therapeutics is introduced into the recombinant cell, e.g., a recombinant E. coli cell, either as a construct or a plasmid.
  • a polynucleotide (or a nucleic acid comprising the polynucleotide) can comprise one or more regulatory regions such as promoters, transcription factor binding sites, operators, activator binding sites, repressor binding sites, enhancers, protein-protein binding domains, RNA binding domains, DNA binding domains, and other control elements known to a person skilled in the art.
  • a polynucleotide encoding a therapeutic of the present disclosure is introduced into the recombinant cell, e.g., a recombinant E. Active 121670349.1 73 Attorney Ref.
  • No.070050.6868 coli cell either as a construct or a plasmid in which it is operably linked to a promoter active in the recombinant cell, e.g., a recombinant E. coli cell, or such that it is inserted into the recombinant cell, e.g., a recombinant E. coli cell, genome at a location where it is operably linked to a suitable promoter.
  • a polynucleotide encoding one or more O-RS and/or O-tRNA is introduced into the recombinant cell, e.g., a recombinant E. coli cell, either as a construct or a plasmid.
  • a polynucleotide (or a nucleic acid comprising the polynucleotide) can comprise one or more regulatory regions such as promoters, transcription factor binding sites, operators, activator binding sites, repressor binding sites, enhancers, protein- protein binding domains, RNA binding domains, DNA binding domains, and other control elements known to a person skilled in the art.
  • a polynucleotide encoding O-RS and/or O-tRNA of the present disclosure is introduced into the recombinant cell, e.g., a recombinant E.
  • coli cell either as a construct or a plasmid in which it is operably linked to a promoter active in the recombinant cell, e.g., a recombinant E. coli cell, or such that it is inserted into the recombinant cell, e.g., a recombinant E. coli cell, genome at a location where it is operably linked to a suitable promoter.
  • a promoter active in the recombinant cell e.g., a recombinant E. coli cell
  • a recombinant E. coli cell e.g., a recombinant E. coli cell
  • a polynucleotide (or a nucleic acid comprising the polynucleotide) encoding one or more of the therapeutics, aminoacyl-tRNA synthetase and/or tRNA can further include a transcription factor for regulation expression of the therapeutic encoded by the nucleic acid.
  • a second nucleic or an additional nucleic acid can be introduced into the cells to express a transcription factor for regulation expression of the therapeutic encoded by the nucleic acid.
  • a polynucleotide (or a nucleic acid comprising the polynucleotide) encoding one or more of the therapeutics, aminoacyl-tRNA synthetase and/or tRNA can be inserted into the genome of the cell, e.g., cell.
  • a nucleic acids encoding a peptide and/or protein therapeutic of the present disclosure e.g., semaglutide
  • one or more nucleic acids encoding a therapeutic, aminoacyl-tRNA synthetase and/or tRNA of the present disclosure can be inserted into a locus of the cell.
  • the one or more nucleic acids can be inserted into one or more loci that minimally affects the cell, e.g., in an intergenic locus or a gene that is not essential and/or does not affect growth, proliferation and cell signaling.
  • Active 121670349.1 74 Attorney Ref. No.070050.6868
  • one or more endogenous genes of the genetically-engineered cells can be knocked out and/or mutated, e.g., knocked out by a genetic engineering system.
  • extra copies of endogenous genes of the genetically-engineered cells can be knocked in, e.g., knocked in by a genetic engineering system.
  • a genetic engineering system Various genetic engineering systems known in the art can be used. Non-limiting examples of such systems include the Clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas system, the zinc-finger nuclease (ZFN) system, the transcription activator-like effector nuclease (TALEN) system, use of yeast endogenous homologous recombination and the use of interfering RNAs.
  • CRISPR Clustered regularly-interspaced short palindromic repeats
  • ZFN zinc-finger nuclease
  • TALEN transcription activator-like effector nuclease
  • a CRISPR/Cas9 system is employed to knock out and/or knock in one or more endogenous genes in the genetically engineered cell.
  • the system includes Cas9 (a protein able to modify DNA utilizing crRNA as its guide), CRISPR RNA (crRNA, contains the RNA used by Cas9 to guide it to the correct section of host DNA along with a region that binds to tracrRNA (generally in a hairpin loop form) forming an active complex with Cas9) and trans-activating crRNA (tracrRNA, binds to crRNA and forms an active complex with Cas9).
  • Cas9 a protein able to modify DNA utilizing crRNA as its guide
  • CRISPR RNA CRISPR RNA
  • tracrRNA trans-activating crRNA
  • guide RNA and “gRNA” refer to any nucleic acid that promotes the specific association (or “targeting”) of an RNA-guided nuclease such as a Cas9 to a target sequence such as a genomic or episomal sequence in a cell.
  • gRNAs can be unimolecular (comprising a single RNA molecule and referred to alternatively as chimeric) or modular (comprising more than one, and typically two, separate RNA molecules, such as a crRNA and a tracrRNA, which are usually associated with one another, for instance by duplexing).
  • a homolog of a nucleotide sequence disclosed herein can be a polynucleotide having changes in one or more nucleotide bases that can result in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide or protein encoded by the nucleotide sequence.
  • Homologs can also include polynucleotides having modifications such as deletion, addition or insertion of nucleotides that do not substantially affect the functional properties of the resulting polynucleotide or transcript. Alterations in a polynucleotide that result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded polypeptide, are well known in the art.
  • a homolog of a peptide, protein or O-RS disclosed herein can be a peptide, protein or O-RS having changes in one or more amino acids but do not affect the functional properties of the therapeutic. Alterations in a peptide, protein or O-RS that do not affect Active 121670349.1 75 Attorney Ref. No.070050.6868 the functional properties of the peptide, protein or O-RS, are well known in the art, e.g., conservative substitutions. It is therefore understood that the disclosure encompasses more than the specific exemplary polynucleotide or amino acid sequences and includes functional equivalents thereof.
  • the cells to be used in the present disclosure can be genetically engineered using recombinant techniques known to those of ordinary skill in the art. Production and manipulation of the polynucleotides described herein are within the skill in the art and can be carried out according to recombinant techniques described, for example, in Sambrook et al.1989. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Innis et al. (eds). 1995. PCR Strategies, Academic Press, Inc., San Diego.
  • the present disclosure further provides compositions of the recombinant cells disclosed herein. In certain embodiments, a composition of the present disclosure includes a plurality of the recombinant cells disclosed herein. V.
  • methods of Production further provides methods for producing a peptide or protein therapeutic with at least one Aib amino acid.
  • methods of the present disclosure include expressing a peptide or protein therapeutic with at least one masked Aib amino acid and purifying the peptide or protein therapeutic with the at least one masked Aib amino acid.
  • methods of the present disclosure further include removing the masking group from the masked Aib amino acid incorporated into the peptide or protein therapeutic to produce at least one peptide or protein therapeutic comprising one or more Aib amino acid (i.e., without the masking group).
  • the present disclosure provides methods using a cell, e.g., a genetically engineered cell, of the present disclosure to produce and/or secrete one protein or peptide therapeutic comprising at least one masked Aib amino acid.
  • methods of the present disclosure use a cell, e.g., a genetically engineered cell, of the present disclosure to produce and/or secrete more than one protein or peptide therapeutic, e.g., two or more protein or peptide therapeutics, three or more protein or peptide therapeutics, four or more therapeutics or five or more therapeutics, where at least one of the one protein or peptide Active 121670349.1 76 Attorney Ref.
  • No.070050.6868 therapeutics comprises one or more masked Aib amino acids.
  • Non-limiting examples of masked Aib amino acids, therapeutic proteins or peptides and recombinant cells are described in Sections II, III, IV and V, respectively.
  • A. Expression The present disclosure provides methods for expressing a peptide or protein therapeutic that comprises one or more masked Aib amino acids.
  • the present disclosure provides methods for expressing a peptide or protein therapeutic that comprises one or more masked Aib amino acids in recombinant cells described herein.
  • Non- limiting examples of masked Aib amino acids, therapeutic proteins or peptides and recombinant cells are described in Sections III, IV and V, respectively.
  • methods of the present disclosure include culturing a recombinant cell that comprises a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon in a cell culture medium comprising a masked Aib amino acid or an analog thereof (e.g., under conditions suitable for growth).
  • the recombinant cell expresses an orthogonal tRNA (O-tRNA) that functions in the recombinant cell and recognizes the selector codon and an orthogonal aminoacyl-tRNA synthetase (O-RS), where the O-RS preferentially aminoacylates the O-tRNA with a masked Aib amino acid.
  • O-tRNA orthogonal tRNA
  • O-RS orthogonal aminoacyl-tRNA synthetase
  • the method further includes incorporating the masked Aib amino acid into the one or more specified positions (e.g., identified by the selector codon) in the peptide or protein therapeutic during translation of the polynucleotide to produce the peptide or protein therapeutic that comprises one or more of the masked Aib amino acids or the analog thereof.
  • the masked Aib amino acid into the one or more specified positions (e.g., identified by the selector codon) in the peptide or protein therapeutic during translation of the polynucleotide to produce the peptide or protein therapeutic that comprises one or more of the masked Aib amino acids or the analog thereof.
  • a method of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture medium under conditions suitable for cell growth.
  • a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture medium under conditions suitable for cell
  • the method of the present disclosure further includes contacting the cell culture medium with a masked Aib amino acid or an analog thereof (e.g., under conditions suitable for incorporation of the masked Aib amino acid or analog thereof into the peptide or protein therapeutic) to produce the peptide or protein therapeutic that comprises one or more of the masked Aib amino acids or the analog thereof at the position indicated by the selector codon.
  • the peptide or protein Active 121670349.1 77 Attorney Ref. No.070050.6868 therapeutic is a GLP-1 receptor agonist.
  • the peptide or protein therapeutic is semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics).
  • the peptide or protein therapeutic is semaglutide.
  • the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14.
  • the O- tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15.
  • the masked Aib has the structure of any one of Formulas I-XVIII.
  • the masked Aib amino acid is added to the cell culture medium at a concentration from about 1 mM to about 50 M, e.g., from about 10 mM to about 25 M, from about 10 mM to about 10 M, from about 10 mM to about 5 M, from about 10 mM to about 1 M, from about 10 mM to about 500 mM, from about 10 mM to about 250 mM, from about 10 mM to about 100 mM or from about 10 mM to about 50 mM.
  • the masked Aib amino acid is added to the cell culture medium at a concentration from about 1 mM to about 1 M.
  • the masked Aib amino acid is added to the cell culture medium at a concentration from about 1 mM to about 500 mM. In certain embodiments, the masked Aib amino acid is added to the cell culture medium at a concentration from about 1 mM to about 100 mM. In certain embodiments, the masked Aib amino acid is added to the cell culture medium at a concentration from about 1 mM to about 50 mM.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • the peptide or protein therapeutic is a GLP-1 receptor agonist.
  • the peptide or protein therapeutic is semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the peptide or protein therapeutic is semaglutide.
  • the O-RS comprises an amino acid sequence that is greater than about 95% homologous to a pyrrolysyl-tRNA synthetase, an Escherichia coli synthetase, or a Saccharomyces cerevisiae synthetase.
  • the O-tRNA comprises a nucleotide sequence of a pyrrolysyl-tRNA, an Escherichia coli tRNA, or a Saccharomyces cerevisiae tRNA.
  • the masked Aib has the structure of any one of Formulas I-XVIII. Active 121670349.1 78 Attorney Ref.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • O-tRNA orthogonal tRNA
  • OF-RS orthogonal aminoacyl-tRNA synthetase
  • the peptide or protein therapeutic is a GLP-1 receptor agonist. In certain embodiments, the peptide or protein therapeutic is semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the peptide or protein therapeutic is semaglutide.
  • the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain embodiments, the O- tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the masked Aib has the structure of any one of Formulas I-XVIII.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • the peptide or protein therapeutic is a GLP-1 receptor agonist.
  • the peptide or protein therapeutic is semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the peptide or protein therapeutic is semaglutide.
  • the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14.
  • the O- tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15.
  • the masked Aib has the structure of Formula IX or Formula X.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • O-tRNA orthogonal tRNA
  • OF-RS orthogonal aminoacyl-tRNA synthetase
  • the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11.
  • the Active 121670349.1 79 Attorney Ref. No.070050.6868 O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14.
  • the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15.
  • the masked Aib has the structure of any one of Formulas I-XVIII.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • O-tRNA orthogonal tRNA
  • OF-RS orthogonal aminoacyl-tRNA synthetase
  • the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11.
  • the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14.
  • the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15.
  • the masked Aib has the structure of any one of Formulas I-XVIII.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • O-tRNA orthogonal tRNA
  • OF-RS orthogonal aminoacyl-tRNA synthetase
  • the peptide or protein therapeutic comprises the amino acid of any one of SEQ ID NOs: 1-3 and 9-11.
  • the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14.
  • the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15.
  • the masked Aib has the structure of Formula IX.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • O-tRNA orthogonal tRNA
  • OF-RS orthogonal aminoacyl-tRNA synthetase
  • the peptide or protein therapeutic comprises the amino acid of any one of SEQ ID NOs: 1-3 and 9-11.
  • the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14.
  • the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15.
  • the masked Aib has the structure of Formula X.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • O-tRNA orthogonal tRNA
  • OF-RS orthogonal aminoacyl-tRNA synthetase
  • the peptide or protein therapeutic comprises the amino acid of any one of SEQ ID NOs: 1-3 and 9-11.
  • the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14.
  • the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15.
  • the masked Aib has the structure of Formula XI.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • O-tRNA orthogonal tRNA
  • OF-RS orthogonal aminoacyl-tRNA synthetase
  • the peptide or protein therapeutic comprises the amino acid of any one of SEQ ID NOs: 1-3 and 9-11.
  • the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14.
  • the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15.
  • the masked Aib has the structure of Formula XII.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • O-tRNA orthogonal tRNA
  • OF-RS orthogonal aminoacyl-tRNA synthetase
  • the peptide or protein therapeutic comprises the amino acid of any one of SEQ ID NOs: 1-3 and 9-11.
  • the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14.
  • the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15.
  • the masked Aib has the structure of Formula XIII.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • O-tRNA orthogonal tRNA
  • OF-RS orthogonal aminoacyl-tRNA synthetase
  • the peptide or protein therapeutic comprises the amino acid of any one of SEQ ID NOs: 1-3 and 9-11.
  • the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14.
  • the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15.
  • the masked Aib has the structure of Formula XIV.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • O-tRNA orthogonal tRNA
  • OF-RS orthogonal aminoacyl-tRNA synthetase
  • the peptide or protein therapeutic comprises the amino acid of any one of SEQ ID NOs: 1-3 and 9-11.
  • the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14.
  • the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15.
  • the masked Aib has the structure of Formula XV.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • O-tRNA orthogonal tRNA
  • OF-RS orthogonal aminoacyl-tRNA synthetase
  • the peptide or protein therapeutic comprises the amino acid of any one of SEQ ID NOs: 1-3 and 9-11.
  • the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14.
  • the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15.
  • the masked Aib has the structure of Formula XVI.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • O-tRNA orthogonal tRNA
  • OF-RS orthogonal aminoacyl-tRNA synthetase
  • the peptide or protein therapeutic comprises the amino acid of any one of SEQ ID NOs: 1-3 and 9-11.
  • the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14.
  • the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15.
  • the masked Aib has the structure of Formula XVII.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • O-tRNA orthogonal tRNA
  • OF-RS orthogonal aminoacyl-tRNA synthetase
  • the peptide or protein therapeutic comprises the amino acid of any one of SEQ ID NOs: 1-3 and 9-11.
  • the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14.
  • the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15.
  • the masked Aib has the structure of Formula XVIII.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1.
  • the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14.
  • the O- Active 121670349.1 83 Attorney Ref. No.070050.6868 tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15.
  • the masked Aib has the structure of any one of Formulas I-XVIII.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • O-tRNA orthogonal tRNA
  • OF-RS orthogonal aminoacyl-tRNA synthetase
  • the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11.
  • the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 4.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 8.
  • the masked Aib has the structure of any one of Formulas I-XVIII.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • O-tRNA orthogonal tRNA
  • OF-RS orthogonal aminoacyl-tRNA synthetase
  • the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11.
  • the O-RS comprises the amino acid of SEQ ID NO: 4.
  • the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 8.
  • the masked Aib has the structure of any one of Formulas I-XVIII.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • O-tRNA orthogonal tRNA
  • OF-RS orthogonal aminoacyl-tRNA synthetase
  • the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11.
  • the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino Active 121670349.1 84 Attorney Ref. No.070050.6868 acid of SEQ ID NO: 5.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 7.
  • the masked Aib has the structure of any one of Formulas I-XVIII.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • O-tRNA orthogonal tRNA
  • OF-RS orthogonal aminoacyl-tRNA synthetase
  • the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11.
  • the O-RS comprises the amino acid of SEQ ID NO: 5.
  • the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 7.
  • the masked Aib has the structure of any one of Formulas I-XVIII.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • O-tRNA orthogonal tRNA
  • OF-RS orthogonal aminoacyl-tRNA synthetase
  • the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11.
  • the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 6.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 7.
  • the masked Aib has the structure of any one of Formulas I-XVIII.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • O-tRNA orthogonal tRNA
  • OF-RS orthogonal aminoacyl-tRNA synthetase
  • the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11.
  • the Active 121670349.1 85 Attorney Ref. No.070050.6868 O-RS comprises the amino acid of SEQ ID NO: 6.
  • the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 7.
  • the masked Aib has the structure of any one of Formulas I-XVIII.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1.
  • the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 4.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 8.
  • the masked Aib has the structure of Formula IX.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1.
  • the O-RS comprises the amino acid of SEQ ID NO: 4.
  • the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 8.
  • the masked Aib has the structure of Formula IX.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1.
  • the O-RS comprises an Active 121670349.1 86 Attorney Ref. No.070050.6868 amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 5.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 7.
  • the masked Aib has the structure of Formula IX.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1.
  • the O-RS comprises the amino acid of SEQ ID NO: 5.
  • the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 7.
  • the masked Aib has the structure of Formula IX.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1.
  • the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 6.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 7.
  • the masked Aib has the structure of Formula IX.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • the peptide or protein therapeutic Active 121670349.1 87 Attorney Ref.
  • No.070050.6868 is semaglutide.
  • the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1.
  • the O-RS comprises the amino acid of SEQ ID NO: 6.
  • the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 7.
  • the masked Aib has the structure of Formula IX.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1.
  • the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 4.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 8.
  • the masked Aib has the structure of Formula X.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1.
  • the O-RS comprises the amino acid of SEQ ID NO: 4.
  • the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 8.
  • the masked Aib has the structure of Formula X.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • the peptide or protein therapeutic Active 121670349.1 88 Attorney Ref.
  • No.070050.6868 comprises the amino acid of SEQ ID NO: 1.
  • the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 5.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 7.
  • the masked Aib has the structure of Formula X.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1.
  • the O-RS comprises the amino acid of SEQ ID NO: 5.
  • the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 7.
  • the masked Aib has the structure of Formula X.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 6.
  • the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 4.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 7.
  • the masked Aib has the structure of Formula X.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one Active 121670349.1 89 Attorney Ref. No.070050.6868 or more of the masked Aib amino acids.
  • the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1.
  • the O-RS comprises the amino acid of SEQ ID NO: 6.
  • the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 7.
  • the masked Aib has the structure of Formula X.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1.
  • the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 4.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 8.
  • the masked Aib has the structure of Formula XI.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1.
  • the O-RS comprises the amino acid of SEQ ID NO: 4.
  • the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 8.
  • the masked Aib has the structure of Formula XI.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one Active 121670349.1 90 Attorney Ref. No.070050.6868 or more of the masked Aib amino acids.
  • the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1.
  • the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 5.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 7.
  • the masked Aib has the structure of Formula XI.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1.
  • the O-RS comprises the amino acid of SEQ ID NO: 5.
  • the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 7.
  • the masked Aib has the structure of Formula XI.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1.
  • the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 6.
  • the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 7.
  • the masked Aib has the structure of Formula XI.
  • methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media Active 121670349.1 91 Attorney Ref. No.070050.6868 comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids.
  • the peptide or protein therapeutic is semaglutide. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1. In certain embodiments, the O-RS comprises the amino acid of SEQ ID NO: 6. In certain embodiments, the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked Aib has the structure of Formula XI. B. Purification The present disclosure further provides methods for isolating or purifying the protein or peptide therapeutic from the cell culture comprising the recombinant cells. In certain embodiments, the present disclosure provides methods for isolating or purifying the protein or peptide therapeutic that comprises one or masked Aib amino acids from the cell culture.
  • the protein or peptide therapeutic that comprises one or masked Aib amino acids is secreted from the cells and is isolated or purified from the cell culture media and/or supernatant.
  • the recombinant cells are lysed and the protein or peptide therapeutic that comprises one or masked Aib amino acids are isolated or purified from the cell lysate.
  • the isolated or purified protein or peptide therapeutic can be further processed to demask the masked Aib amino acids.
  • the isolated or purified protein or peptide therapeutic can be further processed to post-translationally modify the protein or peptide therapeutic comprising the demasked Aib amino acids, e.g., addition of the lipid side chain (see, e.g., FIGs. 1-3).
  • a protein or peptide therapeutic that comprises one or masked Aib amino acids are isolated or purified from the cell lysate, supernatant and/or culture media the includes the protein or peptide therapeutic comprising one or masked Aib amino acids using chromatography.
  • the lysate, supernatant and/or culture media is passed over a chromatography column or solid support, so that the peptide or protein containing masked Aib covalently bonds or non-covalently bonds to the chromatography column or solid support.
  • the chromatography medium is selected to bind covalently or non-covalently to a moiety contained within the protein or peptide.
  • the moiety that binds to the chromatography medium is contained within the masking group of the masked form of Aib present in the protein or peptide. Active 121670349.1 92 Attorney Ref.
  • the moiety contained within the masking group that can be used for isolation and/purification of the peptide or protein is an azide group.
  • an azide group binds covalently with the chromatography column or the solid support.
  • the masking group (or moiety of the masking group) of the masked Aib amino acids covalently binds to the chromatography column or solid support using any suitable reaction.
  • the reaction is a Staudinger ligation reaction, a Cu(I)-catalyzed azide-alkyne cycloaddition reaction, a Strain-promoted azide-alkyne cycloaddition reaction, a Strain-promoted alkyne-nitrone cycloaddition reaction, a Diels-Alder reaction, alkene-azide [3+2] cycloaddition reaction, an Inverse-demand Diels-Alder reaction or an alkene-tetrazine.
  • Staudinger ligation reaction a Cu(I)-catalyzed azide-alkyne cycloaddition reaction
  • a Strain-promoted azide-alkyne cycloaddition reaction a Strain-promoted alkyne-nitrone cycloaddition reaction
  • Diels-Alder reaction alkene-azide [3+2] cyclo
  • the masking group (or moiety of the masking group) of the masked Aib amino acids covalently binds to the chromatography column or solid support using a Staudinger ligation reaction. In certain embodiments, the masking group (or moiety of the masking group) of the masked Aib amino acids covalently binds to the chromatography column or solid support using a Cu(I)-catalyzed azide-alkyne cycloaddition reaction.
  • the masking group (or moiety of the masking group) of the masked Aib amino acids covalently binds to the chromatography column or solid support using a Strain-promoted azide- alkyne cycloaddition reaction. In certain embodiments, the masking group (or moiety of the masking group) of the masked Aib amino acids covalently binds to the chromatography column or solid support using a Strain-promoted alkyne-nitrone cycloaddition reaction.
  • the masking group (or moiety of the masking group) of the masked Aib amino acids covalently binds to the chromatography column or solid support using a Diels-Alder reaction. In certain embodiments, the masking group (or moiety of the masking group) of the masked Aib amino acids covalently binds to the chromatography column or solid support using Alkene-azide [3+2] cycloaddition reaction. In certain embodiments, the masking group (or moiety of the masking group) of the masked Aib amino acids covalently binds to the chromatography column or solid support using an Inverse-demand Diels-Alder reaction.
  • the masking group (or moiety of the masking group) of the masked Aib amino acids covalently binds to the chromatography column or solid support using an alkene-tetrazine Inverse-electron-demand Diels-Alder reaction.
  • Active 121670349.1 93 Attorney Ref. No.070050.6868 When the reaction is a Staudinger ligation or a Strain-promoted azide-alkyne cycloaddition reaction, an azide group of the masking group will covalently bind to the chromatography column or solid support.
  • reaction When the reaction is an alkene-azide [3+2] cycloaddition reaction, an alkene or an azide group will covalently bind to the chromatography column or support.
  • reaction When the reaction is a Strain-promoted alkyne-nitrone cycloaddition reaction, an alkyne or nitrone group of the masking group will covalently bind to the chromatography column or solid support.
  • the reaction is a Cu(I)-catalyzed azide-alkyne cycloaddition reaction, an azide or an alkyne group of the masking group will covalently bind to the chromatography column or solid support.
  • methods of the present disclosure can further include removing the masking group from the masked Aib amino acids in the peptide or protein which contains the masked Aib amino acid.
  • the masking group can be removed using any suitable demasking reaction.
  • the reaction is a desulfurization reaction or a deselenization reaction.
  • methods of the present disclosure can include removing the masking group from the masked Aib amino acids in the peptide or protein which contains the masked Aib amino acids using a desulfurization reaction. In certain embodiments, methods of the present disclosure can include removing the masking group from the masked Aib amino acids in the peptide or protein which contains the masked Aib amino acids using a deselenization reaction. Any suitable catalyst can be used for the demasking reaction, including, but not limited to, metal reagent(s).
  • the catalyst used for the demasking reaction is Pd/Al2O3, Pd/Carbon, Raney Nickel, Pd/BaSO4, PdO, Ni(0), palladium catalyst, Pd(TPPS)4, nickel boride, tin hydride, or combinations thereof.
  • the demasking reaction is a desulfurization reaction, which is performed using a Raney nickel, sodium hypophosphite, acetate buffer system, as shown in Scheme 11.
  • the demasking reaction is a desulfurization reaction, which is performed with UV light, as shown in Scheme 12.
  • the demasking reaction is a desulfurization reaction, which is performed with electrolysis, as shown in Scheme 13.
  • the demasking reaction is a deselenization reaction, which is performed using a Raney nickel, sodium hypophosphite, acetate buffer system, as shown in Scheme 14.
  • the demasking reaction is a Active 121670349.1 94 Attorney Ref. No.070050.6868 deselenization reaction, which is performed with UV light, as shown in Scheme 15.
  • the demasking reaction is a deselenization reaction, which is performed with electrolysis, as shown in Scheme 16.
  • the demasking reaction comprises a allyl removal reaction with a suitable palladium catalyst followed by a desulfurization or deselenization reaction.
  • the demasking reaction comprises a propargyl removal reaction with a Pd(TPPS) 4 catalyst followed by a desulfurization or deselenization reaction.
  • the product of the demasking reaction is a protein or peptide containing unmasked Aib amino acids.
  • the resulting protein or peptide containing Aib residues can be analyzed by any suitable analytical method.
  • Such analytical methods include, but are not limited to, Nuclear Magnetic Resonance (NMR) or Mass Spectrometry (MS). When the analytical method MS is used, the appropriate fragmentation pattern will be observed in the resulting spectra, confirming the presence of the polypeptide or peptide containing native Aib residues.
  • the protein or peptide therapeutic containing unmasked-Aib amino acid residues (i.e., native Aib amino acid residues) produced by a method of the present disclosure can be formulated into a pharmaceutical composition.
  • the present disclosure provides a pharmaceutical composition comprising a therapeutic protein or peptide, where the therapeutic protein or peptide can treat diabetes (e.g., type 2 diabetes), treat obesity, or be used for weight loss and/or reducing the risk of cardiovascular events.
  • the resulting peptide is semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics).
  • the resulting peptide is semaglutide.
  • a pharmaceutical composition of the present disclosure includes a protein or peptide therapeutic produced by the methods disclosed herein and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier includes any carrier which does not interfere with the effectiveness of the biological activity of the active ingredients, e.g., the protein or peptide therapeutic, and that is not toxic to the patient to whom it is administered.
  • Non-limiting examples of suitable pharmaceutical carriers include phosphate- buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting Active 121670349.1 95 Attorney Ref. No.070050.6868 agents and sterile solutions.
  • Additional non-limiting examples of pharmaceutically acceptable carriers can include polymers, gels, bioabsorbable matrix materials, implantation elements containing the yeast and/or any other suitable vehicle, delivery or dispensing means or material. Such carriers can be formulated by conventional methods and can be administered to the subject.
  • the pharmaceutical composition can be formulated for oral administration, intravenous administration, subcutaneous administration or parenteral administration.
  • the kit and/or system of the present disclosure includes one or more recombinant cell compositions and/or one or more masked forms of Aib amino acids.
  • Non-limiting examples of recombinant cells and/or masked forms of Aib amino acids for use in the kits and systems disclosed herein are provided in Section II and IV.
  • the kit and/or system of the present disclosure includes a container containing one or more masked forms of Aib or analogs thereof.
  • the present disclosure provides a kit and/or system comprising at least one masked Aib or amino acid analog having a structure of any one of Formulas I-XVIII (or a composition thereof).
  • the least one masked Aib or amino acid analog having a structure of any one of Formulas I-XVIII or a composition thereof is included in a container.
  • a kit and/or system of the present disclosure can include a container comprising at least one masked Aib or amino acid analog having the structure of Formula IX or a composition thereof.
  • a kit and/or system of the present disclosure can include a container comprising at least one masked Aib or amino acid analog having the structure of Formula X or a composition thereof.
  • a kit and/or system of the present disclosure can include a container comprising at least one masked Aib or amino acid analog having the structure of Formula XI or a composition thereof. In certain embodiments, a kit and/or system of the present disclosure can include a container comprising at least one masked Aib or amino acid analog having the structure of Formula XII or a composition thereof. In certain embodiments, a kit and/or system of the present disclosure can include a container comprising at least one masked Aib or amino acid analog having the structure of Formula XIII or a composition thereof. In certain Active 121670349.1 96 Attorney Ref.
  • a kit and/or system of the present disclosure can include a container comprising at least one masked Aib or amino acid analog having the structure of Formula XIV. In certain embodiments, a kit and/or system of the present disclosure can include a container comprising at least one masked Aib or amino acid analog having the structure of Formula XV. In certain embodiments, a kit and/or system of the present disclosure can include a container comprising at least one masked Aib or amino acid analog having the structure of Formula XVI or a composition thereof.
  • a kit and/or system of the present disclosure can include a container comprising at least one masked Aib or amino acid analog having the structure of Formula XVII or a composition thereof.
  • a kit and/or system of the present disclosure can include a container comprising at least one masked Aib or amino acid analog having the structure of Formula XVIII or a composition thereof.
  • a kit and/or system of the present disclosure can include a cell culture medium comprising one or more masked forms of Aib or analogs thereof of any one of Formulas I-XVIII, e.g., in a container.
  • a kit and/or system of the present disclosure can include a cell culture medium comprising at least one masked Aib or amino acid analog having the structure of Formula IX. In certain embodiments, a kit and/or system of the present disclosure can include a cell culture medium comprising at least one masked Aib or amino acid analog having the structure of Formula X. In certain embodiments, a kit and/or system of the present disclosure can include a cell culture medium comprising at least one masked Aib or amino acid analog having the structure of Formula XI. In certain embodiments, a kit and/or system of the present disclosure can include a cell culture medium comprising at least one masked Aib or amino acid analog having the structure of Formula XII.
  • a kit and/or system of the present disclosure can include a cell culture medium comprising at least one masked Aib or amino acid analog having the structure of Formula XIII. In certain embodiments, a kit and/or system of the present disclosure can include a cell culture medium comprising at least one masked Aib or amino acid analog having the structure of Formula XIV. In certain embodiments, a kit and/or system of the present disclosure can include a cell culture medium comprising at least one masked Aib or amino acid analog having the structure of Formula XVI. In certain embodiments, a kit and/or system of the present disclosure can include a cell culture medium comprising at least one masked Aib or amino acid analog having the structure of Formula XVII.
  • a kit and/or system of the present disclosure can include a cell culture medium Active 121670349.1 97 Attorney Ref. No.070050.6868 comprising at least one masked Aib or amino acid analog having the structure of Formula XVIII.
  • the kit and/or system of the present disclosure includes recombinant cells for expressing a protein or peptide therapeutic, e.g., in a container.
  • the kit and/or system of the present disclosure includes recombinant cells that have been genetically engineered to produce a GLP-1 receptor agonist.
  • the recombinant cells of the present disclosure have been genetically engineered to express semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics).
  • the recombinant cells and compositions thereof have been genetically engineered to express semaglutide.
  • the recombinant cells and compositions thereof have been genetically engineered to express tirzepatide.
  • the recombinant cells and compositions thereof have been genetically engineered to express retatrutide.
  • the recombinant cells that have been genetically engineered to further express one or more aminoacyl-tRNA synthetases e.g., comprises an amino acid sequence that is greater than about 95% homologous to a synthetase known in the art.
  • the recombinant cells that have been genetically engineered to express an aminoacyl-tRNA synthetases that is greater than about 95% homologous to a synthetase is selected from pyrrolysyl-tRNA synthetase, an Escherichia coli synthetase, or a Saccharomyces cerevisiae synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 95% homologous to a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to a Methanococcus jannaschii pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to a Methanosarcina barkeri pyrrolysyl-tRNA synthetase.
  • the O-RS comprises an amino acid sequence that is greater than about 95% homologous to a Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the recombinant cells have been genetically engineered to express one Active 121670349.1 98 Attorney Ref.
  • the kit and/or system further includes a container containing a chromatography medium for purifying a protein or a peptide comprising at least one masked form of Aib.
  • suitable containers include, but are not limited to, bottles, test tubes, vials and microtiter plates. The containers can be formed from a variety of materials such as glass or plastic.
  • the kit and/or system can further include instructions for using the components disclosed herein.
  • the kit and/or system can further include instructions for using the components of the kit and/or system in a method of the present disclosure.
  • the present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula I: wherein: the combination of X and R3 are optional if the combination of Y and R4 are present, but are otherwise required; the combination of Y and R4 are optional if the combination of X and R3 are present, but are otherwise required; R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; Active 121670349.1 99 Attorney Ref.
  • each of X and Y when present is independently S or Se; each of R 3 and R 4 when present is independently an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, - (alkylene or substituted alkylene)-ON(R”) 2 , -(alkylene or substituted alkylene)-C(O)SR′′, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R′′, —C(O)OR′′, —C(O)N(R′′) 2 , or -L
  • the present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula II: wherein: the combination of X, A, and R3 are optional if the combination of Y, B, and R4 are present, but are otherwise required; the combination of Y, B, and R4 are optional if the combination of X, A and R3 are present, but are otherwise required; R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; Active 121670349.1 101 Attorney Ref.
  • each of X and Y when present is independently S or Se; each of A and B are optional, and when present are independently an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”) 2 , -(alkylene or substituted alkylene)-C(O)SR′′, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), — C(O)R′′, —C(O)OR′′, —C(O)N(R′′) 2 , or
  • the present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula III: wherein: the combination of Y and B are optional; R 1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R 2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; Active 121670349.1 103 Attorney Ref.
  • No.070050.6868 X is S or Se; Y when present is S or Se; each of A and B are optional, and when present are independently an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR′′, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), — C(O)R′′, —C(O)OR′′, —C(O)N(R′′) 2 , or
  • No.070050.6868 (alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′- (alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, — N(R′)CO— (alkylene or substituted alkylene)-, —N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N
  • the present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula IV: wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; Active 121670349.1 105 Attorney Ref.
  • No.070050.6868 N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′) ⁇ N—, — N ⁇ C(R′)—, —N ⁇ N—, —C(R′) ⁇ N—N(R′)—, —C(R′)2—N ⁇ N—, or —C(R′)2— N(R′)—N(R′)—; and where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula V: wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; each of X and Y is independently S or Se; A is optional, and when present is an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R
  • each R′′ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R′′ group is present, two R′′ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene,
  • F. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula VI: wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; X is S or Se; R3 is an H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroary
  • No.070050.6868 Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene,
  • the present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula VII: Active 121670349.1 110 Attorney Ref. No.070050.6868 wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R 2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; X is S or Se; A is optional, and when present is an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(
  • No.070050.6868 nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O—, —O-(alkylene or substituted alkylene)-, —S(O) k —, —S(O) k (alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene
  • the present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula VIII: Active 121670349.1 112 Attorney Ref. No.070050.6868 wherein: R 1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R 2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; and X is S or Se; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • R 1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide
  • R 2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide
  • X is S or Se; or a pharmaceutically acceptable salt, active metabolite,
  • the present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula IX: Formula IX or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula X: Active 121670349.1 113 Attorney Ref. No.070050.6868 or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XI: or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • L. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XII: or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XIII: Active 121670349.1 114 Attorney Ref. No.070050.6868 or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. N.
  • the present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XIV: or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XV: or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • P. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XVI: Active 121670349.1 115 Attorney Ref. No.070050.6868 or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XVI: or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • R. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XVII: or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof.
  • the present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of any one of A-R. S1.
  • the present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of A. Active 121670349.1 116 Attorney Ref. No.070050.6868 S2.
  • the present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of B. S3.
  • the present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of C. S4.
  • the present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of D. S5.
  • the present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of E. S6.
  • the present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of F. S7.
  • the present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of G. S8.
  • the present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of H. S9.
  • the present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of I. S10.
  • the present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of J. S11.
  • the present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of K. S12.
  • the present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of L. S13.
  • the present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of M. Active 121670349.1 117 Attorney Ref. No.070050.6868 S14.
  • the present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of N. S15.
  • the present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of O. S16.
  • the present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of P. S17.
  • the present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of Q. S18.
  • the present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of R. T.
  • the present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of any one of A-R. T1.
  • the present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of A. T2.
  • the present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of B. T3.
  • the present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of C. T4.
  • the present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of D. T5.
  • the present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of E. T6.
  • the present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of F. Active 121670349.1 118 Attorney Ref. No.070050.6868 T7.
  • the present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of G. T8.
  • the present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of H. T9.
  • the present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of I. T10.
  • the present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of J. T11.
  • the present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of K. T12.
  • the present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of L. T13.
  • the present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of M. T14.
  • the present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of N. T15.
  • the present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of O. T17.
  • the present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of P. T18.
  • the present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of Q. T19.
  • the present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of R. Active 121670349.1 119 Attorney Ref. No.070050.6868 U.
  • the present disclosure provides a method of producing a peptide or protein comprising one or more 2-aminoisobutyric acid (Aib) amino acid residues in a recombinant cell, wherein the method comprises: (a) culturing the recombinant cell in a cell culture media, wherein the recombinant cell comprises (i) a polynucleotide encoding the peptide or protein and comprising at least one selector codon, wherein the recombinant cell expresses: an orthogonal tRNA (O-tRNA) that functions in the recombinant cell and recognizes the selector codon; and an orthogonal aminoacyl-tRNA synthetase (O-RS), wherein the O-RS preferentially aminoacylates the O-tRNA
  • the method of U further comprising contacting a cell extract and/or supernatant obtained from the recombinant cell with a chromatography column or solid support, wherein the masking group of the masked Aib amino acid residues incorporated in the peptide or protein binds covalently to the chromatography column or solid support.
  • U2. The method of U further comprising contacting a cell extract and/or supernatant obtained from the recombinant cell with a chromatography column or solid support, wherein the masking group of the masked Aib amino acid residues incorporated in the peptide or protein binds non- covalently to the chromatography column or solid support.
  • Active 121670349.1 120 Attorney Ref.
  • No.070050.6868 U3.
  • the method of any one of U-U2 further comprising performing one or more chemical reactions to remove the masking group from the one or more masked Aib amino acid residues present in the peptide or protein to generate the peptide or protein with native Aib residues.
  • U4 The method of U1 or U3, wherein the masking group of the masked Aib binds covalently to the column or solid support via a Staudinger ligation reaction.
  • U5. The method of U1 or U3, wherein the masking group of the masked Aib binds covalently to the column or solid support via a Cu(I)-catalyzed azide-alkyne cycloaddition reaction.
  • U6 The method of any one of U-U2 further comprising performing one or more chemical reactions to remove the masking group from the one or more masked Aib amino acid residues present in the peptide or protein to generate the peptide or protein with native A
  • the method of U3, wherein the chemical reaction is a desulfurization reaction.
  • U13. The method of U3, wherein the chemical reaction is a deselenization reaction.
  • U14. The method of U3, wherein the catalyst used for the chemical reaction is a metal reagent.
  • U15. The method of U3, wherein the catalyst used for the chemical reaction is Pd/Al 2 O 3 . Active 121670349.1 121 Attorney Ref. No.070050.6868 U16.
  • the method of U3, wherein the catalyst used for the chemical reaction is Pd/Carbon.
  • U17. The method of U3, wherein the catalyst used for the chemical reaction is Raney nickel.
  • U18. The method of U3, wherein the catalyst used for the chemical reaction is Pd/BaSO 4 .
  • the method of U3, wherein the catalyst used for the chemical reaction is PdO.
  • U20. The method of U3, wherein the catalyst used for the chemical reaction is a Ni(0) catalyst.
  • U21. The method of U3, wherein the catalyst used for the chemical reaction is Nickel boride.
  • U22. The method of U3, wherein the catalyst used for the chemical reaction is Tin hydride.
  • U23. The method of U3, wherein the chemical reaction is performed using a Raney nickel – sodium hypophosphite – acetate buffer system.
  • the method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of L. V13.
  • the method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of M. V14.
  • the method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of N. V15.
  • the method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of O. V16.
  • W1 The method of W, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to a pyrrolysyl-tRNA synthetase.
  • W3 The method of W2, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to a pyrrolysyl-tRNA synthetase derived from a species of the Methanococcus genus.
  • any one of U, U1-U23, V, V1-V18, or W wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanosarcina mazei pyrrolysyl-tRNA synthetase.
  • W5. The method of W4, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanosarcina mazei pyrrolysyl-tRNA synthetase.
  • W6 The method of any one of U, U1-U23, V, V1-V18, or W, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanosarcina mazei pyrrolysyl-tRNA synthetase.
  • W6 The method of W6, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanosarcina barkeri pyrrolysyl-tRNA synthetase.
  • W7 The method of any one of U, U1-U23, V, V1-V18, or W wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanosarcina barkeri pyrrolysyl-tRNA synthetase.
  • W15 The method of any one of U, U1-U23, V, V1-V18, or W wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Escherichia coli glutaminyl-tRNA synthetase.
  • W17 The method of W15 or W16, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 4.
  • W18 The method of any one of U, U1-U23, V, V1-V18, or W wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the sequence of any one of SEQ ID NOs: 4-6 and 12-14.
  • Active 121670349.1 125 Attorney Ref. No.070050.6868 W18.
  • W19. The method of any one of U, U1-U23, V, V1-V18, or W, wherein the O-tRNA comprises a nucleotide sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the sequence of any one of SEQ ID Nos: 7-8 and 15. W20.
  • the method of W19 or W20, wherein the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 8. W23.
  • W26 The method of any one of U, U1-U23, V, V1-V18, W, or W1-W23, wherein the protein or peptide is a GLP-1 receptor agonist.
  • W27 The method of any one of U, U1-U23, V, V1-V18, W, or W1-W23, wherein the protein or peptide is a dual GLP-1/GIP receptor agonist.
  • W28 The method of any one of U, U1-U23, V, V1-V18, W, or W1-W23, wherein the peptide is selected from the group consisting of semaglutide, tirzepatide, retatrutide, VK2735 and a combination thereof.
  • Active 121670349.1 126 Attorney Ref. No.070050.6868 W29.
  • the method of W28, wherein the peptide is semaglutide.
  • W30. The method of W28, wherein the peptide is tirzepatide.
  • W31. The method of W28, wherein the peptide is retatrutide.
  • W32. The method of any one of U, U1-U23, V, V1-V18, W, or W1-W31, wherein the recombinant cell is a bacterial cell.
  • the method of W32, wherein the bacterial cells is Escherichia coli.
  • the present disclosure provides a kit comprising the composition of any one of S or S1- S18. X2.
  • the present disclosure provides a kit comprising the cell culture media of any one of T or T1-T18.
  • the present disclosure provides a kit for performing the method of any one of U, U1-U23, V, V1-V18, W, or W1-W37. Y.
  • the present disclosure provides a system comprising the masked Aib amino acid or analog thereof of any one of A-R Active 121670349.1 127 Attorney Ref. No.070050.6868 Y1.
  • the present disclosure provides a system comprising the composition of any one of S or S1-S18 Y2.
  • the present disclosure provides a system comprising the cell culture media of any one of T or T1-T18. Y3.
  • the present disclosure provides a system for performing the method of any one of U, U1- U23, V, V1-V18, W, or W1-W37.
  • EXAMPLES The following examples are merely illustrative of the presently disclosed subject matter and should not be considered as limitations in any way.
  • Example 1 Synthesis of Masked Aib Amino Acids This example provides schemes for synthesizing masked Aib amino acids, which can be used in the methods of the present disclosure. 1.1.
  • the solid mixture was treated with NH 3 (7N) in MeOH (100mL) at 0 °C and allowed to react at room temperature for additional 16 h under inert atmosphere.
  • the solvent was evaporated under reduced pressure at 30 °C in the bath and triturated with ether (1x100 mL), acetone (2x50 mL) and ice-cold water (HPLC grade, Milli-Q Active 121670349.1 128 Attorney Ref. No.070050.6868 1x6 mL, stirred for 30 min – fluffy precipitate formed from the suspension).
  • Example 2 Incorporation of a masked Aib amino acid in a protein expressed in recombinant cells This example discloses the culturing of the recombinant E. coli cells described herein to generate a peptide therapeutic that includes a masked Aib amino acid. 2.1.
  • coli (Thermofisher scientific #EC0113)) are transformed with (1) plasmids encoding the PylRS/tRNA Pyl pairs from Methanosarcina mazei (Mm) using pAIB003_pBK_Mm_Pyl_RS_WT and pAIB005_pALS2_Mm-tRNApyl-GFP- WT, (2) plasmids encoding the pyrrolysyl-tRNA synthetase (PylRS)/tRNA Pyl pairs from Methanosarcina mazei (Mm) using pAIB003_pBK_Mm_Pyl_RS_WT and pAIB005_pALS2_Mm-tRNApyl-GFP-150TAG, (3) plasmids encoding the PylRS/tRNA Pyl pair from Candidatus Methanomethylophilus alvus Mx1201 (Ma) using pBK_Ma_
  • M. mazei mkRS1 mutant was previously shown to incorporate CbzK. Active 121670349.1 131 Attorney Ref. No.070050.6868 10 ⁇ L of each plasmid were prepared and then 1:10 serial dilutions (500 ng total, 50 ng total, 5 ng total, 0.5 ng total) were performed.
  • the four DNA amounts were transformed into four Eppendorfs of chemically competent DH10b cells, plated on Spectinomycin and Kanamycin plates and incubated overnight at 37 o C. Five colonies were then inoculated into 5x500 ⁇ L of Non- inducing Media (NIM) culture (Table 4) containing kanamycin (50 ⁇ g/mL) and spectinomycin (100 ⁇ g/mL) in a 96 well block. The cells were grown for 16 h at 37 o C, 300 RPM.
  • NIM Non- inducing Media
  • coli H10B Chemically Competent E. coli (Thermofisher scientific #EC0113) are transformed with (1) plasmids encoding the PylRS/tRNA Pyl pairs from Methanosarcina mazei (Mm) using pAIB003_pBK_Mm_Pyl_RS_WT and pAIB005_pALS2_Mm-tRNApyl-GFP- WT, (2) plasmids encoding the pyrrolysyl-tRNA synthetase (PylRS)/tRNA Pyl pairs from Methanosarcina mazei (Mm) using pAIB003_pBK_Mm_Pyl_RS_WT and pAIB005_pALS2_Mm-tRNApyl-GFP-150TAG, (3) plasmids encoding the the PylRS/tRNA Pyl pair from Candidatus Methanomethylophilus alvus Mx1201 (
  • a pH 5.5 version of the AIM buffer is prepared to perform the test expression in. Colonies are inoculated into 50 mL of defined AIM media (adjusted to pH 5.5 w/ HCl) + Kan (50 ⁇ g/mL) + Spec (50 ⁇ g/mL) + ncAA (10 mM) for culturing the cells.
  • binding buffer (1x DPBS without Ca2+, Mg2+ + 20 mM imidazole) is generated.
  • 3.5 mL of 2M imidazole solution is added to 350 mL of 1x DPBS.
  • 20 mL of elution buffer (1x DPBS without Ca2+, Mg2+ + 500 mM imidazole) is made.
  • 15 mL of 2M imidazole solution is added to 15 mL of 1x DPBS.
  • Cell pellet is resuspended in 30 mL of binding buffer, then transferred to a new 50 mL falcon tube.
  • GFP protein from the cell culture is bound to a metal affinity resin.
  • the resin is washed with 20–50 column volumes of binding buffer, and eluted with elution buffer.
  • Proteins are desalted using a PD-10 column (GE Healthcare) into 50 mM ammonium bicarbonate and analyzed via mass spectroscopy. The deconvoluted masses were obtained by using Waters MassLynx MaxEnt1 software. The following conditions are analyzed by mass spectrometry.
  • the amino acids are used at a concentration of 10 mM. 1.
  • GFP150TAG + M. alvus WT + Compound 1 5.
  • GFP150TAG + M. mazei WT + Compound 1 6.
  • GFP150TAG + M. mazei mkRS1 + Compound 2 3.1. Synthesis of masked Aib-containing peptide 3.1.1.
  • Raney®2400, slurry, in H2O, active catalyst masked Aib-containing peptide 31 (1 equiv.) is added at room temperature and sparged nitrogen for 15 min followed by hydrogen gas (1 mL/min) overnight to yield demasked Aib containing peptide.
  • the isolated and purified peptide 32 is further subjected to testing (see Yan, L.Z. and Dawson, P.E. J. Am. Chem. Soc. 2001:526-533; Mozingo et al., J. Am. Chem. Soc. 1943, 65: 1013–1016,) 3.2.2. Desulfurization with UV light Scheme 12 Active 121670349.1 137 Attorney Ref.
  • No.070050.6868 Aib-containing peptide 31 is suspended in a solvent and irradiated with UV-light.
  • the product 32 is isolated and characterized (see Wan, Q. and Danishefsky, S, J. Angew. Chem., Int. Ed., 46:9248-9252 (2007); Venneti, N.M. et al., J. Am. Chem. Soc.145:1053-1061 (2023)).
  • 3.2.3. Electrochemical Desulfurization Scheme 13 A peptide of interest 31 is dissolved to a concentration of 1 mg mL ⁇ 1 in a buffered TCEP solution comprised of 0.15 M TCEP and 0.1 M pH 5.8 sodium phosphate buffer.
  • Electrochemical Deselenization Scheme 16 A peptide of interest 33 is dissolved to a concentration of 1 mg mL ⁇ 1 in a buffered TCEP solution comprised of 0.15 M TCEP and 0.1 M pH 5.8 sodium phosphate buffer. To this a small volume of acetonitrile is added (1 mL) to ensure the peptide was fully dissolved. The solution is transferred to ElectraSyn 2.0 vial equipped with the platinum electrodes. The solution is stirred vigorously at room temperature and a constant potential of 1.0 V is supplied. Deselenization progress is monitored by LC-MS until full consumption of the starting material.

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Abstract

The present disclosure provides methods for the generation and purification of proteins and peptides comprising at least one 2-aminoisobutyric acid (Aib) and/or at least one masked form of Aib. The present disclosure further provides masked forms of Aib (e.g., of Formulas I-XVIII) and compositions and cell culture media that include such masked forms of Aib. The present disclosure further provides kits and/or systems for performing the methods described herein.

Description

Attorney Ref. No.070050.6868 MASKED 2-AMINOISOBUTYRIC ACID AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63/581,606, filed September 8, 2023, the contents of which are incorporated by reference herein in its entirety. TECHNICAL FIELD The present disclosure relates to methods for the generation and purification of proteins and peptides comprising one or more 2-aminoisobutyric acid (Aib) amino acids and/or at least one masked form of Aib. BACKGROUND Unnatural amino acids (UAAs) are amino acids derivatives that are not naturally used by the translational machinery available. Such UAAs can be used to increase biological activity or improve the stability of desired proteins and peptides. For example, when applied to therapeutic proteins and peptides, UAAs are known to increase the potency and stability of the therapeutic proteins and peptides. UAAs have been used in anti-obesity drugs, such as Glucagon-like Peptide- 1 (GLP-1) Receptor Agonists and dual GLP-1/GIP receptor agonists such as semaglutide, and tirzepatide. Particularly, these receptor agonists comprise the UAA 2-aminoisobutyric acid (Aib), which has the following structure: and differs from alanine by only the substitution of one hydrogen for one methyl group. Incorporation of Aib into semaglutide can, for example, protects semaglutide from enzyme degradation (Mahapatra et al., Rev Endocr Metab Disord.2022:521-539). Semaglutide is typically produced using chemical synthesis (Yu et al., Adv. Drug Del. Rev. 2018:113-130) or hybrid recombinant and chemical synthesis. However, these chemical synthesis routes can have low yields or undesired byproducts, and can be very costly. Recombinant synthesis techniques, on the other hand, are more environmentally friendly and have lower production costs. In addition, methods which can synthesize GLP-1 receptor agonists via recombinant synthesis can increase the efficiency at which the products are created. Active 121670349.1 1 Attorney Ref. No.070050.6868 Therefore, there is a need in the art, therefore, for fully recombinant cell-based methods that produce proteins and peptides comprising at least one Aib amino acid that are more cost-effective and economically friendly. SUMMARY The present disclosure provides methods for the generation and purification of proteins and peptides comprising at least one or more than one 2-aminoisobutyric acid (Aib) and/or at least one masked form of Aib. The present disclosure further provides compositions and cell media compositions comprising one or more masked forms of Aib and/or one or more recombinant cells for use in the disclosed methods. In one aspect, the present disclosure provides masked forms of Aib or analogs thereof. In certain embodiments, the masked form of Aib or analog thereof has the structure of any one of Formulas I-XVIII. In certain embodiments, the masked form of Aib or analog thereof has the structure of Formula IX. In certain embodiments, the masked form of Aib or analog thereof has the structure of Formula X. In certain embodiments, the masked form of Aib or analog thereof has the structure of Formula XI. In certain embodiments, the masked form of Aib or analog thereof has the structure of Formula XII. In certain embodiments, the masked form of Aib or analog thereof has the structure of Formula XIII. In certain embodiments, the masked form of Aib or analog thereof has the structure of Formula XIV. In certain embodiments, the masked form of Aib or analog thereof has the structure of Formula XV. In certain embodiments, the masked form of Aib or analog thereof has the structure of Formula XVI. In certain embodiments, the masked form of Aib or analog thereof has the structure of Formula XVII. In certain embodiments, the masked form of Aib or analog thereof has the structure of Formula XVIII. The present disclosure further provides compositions comprising one or more masked forms of Aib or analogs thereof. In certain embodiments, a composition of the present disclosure includes a cell culture medium and at least one masked form of Aib or analogs thereof, e.g., a masked form Aib or analog thereof that has the structure of any one of Formulas I-XVIII. In certain embodiments, a composition of the present disclosure includes a buffer and at least one masked form of Aib or analogs thereof, e.g., a masked form Aib or analog thereof that has the structure of any one of Formulas I-XVIII. In a further aspect, the present disclosure provides methods for generating polypeptides, e.g., proteins, and peptides that include at least one or more than one Aib and/or at least one masked Active 121670349.1 2 Attorney Ref. No.070050.6868 form of Aib. In certain embodiments, a method of the present disclosure includes providing a recombinant cell comprising a polynucleotide that includes at least one selector codon and encodes a protein or peptide. In certain embodiments, the recombinant cell further comprises at least one or more polynucleotides encoding an orthogonal tRNA (O-tRNA) (e.g., that functions in the recombinant cell and recognizes the selector codon contained within the polynucleotide that encodes the protein or peptide) and encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) (e.g., where the O-RS aminoacylates the O-tRNA with a masked form of Aib). In certain embodiments, the method further includes culturing the recombinant cell in a medium and expressing the protein or peptide that includes at least one masked form of Aib. In certain embodiments, a method of producing a peptide or protein comprising one or more 2- aminoisobutyric acid (Aib) amino acid residues in a recombinant cell includes (a) culturing the recombinant cell in a cell culture media, wherein the recombinant cell comprises (i) a polynucleotide encoding the peptide or protein and comprising at least one selector codon (e.g., where the recombinant cell expresses: an orthogonal tRNA (O-tRNA) that functions in the recombinant cell and recognizes the selector codon; and an orthogonal aminoacyl-tRNA synthetase (O-RS), wherein the O-RS preferentially aminoacylates the O-tRNA with a masked Aib amino acid or analog thereof); (b) providing the masked Aib amino acid or analog thereof to the cell culture media; and (c) incorporating the masked Aib amino acid into one or more specified positions in the peptide or protein during translation of the polynucleotide encoding the peptide or protein and comprising the at least one selector codon to produce the peptide or protein comprising the one or more masked Aib amino acid residues. In certain embodiments, the medium for culturing the recombinant cells comprises the masked form of Aib. In certain embodiments, the masked form of Aib present in the cell culture medium has the structure of any one of Formulas I-XVIII. In certain embodiments, expressing the protein or peptide includes incorporating the masked form of Aib into one or more specified positions in the peptide or protein during translation of the polypeptide with the at least one selector codon. In certain embodiments, the protein or peptide that includes the at least one masked form of Aib is secreted from the recombinant cell. Alternatively, the protein or peptide that includes the at least one masked form of Aib is not secreted from the recombinant cell. In certain embodiments, the method further includes purifying the protein or peptide comprising the at least Active 121670349.1 3 Attorney Ref. No.070050.6868 one masked form of Aib from the medium, supernatant and/or recombinant cell extract, e.g., using chromatography as described herein. In a further aspect, the present disclosure further provides methods for purifying a protein or peptide comprising at least one masked form of Aib. In certain embodiments, a purification method of the present disclosure includes contacting a chromatography medium (e.g., within a chromatography column) or a solid support with a solution containing the protein or peptide comprising the at least one masked Aib. In certain embodiments, the solution is a culture medium, supernatant and/or extract from recombinant cells. In certain embodiments, the chromatography medium specifically binds to (e.g., non-covalently binds to or covalently binds to) a moiety (e.g., an azide group) contained within the masking group of the masked form of Aib present in the protein or peptide. Non-limiting examples of reactions for binding the masking group to the chromatography medium or solid support are provided herein. In certain embodiments, a method of the present disclosure can further include removing the masking group from the masked Aib present in the protein or peptide (e.g., purified protein or peptide), e.g., by performing a chemical reaction. Non-limiting examples of reactions for removing the masking group from the masked Aib present in the protein or peptide and catalysts for performing such reactions are provided herein. In certain embodiments, the chemical reaction is a desulfurization reaction. In certain embodiments, the chemical reaction is a deselenization reaction. In certain embodiments, the catalyst used for the chemical reaction is a metal reagent. In certain embodiments, the catalyst used for the chemical reaction is Pd/Al2O3. In certain embodiments, the catalyst used for the chemical reaction is Pd/Carbon. In certain embodiments, the catalyst used for the chemical reaction is Raney nickel. In certain embodiments, the catalyst used for the chemical reaction is Pd/BaSO4. In certain embodiments, the catalyst used for the chemical reaction is PdO. In certain embodiments, the catalyst used for the chemical reaction is a Ni(0) catalyst. In certain embodiments, the catalyst used for the chemical reaction is Nickel boride. In certain embodiments, the catalyst used for the chemical reaction is Tin hydride. In certain embodiments, the chemical reaction is performed using a Raney nickel – sodium hypophosphite – acetate buffer system. The present disclosure further provides recombinant cells and compositions thereof for use in the disclosed methods. In certain embodiments, the recombinant cell comprises a polynucleotide that includes at least one selector codon and encodes a protein or peptide. In certain embodiments, Active 121670349.1 4 Attorney Ref. No.070050.6868 the recombinant cell further comprises at least one or more polynucleotides encoding an orthogonal tRNA (O-tRNA) (e.g., that functions in the recombinant cell and recognizes the selector codon of the polynucleotide encoding the protein or peptide) and encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) (e.g., where the O-RS aminoacylates the O-tRNA with a masked Aib amino acid). In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl-tRNA synthetase derived from a species of the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to a pyrrolysyl-tRNA synthetase derived from a species of the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanomethylophilus alvus tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanomethylophilus alvus tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous Active 121670349.1 5 Attorney Ref. No.070050.6868 to the Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O- RS comprises an amino acid sequence that is greater than 95% homologous to the Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, the recombinant cell is a genetically modified bacterial cell. In certain embodiments, the recombinant cell is a genetically modified Escherichia coli cell. In certain embodiments, the recombinant cell is a genetically modified fungal cell. In certain embodiments, the recombinant cell is a genetically modified Saccharomyces cerevisiae cell. In certain embodiments, the recombinant cell is a genetically modified mammalian cell. The present disclosure further provides kits and systems for use in the disclosed methods. In certain embodiments, a kit and/or system of the present disclosure can include one more recombinant cell compositions and/or one or more masked forms of Aib or analogs thereof (or compositions thereof). In certain embodiments, a kit and/or system of the present disclosure can further include a chromatography medium for purifying a protein or peptide comprising at least one masked form of Aib. Active 121670349.1 6 Attorney Ref. No.070050.6868 In certain embodiments, the peptide or protein generated and/or purified by the methods of the present disclosure is a therapeutic protein or peptide, e.g., a therapeutic protein or peptide for treating diabetes (e.g., type 2 diabetes), for treating obesity, for weight loss and/or for reducing the risk of cardiovascular events (e.g., heart attack and/or stroke). In certain embodiments, the therapeutic protein or peptide can be a glucagon-like peptide-1 (GLP-1) agonist, a GLP-1 analog, GLP-1 receptor agonist, a glucose-dependent insulinotropic polypeptide (GIP) agonist, a GIP receptor agonist or a dual GLP-1/GIP receptor agonist. In certain embodiments, the GLP-1 receptor agonist can be semaglutide. In certain embodiments, the protein or peptide is a GLP-1 agonist. In certain embodiments, the protein or peptide is a GLP-1 analog. In certain embodiments, the protein or peptide is a GLP-1 receptor agonist. In certain embodiments, the protein or peptide is a dual GLP-1/GIP receptor agonist. In certain embodiments, the peptide is selected from the group consisting of semaglutide, tirzepatide, retatrutide, VK2735 and a combination thereof. In certain embodiments, the peptide is semaglutide. In certain embodiments, the peptide is tirzepatide. In certain embodiments, the peptide is retatrutide. In certain embodiments, the peptide is VK2735. In certain embodiments, a composition of the present disclosure can include a cell culture medium and one or more recombinant cells. In certain embodiments, a composition of the present disclosure can include lyophilized recombinant cells. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the amino acid structure of semaglutide. FIG. 2 shows the amino acid structure of tirzepatide. FIG. 3 shows the amino acid structure of retatrutide. FIG.4A shows the amber suppression in the presence of no amino acids or in the presence of unnatural amino acids with Methanomethylophilus alvus PylRS/tRNApyl. FIG.4B shows the amber suppression in the presence of no amino acids or in the presence of unnatural amino acids and masked amino acids with Methanosarcina mazei PylRS/tRNApyl. FIG. 4C shows the chemical structures of BocK and CbzK. FIG. 5 shows the green fluorescent protein (GFP) amber suppression with the synthetases M. mazei WT and M. mazei mkRS1 mutant in the presence of no amino acids, S-(2-((tert- butoxycarbonyl)amino)ethyl)-L-cysteine, S-(2-(((benzyloxy)carbonyl)amino)ethyl)-L-cysteine or Active 121670349.1 7 Attorney Ref. No.070050.6868 (R)-2-amino-3-((2-(((benzyloxy)carbonyl)amino)ethyl)thio)-2-methylpropanoic acid (Compound 2). FIG.6A compares the GFP fluorescence in the presence of no amino acids or (R)-2-amino- 3-((2-(((benzyloxy)carbonyl)amino)ethyl)thio)-2-methylpropanoic acid (Compound 2). FIG. 6B compares the GFP fluorescence under increasing concentration conditions that include no amino acids or (R)-2-amino-3-((2-(((benzyloxy)carbonyl)amino)ethyl)thio)-2- methylpropanoic acid (Compound 2). FIG. 7 provides an exemplary schematic of a method of the present disclosure for generating a peptide or protein comprising one or more Aib amino acids. DESCRIPTION The presently disclosed subject matter for the synthesis and further purification of proteins and peptides which comprise at least one 2-aminoisobutyric acid (Aib) amino acid (or derivative thereof) and/or at least one masked form of Aib (or derivative thereof) in cells. The presently disclosed subject matter further provides compositions comprising the proteins and peptides comprising Aib and/or masked Aib and kits and systems and methods of using the proteins and peptides comprising Aib and/or masked Aib. As described herein, incorporation of Aib into peptide and protein therapeutics can affect the stability and/or efficacy of the therapeutic. For example, but not by way of limitation, GLP-1 agonists that include one or more αα-disubstituted unnatural amino such as Aib have been shown to have increased stability and efficacy. In certain embodiments, Aib and other αα-disubstituted amino acids constrain the structure of the peptide to particular secondary structures affecting both binding and stimulation of receptors. For example, incorporation of Aib at position 8 in GLP-1 reinforces the alpha-helical structure of the peptide, which is important for its biological activity. Incorporation of Aib in position 8 of GLP-1 also renders the polypeptide resistant to hydrolysis by DPP-IV, which otherwise hydrolyzes and inactivates GLP-1. For clarity, but not by way of limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections: I. Definitions; II. Masked Aib Amino Acids and Compositions Thereof; III. Therapeutics; Active 121670349.1 8 Attorney Ref. No.070050.6868 IV. Recombinant Cells; V. Methods of Production; VI. Kits and Systems; and VII. Exemplary Non-Limiting Embodiments. I. Definitions The terms used in this specification generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them. As used herein, the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms or words that do not preclude additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. The terms “expression” or “expresses,” as used herein, refer to transcription and translation occurring within a cell, e.g., a recombinant cell, e.g., a recombinant E. coli cell. The level of expression of a gene and/or nucleic acid in a cell can be determined on the basis of either the amount of corresponding mRNA that is present in the cell or the amount of the protein encoded Active 121670349.1 9 Attorney Ref. No.070050.6868 by the gene and/or nucleic acid that is produced by the cell. For example, mRNA transcribed from a gene and/or nucleic acid is desirably quantitated by northern hybridization. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 7.3-7.57 (Cold Spring Harbor Laboratory Press, 1989). Protein encoded by a gene and/or nucleic acid can be quantitated either by assaying for the biological activity of the protein or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay using antibodies that are capable of reacting with the protein. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 18.1-18.88 (Cold Spring Harbor Laboratory Press, 1989). As used herein, “polypeptide” refers generally to peptides and proteins having about three or more amino acids. In certain embodiments, the polypeptide can be endogenous to the cell, or preferably, can be exogenous, meaning that it is heterologous, i.e., foreign, to the cell being utilized. The term “protein” as used herein refers to a sequence of amino acids for which the chain length is sufficient to produce the higher levels of tertiary and/or quaternary structure. This is to distinguish from “peptides” that typically do not have such structure. Typically, the protein herein will have a molecular weight of at least about 4-100 kD, e.g., closer to about 15 kD. In certain embodiments, a protein can include at least about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400 or about 500 amino acids. As used herein the terms “amino acid,” “amino acid monomer” or “amino acid residue” refer to organic compounds composed of amine and carboxylic acid functional groups, along with a side-chain specific to each amino acid. In particular, alpha- or α-amino acid refers to organic compounds in which the amine (-NH2) is separated from the carboxylic acid (-COOH) by a methylene group (-CH2), and a side-chain specific to each amino acid connected to this methylene group (-CH2) which is alpha to the carboxylic acid (-COOH). Different amino acids have different side chains and have distinctive characteristics, such as charge, polarity, aromaticity, reduction potential, hydrophobicity and pKa. Amino acids can be covalently linked to form a polymer through peptide bonds by reactions between the carboxylic acid group of the first amino acid and the amine group of the second amino acid. Amino acid in the sense of the disclosure refers to any of the twenty plus naturally occurring amino acids, non-natural amino acids, and includes both D and L optical isomers. The term “nucleic acid,” “nucleic acid molecule” or “polynucleotide” as used herein refers Active 121670349.1 10 Attorney Ref. No.070050.6868 to any compound and/or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Often, the nucleic acid molecule is described by the sequence of bases, whereby the bases represent the primary structure (linear structure) of a nucleic acid molecule. The sequence of bases is typically represented from 5’ to 3’. Herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including, e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule can be linear or circular. In addition, the term nucleic acid molecule includes both, sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid molecule can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of a nucleic acid of the disclosure in vitro and/or in vivo, e.g., in a bacterial cell. For example, but not by way of limitation, a nucleic acid of the present disclosure can encode a therapeutic. In certain embodiments, a nucleic acid of the present disclosure can encode an aminoacyl-tRNA synthetase. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or expression of the encoded molecule. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. As used herein, the term “recombinant cell” refers to cells which have some genetic modification from the original parent cells from which they are derived. Such cells can also be referred to as “genetically-engineered cells.” Such genetic modification can be the result of an introduction of a heterologous gene (or nucleic acid) for expression of an aminoacyl transferase, e.g., an aminoacyl-tRNA synthetase. As used herein, the term “recombinant protein” refers generally to peptides and proteins. Such recombinant proteins are “heterologous,” i.e., foreign to the cell being utilized, such as a GLP-1 receptor agonist produced by a cell, e.g., a bacterial cell. Active 121670349.1 11 Attorney Ref. No.070050.6868 As used herein, “sequence identity” or “identity” in the context of two polynucleotide or polypeptide sequences makes reference to the nucleotide bases or amino acid residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity or similarity is used in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted with a functionally equivalent residue of the amino acid residues with similar physiochemical properties and therefore do not change the functional properties of the molecule. As used herein, “percentage of sequence identity” or “percentage of identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window can include additions or deletions (gaps) as compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. As understood by those skilled in the art, determination of percent identity between any two sequences can be accomplished using certain well-known mathematical algorithms. Non- limiting examples of such mathematical algorithms are the algorithm of Myers and Miller, the local homology algorithm of Smith et al.; the homology alignment algorithm of Needleman and Wunsch; the search-for-similarity-method of Pearson and Lipman; the algorithm of Karlin and Altschul, modified as in Karlin and Altschul. Computer implementations of suitable mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL, ALIGN, GAP, BESTFIT, BLAST, FASTA, among others identifiable by skilled persons. The term “operative connection,” “operably linked” or “operatively linked,” as used herein, with regard to regulatory sequences of a gene indicate an arrangement of elements in a combination enabling production of an appropriate effect. With respect to genes and regulatory sequences, an operative connection indicates a configuration of the genes with respect to the regulatory sequence allowing the regulatory sequences to directly or indirectly increase or decrease Active 121670349.1 12 Attorney Ref. No.070050.6868 transcription or translation of the genes. In particular, in certain embodiments, regulatory sequences directly increasing transcription of the operatively linked gene, comprise promoters typically located on a same strand and upstream on a DNA sequence (towards the 5’ region of the sense strand), adjacent to the transcription start site of the genes whose transcription they initiate. In certain embodiments, regulatory sequences directly increasing transcription of the operatively linked gene or gene cluster comprise enhancers that can be located more distally from the transcription start site compared to promoters, and either upstream or downstream from the regulated genes, as understood by those skilled in the art. Enhancers are typically short (50-1500 bp) regions of DNA that can be bound by transcriptional activators to increase transcription of a particular gene. Typically, enhancers can be located up to 1 Mbp away from the gene, upstream or downstream from the start site. The term “secretable,” as used herein, means able to be secreted, wherein secretion in the present disclosure generally refers to transport or translocation from the interior of a cell, e.g., within the cytoplasm or cytosol of a cell, to its exterior, e.g., outside the plasma membrane of the cell. Secretion can include several procedures, including various cellular processing procedures such as enzymatic processing of the peptide. In certain embodiments, secretion of a protein, e.g., a protein disclosed herein, can be continuous or induced. The terms “detect” or “detection,” as used herein, indicates the determination of the existence and/or presence of a target in a limited portion of space, including but not limited to a sample, a reaction mixture, a molecular complex and a substrate. The “detect” or “detection” as used herein can comprise determination of chemical and/or biological properties of the target, including but not limited to ability to interact, and in particular bind, other compounds, ability to activate another compound and additional properties identifiable by a skilled person upon reading of the present disclosure. The detection can be quantitative or qualitative. A detection is “quantitative” when it refers, relates to, or involves the measurement of quantity or amount of the target or signal (also referred as quantitation), which includes but is not limited to any analysis designed to determine the amounts or proportions of the target or signal. A detection is “qualitative” when it refers, relates to, or involves identification of a quality or kind of the target or signal in terms of relative abundance to another target or signal, which is not quantified. The term “derived” or “derive” is used herein to mean to obtain from a specified source. The term “molecule,” as used herein, refers a group of atoms bonded together, representing Active 121670349.1 13 Attorney Ref. No.070050.6868 the smallest fundamental unit of a chemical compound that can take part in a chemical reaction. The term “derivative” or “analog” as used herein with reference to a protein or amino acid refers to a modified form of the protein or amino acid. Non-limiting examples of such derivatives include mutated forms of the protein. “Pharmaceutically acceptable carrier,” as used herein, refers to a pharmaceutically acceptable material, composition or vehicle that is involved in carrying or transporting a compound or composition of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier can be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it can come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits. As used herein the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be recipient of a particular treatment. As used herein, the term “modified” when referencing an organism, e.g., a cell, refers to an organism that does not exist in nature. The term is used interchangeably with “recombinant” or “engineered.” A “therapeutically effective amount” or a “therapeutically effective level” refers to an amount of a protein or peptide produced by the methods of the present disclosure that prevents, decreases, alleviates or eliminates one or more symptoms of a condition, e.g., obesity. As used herein, “orthogonal” refers either to a tRNA molecule and/or to an aminoacyl synthetase molecule which reacts with reduced efficiency with the endogenous components of a translation system, either in vivo or in vitro. In certain embodiments, an orthogonal tRNA in a translation system of interest e.g., in the translational system of E. coli, is aminoacylated by any endogenous aminoacyl synthetase of the translation system with reduced or even zero efficiency when compared to aminoacylation of an endogenous tRNA by the endogenous aminoacyl synthetase of the translation system. In certain embodiments, an orthogonal aminoacyl synthetase aminoacylates any endogenous tRNA in the translation system of interest, e.g., in the translational system of E. coli, with reduced or even zero efficiency as Active 121670349.1 14 Attorney Ref. No.070050.6868 compared to aminoacylation of the endogenous tRNA by an endogenous aminoacyl synthetase. “RS,” as used herein, refers to an aminoacyl-tRNA synthetase. “O-RS,” as used herein, refers to an orthogonal aminoacyl-tRNA synthetase. “O-tRNA,” as used herein, refers to an orthogonal tRNA. “Selector codon,” as used herein, refers to a codon (i.e., a series of 3 or more nucleic acids) recognized by an O-tRNA in the translation process and not recognized by an endogenous tRNA. The O-tRNA anticodon loop recognizes the selector codon on an mRNA so that the amino acid it carries, e.g., an unnatural amino acid, is incorporated at the site in the peptide encoded by the selector codon. A “suppressor tRNA” is a tRNA that alters the reading of a messenger RNA (mRNA) in a given translation system, e.g., by recognizing a stop codon or other nonsense codon and supplying an amino acid, thereby allowing the translation of codons located 3’ of the stop or nonsense codon. “Unnatural amino acid” means any amino acid, amino acid derivative, amino acid analog, α-hydroxy acid, or other molecule other than a natural amino acid which can be incorporated into a polypeptide chain with an O-tRNA/O-RS pair and which allows extension of the polypeptide chain. In certain embodiments, the term “non-proteogenic amino acid” refers to an unnatural amino acid. As used herein, the term “isolate” can refer to a therapeutic that has been separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting). In certain embodiments, isolated therapeutics, e.g., isolated protein or peptide therapeutics, can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of the other components with which they were initially associated. In certain embodiments, isolated therapeutics, e.g., isolated protein or peptide therapeutics, are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% pure. In certain embodiments, a therapeutic, e.g., a protein or peptide therapeutic, is “pure” if it is substantially free of other components. As used herein, the terms “purify,” “purifying” and “purified,” as used herein, can refer to a protein or peptide therapeutic or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (e.g., whether in nature or in an experimental setting), or during any time after its initial production. In certain Active 121670349.1 15 Attorney Ref. No.070050.6868 embodiments, a protein or peptide therapeutic can be considered purified if it is isolated at or after production, such as from a material or environment containing the protein or peptide therapeutic, and a protein or peptide therapeutic can contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered “isolated.” In certain embodiments, purified protein or peptide therapeutics are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% pure. In the certain embodiments of the compositions described herein, the one or more protein or peptide therapeutics present in the composition can be independently purified from other proteins or peptides present in the material or environment containing the protein or peptide therapeutic comprising the Aib amino acid. It will be understood that although terms such as “first” and “second” are used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another element. Accordingly, an element discussed below could be termed a second element, and similarly, a second element may be termed a first element without departing from the teachings of the present dsclosure. As used herein, the term “alkyl” or “alkyl group” refers to a saturated branched or unbranched carbon chain. In certain embodiments, alkyl refers to a carbon chain with 1 to 12 carbon atoms (C1-C12 alkyl), 1 to 8 carbon atoms (C1-C8 alkyl), 1 to 6 carbon atoms (C1-C6 alkyl), or 1 to 4 carbon atoms (C1-C4 alkyl). Examples of “alkyl groups” include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, 1-methylbutyl, 2- methylbutyl, 3-methylbutyl, neopentyl, 3,3-dimethylpropyl, hexyl, 2-methylpentyl, and the like. The alkyl group can be optionally substituted. The term “alkylene” refers to an alkyl group as defined herein having two free radicals which can bond with another group. The term “alkaryl” refers to an alkyl group substituted with an aryl radical. As used herein, the term “alkenyl” refers to a branched or unbranched carbon chain having at least one carbon-carbon double bond. In certain embodiments, alkenyl refers to a carbon chain with at least one carbon-carbon double-bond having 1 to 12 carbon atoms (C1-C12 alkenyl), 1 to 8 carbon atoms (C1-C8 alkenyl), 1 to 6 carbon atoms (C1-C6 alkenyl), or 1 to 4 carbon atoms (C1-C4 alkenyl). The alkenyl group can be optionally substituted. Representative examples of alkenyl Active 121670349.1 16 Attorney Ref. No.070050.6868 include vinyl, 1-propenyl, 2-propenyl, 1-methyl-1-propenyl, 1-methyl-2-propenyl, 2-methyl-1- propenyl, 2-methyl-2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, and so on. As used herein, the term “alkynyl” refers to a branched or unbranched carbon chain derived having at least one carbon-carbon triple bond. In certain embodiments, alkynyl refers to a carbon chain with at least one carbon-carbon triple-bond having 1 to 12 carbon atoms (C1-C12 alkynyl), 1 to 8 carbon atoms (C1-C8 alkynyl), 1 to 6 carbon atoms (C1-C6 alkynyl), or 1 to 4 carbon atoms (C1-C4 alkynyl). The alkynyl group can be optionally substituted. The alkynyl group can be unbranched or branched. Representative examples include ethynyl, propynyl, butyn-1-yl, and butyn-2-yl. As used herein, the term “alkoxy” refers to –O–alkyl, wherein alkyl is as defined herein. In certain embodiments, alkoxy refers to a carbon chain with –O–alkyl having 1 to 12 carbon atoms (C1-C12 alkoxy), 1 to 8 carbon atoms (C1-C8 alkoxy), 1 to 6 carbon atoms (C1-C6 alkoxy), or 1 to 4 carbon atoms (C1-C4 alkoxy). Representative examples of alkoxy include, but are not limited to, methoxy (C1 alkoxy), ethoxy (C2 alkoxy), propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, cyclopropyloxy-, cyclohexyloxy- and the like. The alkoxy group can be optionally substituted. As used herein, the term “arylalkoxy” refers to an alkoxy group substituted with an aryl group. In certain embodiments, the arylalkoxy group can be optionally substituted. As used herein, the term “cycloalkyl” or “carbocyclic ring” refers to nonaromatic rings that are either partially or fully hydrogenated and may exist as a single ring of 3 to 8 members or bicyclic ring of 9 to 14 members. Unless specified otherwise, the carbocyclic ring is generally a 3- to 8-membered ring (C3-C8 cycloalkyl) or 3- to 6-membered ring (C3-C6 cycloalkyl). For example, a cycloalkyl groups includes cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclpentenyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, norbornyl, norbornenyl, and the like. The term “cycloalkylene” refers to cycloalkyl as defined herein having two free radicals which can bond with another group. As used herein, the term “aryl” refers to a monocyclic, bicyclic, or tricyclic aromatic ring. An aryl group contains 6 to 10 carbon atoms. Examples of an aryl group include phenyl (C6H6, Ph, or Ar) or naphthyl. The term “arylene” refers to aryl as defined herein having a free radical which can bond with another group. The term “aralkyl” refers to an aryl group substituted with an alkyl radical. Active 121670349.1 17 Attorney Ref. No.070050.6868 As used herein, a “heterocycle” or “heterocycloalkyl” refers to a monocyclic, bicyclic, or tricyclic aromatic ring system having 1 to 8 heteroatoms selected from N, O, or S. In certain embodiments, a heterocycloalkyl is a 3-14 membered monocyclic, bicyclic or tricyclic ring system preferably containing 1 to 3 heteroatoms each independently selected from O, N, or S. Examples of hereocycloalkyl groups include oxiranyl, piperidinyl, tetrahydrofuranyl, 1,4-dioxanyl, azetidinyl, or tetrahydrothiophenyl. As used herein, the term “heterocycloalkylene” refers to heterocycloalkyl as defined herein having two free radicals available for bonding. As used herein, the term “heteroaryl” refers to a monocyclic, bicyclic, or tricyclic aromatic ring system having 1 to 8 heteroatoms selected from N, O, or S. In certain embodiments, a heteroaryl is a 5-14 membered aromatic ring system preferably containing 1 to 4 heteroatoms independently selected from O, N, or S. Examples of a heteroaryl group includes 2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrssolyl, 2-, 4-, or 5-imidazolyl, 3-, 4-, or 5- pyrazolyl, 2-, 4-, or 5-thiazolyl, 3-, 4-, or 5-isothiazolyl, 2-, 4-, or 5-oxazolyl, 3-, 4-, or 5-isoxazolyl, 3- or 5-1,2,4-triazolyl, 4- or 5-1,2, 3-triazolyl, tetrazolyl, 2-, 3-, or 4-pyridyl, 3- or 4-pyridazinyl, 3-, 4-, or 5-pyrazinyl, 2- pyrazinyl, and 2-, 4-, or 5-pyrimidinyl. As used herein, the term “heteroarylene” refers to heteroaryl as defined herein having two free radicals available for bonding. As used herein, the term “halogen” or “halo” refers to fluoro, chloro, bromo, or iodo. As used herein, the term “masking group” refers to a group that is introduced temporarily at a desired position of a molecule to “mask” the position so that a reaction does not occur at further synthetic steps. The masking group can be removed via a “demasking” reaction. Any suitable masking group can be used. Examples of masking groups include, but are not limited, to thioethers (R-S-R) or selenoethers (R-Se-R), also called selenides. In certain embodiments, the masking group is a thioether. In certain embodiments, the masking group is a selenoether. As used herein, the term “protecting group” or “protective group” refers to a group that is introduced temporarily at a desired position of a molecule to “protect” the position so that a reaction does not occur at further synthetic steps. The protecting group can be removed via a “deprotecting” reaction. Any suitable protecting group can be used. Examples of protecting groups to protect amines, “amino protecting groups”, include, but are not limited to, t-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), 9-fluorenylmethoxycarbonyl (FMoc), or allyloxycarbonyl (Alloc). Examples of protecting groups to protect carboxylic acids, include, but are not limited to, tert- butyl (tBu), trityl (Trt), 2,4-dimethoxybenzyl (Dmb), 9-fluoromethyl (Fm), or benzyl (Bn). Active 121670349.1 18 Attorney Ref. No.070050.6868 Examples of protecting groups to protect hydroxyl groups, include, but are not limited to, tert- butyldimethylsilyl (TBDMS), allyl (Al), acetate (Ac) or o-nitrobenzyl (ONB). Common ester protecting groups include, but are not limited to, acetate (Ac), pivalate (PV), and benzoate (Bz). II. Masked Aib Amino Acids and Compositions Thereof The present disclosure provides masked forms of Aib and/or analogs thereof. In certain embodiments, the disclosed masked forms of Aib and/or analogs thereof can be used in a method described herein. For example, but not by way of limitation, a masked form of Aib and/or analog thereof can be incorporated into a therapeutic peptide of the present disclosure. In certain embodiments, a masked form of Aib and/or analog thereof can be used in a method of the present disclosure for preparing a therapeutic peptide that incorporates one or more Aib amino acids and/or the one or more masked forms of Aib. In certain embodiments, the present disclosure provides an Aib amino acid or analog thereof that includes a masking group. In certain embodiments, the masking group is selected from a thioether or a selenoether. In certain embodiments, the masking group is a thioether. In certain embodiments, the masking group is a selenoether. In certain embodiments, the present disclosure provides an Aib amino acid coupled to a thioether masking group or a selenoether masking group. In certain embodiments, the present disclosure provides an Aib amino acid coupled to a thioether masking group. In certain embodiments, the present disclosure provides an Aib amino acid coupled to a selenoether masking group. In certain embodiments, the present disclosure provides a masked Aib amino acid or analog thereof that has the structure of Formula I:
Figure imgf000020_0001
wherein: the combination of X and R3 are optional if the combination of Y and R4 are present, but are otherwise required; Active 121670349.1 19 Attorney Ref. No.070050.6868 the combination of Y and R4 are optional if the combination of X and R3 are present, but are otherwise required; R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; each of X and Y when present is independently S or Se; each of R3 and R4 when present is independently an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″, —C(O)OR″, —C(O)N(R″)2, or -L-Z; or R3 and R4 taken together optionally form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl; each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, —O—, —O- (alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, — C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O-(alkylene or Active 121670349.1 20 Attorney Ref. No.070050.6868 substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, — C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, — C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, —N(R′)CO— (alkylene or substituted alkylene)-, — N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, —N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, —N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2—N(R′)— N(R′)—; and where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the present disclosure provides a masked Aib amino acid or analog thereof that has the structure of Formula II:
Figure imgf000022_0001
wherein: the combination of X, A, and R3 are optional if the combination of Y, B, and R4 are present, but are otherwise required; the combination of Y, B, and R4 are optional if the combination of X, A and R3 are present, but are otherwise required; R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; each of X and Y when present is independently S or Se; each of A and B are optional, and when present are independently an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene Active 121670349.1 21 Attorney Ref. No.070050.6868 or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″, —C(O)OR″, —C(O)N(R″)2, or - L-Z; or A and B taken together optionally form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl; each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, —O—, — O— (alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene) —, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O-(alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, — C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, — C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, —N(R′)CO— (alkylene or substituted alkylene)-, — N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, —N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, —N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2—N(R′)— N(R′)—; where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; and Active 121670349.1 22 Attorney Ref. No.070050.6868 each of R3 and R4 when present is independently an azide, tetrazine, substituted tetrazine, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, trans-cyclooctene, substituted trans-cyclooctene, cyclooctyne, or substituted cyclooctyne; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the present disclosure provides a masked Aib amino acid or analog thereof that has the structure of Formula III:
Figure imgf000024_0001
wherein: the combination of Y and B are optional; R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; X is S or Se; Y when present is S or Se; each of A and B are optional, and when present are independently an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″, —C(O)OR″, —C(O)N(R″)2, or - L-Z; or A and B taken together optionally form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl; each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted Active 121670349.1 23 Attorney Ref. No.070050.6868 heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, —O—, —O- (alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, — C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O-(alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, — C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, — C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, —N(R′)CO— (alkylene or substituted alkylene)-, — N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, —N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, —N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2—N(R′)— N(R′)—; where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; and R3 is an azide, tetrazine, substituted tetrazine, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, trans-cyclooctene, substituted trans-cyclooctene, cyclooctyne, or substituted cyclooctyne; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the present disclosure provides a masked Aib amino acid or analog thereof that has the structure of Formula IV: Active 121670349.1 24 Attorney Ref. No.070050.6868
Figure imgf000026_0001
wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; each of X and Y is independently S or Se; R3 is an H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″, —C(O)OR″, —C(O)N(R″)2, or -L-Z; each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, —O—, —O- (alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, — Active 121670349.1 25 Attorney Ref. No.070050.6868 C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O-(alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, — C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, — C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, —N(R′)CO— (alkylene or substituted alkylene)-, — N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, —N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, —N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2—N(R′)— N(R′)—; and where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the present disclosure provides a masked Aib amino acid or analog thereof that has the structure of Formula V:
Figure imgf000027_0001
wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; each of X and Y is independently S or Se; A is optional, and when present is an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)- ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S- (aryl or substituted aryl), —C(O)R″, —C(O)OR″, —C(O)N(R″)2, or -L-Z; Active 121670349.1 26 Attorney Ref. No.070050.6868 each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, —O—, —O- (alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, — C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O-(alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, — C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, — C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, —N(R′)CO— (alkylene or substituted alkylene)-, — N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, —N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, —N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2—N(R′)— N(R′)—; where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; and R3 is an azide, tetrazine, substituted tetrazine, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, trans-cyclooctene, substituted trans-cyclooctene, cyclooctyne, or substituted cyclooctyne; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the present disclosure provides a masked Aib amino acid or analog thereof that has the structure of Formula VI: Active 121670349.1 27 Attorney Ref. No.070050.6868
Figure imgf000029_0001
wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; X is S or Se; R3 is an H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″, —C(O)OR″, —C(O)N(R″)2, or -L-Z; each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, —O—, —O- Active 121670349.1 28 Attorney Ref. No.070050.6868 (alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, — C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O-(alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, — C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, — C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, —N(R′)CO— (alkylene or substituted alkylene)-, — N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, —N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, —N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2—N(R′)— N(R′)—; and where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the present disclosure provides a masked Aib amino acid or analog thereof that has the structure of Formula VII:
Figure imgf000030_0001
wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; X is S or Se; A is optional, and when present is an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)- Active 121670349.1 29 Attorney Ref. No.070050.6868 ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S- (aryl or substituted aryl), —C(O)R″, —C(O)OR″, —C(O)N(R″)2, or -L-Z; each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, —O—, —O- (alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, — C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O-(alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, — C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, — C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, —N(R′)CO— (alkylene or substituted alkylene)-, — N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, —N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, —N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2—N(R′)— N(R′)—; where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; and R3 is an azide, tetrazine, substituted tetrazine, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, trans-cyclooctene, substituted trans-cyclooctene, cyclooctyne, or substituted cyclooctyne; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. Active 121670349.1 30 Attorney Ref. No.070050.6868 In certain embodiments, the present disclosure provides a masked Aib amino acid or analog thereof that has the structure of Formula VIII:
Figure imgf000032_0001
wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; and X is S or Se; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the present disclosure provides a masked Aib amino acid or amino acid analog of Formula III, wherein R1 is H, R2 is OH, X is S, and R3 is alkylalkoxy or substituted alkylalkoxy. In certain embodiments, the present disclosure provides a masked Aib amino acid or amino acid analog of Formula III, wherein R1 is H, R2 is OH, X is S, and R3 is substituted alkylalkoxy. In certain embodiments, the masked Aib amino acid or amino acid analog of Formula III is (R)-2-amino-3-((2-(((benzyloxy)carbonyl)amino)ethyl)thio)-2-methylpropanoic acid, Compound A, with the structure of Formula IX:
Figure imgf000032_0002
Formula IX or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. Active 121670349.1 31 Attorney Ref. No.070050.6868 In certain embodiments, the present disclosure provides a masked Aib amino acid or amino acid analog of Formula III, wherein R1 is H, R2 is OH, X is S, and R3 is alkylalkoxy. In certain embodiments, the masked Aib amino acid or amino acid analog of Formula III is (R)-2-amino-3-((2-((tert-butoxycarbonyl)amino)ethyl)thio)-2-methylpropanoic acid, Compound B, with the structure of Formula X:
Figure imgf000033_0001
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the present disclosure provides a masked Aib amino acid or amino acid analog of Formula VIII, wherein R1 is H, R2 is OH, and X is S. In certain embodiments, the masked Aib amino acid or amino acid analog of Formula VIII is (R)-2-amino-3-((2-((((2-azidobenzyl)oxy)carbonyl)amino)ethyl)thio)-2-methylpropanoic acid, and has the structure of Formula XI:
Figure imgf000033_0002
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the present disclosure provides a masked Aib amino acid or amino acid analog of Formula III, wherein R1 is H, R2 is OH, X is Se, and R3 is alkylalkoxy or substituted alkylalkoxy. In certain embodiments, the present disclosure provides a masked Aib amino acid or amino acid analog of Formula III, wherein R1 is H, R2 is OH, X is Se, and R3 is alkylalkoxy. In certain embodiments, the masked Aib amino acid or amino acid analog of Formula III is (R)-2-amino-3-((2-(((benzyloxy)carbonyl)amino)ethyl)selanyl)-2-methylpropanoic acid, Compound D, with the structure of Formula XII: Active 121670349.1 32 Attorney Ref. No.070050.6868
Figure imgf000034_0001
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the present disclosure provides a masked Aib amino acid or amino acid analog of Formula III, wherein R1 is H, R2 is OH, X is Se, and R3 is substituted alkylalkoxy. In certain embodiments, the masked Aib amino acid or amino acid analog of Formula III is (R)-2-amino-3-((2-((tert-butoxycarbonyl)amino)ethyl)selanyl)-2-methylpropanoic acid, Compound E, with the structure of Formula XIII:
Figure imgf000034_0002
Formula XIII or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the present disclosure provides a masked Aib amino acid or amino acid analog of Formula VIII, wherein R1 is H, R2 is OH, and X is Se. In certain embodiments, the masked Aib amino acid or amino acid analog of Formula VIII is (R)-2-amino-3-((2-((((2-azidobenzyl)oxy)carbonyl)amino)ethyl)selanyl)-2-methylpropanoic acid, Compound F, and has the structure of Formula XIV:
Figure imgf000034_0003
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the present disclosure provides a masked Aib amino acid or amino acid analog of Formula III, wherein R1 is H, R2 is OH, X is S, and R3 is alkenyl. Active 121670349.1 33 Attorney Ref. No.070050.6868 In certain embodiments, the masked Aib amino acid or amino acid analog of Formula III is (R)-3-(allylthio)-2-amino-2-methylpropanoic acid, Compound G, and has the structure of Formula XV:
Figure imgf000035_0001
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the present disclosure provides a masked Aib amino acid or amino acid analog of Formula III, wherein R1 is H, R2 is OH, X is Se, and R3 is alkenyl. In certain embodiments, the masked Aib amino acid or amino acid analog of Formula III is (R)-3-(allylselanyl)-2-amino-2-methylpropanoic acid, Compound H, and has the structure of Formula XVI:
Figure imgf000035_0002
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the present disclosure provides a masked Aib amino acid or amino acid analog of Formula III, wherein R1 is H, R2 is OH, X is S, and R3 is alkynyl. In certain embodiments, the masked Aib amino acid or amino acid analog of Formula III is (R)-2-amino-2-methyl-3-(prop-2-yn-1-ylthio)propanoic acid, Compound I, and has the structure of Formula XVII:
Figure imgf000035_0003
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the present disclosure provides a masked Aib amino acid or amino acid analog of Formula III, wherein R1 is H, R2 is OH, X is Se, and R3 is alkynyl. Active 121670349.1 34 Attorney Ref. No.070050.6868 In certain embodiments, the masked Aib amino acid or amino acid analog of Formula III is (R)-2-amino-2-methyl-3-(prop-2-yn-1-ylselanyl)propanoic acid, Compound J, and has the structure of Formula XVIII:
Figure imgf000036_0001
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof of the present disclosure has the structure of any one of Formulas I-XVIII or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formula I or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formula II or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formula III or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formula IV or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formula V or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formula VI or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formula VII. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formula VIII or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formula IX. In certain embodiments, the masked Aib Active 121670349.1 35 Attorney Ref. No.070050.6868 amino acid or analog thereof has the structure of Formula X or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formula XI or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formula XII or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formula XIII or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formula XIV or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formula XV or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formula XVI or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formula XVII or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formula XVIII or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formulas IX-XVIII or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formulas IX, X, XI, XII, XIII, XIV, XV, XVI, XVII or XVIII or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. In certain embodiments, the masked Aib amino acid or analog thereof has the structure of Formulas IX, X or XI or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. Active 121670349.1 36 Attorney Ref. No.070050.6868 In particular embodiments, the masked Aib amino acid or analog of Formulas I-XVIII is an (R) or (S) enantiomer. In particular embodiments, the masked Aib amino acid or analog of Formulas I-XIII is an (R) enantiomer. In particular embodiments, the masked Aib amino acid or analog of Formulas I-XIII is an (S) enantiomer. The present disclosure further provides compositions comprising one or more masked Aib amino acid or an analog thereof described herein. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof that has a structure selected from the group consisting of Formulas I-XVIII. In certain embodiments, a composition of the present disclosure includes comprises one compound, two compounds, three compounds, four compounds, five compounds, six compounds, seven compounds, eight compounds, nine compounds, or ten compounds selected from the group consisting of Formulas I-XVIII. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof that has the structure of Formula I. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof that has the structure of Formula II. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula III. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula IV. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula V. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula VI. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula VII. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula VIII. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula IX. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula X. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula XI. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula XII. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the Active 121670349.1 37 Attorney Ref. No.070050.6868 structure of Formula XIII. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula XIV. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula XV. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula XVI. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula XVII. In certain embodiments, a composition of the present disclosure includes a masked Aib amino acid analog thereof having the structure of Formula XVIII. The present disclosure further provides a cell culture medium comprising one or more of the masked Aib amino acids or analogs thereof disclosed herein. For example, but not by way of limitation, a cell culture medium comprising two or more, three or more, five or more, six or more, seven or more, eight or more, nine or more or ten or more masked Aib amino acids or analogs thereof of the present disclosure. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog thereof described herein. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having a structure of any one of Formulas I-XVIII. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having a structure of any one of Formulas I-XVIII. For example, but not by way of limitation, a cell culture medium of the present disclosure comprises one masked Aib amino acid, two masked Aib amino acids, three masked Aib amino acids, four masked Aib amino acids, five masked Aib amino acids, six masked Aib amino acids, seven masked Aib amino acids, eight masked Aib amino acids, nine masked Aib amino acids or ten masked Aib amino acids selected from the group consisting of Formulas I-XVIII. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula I. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula II. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula III. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula IV. In certain embodiments, a Active 121670349.1 38 Attorney Ref. No.070050.6868 culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula V. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula VI. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula VII. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula VIII. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula IX. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula X. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula XI. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula XII. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula XIII. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula XIV. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula XV. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula XVI. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula XVII. In certain embodiments, a culture medium of the present disclosure includes at least one masked Aib or amino acid analog having the structure of Formula XVIII. The present disclosure further provides a composition comprising a buffer and a masked Aib amino acid or analog thereof described herein. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having a structure of any one of Formulas I-XVIII. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula I. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula II. In certain Active 121670349.1 39 Attorney Ref. No.070050.6868 embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula III. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula IV. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula V. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula VI. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula VII. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula VIII. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula IX. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula X. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula XI. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula XII. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula XIII. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula XIV. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula XV. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula XVI. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula XVII. In certain embodiments, a composition of the present disclosure includes a buffer and a masked Aib amino acid or analog thereof having the structure of Formula XVIII. The masked Aib amino acids disclosed herein can be synthesized using synthesis strategies known in the art. In certain embodiments, the masked Aib amino acids disclosed herein can be generated using the synthesis schemes provided in Example 1. For example, but not by way of Active 121670349.1 40 Attorney Ref. No.070050.6868 limitation, a masked Aib amino acid disclosed herein can be synthesized using scheme 1, scheme 2, scheme 3 or scheme 4 shown in Example 1. In certain embodiments, a masked Aib amino acid disclosed herein can be synthesized using scheme 1 shown in Example 1. In certain embodiments, a masked Aib amino acid disclosed herein can be synthesized using scheme 2 shown in Example 1. In certain embodiments, a masked Aib amino acid disclosed herein can be synthesized using scheme 3 shown in Example 1. In certain embodiments, a masked Aib amino acid disclosed herein can be synthesized using scheme 4 shown in Example 1. In certain embodiments, compound A (i.e., Formula IX) can be synthesized using scheme 1 shown in Example 1. In certain embodiments, compound B (i.e., Formula X) can be synthesized using scheme 2 shown in Example 1. III. Therapeutics The present disclosure provides methods for producing a therapeutic including one or more of the Aib amino acids and/or masked Aib amino acids disclosed herein. In certain embodiments, the present disclosure provides methods for producing a peptide or protein therapeutic including one or more of the Aib amino acids and/or masked Aib amino acids disclosed herein in recombinant cells. In certain embodiments, the peptide or protein therapeutic includes one or more Aib amino acids and/or masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic includes two or more Aib amino acids and/or masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic includes three or more Aib amino acids and/or masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic includes four or more Aib amino acids and/or masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic includes five or more Aib amino acids and/or masked Aib amino acids. In certain embodiments, the therapeutic can be a peptide or a derivative thereof or a functional fragment thereof. In certain embodiments, the peptide therapeutic can include at least about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90 or about 100 amino acids. In certain embodiments, the peptide therapeutic can include from about 10 to about to about 50 amino acids. In certain embodiments, the peptide therapeutic can include from about 15 to about to about 50 amino acids. In certain embodiments, the peptide therapeutic can Active 121670349.1 41 Attorney Ref. No.070050.6868 include from about 20 to about to about 50 amino acids. In certain embodiments, the peptide therapeutic can include from about 25 to about to about 50 amino acids. In certain embodiments, the peptide therapeutic can include from about 25 to about to about 45 amino acids. In certain embodiments, the peptide therapeutic can include from about 30 to about to about 45 amino acids. In certain embodiments, the peptide therapeutic can include from about 30 to about to about 40 amino acids. In certain embodiments, the peptide, e.g., peptide therapeutic can have a molecular weight of less than about 5 kD. In certain embodiments, the therapeutic can be a protein or a derivative thereof or a functional fragment thereof. The proteins disclosed herein refer to a sequence of amino acids for which the chain length is sufficient to produce the higher levels of tertiary and/or quaternary structure. This is to distinguish from “peptides” that typically do not have such structure. In certain embodiments, the protein, e.g., protein therapeutic can have a molecular weight of at least about 5-100 kD, e.g., closer to about 15 kD. In certain embodiments, the protein therapeutic can include at least about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500 amino acids, about 1,000 amino acids, about 1,500 amino acids, about 2,000 amino acids, about 2,500 amino acids, about 3,000 amino acids, about 35,000 amino acids or about 40,000 amino acids. Non-limiting examples of protein therapeutics include all proteins, and, in general proteins that contain one or more disulfide bonds, including multi-chain polypeptides comprising one or more inter- and/or intrachain disulfide bonds. In certain embodiments, the peptide or protein therapeutic can include other post- translation modifications including, but not limited to, glycosylation and lipidation. See, e.g., Prabakaran et al., WIREs Syst Biol Med (2012), which is incorporated herein by reference in its entirety. In certain embodiments, the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for treating diabetes, for treating obesity, for weight loss and/or for reducing the risk of cardiovascular events. In certain embodiments, the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for treating diabetes. In certain embodiments, the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for treating type 2 diabetes. Active 121670349.1 42 Attorney Ref. No.070050.6868 In certain embodiments, the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for treating obesity. In certain embodiments, the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for reducing the risk of cardiovascular events. In certain embodiments, the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for reducing the risk of heart attack. In certain embodiments, the peptide or protein generated by the methods of the present disclosure is a therapeutic protein or peptide for reducing the risk of stroke. In certain embodiments, the therapeutic protein or peptide can be a glucagon-like peptide- 1 (GLP-1) agonist, a GLP-1 analog, a GLP-1 receptor agonist, a glucose-dependent insulinotropic polypeptide (GIP) agonist, a GIP receptor agonist or a dual GLP-1/GIP receptor agonist. In certain embodiments, the therapeutic protein or peptide is a GLP-1 analog. In certain embodiments, the therapeutic protein or peptide is a GLP-1 agonist. In certain embodiments, the therapeutic protein or peptide is a GLP-1 receptor agonist. In certain embodiments, the therapeutic protein or peptide is a GIP agonist. In certain embodiments, the therapeutic protein or peptide is a GIP receptor agonist. In certain embodiments, the therapeutic protein or peptide is a dual GLP-1/GIP receptor agonist. In certain embodiments, the therapeutic protein or peptide is semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the GLP-1 receptor agonist can be semaglutide. Semaglutide shares a 94% structural homology with native human GLP-1, and is distinguished by three modifications: 1) amino-acid substitutions at position 8 (alanine to α-aminoisobutyric acid or Aib) and position 34 (lysine to arginine), and acylation of the lysine in position 26 with a spacer consisting of two 8-amino-3,6-dioxaoctanoic acid (ADO) moieties, a glutamic acid moiety, and a C-18 fatty di-acid side chain (as shown in FIG.1) (see Lau et al. Journal of Medicinal Chemistry 58(18): 7370–7380 (2015)). In certain embodiments, the peptide therapeutic can be tirzepatide. In certain embodiments, tirzepatide has the amino acid structure shown in FIG.2. In certain embodiments, the peptide therapeutic can be retatrutide. In certain embodiments, retatrutide has the amino acid structure shown in FIG.3. Active 121670349.1 43 Attorney Ref. No.070050.6868 In certain embodiments, the peptide therapeutic can be VK2735 (Viking Therapeutics). In certain embodiments, retatrutide has the amino acid structure shown in Table 1. In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous to a sequence comprising the sequence of any one of the above-noted peptides or proteins. In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous to a sequence comprising the sequence of any one of the therapeutics disclosed herein. In certain embodiments, the therapeutic comprises an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% or 100% homologous to a sequence provided in Table 1. In certain embodiments, the therapeutic comprises an amino acid sequence that is at least about 97% homologous to a sequence provided in Table 1. In certain embodiments, the therapeutic comprises an amino acid sequence that is at least about 98% homologous to a sequence provided in Table 1. In certain embodiments, the therapeutic comprises an amino acid sequence that is at least about 99% homologous to a sequence provided in Table 1. In certain embodiments, the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence provided in Table 1. In certain embodiments, the therapeutic comprises an amino acid sequence provided in Table 1. Table 1
Figure imgf000045_0001
Active 121670349.1 44 Attorney Ref. No.070050.6868
Figure imgf000046_0001
In certain embodiments, VK2735 comprises the amino acid structure of Tirzepatide, represented in FIG.3 and SEQ ID NO: 3. In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous, e.g., about 97% to about 100% homologous, to a sequence comprising the sequence of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the therapeutic comprises an amino acid sequence that is at least about 97% homologous to a sequence of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the therapeutic comprises an amino acid sequence that is at least about 98% homologous to a sequence of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the therapeutic comprises an amino acid sequence that is at least about 99% homologous to a sequence of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the therapeutic comprises an amino acid sequence of SEQ ID NOs: 1-3 and 9-11. Active 121670349.1 45 Attorney Ref. No.070050.6868 In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous, e.g., about 97% to about 100% homologous, to a sequence comprising the sequence of SEQ ID NO: 1. In certain embodiments, the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NO: 1. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous, e.g., about 97% to about 100% homologous, to a sequence comprising the sequence of SEQ ID NO: 2. In certain embodiments, the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NO: 2. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous, e.g., about 97% to about 100% homologous, to a sequence comprising the sequence of SEQ ID NO: 3. In certain embodiments, the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NO: 3. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at Active 121670349.1 46 Attorney Ref. No.070050.6868 least about 97%, at least about 98%, at least about 99% or 100% homologous, e.g., about 97% to about 100% homologous, to a sequence comprising the sequence of SEQ ID NO: 9. In certain embodiments, the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NO: 9. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 9, where one or more amino acids of the therapeutic have been substituted by an Aib amino acid. In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous, e.g., about 97% to about 100% homologous, to a sequence comprising the sequence of SEQ ID NO: 10. In certain embodiments, the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NO: 10. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the therapeutic is a peptide or protein comprising an amino acid sequence that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous, e.g., about 97% to about 100% homologous, to a sequence comprising the sequence of SEQ ID NO: 11. In certain embodiments, the therapeutic comprises an amino acid sequence that is from about 98% to about 100% homologous to a sequence of SEQ ID NO: 11. In certain embodiments, the therapeutic comprises the amino acid sequence of SEQ ID NO: 11. IV. Recombinant Cells The present disclosure further provides recombinant cells and compositions thereof for use in the disclosed methods. For example, but not by way of limitation, the present disclosure provides recombinant cells and compositions thereof for synthesizing a peptide or protein therapeutic comprising one or more masked one or more masked Aib amino acids (or derivatives thereof). Active 121670349.1 47 Attorney Ref. No.070050.6868 In certain embodiments, the present disclosure provides recombinant cells and compositions thereof for synthesizing a glucagon-like peptide-1 (GLP-1) agonist, a GLP-1 analog, a GLP-1 receptor agonist, a glucose-dependent insulinotropic polypeptide (GIP) agonist, a GIP receptor agonist or a dual GLP-1/GIP receptor agonist that includes one or more one or more masked Aib amino acids (or derivatives thereof). In certain embodiments, the present disclosure provides recombinant cells and compositions thereof for synthesizing a GLP-1 analog. In certain embodiments, the present disclosure provides recombinant cells and compositions thereof for synthesizing a GLP-1 receptor agonist. In certain embodiments, the present disclosure provides recombinant cells and compositions thereof for synthesizing semaglutide. In certain embodiments, the present disclosure provides recombinant cells and compositions thereof for synthesizing tirzepatide. In certain embodiments, the present disclosure provides recombinant cells and compositions thereof for synthesizing retatrutide. In certain embodiments, the present disclosure provides recombinant cells and compositions thereof for synthesizing VK2735 (Viking Therapeutics). In certain embodiments, the recombinant cells for use in expressing a protein or protein therapeutic that comprises one or more masked Aib amino acids (or derivatives thereof) can be a mammalian cell, a plant cell, a bacterial cell or a fungal cell. For example, but not by way of limitation, the cell can be a mammalian cell, e.g., a genetically engineered mammalian cell. In certain embodiments, the cell can be a plant cell, e.g., a genetically engineered plant cell. In certain embodiments, the cell can be a bacterial cell, e.g., a genetically engineered bacterial cell. In certain embodiments, the cell can be a fungal cell, e.g., a genetically engineered fungal cell. In certain embodiments, the recombinant cell of the present disclosure is a bacterial cell. Non-limiting examples of bacteria include Caulobacter crescentus, Rodhobacter sphaeroides, Pseudoalteromonas haloplanktis, Shewanella sp. strain Ac10, Pseudomonas fluorescens, Pseudomonas aeruginosa, Halomonas elongata, Chromohalobacter salexigens, Streptomyces lividans, Streptomyces griseus, Nocardia lactamdurans, Mycobacterium smegmatis, Corynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacterium lactofermentum, Bacillus subtilis, Bacillus brevis, Bacillus megaterium, Bacillus licheniformis, Bacillus Active 121670349.1 48 Attorney Ref. No.070050.6868 amyloliquefaciens, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus gasseri and Escherichia coli. In certain embodiments, the bacteria cell is Escherichia coli. In certain embodiments, the cell can be a fungal cell, e.g., a genetically engineered fungal cell. In certain embodiments, the genetically engineered fungal cell of the present disclosure is a species of phylum Ascomycota. In certain embodiments, the species of the phylum Ascomycota is selected from Saccharomyces cerevisiae, Saccharomyces castellii, Saccharomyces var boulardii, Vanderwaltozyma polyspora, Torulaspora delbrueckii, Saccharomyces kluyveri, Kluyveromyces lactis, Zygosaccharomyces rouxii, Zygosaccharomyces bailii, Candida glabrata, Ashbya gossypii, Scheffersomyces stipites, Komagataella (Pichia) pastoris, Candida (Pichia) guilliermondii, Candida parapsilosis, Candida auris, Yarrowia lipolytica, Candida (Clavispora) lusitaniae, Candida albicans, Candida tropicalis, Candida tenuis, Lodderomyces elongisporous, Geotrichum candidum, Baudoinia compniacensis, Schizosaccharomyces octosporus, Tuber melanosporum, Aspergillus oryzae, Schizosaccharomyces pombe, Aspergillus (Neosartorya) fischeri, Pseudogymnoascus destructans, Schizosaccharomyces japonicus, Paracoccidioides brasiliensis, Mycosphaerella graminicola, Penicillium chrysogenum, Aspergillus nidulans, Phaeosphaeria nodorum, Hypocrea jecorina, Botrytis cinereal, Beauvaria bassiana, Neurospora crassa, Sporothrix scheckii, Magnaporthe oryzea, Dactylellina haptotyla, Fusarium graminearum, Capronia coronate and combinations thereof. In certain embodiments, the genetically-engineered cell of the present disclosure is a species of the Saccharomyces genus. In certain embodiments, the recombinant cell of the present disclosure is a species of the Saccharomyces genus. In certain embodiments, the species from the Saccharomyces genus is selected from the group consisting of Saccharomyces bayanus, Saccharomyces boulardii, Saccharomyces castellii, Saccharomyces cerevisiae, Saccharomyces dairenensis and Saccharomyces mikatae. In certain embodiments, the genetically-engineered cell of the present disclosure is Saccharomyces cerevisiae. In certain embodiments, the genetically-engineered cell of the present disclosure is Saccharomyces boulardii. In certain embodiments, the recombinant cell of the present disclosure is a species from the Pichia genus. In certain embodiments, species from the Pichia genus is selected from the group consisting of Pichia acacia, Pichia alni, Pichia americana, Pichia amethionina, Pichia amylophila, Pichia angophorae, Pichia angusta, Pichia anomala, Pichia antillensis, Pichia Active 121670349.1 49 Attorney Ref. No.070050.6868 barkeri, Pichia besseyi, Pichia bimundalis, Pichia bispora, Pichia bovis, Pichia cactophila, Pichia canadensis, Pichia capsulate, Pichia caribaea, Pichia castillae, Pichia chambardii, Pichia ciferrii, Pichia delftensis, Pichia deserticola, Pichia dryadoides, Pichia euphorbiae, Pichia euphorbiiphila, Pichia fabianii, Pichia farinose, Pichia fermentans, Pichia finlandica, Pichia fluxuum, Pichia galaeiformis, Pichia glucozyma, Pichia guilliermondii, Pichia hampshirensis, Pichia haplophila, Pichia heedii, Pichia heimii, Pichia henricii, Pichia holstii, Pichia inositovora, Pichia jadinii, Pichia japonica, Pichia kluyveri, Pichia kodamae, Pichia lynferdii, Pichia maganishii, Pichia media, Pichia membranifaciens, Pichia methanolica, Pichia methylivoria, Pichia Mexicana, Pichia meyerae, Pichia minuta, Pichia mississippiensis, Pichia nakasei, Pichia nakazawae, Pichia norvegensis, Pichia ofunaensis, Pichia ohmeri, Pichia onychis, Pichia opuntiae, Pichia pastoris, Pichia petersonii, Pichia philodendra, Pichia philogaea, Pichia pijperi, Pichia pini, Pichia populi, Pichia pseudocactophila, Pichia quercuum, Pichia rabaulensis, Pichia rhodanensis, Pichia salicaria, Pichia scolyti, Pichia segobiensis, Pichia silvicola, Pichia spartinae, Pichia stipites, Pichia strasburgensis, Pichia subpelliculosa, Pichia sydowiorum, Pichia tannicola, Pichia thermotolerans, Pichia toletana, Pichia trehalophila, Pichia triangularis, Pichia veronae, Pichia wickerhamii and Pichia xylosa. In certain embodiments, the genetically- engineered cell of the present disclosure is Pichia pastoris. In certain embodiments, the recombinant cell of the present disclosure is a species of the Kluyveromyces genus. In certain embodiments, the genetically-engineered cell of the present disclosure is Kluyveromyces lactis. In certain embodiments, the recombinant cell of the present disclosure is a mammalian cell. Non-limiting examples of mammalian cells include monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; FS4 cells; MCF-7 cells; 3T3 cells; U2SO cells; Chinese hamster ovary (CHO) cells and myeloma cell lines such as Y0, NS0 and Sp2/0. In certain embodiments, the recombinant cell of the present disclosure is not a mammalian Active 121670349.1 50 Attorney Ref. No.070050.6868 cell. In certain embodiments, the recombinant cells express and/or secrete a protein or peptide therapeutic that includes an Aib amino acid (or derivative thereof) or a masked Aib amino acid (or derivative thereof) at high levels. In certain embodiments, the recombinant cells express and/or secrete a protein or peptide therapeutic that includes an Aib amino acid (or derivative thereof) or a masked Aib amino acid (or derivative thereof) at levels greater than about 10 mg/L, e.g., greater than about 50 mg/L, greater than about 100 mg/L, greater than about 250 mg/L, greater than about 500 mg/L, greater than about 750 mg/L, greater than about 1 g/L, greater than about 10 g/L, greater than about 25 g/L, greater than about 50 g/L or greater than about 100 g/L. In certain embodiments, the recombinant cells express and/or secrete a protein or peptide therapeutic that includes an Aib amino acid (or derivative thereof) or a masked Aib amino acid (or derivative thereof) at levels from about 10 mg/L to about 100 g/L. In certain embodiments, the recombinant cells that have been genetically engineered to express one or more protein and/or peptide therapeutics. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express two or more, three or more, five or more, six or more, seven or more, eight or more, nine or more or ten or more protein and/or peptide therapeutics. For example, but not by way of limitation, a cell can be genetically engineered to express a protein or protein therapeutic that comprises one or more masked Aib amino acids (or derivatives thereof). In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics disclosed in Table 1. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express semaglutide. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express tirzepatide. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express retatrutide. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express VK2735 (Viking Therapeutics). In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of Active 121670349.1 51 Attorney Ref. No.070050.6868 any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 9 (e.g., where one or more amino acids of the therapeutic have been substituted by an Aib amino acid). In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express one or more therapeutics comprising the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the recombinant cell comprises a polynucleotide that encodes a protein or peptide therapeutic and includes at least one selector codon. For example, but not by way of limitation, a cell can be genetically engineered to express a protein or protein therapeutic that comprises one or more masked Aib amino acids (or derivatives thereof). As described herein, incorporation of the masked amino acid or analog thereof will occur during translation of the nucleic acid that comprises at least one selector codon and encodes a protein. In certain embodiments, the masked Aib amino acid is incorporated into the one or more specified positions in the peptide or protein during translation of the nucleic acid that comprises at least one selector codon and encodes a protein. In certain embodiments, the selector codon is a stop codon. In certain embodiments, the selector codon is selected from the group consisting of UAG, UAA and UGA. In certain embodiments, the selector codon is UAG. In certain embodiments, the selector codon is UGA. In certain embodiments, the selector codon is UAA. In certain embodiments, the selector codon is a 4-base codon. Non-limiting examples of 4-base codons include AGGA, AGUA, AGGU, CGGU, CCCU, CUCU, CUAU, CUAG and GGGU (see, also Shandell et al., Biochemistry 60(46): 3455–3469 (2021), the contents of which are incorporated herein by reference in its entirety). In certain embodiments, the selector codon is Active 121670349.1 52 Attorney Ref. No.070050.6868 AGGU. In certain embodiments, the selector codon is CGGU. In certain embodiments, the selector codon is CCCU. In certain embodiments, the selector codon is CUCU. In certain embodiments, the selector codon is CGGU. In certain embodiments, the selector codon is CUAU. In certain embodiments, the selector codon is CGGU. In certain embodiments, the selector codon is GGGU. In certain embodiments, the selector codon is selected from the group consisting of UUU, UUA, UUG, UCA, CUG, AAA, UCG, UAU, GCA, UCU, CUG, AUG, UUA, AGA, AGG, CGU, CGC, CGA and CGG. Additional non-limiting of examples of codons that can be used as selector codons in the methods of the present disclosure can be found in Fredens et al. Nature 569:514– 518 (2019) and Mukai et al., Annu Rev Microbiol. 71: 557–577 (2017) (see Supplemental Table 1), the contents of each of which is incorporated herein in their entireties. In certain embodiments, the recombinant cells that have been genetically engineered to further express one or more aminoacyl-tRNA synthetases. For example, but not by way of limitation, the recombinant cells that have been genetically engineered to further express one or more orthogonal aminoacyl-tRNA synthetase (O-RS). In certain embodiments, the recombinant cell further comprises at least one or more polynucleotides encoding an orthogonal tRNA (O- tRNA). In certain embodiments, the polynucleotide encoding the orthogonal tRNA (O-tRNA) and the polynucleotide encoding the orthogonal aminoacyl-tRNA synthetase (O-RS) are included in a single nucleic acid. Alternatively or additionally, the polynucleotide encoding the orthogonal tRNA (O-tRNA) is included in a first nucleic acid and the polynucleotide encoding the orthogonal aminoacyl-tRNA synthetase (O-RS) is included in a second nucleic acid. In certain embodiments, the O-tRNA functions in the recombinant cell and recognizes the selector codon of the polynucleotide encoding the protein or peptide therapeutic and the O-RS aminoacylates the O- tRNA with a masked Aib amino acid described herein. In certain embodiments, the polynucleotides (e.g., the polynucleotide encoding the orthogonal tRNA (O-tRNA) and/or the polynucleotide encoding the orthogonal aminoacyl-tRNA synthetase (O-RS) are integrated into the chromosome, e.g., for stability. In certain embodiments, the O-RS can be homologous to a synthetase known in the art. In certain embodiments, a synthetase useful in the present disclosure includes one or more, two or more, three or more or all four of the following features: lacks an editing domain, low selectivity for anticodon, has only minor interactions with amine and can accommodate diverse side chains. Active 121670349.1 53 Attorney Ref. No.070050.6868 In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95%, or 99% homologous to a synthetase known in the art. In certain embodiments, the synthetase is selected from pyrrolysyl-tRNA synthetase, a tyrosyl-tRNA synthetase, a glutaminyl-tRNA synthetase and a leucyl-tRNA synthetase. In certain embodiments, the synthetase is an Escherichia coli synthetase or a Saccharomyces cerevisiae synthetase. In certain embodiments, the pyrrolysyl-tRNA synthetase is derived from the Methanococcus genus, e.g., Methanosarcina mazei, Methanosarcina barkeri or Methanococcus jannaschii. In certain embodiments, the Escherichia coli synthetase is Escherichia coli glutaminyl-tRNA synthetase or a tyrosyl-tRNA synthetase or a leucyl-tRNA synthetase. In certain embodiments, the Saccharomyces cerevisiae synthetase is a Saccharomyces cerevisiae glutaminyl-tRNA synthetase. Additional non-limiting examples of tRNA synthetases (and O-RS/O-tRNA pairs for use in the present disclosure) are provided in Shandell et al., Biochemistry 60(46): 3455–3469 (2021) and Dumas et al., Chem Sci 6(1):50-69 (2015), the contents of each of which are incorporated herein by reference in their entireties. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl-tRNA synthetase, a tyrosyl-tRNA synthetase, a leucyl-tRNA synthetase or a glutaminyl-tRNA synthetase (e.g., an Escherichia coli glutaminyl-tRNA synthetase or a Saccharomyces cerevisiae glutaminyl-tRNA synthetase). In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a pyrrolysyl-tRNA synthetase. Active 121670349.1 54 Attorney Ref. No.070050.6868 In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a pyrrolysyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a pyrrolysyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a pyrrolysyl- tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a pyrrolysyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a pyrrolysyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises the amino acid sequence of a pyrrolysyl-tRNA synthetase derived from the Methanococcus genus. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of a Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the Methanosarcina mazei pyrrolysyl- tRNA synthetase comprises the amino acid sequence set forth in SEQ ID NO: 5, 6 or 12. In certain embodiments, the Methanosarcina mazei pyrrolysyl-tRNA synthetase comprises the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the Methanosarcina mazei pyrrolysyl-tRNA synthetase comprises the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the Methanosarcina mazei pyrrolysyl-tRNA synthetase comprises the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Methanosarcina mazei Active 121670349.1 55 Attorney Ref. No.070050.6868 pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the Methanosarcina barkeri pyrrolysyl-tRNA synthetase comprises the amino acid sequence set forth in SEQ ID NO: 13 or 14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanococcus jannaschii pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a Methanococcus jannaschii pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Methanococcus jannaschii pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Methanococcus jannaschii pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Methanococcus jannaschii pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater Active 121670349.1 56 Attorney Ref. No.070050.6868 than about 99% homologous to the sequence of a Methanococcus jannaschii pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a Methanococcus jannaschii pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase. In certain embodiments, the Methanomethylophilus alvus pyrrolysyl-tRNA synthetase comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a glutaminyl-tRNA synthetase. Active 121670349.1 57 Attorney Ref. No.070050.6868 In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to an Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of an Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of an Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of an Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of an Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of an Escherichia coli glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of an Escherichia coli glutaminyl- tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a Saccharomyces cerevisiae glutaminyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% Active 121670349.1 58 Attorney Ref. No.070050.6868 homologous to the sequence of a tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a Methanococcus jannaschii tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to an E. coli tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of an E. coli tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of an E. coli tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of an E. coli tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence Active 121670349.1 59 Attorney Ref. No.070050.6868 that is greater than about 95% homologous to the sequence of an E. coli tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of an E. coli tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of an E. coli tyrosyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Saccharomyces cerevisiae synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a Saccharomyces cerevisiae synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a Saccharomyces cerevisiae synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a Saccharomyces cerevisiae. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a Saccharomyces cerevisiae. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a Saccharomyces cerevisiae. In certain embodiments, the O-RS comprises the amino acid sequence of a Saccharomyces cerevisiae. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of a leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of a leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of a leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of a leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of a leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of a leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to an Escherichia coli leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about Active 121670349.1 60 Attorney Ref. No.070050.6868 70% homologous to the sequence of an Escherichia coli leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of an Escherichia coli leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of an Escherichia coli leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of an Escherichia coli leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of an Escherichia coli leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises the amino acid sequence of an Escherichia coli leucyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a sequence provided in Table 2. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to a sequence provided in Table 2. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to a sequence provided in Table 2. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to a sequence provided in Table 2. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to a sequence provided in Table 2. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to a sequence provided in Table 2. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than Active 121670349.1 61 Attorney Ref. No.070050.6868 about 99% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises the amino acid sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the O-RS comprises the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 12. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 12. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 12. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 12. In certain embodiments, the O-RS comprises Active 121670349.1 62 Attorney Ref. No.070050.6868 an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 12. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 12. In certain embodiments, the O-RS comprises the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 13. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 13. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 13. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 13. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 13. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 13. In certain embodiments, the O-RS comprises the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 14. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 14. In certain embodiments, the O-RS comprises the amino acid sequence of SEQ ID NO: 14. Table 2
Figure imgf000064_0001
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Figure imgf000065_0001
Active 121670349.1 64 Attorney Ref. No.070050.6868
Figure imgf000066_0001
In certain embodiments, the recombinant cells that have been genetically engineered to express one or more O-tRNAs. In certain embodiments, the O-tRNA can be homologous to a tRNA known in the art. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70%, 80%, 90%, 95%, or 99% homologous to a tRNA known in the art. In certain embodiments, the tRNA is selected from a tRNATyr, a tRNALeu, a tRNAGln and a tRNAPyl. In certain embodiments, the tRNA is an Escherichia coli tRNA or a Saccharomyces cerevisiae tRNA. In certain embodiments, the tRNA is derived from the Methanococcus genus, e.g., Methanosarcina mazei, Methanosarcina barkeri or Methanococcus jannaschii. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a sequence provided in Table 3. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70% homologous to a sequence provided in Table 3. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 80% homologous to a sequence provided in Table 3. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 90% homologous to a sequence provided in Table 3. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to a sequence provided in Table 3. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 99% homologous to a sequence provided in Table 3. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a sequence of any one of SEQ ID NOs: 7-8 Active 121670349.1 65 Attorney Ref. No.070050.6868 and 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70% homologous to a sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 80% homologous to a sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 90% homologous to a sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to a sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 99% homologous to a sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, the O-tRNA comprises a nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is Active 121670349.1 66 Attorney Ref. No.070050.6868 greater than about 99% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, the O-tRNA comprises a nucleotide sequence of SEQ ID NO: 8. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 70% homologous to the sequence of SEQ ID NO: 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 80% homologous to the sequence of SEQ ID NO: 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 90% homologous to the sequence of SEQ ID NO: 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 15. In certain embodiments, the O-tRNA comprises a nucleotide sequence of SEQ ID NO: 15. Table 3
Figure imgf000068_0001
In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanosarcina mazei pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanosarcina mazei tRNA. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 95% homologous to a Methanosarcina mazei pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 95% homologous to a Methanosarcina mazei tRNA. In certain embodiments, a recombinant cell of the present Active 121670349.1 67 Attorney Ref. No.070050.6868 disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 99% homologous to a Methanosarcina mazei pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 99% homologous to a Methanosarcina mazei tRNA. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic, e.g., a peptide or protein therapeutic disclosed in Table 1. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics) that comprises a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding semaglutide that comprises a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding tirzepatide that comprises a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding retatrutide that comprises a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding VK2735 that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising an amino acid of any one of SEQ ID NOs: 1-3 and 9- 11 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 1 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 2 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 3 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 9 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 10 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 11 and a selector codon. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 5 or 6 and (b) a Active 121670349.1 68 Attorney Ref. No.070050.6868 polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 5 or 6 and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 5 or 6 and (b) a polynucleotide encoding an O- tRNA comprising a nucleotide sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 7. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic, e.g., a peptide or protein therapeutic disclosed in Table 1 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the recombinant cell further comprises a polynucleotide encoding semaglutide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding tirzepatide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding retatrutide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding VK2735 that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising an amino acid of any one of SEQ ID NOs: 1-3 and 9-11 and comprises a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 1 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 2 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 3 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 9 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a Active 121670349.1 69 Attorney Ref. No.070050.6868 peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 10 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 11 and a selector codon. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanomethylophilus alvus tRNA. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 95% homologous to a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 95% homologous to a Methanomethylophilus alvus tRNA. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 99% homologous to a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 99% homologous to a Methanomethylophilus alvus tRNA. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic, e.g., a peptide or protein therapeutic disclosed in Table 1 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the recombinant cell further comprises a polynucleotide encoding semaglutide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding tirzepatide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding retatrutide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding VK2735 that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising an amino acid of any one of SEQ ID NOs: 1-3 and 9-11 and comprises a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 1 and a selector codon. In certain embodiments, the Active 121670349.1 70 Attorney Ref. No.070050.6868 recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 2 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 3 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 9 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 10 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 11 and a selector codon. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 4 and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 4 and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 4 and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 8. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanosarcina barkeri pyrrolysyl-tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 70%, 80%, 90%, 95% or 99% homologous to a Methanosarcina barkeri tRNA. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 95% homologous to a Methanosarcina barkeri pyrrolysyl-tRNA synthetase and (b) a Active 121670349.1 71 Attorney Ref. No.070050.6868 polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 95% homologous to a Methanosarcina barkeri tRNA. In certain embodiments, a recombinant cell of the present disclosure comprises (a) a polynucleotide encoding an O-RS comprising an amino acid sequence that is greater than about 99% homologous to a Methanosarcina barkeri pyrrolysyl- tRNA synthetase and (b) a polynucleotide encoding an O-tRNA comprising a nucleotide sequence that is greater than about 99% homologous to a Methanosarcina barkeri tRNA. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic, e.g., a peptide or protein therapeutic disclosed in Table 1 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the recombinant cell further comprises a polynucleotide encoding semaglutide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding tirzepatide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding retatrutide that includes a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising an amino acid of any one of SEQ ID NOs: 1-3 and 9-11 and comprises a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 1 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 2 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 3 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 9 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 10 and a selector codon. In certain embodiments, the recombinant cell further comprises a polynucleotide encoding a peptide or protein therapeutic comprising the amino acid of SEQ ID NO: 11 and a selector codon. In certain embodiments, one or more polynucleotides (or nucleic acids comprising one or more polynucleotides) of the present disclosure can be introduced into cells, e.g., bacteria cells, Active 121670349.1 72 Attorney Ref. No.070050.6868 using vectors, such as plasmid vectors and cell transformation techniques such as electroporation, heat shock and others known to those skilled in the art and described herein. In certain embodiments, one or more polynucleotides encoding an O-RS (or nucleic acids comprising one or more polynucleotides) of the present disclosure can be introduced into cells, e.g., bacteria cells, using vectors, such as plasmid vectors and cell transformation techniques such as electroporation, heat shock and others known to those skilled in the art and described herein. In certain embodiments, one or more polynucleotides encoding an O-tRNA (or nucleic acids comprising one or more polynucleotides) of the present disclosure can be introduced into cells, e.g., bacteria cells, using vectors, such as plasmid vectors and cell transformation techniques such as electroporation, heat shock and others known to those skilled in the art and described herein. In certain embodiments, one or more polynucleotides encoding a peptide or protein therapeutic (or nucleic acids comprising one or more polynucleotides) of the present disclosure can be introduced into cells, e.g., bacteria cells, using vectors, such as plasmid vectors and cell transformation techniques such as electroporation, heat shock and others known to those skilled in the art and described herein. In certain embodiments, the genetic molecular components are introduced into the cell to persist as a plasmid or integrate into the genome. For example, but not by way of limitation, the nucleic acid can be incorporated into the genome of the genetically-engineered cell. In certain embodiments, the cells can be engineered to chromosomally integrate a polynucleotide of one or more genetic molecular components described herein, using methods identifiable to skilled persons upon reading the present disclosure. In certain embodiments, one or more polynucleotides (or nucleic acids comprising one or more polynucleotides) can be inserted into the genome of a genetically engineered cell using a CRISPR/Cas9 system. In certain embodiments, a polynucleotide encoding one or more therapeutics is introduced into the recombinant cell, e.g., a recombinant E. coli cell, either as a construct or a plasmid. In certain embodiments, a polynucleotide (or a nucleic acid comprising the polynucleotide) can comprise one or more regulatory regions such as promoters, transcription factor binding sites, operators, activator binding sites, repressor binding sites, enhancers, protein-protein binding domains, RNA binding domains, DNA binding domains, and other control elements known to a person skilled in the art. For example, but not by way of limitation, a polynucleotide encoding a therapeutic of the present disclosure is introduced into the recombinant cell, e.g., a recombinant E. Active 121670349.1 73 Attorney Ref. No.070050.6868 coli cell, either as a construct or a plasmid in which it is operably linked to a promoter active in the recombinant cell, e.g., a recombinant E. coli cell, or such that it is inserted into the recombinant cell, e.g., a recombinant E. coli cell, genome at a location where it is operably linked to a suitable promoter. In certain embodiments, a polynucleotide encoding one or more O-RS and/or O-tRNA is introduced into the recombinant cell, e.g., a recombinant E. coli cell, either as a construct or a plasmid. In certain embodiments, a polynucleotide (or a nucleic acid comprising the polynucleotide) can comprise one or more regulatory regions such as promoters, transcription factor binding sites, operators, activator binding sites, repressor binding sites, enhancers, protein- protein binding domains, RNA binding domains, DNA binding domains, and other control elements known to a person skilled in the art. For example, but not by way of limitation, a polynucleotide encoding O-RS and/or O-tRNA of the present disclosure is introduced into the recombinant cell, e.g., a recombinant E. coli cell, either as a construct or a plasmid in which it is operably linked to a promoter active in the recombinant cell, e.g., a recombinant E. coli cell, or such that it is inserted into the recombinant cell, e.g., a recombinant E. coli cell, genome at a location where it is operably linked to a suitable promoter. In certain embodiments, a polynucleotide (or a nucleic acid comprising the polynucleotide) encoding one or more of the therapeutics, aminoacyl-tRNA synthetase and/or tRNA can further include a transcription factor for regulation expression of the therapeutic encoded by the nucleic acid. Alternatively and/or additionally, a second nucleic or an additional nucleic acid can be introduced into the cells to express a transcription factor for regulation expression of the therapeutic encoded by the nucleic acid. In certain embodiments, a polynucleotide (or a nucleic acid comprising the polynucleotide) encoding one or more of the therapeutics, aminoacyl-tRNA synthetase and/or tRNA can be inserted into the genome of the cell, e.g., cell. For example, but not by way of limitation, one or more nucleic acids encoding a peptide and/or protein therapeutic of the present disclosure, e.g., semaglutide, can be inserted into a locus of the cell. Additionally or alternatively, one or more nucleic acids encoding a therapeutic, aminoacyl-tRNA synthetase and/or tRNA of the present disclosure can be inserted into a locus of the cell. In certain embodiments, the one or more nucleic acids can be inserted into one or more loci that minimally affects the cell, e.g., in an intergenic locus or a gene that is not essential and/or does not affect growth, proliferation and cell signaling. Active 121670349.1 74 Attorney Ref. No.070050.6868 In certain embodiments, one or more endogenous genes of the genetically-engineered cells can be knocked out and/or mutated, e.g., knocked out by a genetic engineering system. Alternatively or additionally, extra copies of endogenous genes of the genetically-engineered cells can be knocked in, e.g., knocked in by a genetic engineering system. Various genetic engineering systems known in the art can be used. Non-limiting examples of such systems include the Clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas system, the zinc-finger nuclease (ZFN) system, the transcription activator-like effector nuclease (TALEN) system, use of yeast endogenous homologous recombination and the use of interfering RNAs. In certain non-limiting embodiments, a CRISPR/Cas9 system is employed to knock out and/or knock in one or more endogenous genes in the genetically engineered cell. When utilized for genome editing, the system includes Cas9 (a protein able to modify DNA utilizing crRNA as its guide), CRISPR RNA (crRNA, contains the RNA used by Cas9 to guide it to the correct section of host DNA along with a region that binds to tracrRNA (generally in a hairpin loop form) forming an active complex with Cas9) and trans-activating crRNA (tracrRNA, binds to crRNA and forms an active complex with Cas9). The terms “guide RNA” and “gRNA” refer to any nucleic acid that promotes the specific association (or “targeting”) of an RNA-guided nuclease such as a Cas9 to a target sequence such as a genomic or episomal sequence in a cell. gRNAs can be unimolecular (comprising a single RNA molecule and referred to alternatively as chimeric) or modular (comprising more than one, and typically two, separate RNA molecules, such as a crRNA and a tracrRNA, which are usually associated with one another, for instance by duplexing). In certain embodiments, a homolog of a nucleotide sequence disclosed herein can be a polynucleotide having changes in one or more nucleotide bases that can result in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide or protein encoded by the nucleotide sequence. Homologs can also include polynucleotides having modifications such as deletion, addition or insertion of nucleotides that do not substantially affect the functional properties of the resulting polynucleotide or transcript. Alterations in a polynucleotide that result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded polypeptide, are well known in the art. In certain embodiments, a homolog of a peptide, protein or O-RS disclosed herein can be a peptide, protein or O-RS having changes in one or more amino acids but do not affect the functional properties of the therapeutic. Alterations in a peptide, protein or O-RS that do not affect Active 121670349.1 75 Attorney Ref. No.070050.6868 the functional properties of the peptide, protein or O-RS, are well known in the art, e.g., conservative substitutions. It is therefore understood that the disclosure encompasses more than the specific exemplary polynucleotide or amino acid sequences and includes functional equivalents thereof. The cells to be used in the present disclosure can be genetically engineered using recombinant techniques known to those of ordinary skill in the art. Production and manipulation of the polynucleotides described herein are within the skill in the art and can be carried out according to recombinant techniques described, for example, in Sambrook et al.1989. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Innis et al. (eds). 1995. PCR Strategies, Academic Press, Inc., San Diego. The present disclosure further provides compositions of the recombinant cells disclosed herein. In certain embodiments, a composition of the present disclosure includes a plurality of the recombinant cells disclosed herein. V. Methods of Production The present disclosure further provides methods for producing a peptide or protein therapeutic with at least one Aib amino acid. For example, but not by way of limitation, methods of the present disclosure include expressing a peptide or protein therapeutic with at least one masked Aib amino acid and purifying the peptide or protein therapeutic with the at least one masked Aib amino acid. In certain embodiments, methods of the present disclosure further include removing the masking group from the masked Aib amino acid incorporated into the peptide or protein therapeutic to produce at least one peptide or protein therapeutic comprising one or more Aib amino acid (i.e., without the masking group). An exemplary schematic of a method of the present disclosure is provided in FIG. 7. In certain embodiments, the present disclosure provides methods using a cell, e.g., a genetically engineered cell, of the present disclosure to produce and/or secrete one protein or peptide therapeutic comprising at least one masked Aib amino acid. In certain embodiments, methods of the present disclosure use a cell, e.g., a genetically engineered cell, of the present disclosure to produce and/or secrete more than one protein or peptide therapeutic, e.g., two or more protein or peptide therapeutics, three or more protein or peptide therapeutics, four or more therapeutics or five or more therapeutics, where at least one of the one protein or peptide Active 121670349.1 76 Attorney Ref. No.070050.6868 therapeutics comprises one or more masked Aib amino acids. Non-limiting examples of masked Aib amino acids, therapeutic proteins or peptides and recombinant cells are described in Sections II, III, IV and V, respectively. A. Expression The present disclosure provides methods for expressing a peptide or protein therapeutic that comprises one or more masked Aib amino acids. For example, but not by way of limitation, the present disclosure provides methods for expressing a peptide or protein therapeutic that comprises one or more masked Aib amino acids in recombinant cells described herein. Non- limiting examples of masked Aib amino acids, therapeutic proteins or peptides and recombinant cells are described in Sections III, IV and V, respectively. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon in a cell culture medium comprising a masked Aib amino acid or an analog thereof (e.g., under conditions suitable for growth). In certain embodiments, the recombinant cell expresses an orthogonal tRNA (O-tRNA) that functions in the recombinant cell and recognizes the selector codon and an orthogonal aminoacyl-tRNA synthetase (O-RS), where the O-RS preferentially aminoacylates the O-tRNA with a masked Aib amino acid. In certain embodiments, the method further includes incorporating the masked Aib amino acid into the one or more specified positions (e.g., identified by the selector codon) in the peptide or protein therapeutic during translation of the polynucleotide to produce the peptide or protein therapeutic that comprises one or more of the masked Aib amino acids or the analog thereof. In certain embodiments, a method of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture medium under conditions suitable for cell growth. In certain embodiments, the method of the present disclosure further includes contacting the cell culture medium with a masked Aib amino acid or an analog thereof (e.g., under conditions suitable for incorporation of the masked Aib amino acid or analog thereof into the peptide or protein therapeutic) to produce the peptide or protein therapeutic that comprises one or more of the masked Aib amino acids or the analog thereof at the position indicated by the selector codon. In certain embodiments, the peptide or protein Active 121670349.1 77 Attorney Ref. No.070050.6868 therapeutic is a GLP-1 receptor agonist. In certain embodiments, the peptide or protein therapeutic is semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the peptide or protein therapeutic is semaglutide. In certain embodiments, the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain embodiments, the O- tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the masked Aib has the structure of any one of Formulas I-XVIII. In certain embodiments, the masked Aib amino acid is added to the cell culture medium at a concentration from about 1 mM to about 50 M, e.g., from about 10 mM to about 25 M, from about 10 mM to about 10 M, from about 10 mM to about 5 M, from about 10 mM to about 1 M, from about 10 mM to about 500 mM, from about 10 mM to about 250 mM, from about 10 mM to about 100 mM or from about 10 mM to about 50 mM. In certain embodiments, the masked Aib amino acid is added to the cell culture medium at a concentration from about 1 mM to about 1 M. In certain embodiments, the masked Aib amino acid is added to the cell culture medium at a concentration from about 1 mM to about 500 mM. In certain embodiments, the masked Aib amino acid is added to the cell culture medium at a concentration from about 1 mM to about 100 mM. In certain embodiments, the masked Aib amino acid is added to the cell culture medium at a concentration from about 1 mM to about 50 mM. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic is a GLP-1 receptor agonist. In certain embodiments, the peptide or protein therapeutic is semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the peptide or protein therapeutic is semaglutide. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to a pyrrolysyl-tRNA synthetase, an Escherichia coli synthetase, or a Saccharomyces cerevisiae synthetase. In certain embodiments, the O-tRNA comprises a nucleotide sequence of a pyrrolysyl-tRNA, an Escherichia coli tRNA, or a Saccharomyces cerevisiae tRNA. In certain embodiments, the masked Aib has the structure of any one of Formulas I-XVIII. Active 121670349.1 78 Attorney Ref. No.070050.6868 In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic is a GLP-1 receptor agonist. In certain embodiments, the peptide or protein therapeutic is semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the peptide or protein therapeutic is semaglutide. In certain embodiments, the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain embodiments, the O- tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the masked Aib has the structure of any one of Formulas I-XVIII. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic is a GLP-1 receptor agonist. In certain embodiments, the peptide or protein therapeutic is semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the peptide or protein therapeutic is semaglutide. In certain embodiments, the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain embodiments, the O- tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the masked Aib has the structure of Formula IX or Formula X. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the Active 121670349.1 79 Attorney Ref. No.070050.6868 O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain embodiments, the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the masked Aib has the structure of any one of Formulas I-XVIII. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain embodiments, the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the masked Aib has the structure of any one of Formulas I-XVIII. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain embodiments, the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the masked Aib has the structure of Formula IX. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain Active 121670349.1 80 Attorney Ref. No.070050.6868 embodiments, the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the masked Aib has the structure of Formula X. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain embodiments, the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the masked Aib has the structure of Formula XI. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain embodiments, the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the masked Aib has the structure of Formula XII. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain Active 121670349.1 81 Attorney Ref. No.070050.6868 embodiments, the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the masked Aib has the structure of Formula XIII. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain embodiments, the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the masked Aib has the structure of Formula XIV. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain embodiments, the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the masked Aib has the structure of Formula XV. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain Active 121670349.1 82 Attorney Ref. No.070050.6868 embodiments, the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the masked Aib has the structure of Formula XVI. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain embodiments, the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the masked Aib has the structure of Formula XVII. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain embodiments, the O-tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the masked Aib has the structure of Formula XVIII. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1. In certain embodiments, the O-RS comprises an amino acid sequence of any one of SEQ ID NOs: 4-6 and 12-14. In certain embodiments, the O- Active 121670349.1 83 Attorney Ref. No.070050.6868 tRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the masked Aib has the structure of any one of Formulas I-XVIII. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 4. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 8. In certain embodiments, the masked Aib has the structure of any one of Formulas I-XVIII. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises the amino acid of SEQ ID NO: 4. In certain embodiments, the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 8. In certain embodiments, the masked Aib has the structure of any one of Formulas I-XVIII. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino Active 121670349.1 84 Attorney Ref. No.070050.6868 acid of SEQ ID NO: 5. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked Aib has the structure of any one of Formulas I-XVIII. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises the amino acid of SEQ ID NO: 5. In certain embodiments, the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked Aib has the structure of any one of Formulas I-XVIII. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 6. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked Aib has the structure of any one of Formulas I-XVIII. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises an amino acid of any one of SEQ ID NOs: 1-3 and 9-11. In certain embodiments, the Active 121670349.1 85 Attorney Ref. No.070050.6868 O-RS comprises the amino acid of SEQ ID NO: 6. In certain embodiments, the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked Aib has the structure of any one of Formulas I-XVIII. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 4. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 8. In certain embodiments, the masked Aib has the structure of Formula IX. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1. In certain embodiments, the O-RS comprises the amino acid of SEQ ID NO: 4. In certain embodiments, the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 8. In certain embodiments, the masked Aib has the structure of Formula IX. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1. In certain embodiments, the O-RS comprises an Active 121670349.1 86 Attorney Ref. No.070050.6868 amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 5. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked Aib has the structure of Formula IX. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1. In certain embodiments, the O-RS comprises the amino acid of SEQ ID NO: 5. In certain embodiments, the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked Aib has the structure of Formula IX. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 6. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked Aib has the structure of Formula IX. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic Active 121670349.1 87 Attorney Ref. No.070050.6868 is semaglutide. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1. In certain embodiments, the O-RS comprises the amino acid of SEQ ID NO: 6. In certain embodiments, the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked Aib has the structure of Formula IX. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 4. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 8. In certain embodiments, the masked Aib has the structure of Formula X. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1. In certain embodiments, the O-RS comprises the amino acid of SEQ ID NO: 4. In certain embodiments, the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 8. In certain embodiments, the masked Aib has the structure of Formula X. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic Active 121670349.1 88 Attorney Ref. No.070050.6868 comprises the amino acid of SEQ ID NO: 1. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 5. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked Aib has the structure of Formula X. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1. In certain embodiments, the O-RS comprises the amino acid of SEQ ID NO: 5. In certain embodiments, the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked Aib has the structure of Formula X. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 6. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 4. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked Aib has the structure of Formula X. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one Active 121670349.1 89 Attorney Ref. No.070050.6868 or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1. In certain embodiments, the O-RS comprises the amino acid of SEQ ID NO: 6. In certain embodiments, the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked Aib has the structure of Formula X. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 4. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 8. In certain embodiments, the masked Aib has the structure of Formula XI. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1. In certain embodiments, the O-RS comprises the amino acid of SEQ ID NO: 4. In certain embodiments, the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 8. In certain embodiments, the masked Aib has the structure of Formula XI. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one Active 121670349.1 90 Attorney Ref. No.070050.6868 or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 5. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked Aib has the structure of Formula XI. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1. In certain embodiments, the O-RS comprises the amino acid of SEQ ID NO: 5. In certain embodiments, the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked Aib has the structure of Formula XI. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to the amino acid of SEQ ID NO: 6. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked Aib has the structure of Formula XI. In certain embodiments, methods of the present disclosure include culturing a recombinant cell that comprises (a) a polynucleotide encoding a peptide or protein therapeutic and includes at least one selector codon, (b) a polynucleotide encoding an orthogonal tRNA (O-tRNA) and (c) a polynucleotide encoding an orthogonal aminoacyl-tRNA synthetase (O-RS) in a cell culture media Active 121670349.1 91 Attorney Ref. No.070050.6868 comprising a masked Aib amino acid to express the peptide or protein therapeutic comprising one or more of the masked Aib amino acids. In certain embodiments, the peptide or protein therapeutic is semaglutide. In certain embodiments, the peptide or protein therapeutic comprises the amino acid of SEQ ID NO: 1. In certain embodiments, the O-RS comprises the amino acid of SEQ ID NO: 6. In certain embodiments, the O-tRNA comprises the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the masked Aib has the structure of Formula XI. B. Purification The present disclosure further provides methods for isolating or purifying the protein or peptide therapeutic from the cell culture comprising the recombinant cells. In certain embodiments, the present disclosure provides methods for isolating or purifying the protein or peptide therapeutic that comprises one or masked Aib amino acids from the cell culture. For example, but not by way of limitation, the protein or peptide therapeutic that comprises one or masked Aib amino acids is secreted from the cells and is isolated or purified from the cell culture media and/or supernatant. Alternatively or additionally, the recombinant cells are lysed and the protein or peptide therapeutic that comprises one or masked Aib amino acids are isolated or purified from the cell lysate. In certain embodiments, the isolated or purified protein or peptide therapeutic can be further processed to demask the masked Aib amino acids. In certain embodiments, the isolated or purified protein or peptide therapeutic can be further processed to post-translationally modify the protein or peptide therapeutic comprising the demasked Aib amino acids, e.g., addition of the lipid side chain (see, e.g., FIGs. 1-3). In certain embodiments, a protein or peptide therapeutic that comprises one or masked Aib amino acids are isolated or purified from the cell lysate, supernatant and/or culture media the includes the protein or peptide therapeutic comprising one or masked Aib amino acids using chromatography. For example, but not by way of limitation, the lysate, supernatant and/or culture media is passed over a chromatography column or solid support, so that the peptide or protein containing masked Aib covalently bonds or non-covalently bonds to the chromatography column or solid support. In certain embodiments when chromatography column is used for purification, the chromatography medium is selected to bind covalently or non-covalently to a moiety contained within the protein or peptide. In certain embodiments, the moiety that binds to the chromatography medium is contained within the masking group of the masked form of Aib present in the protein or peptide. Active 121670349.1 92 Attorney Ref. No.070050.6868 In certain embodiments, the moiety contained within the masking group that can be used for isolation and/purification of the peptide or protein is an azide group. In particular embodiments, an azide group binds covalently with the chromatography column or the solid support. In certain embodiments, the masking group (or moiety of the masking group) of the masked Aib amino acids covalently binds to the chromatography column or solid support using any suitable reaction. For example, but not by way of limitation, the reaction is a Staudinger ligation reaction, a Cu(I)-catalyzed azide-alkyne cycloaddition reaction, a Strain-promoted azide-alkyne cycloaddition reaction, a Strain-promoted alkyne-nitrone cycloaddition reaction, a Diels-Alder reaction, alkene-azide [3+2] cycloaddition reaction, an Inverse-demand Diels-Alder reaction or an alkene-tetrazine. In certain embodiments, the masking group (or moiety of the masking group) of the masked Aib amino acids covalently binds to the chromatography column or solid support using a Staudinger ligation reaction. In certain embodiments, the masking group (or moiety of the masking group) of the masked Aib amino acids covalently binds to the chromatography column or solid support using a Cu(I)-catalyzed azide-alkyne cycloaddition reaction. In certain embodiments, the masking group (or moiety of the masking group) of the masked Aib amino acids covalently binds to the chromatography column or solid support using a Strain-promoted azide- alkyne cycloaddition reaction. In certain embodiments, the masking group (or moiety of the masking group) of the masked Aib amino acids covalently binds to the chromatography column or solid support using a Strain-promoted alkyne-nitrone cycloaddition reaction. In certain embodiments, the masking group (or moiety of the masking group) of the masked Aib amino acids covalently binds to the chromatography column or solid support using a Diels-Alder reaction. In certain embodiments, the masking group (or moiety of the masking group) of the masked Aib amino acids covalently binds to the chromatography column or solid support using Alkene-azide [3+2] cycloaddition reaction. In certain embodiments, the masking group (or moiety of the masking group) of the masked Aib amino acids covalently binds to the chromatography column or solid support using an Inverse-demand Diels-Alder reaction. In certain embodiments, the masking group (or moiety of the masking group) of the masked Aib amino acids covalently binds to the chromatography column or solid support using an alkene-tetrazine Inverse-electron-demand Diels-Alder reaction. Active 121670349.1 93 Attorney Ref. No.070050.6868 When the reaction is a Staudinger ligation or a Strain-promoted azide-alkyne cycloaddition reaction, an azide group of the masking group will covalently bind to the chromatography column or solid support. When the reaction is an alkene-azide [3+2] cycloaddition reaction, an alkene or an azide group will covalently bind to the chromatography column or support. When the reaction is a Strain-promoted alkyne-nitrone cycloaddition reaction, an alkyne or nitrone group of the masking group will covalently bind to the chromatography column or solid support. When the reaction is a Cu(I)-catalyzed azide-alkyne cycloaddition reaction, an azide or an alkyne group of the masking group will covalently bind to the chromatography column or solid support. When the reaction is a Diels Alder or Inverse-electron-demand Diels-Alder, the alkene group of the masking group will covalently bind to the chromatography column or solid support. In certain embodiments, methods of the present disclosure can further include removing the masking group from the masked Aib amino acids in the peptide or protein which contains the masked Aib amino acid. The masking group can be removed using any suitable demasking reaction. In certain embodiments, the reaction is a desulfurization reaction or a deselenization reaction. In certain embodiments, methods of the present disclosure can include removing the masking group from the masked Aib amino acids in the peptide or protein which contains the masked Aib amino acids using a desulfurization reaction. In certain embodiments, methods of the present disclosure can include removing the masking group from the masked Aib amino acids in the peptide or protein which contains the masked Aib amino acids using a deselenization reaction. Any suitable catalyst can be used for the demasking reaction, including, but not limited to, metal reagent(s). In certain embodiments, the catalyst used for the demasking reaction is Pd/Al2O3, Pd/Carbon, Raney Nickel, Pd/BaSO4, PdO, Ni(0), palladium catalyst, Pd(TPPS)4, nickel boride, tin hydride, or combinations thereof. In certain embodiments, the demasking reaction is a desulfurization reaction, which is performed using a Raney nickel, sodium hypophosphite, acetate buffer system, as shown in Scheme 11. In particular embodiments, the demasking reaction is a desulfurization reaction, which is performed with UV light, as shown in Scheme 12. In certain embodiments, the demasking reaction is a desulfurization reaction, which is performed with electrolysis, as shown in Scheme 13. In certain embodiments, the demasking reaction is a deselenization reaction, which is performed using a Raney nickel, sodium hypophosphite, acetate buffer system, as shown in Scheme 14. In particular embodiments, the demasking reaction is a Active 121670349.1 94 Attorney Ref. No.070050.6868 deselenization reaction, which is performed with UV light, as shown in Scheme 15. In certain embodiments, the demasking reaction is a deselenization reaction, which is performed with electrolysis, as shown in Scheme 16. In certain embodiments, the demasking reaction comprises a allyl removal reaction with a suitable palladium catalyst followed by a desulfurization or deselenization reaction. In certain embodiments, the demasking reaction comprises a propargyl removal reaction with a Pd(TPPS)4 catalyst followed by a desulfurization or deselenization reaction. In certain embodiments, the product of the demasking reaction is a protein or peptide containing unmasked Aib amino acids. In certain embodiments, the resulting protein or peptide containing Aib residues can be analyzed by any suitable analytical method. Such analytical methods include, but are not limited to, Nuclear Magnetic Resonance (NMR) or Mass Spectrometry (MS). When the analytical method MS is used, the appropriate fragmentation pattern will be observed in the resulting spectra, confirming the presence of the polypeptide or peptide containing native Aib residues. C. Pharmaceutical Compositions In certain embodiments, the protein or peptide therapeutic containing unmasked-Aib amino acid residues (i.e., native Aib amino acid residues) produced by a method of the present disclosure can be formulated into a pharmaceutical composition. For example, but not by way of limitation, the present disclosure provides a pharmaceutical composition comprising a therapeutic protein or peptide, where the therapeutic protein or peptide can treat diabetes (e.g., type 2 diabetes), treat obesity, or be used for weight loss and/or reducing the risk of cardiovascular events. In certain embodiments, the resulting peptide is semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the resulting peptide is semaglutide. In certain embodiments, the resulting peptide is tirzepatide. In certain embodiments, the resulting peptide is retatrutide. In certain embodiments, the resulting peptide is VK2735. In certain embodiments, a pharmaceutical composition of the present disclosure includes a protein or peptide therapeutic produced by the methods disclosed herein and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutically acceptable carrier includes any carrier which does not interfere with the effectiveness of the biological activity of the active ingredients, e.g., the protein or peptide therapeutic, and that is not toxic to the patient to whom it is administered. Non-limiting examples of suitable pharmaceutical carriers include phosphate- buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting Active 121670349.1 95 Attorney Ref. No.070050.6868 agents and sterile solutions. Additional non-limiting examples of pharmaceutically acceptable carriers can include polymers, gels, bioabsorbable matrix materials, implantation elements containing the yeast and/or any other suitable vehicle, delivery or dispensing means or material. Such carriers can be formulated by conventional methods and can be administered to the subject. In certain embodiments, the pharmaceutical composition can be formulated for oral administration, intravenous administration, subcutaneous administration or parenteral administration. VI. Kits and Systems The present disclosure further provides kits and systems for performing the methods disclosed herein. In certain embodiments, the kit and/or system of the present disclosure includes one or more recombinant cell compositions and/or one or more masked forms of Aib amino acids. Non-limiting examples of recombinant cells and/or masked forms of Aib amino acids for use in the kits and systems disclosed herein are provided in Section II and IV. In certain embodiments, the kit and/or system of the present disclosure includes a container containing one or more masked forms of Aib or analogs thereof. For example, but not by way of limitation, the present disclosure provides a kit and/or system comprising at least one masked Aib or amino acid analog having a structure of any one of Formulas I-XVIII (or a composition thereof). In certain embodiments, the least one masked Aib or amino acid analog having a structure of any one of Formulas I-XVIII or a composition thereof is included in a container. For example, but not by way of limitation, a kit and/or system of the present disclosure can include a container comprising at least one masked Aib or amino acid analog having the structure of Formula IX or a composition thereof. In certain embodiments, a kit and/or system of the present disclosure can include a container comprising at least one masked Aib or amino acid analog having the structure of Formula X or a composition thereof. In certain embodiments, a kit and/or system of the present disclosure can include a container comprising at least one masked Aib or amino acid analog having the structure of Formula XI or a composition thereof. In certain embodiments, a kit and/or system of the present disclosure can include a container comprising at least one masked Aib or amino acid analog having the structure of Formula XII or a composition thereof. In certain embodiments, a kit and/or system of the present disclosure can include a container comprising at least one masked Aib or amino acid analog having the structure of Formula XIII or a composition thereof. In certain Active 121670349.1 96 Attorney Ref. No.070050.6868 embodiments, a kit and/or system of the present disclosure can include a container comprising at least one masked Aib or amino acid analog having the structure of Formula XIV. In certain embodiments, a kit and/or system of the present disclosure can include a container comprising at least one masked Aib or amino acid analog having the structure of Formula XV. In certain embodiments, a kit and/or system of the present disclosure can include a container comprising at least one masked Aib or amino acid analog having the structure of Formula XVI or a composition thereof. In certain embodiments, a kit and/or system of the present disclosure can include a container comprising at least one masked Aib or amino acid analog having the structure of Formula XVII or a composition thereof. In certain embodiments, a kit and/or system of the present disclosure can include a container comprising at least one masked Aib or amino acid analog having the structure of Formula XVIII or a composition thereof. In certain embodiments, a kit and/or system of the present disclosure can include a cell culture medium comprising one or more masked forms of Aib or analogs thereof of any one of Formulas I-XVIII, e.g., in a container. In certain embodiments, a kit and/or system of the present disclosure can include a cell culture medium comprising at least one masked Aib or amino acid analog having the structure of Formula IX. In certain embodiments, a kit and/or system of the present disclosure can include a cell culture medium comprising at least one masked Aib or amino acid analog having the structure of Formula X. In certain embodiments, a kit and/or system of the present disclosure can include a cell culture medium comprising at least one masked Aib or amino acid analog having the structure of Formula XI. In certain embodiments, a kit and/or system of the present disclosure can include a cell culture medium comprising at least one masked Aib or amino acid analog having the structure of Formula XII. In certain embodiments, a kit and/or system of the present disclosure can include a cell culture medium comprising at least one masked Aib or amino acid analog having the structure of Formula XIII. In certain embodiments, a kit and/or system of the present disclosure can include a cell culture medium comprising at least one masked Aib or amino acid analog having the structure of Formula XIV. In certain embodiments, a kit and/or system of the present disclosure can include a cell culture medium comprising at least one masked Aib or amino acid analog having the structure of Formula XVI. In certain embodiments, a kit and/or system of the present disclosure can include a cell culture medium comprising at least one masked Aib or amino acid analog having the structure of Formula XVII. In certain embodiments, a kit and/or system of the present disclosure can include a cell culture medium Active 121670349.1 97 Attorney Ref. No.070050.6868 comprising at least one masked Aib or amino acid analog having the structure of Formula XVIII. In certain embodiments, the kit and/or system of the present disclosure includes recombinant cells for expressing a protein or peptide therapeutic, e.g., in a container. In certain embodiments, the kit and/or system of the present disclosure includes recombinant cells that have been genetically engineered to produce a GLP-1 receptor agonist. In certain embodiments, the recombinant cells of the present disclosure have been genetically engineered to express semaglutide, tirzepatide, retatrutide or VK2735 (Viking Therapeutics). In certain embodiments, the recombinant cells and compositions thereof have been genetically engineered to express semaglutide. In certain embodiments, the recombinant cells and compositions thereof have been genetically engineered to express tirzepatide. In certain embodiments, the recombinant cells and compositions thereof have been genetically engineered to express retatrutide. In certain embodiments, the recombinant cells that have been genetically engineered to further express one or more aminoacyl-tRNA synthetases, e.g., comprises an amino acid sequence that is greater than about 95% homologous to a synthetase known in the art. In certain embodiments, the recombinant cells that have been genetically engineered to express an aminoacyl-tRNA synthetases that is greater than about 95% homologous to a synthetase is selected from pyrrolysyl-tRNA synthetase, an Escherichia coli synthetase, or a Saccharomyces cerevisiae synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to a Methanomethylophilus alvus pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to a Methanococcus jannaschii pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to a Methanosarcina barkeri pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 95% homologous to a Methanosarcina mazei pyrrolysyl-tRNA synthetase. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 4. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 5. In certain embodiments, the O-RS comprises an amino acid sequence that is greater than about 99% homologous to the sequence of SEQ ID NO: 6. In certain embodiments, the recombinant cells have been genetically engineered to express one Active 121670349.1 98 Attorney Ref. No.070050.6868 or more O-tRNAs. In certain embodiments, the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to a sequence of any one of SEQ ID NOs: 7-8 and 15. In certain embodiments, the kit and/or system further includes a container containing a chromatography medium for purifying a protein or a peptide comprising at least one masked form of Aib. Suitable containers include, but are not limited to, bottles, test tubes, vials and microtiter plates. The containers can be formed from a variety of materials such as glass or plastic. In certain embodiments, the kit and/or system can further include instructions for using the components disclosed herein. For example, but not by way of limitation, the kit and/or system can further include instructions for using the components of the kit and/or system in a method of the present disclosure. VII. Exemplary Non-Limiting Embodiments A. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula I:
Figure imgf000100_0001
wherein: the combination of X and R3 are optional if the combination of Y and R4 are present, but are otherwise required; the combination of Y and R4 are optional if the combination of X and R3 are present, but are otherwise required; R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; Active 121670349.1 99 Attorney Ref. No.070050.6868 each of X and Y when present is independently S or Se; each of R3 and R4 when present is independently an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, - (alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″, —C(O)OR″, —C(O)N(R″)2, or -L-Z; or R3 and R4 taken together optionally form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl; each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O—, —O-(alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O- (alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted Active 121670349.1 100 Attorney Ref. No.070050.6868 alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′- (alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, — N(R′)CO— (alkylene or substituted alkylene)-, —N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, — N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, — N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2— N(R′)—N(R′)—; where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. B. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula II:
Figure imgf000102_0001
wherein: the combination of X, A, and R3 are optional if the combination of Y, B, and R4 are present, but are otherwise required; the combination of Y, B, and R4 are optional if the combination of X, A and R3 are present, but are otherwise required; R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; Active 121670349.1 101 Attorney Ref. No.070050.6868 each of X and Y when present is independently S or Se; each of A and B are optional, and when present are independently an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), — C(O)R″, —C(O)OR″, —C(O)N(R″)2, or -L-Z; or A and B taken together optionally form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl; each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O—, —O-(alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O- (alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted Active 121670349.1 102 Attorney Ref. No.070050.6868 alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′- (alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, — N(R′)CO— (alkylene or substituted alkylene)-, —N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, — N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, — N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2— N(R′)—N(R′)—; where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; each of R3 and R4 when present is independently an azide, tetrazine, substituted tetrazine, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, trans-cyclooctene, substituted trans-cyclooctene, cyclooctyne, or substituted cyclooctyne; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. C. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula III:
Figure imgf000104_0001
wherein: the combination of Y and B are optional; R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; Active 121670349.1 103 Attorney Ref. No.070050.6868 X is S or Se; Y when present is S or Se; each of A and B are optional, and when present are independently an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), — C(O)R″, —C(O)OR″, —C(O)N(R″)2, or -L-Z; or A and B taken together optionally form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl; each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O—, —O-(alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O- Active 121670349.1 104 Attorney Ref. No.070050.6868 (alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′- (alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, — N(R′)CO— (alkylene or substituted alkylene)-, —N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, — N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, — N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2— N(R′)—N(R′)—; where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; and R3 is an azide, tetrazine, substituted tetrazine, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, trans-cyclooctene, substituted trans-cyclooctene, cyclooctyne, or substituted cyclooctyne; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. D. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula IV:
Figure imgf000106_0001
wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; Active 121670349.1 105 Attorney Ref. No.070050.6868 each of X and Y is independently S or Se; R3 is an H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, - (alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″, —C(O)OR″, —C(O)N(R″)2, or -L-Z; each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O—, —O-(alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O- (alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′- (alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, — N(R′)CO— (alkylene or substituted alkylene)-, —N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, — Active 121670349.1 106 Attorney Ref. No.070050.6868 N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, — N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2— N(R′)—N(R′)—; and where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. E. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula V:
Figure imgf000108_0001
wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; each of X and Y is independently S or Se; A is optional, and when present is an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″, —C(O)OR″, —C(O)N(R″)2, or -L- Z; Active 121670349.1 107 Attorney Ref. No.070050.6868 each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O—, —O-(alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O- (alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′- (alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, — N(R′)CO— (alkylene or substituted alkylene)-, —N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, — N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, — N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2— N(R′)—N(R′)—; where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; and R3 is an azide, tetrazine, substituted tetrazine, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, trans-cyclooctene, substituted trans-cyclooctene, cyclooctyne, or substituted cyclooctyne; Active 121670349.1 108 Attorney Ref. No.070050.6868 or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. F. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula VI:
Figure imgf000110_0001
wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; X is S or Se; R3 is an H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, - (alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″, —C(O)OR″, —C(O)N(R″)2, or -L-Z; each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Active 121670349.1 109 Attorney Ref. No.070050.6868 Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O—, —O-(alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O- (alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′- (alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, — N(R′)CO— (alkylene or substituted alkylene)-, —N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, — N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, — N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2— N(R′)—N(R′)—; and where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. G. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula VII: Active 121670349.1 110 Attorney Ref. No.070050.6868
Figure imgf000112_0001
wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; X is S or Se; A is optional, and when present is an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″, —C(O)OR″, —C(O)N(R″)2, or -L- Z; each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one Active 121670349.1 111 Attorney Ref. No.070050.6868 nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O—, —O-(alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O- (alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′- (alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, — N(R′)CO— (alkylene or substituted alkylene)-, —N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, — N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, — N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2— N(R′)—N(R′)—; where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; R3 is an azide, tetrazine, substituted tetrazine, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, trans-cyclooctene, substituted trans-cyclooctene, cyclooctyne, or substituted cyclooctyne; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. H. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula VIII: Active 121670349.1 112 Attorney Ref. No.070050.6868
Figure imgf000114_0001
wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; and X is S or Se; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. I. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula IX:
Figure imgf000114_0002
Formula IX or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. J. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula X: Active 121670349.1 113 Attorney Ref. No.070050.6868
Figure imgf000115_0001
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. K. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XI:
Figure imgf000115_0002
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. L. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XII:
Figure imgf000115_0003
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. M. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XIII: Active 121670349.1 114 Attorney Ref. No.070050.6868
Figure imgf000116_0001
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. N. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XIV:
Figure imgf000116_0002
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. O. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XV:
Figure imgf000116_0003
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. P. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XVI: Active 121670349.1 115 Attorney Ref. No.070050.6868
Figure imgf000117_0001
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. Q. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XVI:
Figure imgf000117_0002
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. R. The present disclosure provides a masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XVII:
Figure imgf000117_0003
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. S. The present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of any one of A-R. S1. The present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of A. Active 121670349.1 116 Attorney Ref. No.070050.6868 S2. The present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of B. S3. The present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of C. S4. The present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of D. S5. The present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of E. S6. The present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of F. S7. The present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of G. S8. The present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of H. S9. The present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of I. S10. The present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of J. S11. The present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of K. S12. The present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of L. S13. The present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of M. Active 121670349.1 117 Attorney Ref. No.070050.6868 S14. The present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of N. S15. The present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of O. S16. The present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of P. S17. The present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of Q. S18. The present disclosure provides a composition comprising the masked Aib amino acid or analog thereof of R. T. The present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of any one of A-R. T1. The present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of A. T2. The present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of B. T3. The present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of C. T4. The present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of D. T5. The present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of E. T6. The present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of F. Active 121670349.1 118 Attorney Ref. No.070050.6868 T7. The present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of G. T8. The present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of H. T9. The present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of I. T10. The present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of J. T11. The present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of K. T12. The present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of L. T13. The present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of M. T14. The present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of N. T15. The present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of O. T17. The present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of P. T18. The present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of Q. T19. The present disclosure provides a cell culture media comprising the masked Aib amino acid or analog thereof of R. Active 121670349.1 119 Attorney Ref. No.070050.6868 U. The present disclosure provides a method of producing a peptide or protein comprising one or more 2-aminoisobutyric acid (Aib) amino acid residues in a recombinant cell, wherein the method comprises: (a) culturing the recombinant cell in a cell culture media, wherein the recombinant cell comprises (i) a polynucleotide encoding the peptide or protein and comprising at least one selector codon, wherein the recombinant cell expresses: an orthogonal tRNA (O-tRNA) that functions in the recombinant cell and recognizes the selector codon; and an orthogonal aminoacyl-tRNA synthetase (O-RS), wherein the O-RS preferentially aminoacylates the O-tRNA with a masked Aib amino acid or analog thereof; (b) providing the masked Aib amino acid or analog thereof to the cell culture media; and (c) incorporating the masked Aib amino acid into one or more specified positions in the peptide or protein during translation of the polynucleotide encoding the peptide or protein and comprising the at least one selector codon to produce the peptide or protein comprising the one or more masked Aib amino acid residues. U1. The method of U further comprising contacting a cell extract and/or supernatant obtained from the recombinant cell with a chromatography column or solid support, wherein the masking group of the masked Aib amino acid residues incorporated in the peptide or protein binds covalently to the chromatography column or solid support. U2. The method of U further comprising contacting a cell extract and/or supernatant obtained from the recombinant cell with a chromatography column or solid support, wherein the masking group of the masked Aib amino acid residues incorporated in the peptide or protein binds non- covalently to the chromatography column or solid support. Active 121670349.1 120 Attorney Ref. No.070050.6868 U3. The method of any one of U-U2 further comprising performing one or more chemical reactions to remove the masking group from the one or more masked Aib amino acid residues present in the peptide or protein to generate the peptide or protein with native Aib residues. U4. The method of U1 or U3, wherein the masking group of the masked Aib binds covalently to the column or solid support via a Staudinger ligation reaction. U5. The method of U1 or U3, wherein the masking group of the masked Aib binds covalently to the column or solid support via a Cu(I)-catalyzed azide-alkyne cycloaddition reaction. U6. The method of U1 or U3, wherein the masking group of the masked Aib binds covalently to the column or solid support via a Strain-promoted azide-alkyne cycloaddition reaction. U7. The method of U1 or U3, wherein the masking group of the masked Aib binds covalently to the column or solid support via a Strain-promoted alkyne-nitrone cycloaddition reaction. U8. The method of U1 or U3, wherein the masking group of the masked Aib binds covalently to the column or solid support via a Diels-Alder reaction. U9. The method of U1 or U3, wherein the masking group of the masked Aib binds covalently to the column or solid support via an Alkene-azide [3+2] cycloaddition reaction. U10. The method of U1 or U3, wherein the masking group of the masked Aib binds covalently to the column or solid support via an Inverse-electron-demand Diels-Alder reaction. U11. The method of U1 or U3, wherein the masking group of the masked Aib binds covalently to the column or solid support via an alkene-tetrazine Inverse-electron-demand Diels-Alder reaction. U12. The method of U3, wherein the chemical reaction is a desulfurization reaction. U13. The method of U3, wherein the chemical reaction is a deselenization reaction. U14. The method of U3, wherein the catalyst used for the chemical reaction is a metal reagent. U15. The method of U3, wherein the catalyst used for the chemical reaction is Pd/Al2O3. Active 121670349.1 121 Attorney Ref. No.070050.6868 U16. The method of U3, wherein the catalyst used for the chemical reaction is Pd/Carbon. U17. The method of U3, wherein the catalyst used for the chemical reaction is Raney nickel. U18. The method of U3, wherein the catalyst used for the chemical reaction is Pd/BaSO4. U19. The method of U3, wherein the catalyst used for the chemical reaction is PdO. U20. The method of U3, wherein the catalyst used for the chemical reaction is a Ni(0) catalyst. U21. The method of U3, wherein the catalyst used for the chemical reaction is Nickel boride. U22. The method of U3, wherein the catalyst used for the chemical reaction is Tin hydride. U23. The method of U3, wherein the chemical reaction is performed using a Raney nickel – sodium hypophosphite – acetate buffer system. V. The method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of any one of A-R. V1. The method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of A. V2. The method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of B. V3. The method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of C. V4. The method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of D. V5. The method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of E. V6. The method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of F. Active 121670349.1 122 Attorney Ref. No.070050.6868 V7. The method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of G. V8. The method of any one of U or U1-U23., wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of H. V9. The method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of I. V10. The method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of J. V11. The method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of K. V12. The method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of L. V13. The method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of M. V14. The method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of N. V15. The method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of O. V16. The method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of P. V17. The method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of Q. V18. The method of any one of U or U1-U23, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of R. Active 121670349.1 123 Attorney Ref. No.070050.6868 W. The method of any one of U, U1-U23, V, or V1-V18, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl-tRNA synthetase. W1. The method of W, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to a pyrrolysyl-tRNA synthetase. W2. The method of any one of U, U1-U23, V, or V1-V18, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl-tRNA synthetase derived from a species of the Methanococcus genus. W3. The method of W2, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to a pyrrolysyl-tRNA synthetase derived from a species of the Methanococcus genus. W4. The method of any one of U, U1-U23, V, V1-V18, or W, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanosarcina mazei pyrrolysyl-tRNA synthetase. W5. The method of W4, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanosarcina mazei pyrrolysyl-tRNA synthetase. W6. The method of any one of U, U1-U23, V, V1-V18, or W wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanosarcina barkeri pyrrolysyl-tRNA synthetase. W6. The method of W6, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanosarcina barkeri pyrrolysyl-tRNA synthetase. W7. The method of any one of U, U1-U23, V, V1-V18, or W wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanococcus jannaschii tyrosyl-tRNA synthetase. W8. The method of W7, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanomethylophilus alvus tyrosyl-tRNA synthetase. Active 121670349.1 124 Attorney Ref. No.070050.6868 W9. The method of any one of U, U1-U23, V, V1-V18, or W wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanomethylophilus alvus tyrosyl-tRNA synthetase. W10. The method of W9, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanococcus jannaschii tyrosyl-tRNA synthetase. W11. The method of U, U1-U23, V, V1-V18, or W wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Saccharomyces cerevisiae glutaminyl-tRNA synthetase. W12. The method of W11, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Saccharomyces cerevisiae glutaminyl-tRNA synthetase. W13. The method of any one of U, U1-U23, V, V1-V18, or W wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Escherichia coli glutaminyl-tRNA synthetase. W14. The method of W13, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Escherichia coli glutaminyl-tRNA synthetase. W15. The method of any one of U, U1-U23, V, V1-V18, or W wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the sequence of any one of SEQ ID NOs: 4-6 and 12-14. W16. The method of W15, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of any one of SEQ ID NOs: 4-6 and 12-14. W17. The method of W15 or W16, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 4. W18. The method of W12 or W14, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 5. Active 121670349.1 125 Attorney Ref. No.070050.6868 W18. The method of W14 or W15, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 6. W19. The method of any one of U, U1-U23, V, V1-V18, or W, wherein the O-tRNA comprises a nucleotide sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the sequence of any one of SEQ ID Nos: 7-8 and 15. W20. The method of W19, wherein the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of any one of SEQ ID Nos: 7-8 and 15. W21. The method of W19 or W20, wherein the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 7. W22. The method of W19 or W20, wherein the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 8. W23. The method of any one of U, U1-U23, V, V1-V18, W, or W1-W22, wherein the protein or peptide is selected from the group consisting of a GLP-1 agonist, a GLP-1 analog, a GLP-1 receptor agonist, a dual GLP-1/GIP receptor agonist and a combination thereof. W24. The method of any one of U, U1-U23, V, V1-V18, W, or W1-W23, wherein the protein or peptide is a GLP-1 agonist. W25. The method of any one of U, U1-U23, V, V1-V18, W, or W1-W23, wherein the protein or peptide is a GLP-1 analog. W26. The method of any one of U, U1-U23, V, V1-V18, W, or W1-W23, wherein the protein or peptide is a GLP-1 receptor agonist. W27. The method of any one of U, U1-U23, V, V1-V18, W, or W1-W23, wherein the protein or peptide is a dual GLP-1/GIP receptor agonist. W28. The method of any one of U, U1-U23, V, V1-V18, W, or W1-W23, wherein the peptide is selected from the group consisting of semaglutide, tirzepatide, retatrutide, VK2735 and a combination thereof. Active 121670349.1 126 Attorney Ref. No.070050.6868 W29. The method of W28, wherein the peptide is semaglutide. W30. The method of W28, wherein the peptide is tirzepatide. W31. The method of W28, wherein the peptide is retatrutide. W32. The method of any one of U, U1-U23, V, V1-V18, W, or W1-W31, wherein the recombinant cell is a bacterial cell. W33. The method of W32, wherein the bacterial cells is Escherichia coli. W34. The method of any one of U, U1-U23, V, V1-V18, W, or W1-W31, wherein the recombinant cell is a fungal cell. W35. The method of W34, wherein the fungal cell is Saccharomyces cerevisiae. W36. The method of any one of U, U1-U23, V, V1-V18, W, or W1-W31, wherein the recombinant cell is a mammalian cell. W37. The method of W36, wherein the recombinant cells is an immortalized mammalian cell. X. The present disclosure provides a kit comprising the masked Aib amino acid or analog thereof of any one of A-R. X1. The present disclosure provides a kit comprising the composition of any one of S or S1- S18. X2. The present disclosure provides a kit comprising the cell culture media of any one of T or T1-T18. X3. The present disclosure provides a kit for performing the method of any one of U, U1-U23, V, V1-V18, W, or W1-W37. Y. The present disclosure provides a system comprising the masked Aib amino acid or analog thereof of any one of A-R Active 121670349.1 127 Attorney Ref. No.070050.6868 Y1. The present disclosure provides a system comprising the composition of any one of S or S1-S18 Y2. The present disclosure provides a system comprising the cell culture media of any one of T or T1-T18. Y3. The present disclosure provides a system for performing the method of any one of U, U1- U23, V, V1-V18, W, or W1-W37. EXAMPLES The following examples are merely illustrative of the presently disclosed subject matter and should not be considered as limitations in any way. Example 1: Synthesis of Masked Aib Amino Acids This example provides schemes for synthesizing masked Aib amino acids, which can be used in the methods of the present disclosure. 1.1. Synthesis of Compound A: (R)-2-amino-3-((2-(((benzyloxy)carbonyl)amino)ethyl)thio)-2- methylpropanoic acid
Figure imgf000129_0001
Scheme 1 To a 250 mL round-bottom flask equipped with a magnetic stirring bar, (R)-2-amino-3- mercapto-2-methylpropanoic acid, HCl 1 (2.060g, 1 Eq, 12.00 mmol) and benzyl (2- bromoethyl)carbamate 2 (3.252 g, 1.05 Eq, 12.60 mmol) were added under inert atmosphere and flushed with nitrogen at room temperature for 15 minutes. The solid mixture was treated with NH3 (7N) in MeOH (100mL) at 0 °C and allowed to react at room temperature for additional 16 h under inert atmosphere. The solvent was evaporated under reduced pressure at 30 °C in the bath and triturated with ether (1x100 mL), acetone (2x50 mL) and ice-cold water (HPLC grade, Milli-Q Active 121670349.1 128 Attorney Ref. No.070050.6868 1x6 mL, stirred for 30 min – fluffy precipitate formed from the suspension). The solid product was filtered on Schott funnel with a paper filter, and characterized by ESI MS, NMR (1H, 13C), LCMS-TOF to afford 3.56 g (95%) of (R)-2-amino-3-((2- (((benzyloxy)carbonyl)amino)ethyl)thio)-2-methylpropanoic acid (compound A). 1H NMR (500 MHz, CF3CO2D) δ 7.21 (m, 5H), 5.09 (s, 2H), 3.38 – 3.36 (m, 2H), 3.25-3.01 (m, 2H), 2.76-2.65 (m, 2H), 1.68 (s, 3H). HRMS (ESI) m/z calculated for C14H20N2O4S [M+H]+ : 313.1217, found 313.1234. 1.2. Synthesis of Compound B: (R)-2-amino-3-((2-((tert-butoxycarbonyl)amino)ethyl)thio)-2- methylpropanoic acid
Figure imgf000130_0001
Scheme 2 To the mixture of (R)-2-amino-3-mercapto-2-methylpropanoic acid, HCl 3 (564.0 mg, 1 Eq, 3.286 mmol) and tert-butyl (2-bromoethyl)carbamate 4 (773.2 mg, 1.05 Eq, 3.450 mmol) under inert atmosphere (N2 – purge for 15 min) NH3 in MeOH (32 mL, 7N, 10 mL/mmol) was added at 0 °C and stirred for 16 h at rt under the flow of nitrogen. The solvent was evaporated under reduced pressure and the solid crude was triturated in dry ether (1x25 mL), acetone (2x25 mL) and again with ether (1x25 mL) and filtered on a Schott funnel under nitrogen. The solid was collected and dried overnight under reduced pressure to afford 970 mg (106%). The solid was stirred in deionized water (2 mL) for 15 min and filtered on a Schott funnel, thoroughly pressed with a syringe plunger and dried under nitrogen. The collected white solid was lyophilized to afford 549 mg (60% yield). The procedure was repeated with (R)-2-amino-3-mercapto-2- methylpropanoic acid, HCl 3 (2.060 g, 1 Eq, 12.00 mmol) and tert-butyl (2-bromoethyl)carbamate 4 (2.958g, 1.05 Eq, 13.20 mmol) under inert atmosphere (N2 – purge for 15 min) NH3 in MeOH (100 mL, 7N, 10 mL/mmol) to afford 2.0 g (60% yield) of (R)-2-amino-3-((2-((tert- butoxycarbonyl)amino)ethyl)thio)-2-methylpropanoic acid (compound B). 1H NMR (500 MHz, Active 121670349.1 129 Attorney Ref. No.070050.6868 D2O) δ 3.41 – 3.16 (m, 1H), 2.98 (t, J = 17.5 Hz, 1H), 2.82 – 2.61 (m, 1H), 1.62 (s, 1H), 1.43 (s, 3H). HRMS (ESI) m/z calculated for C11H22N2O4S [M+H]+: 279.1373, found 279.1358. 1.3. Synthesis of Masked Aib
Figure imgf000131_0001
Scheme 3 A solution of compound 5 is stirred with thionyl chloride (SOCl2) in Et3N, resulting in compound 6. The resulting derivative, compound 6 and TEMPO is stirred with NaClO and NaClO2 to produce carboxylic acid 7. Next, carboxylic acid 7 undergoes a reaction with a chiral base followed by resolution to afford compound 8. Finally, compound 8 is stirred with compound 9 in the presence of K2CO3 to yield masked Aib amino acid 10. 1.4. Synthesis of Masked Aib
Figure imgf000131_0002
Scheme 4 Active 121670349.1 130 Attorney Ref. No.070050.6868 A solution of compound 11 is stirred with compound 12 and a lipase, resulting in compound 13. The resulting derivative, compound 13 and TEMPO is stirred with NaClO and NaClO2 in the presence of K2CO3 to produce carboxylic acid 14. Next, carboxylic acid 14 undergoes a reaction with HBTU and Et3N to afford compound 15. The product, compound 15 is stirred with compound 16 in the presence of K2CO3 to result in compound 17. Finally, compound 17 is subjected to acid or heat to provide masked Aib amino acid 18. Example 2: Incorporation of a masked Aib amino acid in a protein expressed in recombinant cells This example discloses the culturing of the recombinant E. coli cells described herein to generate a peptide therapeutic that includes a masked Aib amino acid. 2.1. In vivo aminoacylation An in vivo aminoacylation assay using pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs was performed in E. coli. The protocol for performing this assay was derived from Avila-Crump et al., ACS Chem Biol.17(12): 3458–3469 (2022). E. coli (DH10B Chemically Competent E. coli (Thermofisher scientific #EC0113)) are transformed with (1) plasmids encoding the PylRS/tRNAPyl pairs from Methanosarcina mazei (Mm) using pAIB003_pBK_Mm_Pyl_RS_WT and pAIB005_pALS2_Mm-tRNApyl-GFP- WT, (2) plasmids encoding the pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs from Methanosarcina mazei (Mm) using pAIB003_pBK_Mm_Pyl_RS_WT and pAIB005_pALS2_Mm-tRNApyl-GFP-150TAG, (3) plasmids encoding the PylRS/tRNAPyl pair from Candidatus Methanomethylophilus alvus Mx1201 (Ma) using pBK_Ma_Pyl_RS_WT (Addgene No. 197573) and pALS2_Ma-tRNApyl-GFP-150TAG (Addgene No. 197574), (4) plasmids encoding the PylRS/tRNAPyl pair from Candidatus Methanomethylophilus alvus Mx1201 (Ma) using pBK_Ma_Pyl_RS_WT (Addgene No. 197573) and pALS2_Ma-tRNApyl- GFP-WT (Addgene No. 197575) and (5) plasmids encoding the PylRS/tRNAPyl pairs from Methanosarcina mazei (Mm) using pAIB008_pBK_Mm_Pyl_RS_mkRS1 and pAIB005_pALS2_Mm-tRNApyl-GFP-150TAG. M. mazei mkRS1 mutant was previously shown to incorporate CbzK. Active 121670349.1 131 Attorney Ref. No.070050.6868 10 µL of each plasmid were prepared and then 1:10 serial dilutions (500 ng total, 50 ng total, 5 ng total, 0.5 ng total) were performed. The four DNA amounts were transformed into four Eppendorfs of chemically competent DH10b cells, plated on Spectinomycin and Kanamycin plates and incubated overnight at 37 oC. Five colonies were then inoculated into 5x500 µL of Non- inducing Media (NIM) culture (Table 4) containing kanamycin (50 μg/mL) and spectinomycin (100 μg/mL) in a 96 well block. The cells were grown for 16 h at 37 oC, 300 RPM. 10 μL from the NIM culture was added into 500 μL of Auto-inducing Media (AIM) culture (Table 5) containing kanamycin (50 μg/mL) and spectinomycin (100 μg/mL) with or without the relevant UAA (e.g., masked Aib amino acid). The cells were then grown at 37 oC shaking at 300 RPM. Cellfluorescence and OD600 were then measured at 24 hr on a plate reader. Table 4
Figure imgf000133_0002
Table 5
Figure imgf000133_0001
Active 121670349.1 132 Attorney Ref. No.070050.6868 The amino acids derivatives shown in Table 6 were initially tested. As an initial characterization of the amino acids shown in Table 6, their ability to suppress GFP-150TAG as a function of amino acid concentration was analyzed. The results of the in vivo GFP suppression assay show that M. mazei suppression efficiency was lower than M. alvus (FIGs.4A-4B). Table 6
Figure imgf000134_0001
Figure imgf000134_0002
Additional in vivo GFP suppression assays were performed for masked Aib amino acids of Example 1. In vivo GFP suppression assays were performed for Compound 1 (referred to Compound B herein) and Compound 2 (referred to as “AIB2” in Table 8 and Compound A herein) of Example 1. As shown in FIG. 5, Compound 2 showed suppression of GFP compared to the control (i.e., where no unnatural amino acids were added (“None”)). Examination of the efficiency and fidelity of incorporating the masked Aib amino acids at residue 150 of the super folder GFP reporter (i.e., GFP-150TAG) was also analyzed. Three tRNA synthetases were tested (i.e., M. alvus WT, M. mazei WT and M. mazei mkRS1 mutant), and 4 compound conditions were tested (i.e., no compound, 10 mM BocK, 10 mM CbxK and 10 mM compound 2). As shown in FIG. 6A, masked Aib of compound 2 was incorporated into the GFP reporter using the M. mazei mkRS1 mutant compared to the control. Similar results were observed under increasing concentration conditions as shown in FIG.6B. Further analysis of the incorporation of masked Aib amino acids of Formulas XI-XVIII is performed as described above. Active 121670349.1 133 Attorney Ref. No.070050.6868 Further analysis of the incorporation of masked Aib amino acids of Formulas IX-XVIII into semaglutide is performed as described above except that a polynucleotide encoding semaglutide and a selector codon for the masked Aib amino acid is transformed into the E. coli cells. 2.2. Mass Spectrometry Mass spectrometry is performed to further confirm that the masked Aib is incorporated into the synthesized peptide or protein (e.g., GFP reporter protein). E. coli (H10B Chemically Competent E. coli (Thermofisher scientific #EC0113)) are transformed with (1) plasmids encoding the PylRS/tRNAPyl pairs from Methanosarcina mazei (Mm) using pAIB003_pBK_Mm_Pyl_RS_WT and pAIB005_pALS2_Mm-tRNApyl-GFP- WT, (2) plasmids encoding the pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs from Methanosarcina mazei (Mm) using pAIB003_pBK_Mm_Pyl_RS_WT and pAIB005_pALS2_Mm-tRNApyl-GFP-150TAG, (3) plasmids encoding the the PylRS/tRNAPyl pair from Candidatus Methanomethylophilus alvus Mx1201 (Ma) using pBK_Ma_Pyl_RS_WT (Addgene No. 197573) and pALS2_Ma-tRNApyl-GFP-150TAG (Addgene No. 197574), (4) plasmids encoding the the PylRS/tRNAPyl pair from Candidatus Methanomethylophilus alvus Mx1201 (Ma) using pBK_Ma_Pyl_RS_WT (Addgene No. 197573) and pALS2_Ma-tRNApyl-GFP-WT (Addgene No. 197575) and (5) plasmids encoding the the PylRS/tRNAPyl pairs from Methanosarcina mazei (Mm) using pAIB008_pBK_Mm_Pyl_RS_mkRS1 and pAIB005_pALS2_Mm-tRNApyl-GFP-150TAG. A pH 5.5 version of the AIM buffer is prepared to perform the test expression in. Colonies are inoculated into 50 mL of defined AIM media (adjusted to pH 5.5 w/ HCl) + Kan (50 μg/mL) + Spec (50 μg/mL) + ncAA (10 mM) for culturing the cells. To purify the GFP reporter protein from the cell cultures, 350 mL of binding buffer (1x DPBS without Ca2+, Mg2+ + 20 mM imidazole) is generated. 3.5 mL of 2M imidazole solution is added to 350 mL of 1x DPBS. 20 mL of elution buffer (1x DPBS without Ca2+, Mg2+ + 500 mM imidazole) is made. 15 mL of 2M imidazole solution is added to 15 mL of 1x DPBS. Cell pellet is resuspended in 30 mL of binding buffer, then transferred to a new 50 mL falcon tube. One complete Mini, EDTA-free protease inhibitor is added and rocked up and down gently. 6 mg of lysozyme (L-7651), 0.6 mg of DNase I and 15 µl of 2M MgCl2 solution is added, and incubated Active 121670349.1 134 Attorney Ref. No.070050.6868 at RT for 10 min, then on an end-to-end shaker for 45 min. Sonication is performed for 3 cycles total with intervening 2.5 min rest periods (1 cycle = 5s ON, 15s OFF x 6), and then transferred to the high speed 38 mL centrifuge tubes and centrifuged at 15,000xg for 45 min in a precooled centrifuge. GFP protein from the cell culture is bound to a metal affinity resin. The resin is washed with 20–50 column volumes of binding buffer, and eluted with elution buffer. Proteins are desalted using a PD-10 column (GE Healthcare) into 50 mM ammonium bicarbonate and analyzed via mass spectroscopy. The deconvoluted masses were obtained by using Waters MassLynx MaxEnt1 software. The following conditions are analyzed by mass spectrometry. The amino acids are used at a concentration of 10 mM. 1. WT GFP + M. alvus WT + None 2. GFP150TAG + M. alvus WT + BocK 3. GFP150TAG + M. alvus WT + aMe-BocK 4. GFP150TAG + M. alvus WT + Compound 1 5. GFP150TAG + M. mazei WT + Compound 1 6. GFP150TAG + M. mazei mkRS1 + Compound 2
Figure imgf000136_0001
3.1. Synthesis of masked Aib-containing peptide 3.1.1. Synthesis of Aib-containing peptide with Compound A
Figure imgf000136_0002
Scheme 5 To the solution of Na-(tert-butoxycarbonyl)-Nt-trityl-L-histidine 19 (199 mg, 1 Eq, 400 μmol) and 2,3,4,5,6-pentafluorophenol 20 (81.0 mg, 1.1 Eq, 440 μmol) DCM (2 mL) dicyclohexylmethanediimine 21 (86.6 mg, 1.05 Eq, 420 μmol) was added at 0 °C. The reaction was allowed to stir at room temperature overnight. The precipitate was filtered off, and the filtrate concentrated under reduced pressure to afford 265 mg (100% yield) of the pure product 22. ESI Active 121670349.1 135 Attorney Ref. No.070050.6868 [M+H]+: 664.22 expected, 664.00 found; [2M+Na]+: 1349.41 expected, 1349.09 found. The product was isolated as white foam and used without further purification.
Figure imgf000137_0001
Scheme 6 To the solution of perfluorophenyl Na-(tert-butoxycarbonyl)-Nt-trityl-L-histidinate 22 (265 mg, 1 Eq, 0.400 mmol) was treated with Compound A (164 mg, 84% Wt, 1.1Eq, 440 μmol) in DMF (2 mL) at 0 °C, and 4-methylmorpholine 23 (44.5 mg, 49 μL, 1.1 Eq, 440 μmol) was added in 2 equal portions. The resulting mixture was allowed to stir for 1 h at room temperature. The ESI of the reaction mixture was recorded to confirm the formation of the peptide 24. ESI [M+H]+: 792.34 expected, 792.27 found; [M-H]- 790.32 expected, 790.36 found. Solvent was removed under reduced pressure. The oily residue was purified by flash column chromatography (SiO2, gradient: 1:45:55 MeOH/Hex/EA to 5:12:45:55 AcOH/MeOH/Hex/EA). The isolated peptide was characterized by LRMS. LRMS (ESI) m/z C44H49N5O7S [M+H]+: 792.34 expected, 792.18 found.
Figure imgf000137_0002
Scheme 7 To Fmoc-GLP-1(9-36)-NH225 is added a solution of Boc-His(Trt)-Aib(MaskCbz)-OH 24 in DMF pre-activated with hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU) and N-ethyl-N-isopropylpropan-2-amine (DIPEA) for 2 min. The mixture is agitated for 1 h and rinsed with DMF followed by DCM. TIPS , H2O and TFA are added to the resulting mixture, which is then stirred at room temperature for 1 h. The solvent is evaporated under reduced pressure and dried overnight under high vacuum. Active 121670349.1 136 Attorney Ref. No.070050.6868 3.2. Demasking of Masked Aib Amino Acids: Desulfurization This example describes the techniques to be used to demask the peptides or proteins that include a masked Aib amino acid. In particular, the techniques provided below can be used to demask the peptides or proteins that include a masked Aib amino acid of Example 1 (i.e., Compound A and Compound B) and compounds of Formulas IX-XVIII. 3.2.1. Desulfurization with Raney Nickel
Figure imgf000138_0001
Scheme 11 To the aqueous solution of acetic acid (0.01 M solution) containing Raney Ni (0.1 equiv, W.R. Grace and Co. Raney®2400, slurry, in H2O, active catalyst) masked Aib-containing peptide 31 (1 equiv.) is added at room temperature and sparged nitrogen for 15 min followed by hydrogen gas (1 mL/min) overnight to yield demasked Aib containing peptide. The isolated and purified peptide 32 is further subjected to testing (see Yan, L.Z. and Dawson, P.E. J. Am. Chem. Soc. 2001:526-533; Mozingo et al., J. Am. Chem. Soc. 1943, 65: 1013–1016,) 3.2.2. Desulfurization with UV light
Figure imgf000138_0002
Scheme 12 Active 121670349.1 137 Attorney Ref. No.070050.6868 Aib-containing peptide 31 is suspended in a solvent and irradiated with UV-light. The product 32 is isolated and characterized (see Wan, Q. and Danishefsky, S, J. Angew. Chem., Int. Ed., 46:9248-9252 (2007); Venneti, N.M. et al., J. Am. Chem. Soc.145:1053-1061 (2023)). 3.2.3. Electrochemical Desulfurization
Figure imgf000139_0001
Scheme 13 A peptide of interest 31 is dissolved to a concentration of 1 mg mL−1 in a buffered TCEP solution comprised of 0.15 M TCEP and 0.1 M pH 5.8 sodium phosphate buffer. To this a small volume of acetonitrile is added (1 mL) to ensure the peptide was fully dissolved. The solution is transferred to ElectraSyn 2.0 vial equipped with the platinum electrodes. The solution is stirred vigorously at room temperature and a constant potential of 1.0 V is supplied. Desulfurization progress is monitored by LC-MS until full consumption of the starting material. The desulfurized peptide is purified directly from the solution via preparative HPLC and the eluted fractions freeze-dried to produce desulfurized peptide 32. (Macmillan, D. et al., Org. Biomol. Chem., 2022:7343-7350.) Active 121670349.1 138 Attorney Ref. No.070050.6868 3.3 Demasking of Masked Aib Amino Acids: Deselenization 3.3.1. Deselenization with Raney Nickel
Figure imgf000140_0001
Scheme 14 To the aqueous solution of acetic acid (0.01 M solution) containing Raney Ni (0.1 equiv, W.R. Grace and Co. Raney®2400, slurry, in H2O, active catalyst) masked Aib-containing peptide 33 (1 equiv.) is added at room temperature and sparged nitrogen for 15 min followed by hydrogen gas (1 mL/min) overnight to yield demasked Aib containing peptide. The isolated and purified peptide 32 is further subjected to testing (see Yan, L.Z. and Dawson, P.E. J. Am. Chem. Soc. 2001:526-533; Mozingo et al., J. Am. Chem. Soc. 1943, 65: 1013–1016,) 3.3.2. Deselenization with UV light
Figure imgf000140_0002
Scheme 15 Aib-containing peptide 33 is suspended in a solvent and irradiated with UV-light. The product 32 is isolated and characterized (see Wan, Q. and Danishefsky, S, J. Angew. Chem., Int. Ed., 46:9248-9252 (2007); Venneti, N.M. et al., J. Am. Chem. Soc.145:1053-1061 (2023)). Active 121670349.1 139 Attorney Ref. No.070050.6868 3.3.3. Electrochemical Deselenization
Figure imgf000141_0001
Scheme 16 A peptide of interest 33 is dissolved to a concentration of 1 mg mL−1 in a buffered TCEP solution comprised of 0.15 M TCEP and 0.1 M pH 5.8 sodium phosphate buffer. To this a small volume of acetonitrile is added (1 mL) to ensure the peptide was fully dissolved. The solution is transferred to ElectraSyn 2.0 vial equipped with the platinum electrodes. The solution is stirred vigorously at room temperature and a constant potential of 1.0 V is supplied. Deselenization progress is monitored by LC-MS until full consumption of the starting material. The desulfurized peptide is purified directly from the solution via preparative HPLC and the eluted fractions freeze-dried to produce desulfurized peptide 32.(Macmillan, D. et al., Org. Biomol. Chem., 2022:7343-7350.) * * * * * * * * Although the presently disclosed subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the present disclosure. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, and composition of matter, means, methods and steps described in the specification. Accordingly, the appended claims are intended to comprise within their scope such processes, machines, manufacture, compositions of matter, means, methods or steps. Various patents, patent applications, publications, product descriptions and protocols are cited throughout this application, the disclosure of which are incorporated herein by reference in their entireties for all purposes. Active 121670349.1 140

Claims

Attorney Ref. No.070050.6868 What is claimed is: 1. A masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula I:
Figure imgf000142_0001
wherein: the combination of X and R3 are optional if the combination of Y and R4 are present, but are otherwise required; the combination of Y and R4 are optional if the combination of X and R3 are present, but are otherwise required; R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; each of X and Y when present is independently S or Se; each of R3 and R4 when present is independently an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, - (alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″, —C(O)OR″, —C(O)N(R″)2, or -L-Z; Active 121670349.1 141 Attorney Ref. No.070050.6868 or R3 and R4 taken together optionally form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl; each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O—, —O-(alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O- (alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′- (alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, — N(R′)CO— (alkylene or substituted alkylene)-, —N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, — N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, — N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2— N(R′)—N(R′)—; where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; Active 121670349.1 142 Attorney Ref. No.070050.6868 or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. 2. A masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula II:
Figure imgf000144_0001
wherein: the combination of X, A, and R3 are optional if the combination of Y, B, and R4 are present, but are otherwise required; the combination of Y, B, and R4 are optional if the combination of X, A and R3 are present, but are otherwise required; R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; each of X and Y when present is independently S or Se; each of A and B are optional, and when present are independently an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), — C(O)R″, —C(O)OR″, —C(O)N(R″)2, or -L-Z; Active 121670349.1 143 Attorney Ref. No.070050.6868 or A and B taken together optionally form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl; each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O—, —O-(alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O- (alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′- (alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, — N(R′)CO— (alkylene or substituted alkylene)-, —N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, — N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, — N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2— N(R′)—N(R′)—; where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; Active 121670349.1 144 Attorney Ref. No.070050.6868 each of R3 and R4 when present is independently an azide, tetrazine, substituted tetrazine, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, trans-cyclooctene, substituted trans-cyclooctene, cyclooctyne, or substituted cyclooctyne; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. 3. A masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula III:
Figure imgf000146_0001
wherein: the combination of Y and B are optional; R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; X is S or Se; Y when present is S or Se; each of A and B are optional, and when present are independently an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted Active 121670349.1 145 Attorney Ref. No.070050.6868 alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), — C(O)R″, —C(O)OR″, —C(O)N(R″)2, or -L-Z; or A and B taken together optionally form a cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl; each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O—, —O-(alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O- (alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′- (alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, — N(R′)CO— (alkylene or substituted alkylene)-, —N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, — N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, — Active 121670349.1 146 Attorney Ref. No.070050.6868 N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2— N(R′)—N(R′)—; where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; and R3 is an azide, tetrazine, substituted tetrazine, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, trans-cyclooctene, substituted trans-cyclooctene, cyclooctyne, or substituted cyclooctyne; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. 4. A masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula IV:
Figure imgf000148_0001
wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; each of X and Y is independently S or Se; R3 is an H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, - Active 121670349.1 147 Attorney Ref. No.070050.6868 (alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″, —C(O)OR″, —C(O)N(R″)2, or -L-Z; each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O—, —O-(alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O- (alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′- (alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, — N(R′)CO— (alkylene or substituted alkylene)-, —N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, — N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, — N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2— N(R′)—N(R′)—; and where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; Active 121670349.1 148 Attorney Ref. No.070050.6868 or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. 5. A masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula V:
Figure imgf000150_0001
wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; each of X and Y is independently S or Se; A is optional, and when present is an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″, —C(O)OR″, —C(O)N(R″)2, or -L- Z; each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Active 121670349.1 149 Attorney Ref. No.070050.6868 Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O—, —O-(alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O- (alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′- (alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, — N(R′)CO— (alkylene or substituted alkylene)-, —N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, — N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, — N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2— N(R′)—N(R′)—; where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; and R3 is an azide, tetrazine, substituted tetrazine, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, trans-cyclooctene, substituted trans-cyclooctene, cyclooctyne, or substituted cyclooctyne; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. Active 121670349.1 150 Attorney Ref. No.070050.6868 6. A masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula VI:
Figure imgf000152_0001
wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; X is S or Se; R3 is an H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, - (alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″, —C(O)OR″, —C(O)N(R″)2, or -L-Z; each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one Active 121670349.1 151 Attorney Ref. No.070050.6868 nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O—, —O-(alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O- (alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′- (alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, — N(R′)CO— (alkylene or substituted alkylene)-, —N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, — N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, — N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2— N(R′)—N(R′)—; and where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. 7. A masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula VII: Active 121670349.1 152 Attorney Ref. No.070050.6868
Figure imgf000154_0001
wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; X is S or Se; A is optional, and when present is an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″, —C(O)OR″, —C(O)N(R″)2, or -L- Z; each R″ is independently H, a protecting group, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl or heteroaryl; Z is selected from the group consisting of a water-soluble polymer; a polyalkylene oxide; a polyethylene glycol; a derivative of polyethylene glycol; a photocrosslinker; at least one amino acid; at least one sugar group; at least one nucleotide; at least one Active 121670349.1 153 Attorney Ref. No.070050.6868 nucleoside; a ligand; biotin; a biotin analogue; a detectable label; and any combination thereof; L is optional, and when present is a bond, alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, heteroalkylene, substituted heteroalkylene, heterocycloalkylene, substituted heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, substituted aralkylene, — O—, —O-(alkylene or substituted alkylene)-, —S(O)k—, —S(O)k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(O)O—, —C(O)O- (alkylene or substituted alkylene)-, —OC(O)—, —OC(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′- (alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—, — N(R′)CO— (alkylene or substituted alkylene)-, —N(R′)CS—, —N(R′)CS— (alkylene or substituted alkylene)-, —N(R′)C(O)O—, OC(O)N(R′)—, —S(O)kN(R′)—, — N(R′)S(O)k—, —N(R′)C(O)N(R′)—, —N(R′)S(O)kN(R′)—, —C(R′)═N—, — N═C(R′)—, —N═N—, —C(R′)═N—N(R′)—, —C(R′)2—N═N—, or —C(R′)2— N(R′)—N(R′)—; where k is 0, 1 or 2 and each R′ is independently H, alkyl, or substituted alkyl; R3 is an azide, tetrazine, substituted tetrazine, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, trans-cyclooctene, substituted trans-cyclooctene, cyclooctyne, or substituted cyclooctyne; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. 8. A masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula VIII: Active 121670349.1 154 Attorney Ref. No.070050.6868
Figure imgf000156_0001
wherein: R1 is H, an amino protecting group, resin, at least one amino acid, or at least one nucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or at least one nucleotide; and X is S or Se; or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. 9. A masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula IX:
Figure imgf000156_0002
Formula IX or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. 10. A masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula X: Active 121670349.1 155 Attorney Ref. No.070050.6868
Figure imgf000157_0001
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. 11. A masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XI:
Figure imgf000157_0002
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. 12. A masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XII:
Figure imgf000157_0003
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. 13. A masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XIII: Active 121670349.1 156 Attorney Ref. No.070050.6868
Figure imgf000158_0001
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. 14. A masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XIV:
Figure imgf000158_0002
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. 15. A masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XV:
Figure imgf000158_0003
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. 16. A masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XVI: Active 121670349.1 157 Attorney Ref. No.070050.6868
Figure imgf000159_0001
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. 17. A masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XVI:
Figure imgf000159_0002
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. 18. A masked Aib amino acid or analog thereof, wherein the masked Aib amino acid or analog thereof has the structure of Formula XVII:
Figure imgf000159_0003
or a pharmaceutically acceptable salt, active metabolite, prodrug, solvate, polymorph, tautomer, stereoisomer or enantiomer thereof. 19. A composition comprising the masked Aib amino acid or analog thereof of any one of claims 1-18. 20. A composition comprising the masked Aib amino acid or analog thereof of claim 1. 21. A composition comprising the masked Aib amino acid or analog thereof of claim 2. Active 121670349.1 158 Attorney Ref. No.070050.6868 22. A composition comprising the masked Aib amino acid or analog thereof of claim 3. 23. A composition comprising the masked Aib amino acid or analog thereof of claim 4. 24. A composition comprising the masked Aib amino acid or analog thereof of claim 5. 25. A composition comprising the masked Aib amino acid or analog thereof of claim 6. 26. A composition comprising the masked Aib amino acid or analog thereof of claim 7. 27. A composition comprising the masked Aib amino acid or analog thereof of claim 8. 28. A composition comprising the masked Aib amino acid or analog thereof of claim 9. 28. A composition comprising the masked Aib amino acid or analog thereof of claim 10. 29. A composition comprising the masked Aib amino acid or analog thereof of claim 11. 30. A composition comprising the masked Aib amino acid or analog thereof of claim 12. 31. A composition comprising the masked Aib amino acid or analog thereof of claim 13. 32. A composition comprising the masked Aib amino acid or analog thereof of claim 14. 33. A composition comprising the masked Aib amino acid or analog thereof of claim 15. 34. A composition comprising the masked Aib amino acid or analog thereof of claim 16. 35. A composition comprising the masked Aib amino acid or analog thereof of claim 17. 36. A composition comprising the masked Aib amino acid or analog thereof of claim 18. 37. A cell culture media comprising the masked Aib amino acid or analog thereof of any one of claims 1-18. 38. A cell culture media comprising the masked Aib amino acid or analog thereof of claim 1. 39. A cell culture media comprising the masked Aib amino acid or analog thereof of claim 2. Active 121670349.1 159 Attorney Ref. No.070050.6868 40. A cell culture media comprising the masked Aib amino acid or analog thereof of claim 3. 41. A cell culture media comprising the masked Aib amino acid or analog thereof of claim 4. 42. A cell culture media comprising the masked Aib amino acid or analog thereof of claim 5. 43. A cell culture media comprising the masked Aib amino acid or analog thereof of claim 6. 44. A cell culture media comprising the masked Aib amino acid or analog thereof of claim 7. 45. A cell culture media comprising the masked Aib amino acid or analog thereof of claim 8. 46. A cell culture media comprising the masked Aib amino acid or analog thereof of claim 9. 47. A cell culture media comprising the masked Aib amino acid or analog thereof of claim 10. 48. A cell culture media comprising the masked Aib amino acid or analog thereof of claim 11. 49. A cell culture media comprising the masked Aib amino acid or analog thereof of claim 12. 50. A cell culture media comprising the masked Aib amino acid or analog thereof of claim 13. 51. A cell culture media comprising the masked Aib amino acid or analog thereof of claim 14. 52. A cell culture media comprising the masked Aib amino acid or analog thereof of claim 15. 53. A cell culture media comprising the masked Aib amino acid or analog thereof of claim 16. 54. A cell culture media comprising the masked Aib amino acid or analog thereof of claim 17. 55. A cell culture media comprising the masked Aib amino acid or analog thereof of claim 18. 56. A method of producing a peptide or protein comprising one or more 2-aminoisobutyric acid (Aib) amino acid residues in a recombinant cell, wherein the method comprises: (a) culturing the recombinant cell in a cell culture media, wherein the recombinant cell comprises (i) a polynucleotide encoding the peptide or protein and comprising at least one selector codon, Active 121670349.1 160 Attorney Ref. No.070050.6868 wherein the recombinant cell expresses: an orthogonal tRNA (O-tRNA) that functions in the recombinant cell and recognizes the selector codon; and an orthogonal aminoacyl-tRNA synthetase (O-RS), wherein the O-RS preferentially aminoacylates the O-tRNA with a masked Aib amino acid or analog thereof; (b) providing the masked Aib amino acid or analog thereof to the cell culture media; and (c) incorporating the masked Aib amino acid into one or more specified positions in the peptide or protein during translation of the polynucleotide encoding the peptide or protein and comprising the at least one selector codon to produce the peptide or protein comprising the one or more masked Aib amino acid residues. 57. The method of claim 56 further comprising contacting a cell extract and/or supernatant obtained from the recombinant cell with a chromatography column or solid support, wherein the masking group of the masked Aib amino acid residues incorporated in the peptide or protein binds covalently to the chromatography column or solid support. 58. The method of claim 56 further comprising contacting a cell extract and/or supernatant obtained from the recombinant cell with a chromatography column or solid support, wherein the masking group of the masked Aib amino acid residues incorporated in the peptide or protein binds non-covalently to the chromatography column or solid support. 59. The method of any one of claims 56-58 further comprising performing one or more chemical reactions to remove the masking group from the one or more masked Aib amino acid residues present in the peptide or protein to generate the peptide or protein with native Aib residues. 60. The method of claim 57 or 59, wherein the masking group of the masked Aib binds covalently to the column or solid support via a Staudinger ligation reaction. Active 121670349.1 161 Attorney Ref. No.070050.6868 61. The method of claim 57 or 59, wherein the masking group of the masked Aib binds covalently to the column or solid support via a Cu(I)-catalyzed azide-alkyne cycloaddition reaction. 62. The method of claim 57 or 59, wherein the masking group of the masked Aib binds covalently to the column or solid support via a Strain-promoted azide-alkyne cycloaddition reaction. 63. The method of claim 57 or 59, wherein the masking group of the masked Aib binds covalently to the column or solid support via a Strain-promoted alkyne-nitrone cycloaddition reaction. 64. The method of claim 57 or 59, wherein the masking group of the masked Aib binds covalently to the column or solid support via a Diels-Alder reaction. 65. The method of claim 57 or 59, wherein the masking group of the masked Aib binds covalently to the column or solid support via an Alkene-azide [3+2] cycloaddition reaction. 66. The method of claim 57 or 59, wherein the masking group of the masked Aib binds covalently to the column or solid support via an Inverse-electron-demand Diels-Alder reaction. 67. The method of claim 57 or 59, wherein the masking group of the masked Aib binds covalently to the column or solid support via an alkene-tetrazine Inverse-electron-demand Diels- Alder reaction. 68. The method of claim 59, wherein the chemical reaction is a desulfurization reaction. 69. The method of claim 59, wherein the chemical reaction is a deselenization reaction. 70. The method of claim 59, wherein the catalyst used for the chemical reaction is a metal reagent. 71. The method of claim 59, wherein the catalyst used for the chemical reaction is Pd/Al2O3. 72. The method of claim 59, wherein the catalyst used for the chemical reaction is Pd/Carbon. Active 121670349.1 162 Attorney Ref. No.070050.6868 73. The method of claim 59, wherein the catalyst used for the chemical reaction is Raney nickel. 74. The method of claim 59, wherein the catalyst used for the chemical reaction is Pd/BaSO4. 75. The method of claim 59, wherein the catalyst used for the chemical reaction is PdO. 76. The method of claim 59, wherein the catalyst used for the chemical reaction is a Ni(0) catalyst. 77. The method of claim 59, wherein the catalyst used for the chemical reaction is Nickel boride. 78. The method of claim 59, wherein the catalyst used for the chemical reaction is Tin hydride. 79. The method of claim 59, wherein the chemical reaction is performed using a Raney nickel – sodium hypophosphite – acetate buffer system. 80. The method of any one of claims 56-79, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of any one of claims 1-18. 81. The method of any one of claims 56-79, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of claim 1. 82. The method of any one of claims 56-79, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of claim 2. 83. The method of any one of claims 56-79, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of claim 3. 84. The method of any one of claims 56-79, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of claim 4. 85. The method of any one of claims 56-79, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of claim 5. Active 121670349.1 163 Attorney Ref. No.070050.6868 86. The method of any one of claims 56-79, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of claim 6. 87. The method of any one of claims 56-79, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of claim 7. 88. The method of any one of claims 56-79, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of claim 8. 89. The method of any one of claims 56-79, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of claim 9. 90. The method of any one of claims 56-79, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of claim 10. 91. The method of any one of claims 56-79, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of claim 11. 92. The method of any one of claims 56-79, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of claim 12. 94. The method of any one of claims 56-79, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of claim 13. 95. The method of any one of claims 56-79, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of claim 14. 96. The method of any one of claims 56-79, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of claim 15. 97. The method of any one of claims 56-79, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of claim 16. 98. The method of any one of claims 56-79, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of claim 17. Active 121670349.1 164 Attorney Ref. No.070050.6868 99. The method of any one of claims 56-79, wherein the masked Aib amino acid or analog thereof is the masked Aib amino acid or analog thereof of claim 18. 100. The method of any one of claims 56-99, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl- tRNA synthetase. 101. The method of claim 100, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to a pyrrolysyl-tRNA synthetase. 102. The method of any one of claims 56-99, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to a pyrrolysyl- tRNA synthetase derived from a species of the Methanococcus genus. 103. The method of claim 102, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to a pyrrolysyl-tRNA synthetase derived from a species of the Methanococcus genus. 104. The method of any one of claims 56-100, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanosarcina mazei pyrrolysyl-tRNA synthetase. 105. The method of claim 102, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanosarcina mazei pyrrolysyl-tRNA synthetase. 106. The method of any one of claims 56-100, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanosarcina barkeri pyrrolysyl-tRNA synthetase. 106. The method of claim 104, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanosarcina barkeri pyrrolysyl-tRNA synthetase. 107. The method of any one of claims 56-100, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanococcus jannaschii tyrosyl-tRNA synthetase. Active 121670349.1 165 Attorney Ref. No.070050.6868 108. The method of claim 106, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanomethylophilus alvus tyrosyl-tRNA synthetase. 109. The method of any one of claims 56-100, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Methanomethylophilus alvus tyrosyl-tRNA synthetase. 110. The method of claim 108, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Methanococcus jannaschii tyrosyl-tRNA synthetase. 111. The method of any one of claims 56-100, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Saccharomyces cerevisiae glutaminyl-tRNA synthetase. 112. The method of claim 110, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Saccharomyces cerevisiae glutaminyl-tRNA synthetase. 113. The method of any one of claims 56-100, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the Escherichia coli glutaminyl-tRNA synthetase. 114. The method of claim 112, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the Escherichia coli glutaminyl-tRNA synthetase. 115. The method of any one of claims 56-100, wherein the O-RS comprises an amino acid sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the sequence of any one of SEQ ID NOs: 4-6 and 12-14. 116. The method of claim 114, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of any one of SEQ ID NOs: 4-6 and 12-14. 117. The method of claim 114 or 115, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 4. Active 121670349.1 166 Attorney Ref. No.070050.6868 118. The method of claim 112 or 114, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 5. 118. The method of claim 114 or 115, wherein the O-RS comprises an amino acid sequence that is greater than 95% homologous to the sequence of SEQ ID NO: 6. 119. The method of any one of claims 56-100, wherein the O-tRNA comprises a nucleotide sequence that is greater than 70%, 75%, 80%, 90%, 95% or 99% homologous to the sequence of any one of SEQ ID NOs: 7-8 and 15. 120. The method of claim 119, wherein the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of any one of SEQ ID NOs: 7-8 and 15. 121. The method of claim4119 or 120, wherein the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 7. 122. The method of 1119 or 120, wherein the O-tRNA comprises a nucleotide sequence that is greater than about 95% homologous to the sequence of SEQ ID NO: 8. 123. The method of any one of claims 56-122, wherein the protein or peptide is selected from the group consisting of a GLP-1 agonist, a GLP-1 analog, a GLP-1 receptor agonist, a dual GLP- 1/GIP receptor agonist and a combination thereof. 124. The method of any one of claims 56-123, wherein the protein or peptide is a GLP-1 agonist. 125. The method of any one of claims 56-123, wherein the protein or peptide is a GLP-1 analog. 126. The method of any one of claims 56-123, wherein the protein or peptide is a GLP-1 receptor agonist. 127. The method of any one of claims 56-123, wherein the protein or peptide is a dual GLP- 1/GIP receptor agonist. 128. The method of any one of claims 56-123, wherein the peptide is selected from the group consisting of semaglutide, tirzepatide, retatrutide, VK2735 and a combination thereof. Active 121670349.1 167 Attorney Ref. No.070050.6868 129. The method of claim 128, wherein the peptide is semaglutide. 130. The method of claim 128, wherein the peptide is tirzepatide. 131. The method of claim 128, wherein the peptide is retatrutide. 132. The method of any one of claims 56-131, wherein the recombinant cell is a bacterial cell. 133. The method of claim 132, wherein the bacterial cells is Escherichia coli. 134. The method of any one of claims 56-131, wherein the recombinant cell is a fungal cell. 135. The method of claim 134, wherein the fungal cell is Saccharomyces cerevisiae. 136. The method of any one of claims 56-131, wherein the recombinant cell is a mammalian cell. 137. The method of claim 136, wherein the recombinant cells is an immortalized mammalian cell. 138. A kit comprising the masked Aib amino acid or analog thereof of any one of claims 1-18. 139. A kit comprising the composition of any one of claims 19-36. 140. A kit comprising the cell culture media of any one of claims 36-55. 141. A kit for performing the method of any one of claims 56-117. 142. A system comprising the masked Aib amino acid or analog thereof of any one of claims 1- 18. 143. A system comprising the composition of any one of claims 19-36. 144. A system comprising the cell culture media of any one of claims 37-55. 145. A system for performing the method of any one of claims 56-117. Active 121670349.1 168
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