EP3265469A1 - New synthetic methods - Google Patents

Abstract

New methods for organic synthesis are disclosed. The invention relates to the rapid synthesis of peptides and proteins.

Description

New Synthetic Methods

Technical Field

[1] The disclosure relates to organic synthesis. In particular, the disclosure relates to the synthesis of peptides and proteins.

Cross-reference to related applications

[2] This application claims priority to Australian Provisional Patent Application Nos. 2015900739, filed 3 March 2015, and 2015902928, filed 23 July 2015, the disclosures of which are incorporated by reference in their entirety.

Background

[3] The ability to prepare native, correctly folded proteins is essential for the investigation of their structure and function and for the development of protein-based therapeutics. Recombinant expression, while enabling successful production of native sequences, is generally limited to the incorporation of the 20 genetically encoded amino acids. Using this technique it is therefore impossible to homogeneously replicate the many enzyme-mediated post-translational modifications (PTMs) displayed by over 70% of proteins expressed in humans. Such modifications play an important role in a myriad of vital biological processes including structure, function, folding and recognition. The influence of the many PTMs can therefore not be elucidated using recombinant expression. Moreover, the lack of modifications may severely limit or even completely negate the protein's natural activity. To this end, synthetic chemistry can provide the tools necessary to access homogeneously modified, native proteins either in combination with biological expression or solely through chemical synthesis via the convergent assembly of peptide fragments using native chemical ligation (NCL). [4] Despite numerous advances to the native chemical ligation method, the reaction is limited by the prohibitive rate of ligation at sterically hindered C-terminal esters. With reaction rates between 24->48 h, a large excess of thioester must be employed. In many cases the ligation will not reach completion and so such hindered junctions are therefore generally avoided.

[5] Thus, there is a need for the development of chemical synthesis methods that allow for the rapid and facile preparation of native peptides and proteins and furthermore are clean, robust, scalable and are suitable for deployment on solid-phase.

Summary

[6] The present disclosure relates to a method of preparing an amide containing compound comprising the step of reacting an acyl donor with a diselenide bearing an amino group. In one example, the acyl donor is an acyl halide. In one example, the acyl donor is an anhydride. In one example, the acyl halide is an acyl bromide. In one example, the acyl halide is an acyl chloride. The present disclosure relates to a method of preparing an amide containing compound comprising the step of reacting an ester with a diselenide bearing an amino group. Preferably, the reaction proceeds in the absence of an additive. The amide containing compound is preferably a peptide.

[7] Preferably, the peptide is defined by formula (I):

I the ester is defined by formula (II):

II and the diselenide is defined by formula (III)

2

H₂N'^y^^li9~~r|^(^AA)n'-^Cterm

O

III, wherein:

[8] Nterm is the N-terminus of the peptide;

[9] Cterm is the C-terminus of the peptide;

[ 10] AA is an amino acid;

[ 1 1] n is an integer;

[ 12] (AA)n represents a peptide comprising n number of amino acid monomers;

[ 13] DG is a displaceable group;

site. [15] Preferab is Sec. The DG is preferably a selenoate or a leaving group (LG). Preferably, the selenoate is an aryl selenoate. The aryl selenoate is preferably phenyl selenolate.

[16] Preferably, the peptide is defined by formula (IV):

IV

[17] the ester is defined by formula (V):

O

Nterm-AAa-AAb-AAc-AAd-AAe-X SePh . ; and

V

[18] the diselenide is defined by formula (VI):

VI,

[19] wherein AAa-e, AAl-5 and X are amino acids; [20] wherein * ^{AAl AA2 AAs AA<}* ^ΑΑδ< represents a peptide comprising the five amino acid residues AAi, AA₂, AA₃, AA₄, and AA₅;

[21] wherein « ^{AAa AAb AAc AAd AAe~} represents a peptide comprising the five amino acid residues AAa, AAb, AAc, AAd, and AAe.

[22] Preferably, AAa is L; AAb is Y; AAc is R; AAd is A; AAe is N. X is preferably selected from the group consisting of: Ala, Ser, Thr, Leu, He, Val, Phe, Met and Lys. AAu_g is preferably U; AAi is S; AA₂ is P; AA is G; AA₄ is Y; AA₅ is S.

[23] Preferably, the reaction is conducted in an aqueous solution. Preferably, the aqueous solution has a pH in the range of about 2 to 14. In one embodiment, the aqueous solution has a pH in the range of about 2 to 8 when the reaction is run in the absence of an additive. In another embodiment, when the reaction is run in the presence of an additive the pH can be in the range of about 8 to 14. The aqueous solution is preferably a buffer comprising a denaturing agent and an aqueous solution of Na₂HP0₄. Preferably, the denaturing agent is 6 M guanidine hydrochloride. The aqueous solution of Na₂HP0₄ preferably has a concentration of about 100 mM. Preferably, the buffer is at a pH of about 7.2 when the reaction is run in the absence of an additive.

[24] Preferably, the ester and the diselenide are dissolved in the buffer before the reaction step at a concentration of about 10 mM. The reaction is preferably commenced by combining the solutions of the ester and the diselenide and then allowed to proceed to completion as measured by an analytical technique. Preferably, the pH of the combined solution is at a pH of about 6.5 when the reaction is run in the absence of an additive and the final concentration of the reaction with respect to the ester peptide fragment 5 mM.

The reaction is preferably complete within about 60 seconds as measured by the consumption of the ester by HPLC excepting those reactions where X is selected from He or Val wherein the reaction is complete within about 10 minutes as measured by HPLC.

[26] Preferably, the method additionally comprises the step of deselenizing the peptide. The peptide preferably comprises a cysteine residue and the deselenisation step comprises selectively deselenizing the peptide so as not to desulfurize the cysteine residue. The deselenization preferably comprises reacting the peptide with a reducing agent. In one example, the reducing agent may be a mild reducing agent. Preferably, the reducing agent comprises a phosphine. The phosphine is preferably water soluble. Preferably, the phosphine is tris-(2-carboxyethyl)phosphine (TCEP). The reducing agent preferably additionally comprises a thiol. Preferably, the thiol is dithiothreitol.

[27] The deselenization preferably comprises reacting the peptide with a reducing agent and an oxidizing agent. In one example, the reducing agent may be a mild reducing agent. In one example, the oxidizing agent may be a mild oxidizing agent. Preferably, the reducing agent comprises a phosphine. The phosphine is preferably water soluble. Preferably, the phosphine is tris-(2-carboxyethyl)phosphine (TCEP). The oxidising agent is preferably potassium peroxy monosulfate. Preferably, the deselenization is conducted at a pH of about 4 to 5. The reaction step and the deselenization step are preferably conducted in a one-pot reaction.

[28] The present disclosure also relates to a method for oxidatively deselenizing a seleno functionalized amino acid residue in a peptide, said method comprising exposing the peptide to a mild reducing agent and a mild oxidizing agent. The mild reducing agent preferably comprises a phosphine. Preferably, the phosphine is water soluble. The phosphine is preferably tris-(2-carboxyethyl)phosphine (TCEP). Preferably, the oxidizing agent is potassium peroxy monosulfate. The deselenization is preferably conducted at a pH of about 4 to 5.

[29] The disclosure also relates to a method of preparing an ester containing compound comprising the step of reacting an ester reagent with a diselenide bearing a hydroxyl group. Preferably, the ester reagent is a selenoester reagent. [30] The disclosure also relates to a method of preparing a hydrazide containing compound comprising the step of reacting an ester reagent with a diselenide bearing a hydrazine group. Preferably, the ester reagent is a selenoester reagent.

[31] The disclosure also relates to a method of preparing an amide containing compound comprising the step of reacting an ester with a dithiol bearing an amino group, wherein the reaction proceeds in the absence of an additive. Preferably, the ester reagent is a selenoester reagent.

[32] The disclosure also relates to a method of preparing an ester containing compound comprising the step of reacting an ester with a dithiol bearing a hydroxyl group, wherein the reaction proceeds in the absence of an additive. Preferably, the ester reagent is a selenoester reagent.

[33] The disclosure also relates to a method of preparing a hydrazide containing compound comprising the step of reacting an ester with a dithiol bearing a hydrazine group, wherein the reaction proceeds in the absence of an additive. Preferably, the ester reagent is a selenoester reagent.

[34] The disclosure also relates to a method for preparing a phenylselenoester, the method comprising the step of treating a carboxylic acid compound with diphenyldiselenide (DPDS) followed by Bu₃P. Preferably, the selenoester is a peptide selenoester and the carboxylic acid is a peptide carboxylic acid.

[35] The methods disclosed herein may also be carried out on a solid support.

[36] Also disclosed herein are amides, esters, hydrazides and peptides.

[37] The disclosure also relates to a peptide of Formula (I) as described above.

[38] The disclosure also relates to a peptide of Formula (IV) as described above. [39] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Thus, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly indicates otherwise. For example, reference to "a protein" includes a plurality of proteins; reference to "a cell" includes populations of a plurality of cells.

[40] In the present specification, the term "additive" refers to any means for promoting a reaction and/or preventing a side reaction that is added separately, from an external source, to a reaction mixture. The reaction mixture contains reagents. The reaction mixture may include a solvent. The skilled addressee would appreciate that the solvent may include an aqueous solution. The aqueous solution may include buffering salts and a denaturing agent. By way of illustration an additive may refer to an exogenous molecule that is added to the reaction mixture. An additive may also refer to the addition of electrons as required in an electrochemical reduction. Some non-limiting examples of additives are nucleophiles and reductants. For illustration, some thiol group containing reductants that have been traditionally used in NCL are MPAA, thiophenol and MESNa. Reductants that may be used with the selenocystine- selenoester ligation methodology disclosed herein include, but are not limited to, TCEP (tris(2-carboxyethyl)phosphine), THPP (tris(3-hydroxypropyl)phosphine), DTT (dithiothreitol), NaBH₄, NaHBH₃CN and ascorbic acid. Alternatively, selenol-based nucleophiles and reductants may also be used. Additives to suppress side reactions such as deselenization during ligation include, but are not limited to, diphenyldiselenide (DPDS) or ascorbic acid or a salt thereof. The methods disclosed herein may be performed in the absence of an additive. Preferably, the additive is ascorbic acid or a salt thereof. Preferably, the additive is sodium ascorbate.

[41] In the present specification, the term "amino acid" refers to a molecule containing both an amino group and a carboxy group. For example, in an a- amino acid, there is an "a-amino group" attached directly to the carbon atom bearing both an amino and a carboxyl group and an "a-carboxyl group" attached directly to the carbon atom bearing both an amino and a carboxyl group. The term "carboxyl" may refer to either a -COOH group or a -COO^" group, oc-amino acids are of the general form ¾N- CHR-COOH, where R is a side chain or H. The side chain in general is an alkyl chain, which is optionally substituted, commonly but not necessarily at its distal end. The N terminus of the amino acid (or of a peptide) is that end at which the amine functionality (optionally ionised or substituted/protected) is located, and the C terminus is the end at which the carboxyl functionality (optionally ionised or substituted/protected) is located.

[42] As used herein, the term "peptide" refers to a chain comprising (or consisting of) at least two amino acid residues joined by amide bond(s). They may be dipeptides, oligopeptides, polypeptides, proteins, glycopeptides, glycoproteins etc. The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and include a molecular chain of two or more amino acids linked covalently through peptide bonds. The terms do not refer to a specific length of the product. The terms include post- translational modifications of the peptide, for example, glycosylations, acetylations, biotinylations, 4-pentynoylations, PEGylations, phosphorylations, sulfations and the like. In addition, protein fragments, analogs, mutated or variant proteins, fusion proteins and the like are included within the meaning of polypeptide. The terms also include molecules in which one or more amino acid analogs or non-canonical or unnatural amino acids are included. In addition, peptides can be derivatized as described herein by well-known organic chemistry techniques set forth, for example, in Smith, M. B. March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Seventh Edition; John Wiley & Sons, Inc.: Hoboken, NJ, 2013; Fmoc Solid Phase Peptide Synthesis, A Practical Approach; Chan, W.C., White, P. D., Eds.; Oxford University Press, 2000 (chapter 6, pages 137-178; chapter 9, pages 215-227, chapter 11, pages 243-262); and Peptide Synthesis and Applications, Second Edition (Methods in Molecular Biology); Jensen, K. J., Shelton, P. T., Pedersen, S. L., Eds.; Humana Press, 2013 (chapter 8, pages 119-130).

[43] The term "aryl", alone or in combination, means a carbocyclic aromatic moiety containing one, two or even three rings wherein such rings may be attached together in a fused manner. Thus the term "aryl" embraces aromatic radicals such as phenyl, naphthyl, indenyl, tetrahydronaphthyl, anthracenyl, and indanyl. Said "aryl" group may have 1 or more substituents such as lower alkyl, hydroxyl, halo, haloalkyl, nitro, cyano, alkoxy and lower alkylamino, and the like. Phenyl substituted with -O- CH₂-O- forms an aryl benzodioxolyl substituent. Aryl as used herein, implies a fully unsaturated ring.

[44] Groups that are displaceable, "displaceable groups", generally refer to groups that are displaceable from a molecule during the course of a reaction.

[45] "Leaving groups" generally refer to groups that are displaceable by a nucleophile. Such leaving groups are known in the art. Examples of leaving groups include, but are not limited to, halides (e.g., I, Br, F, CI), sulfonates (e.g., mesylate, tosylate), sulfides (e.g., SCH₃), thiolate, selenoates, N-hydroxysuccinimide, N- hydroxybenzotriazole, and the like.

[46] "Nucleophiles" are species that are capable of attacking a molecule at the point of attachment of the leaving group causing displacement of the leaving group. Nucleophiles are known in the art. Examples of nucleophilic groups include, but are not limited to, amines, thiols, alcohols, selenols, Grignard reagents, anionic species (e.g., alkoxides, amides, carbanions) and the like.

[47] In further describing the peptides described herein, a one-letter abbreviation system is frequently applied to designate the identities of the twenty "canonical" or proteogenic amino acid residues generally incorporated into naturally occurring peptides and proteins (Table 1). Such one-letter abbreviations are entirely interchangeable in meaning with three-letter abbreviations, or non-abbreviated amino acid names.

[48] Non-canonical or non-proteogenic amino acid residues can be incorporated into a peptide by employing the techniques disclosed herein. The term "non-canonical amino acid residue" refers to amino acid residues in D- or L-form that are not among the 20 canonical amino acids generally incorporated into naturally occurring proteins, for example, β-amino acids, homoamino acids, cyclic amino acids, seleno amino acids, thio amino acids, and amino acids with derivatized side chains such as those described in US 2015/0023988.

[49] Table 1. Three and one-letter abbreviations for the twenty canonical/naturally- occurring amino acids and the twenty first amino acid Selenocysteine. [50] Nomenclature and Symbolism for Amino Acids and Peptides by the IUPAC- IUB Joint Commission on Biochemical Nomenclature (JCBN) have been published in the following documents: Biochem. J., 1984, 219, 345-373; Eur. J. Biochem., 1984, 138, 9-37; 1985, 152, 1; 1993, 213, 2; Internat. J. Pept. Prot. Res., 1984, 24, following page 84; J. Biol. Chem., 1985, 260, 14-42; Pure Appl. Chem., 1984, 56, 595-624; Amino Acids and Peptides, 1985, 16, 387-410; Biochemical Nomenclature and Related Documents, 2nd edition, Portland Press, 1992, pages 39-69 which are all referenced herein by their entirety.

[51] As stated herein above, in accordance with the present disclosure, the peptides described can also be chemically derivatized at one or more amino acid residues by known organic chemistry techniques. "Derivative" or "derivatized" refers to a subject peptide having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized molecules include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-benzylhistidine. Also included as chemical derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty canonical amino acids, whether in L- or D-form. For example, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.

[52] Useful derivatizations include modification of an N-terminal free amino group for attachment of an imaging agent, e.g. a fluorescent dye or a therapeutic agent whose activity adds to the potential therapeutic activity of the peptide. The N-terminus can be acylated or modified to a substituted amine, or derivatized with another functional group, such as an aromatic moiety (e.g., an indole acid, benzyl (Bzl or Bn), dibenzyl (DiBzl or Bn₂), or benzyloxycarbonyl (Cbz or Z)), Ν,Ν-dimethylglycine or creatine. For example, in some embodiments, an acyl moiety, such as, but not limited to, a formyl, acetyl (Ac), propanoyl, butanyl, pentanyl, heptanyl, hexanoyl, octanoyl, or nonanoyl, can be covalently linked to the N-terminal end of the peptide. Other exemplary N-terminal derivative groups include -NRRi (other than -NH₂), -NRC(0)Ri, -NRC(0)ORi, -NRS(0)₂Ri, -NHC(0)NHRi, succinimide, or benzyloxycarbonyl-NH- (Cbz-NH-), wherein R and Ri are each independently hydrogen or lower alkyl or phenyl and wherein the phenyl ring may be substituted with 1 to 5 substituents selected from Ci-C₄ alkyl, Ci-C₄ alkoxy, chloro, and bromo.

[53] In some embodiments, one or more peptidyl [-C(0)NR-] linkages (bonds) between amino acid residues can be replaced by a non-peptidyl linkage. Exemplary non-peptidyl linkages are -CH₂-carbamate [-CH₂-OC(0)NR-], phosphonate, -CH₂- sulfonamide [-CH2-S(0)₂NR-], thiourea [-NHC(S)NH-], urea [-NHC(0)NH-], -CH₂- secondary amine, and alkylated peptide [-C(0)NR₆, wherein R₆ is lower alkyl] .

[54] The above examples of derivatizations are not intended to be an exhaustive treatment, but merely illustrative. The skilled addressee would appreciate that one or more individual amino acids can be derivatized by well-known organic chemistry techniques as described for example in Smith, M. B. March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Seventh Edition; John Wiley & Sons, Inc.: Hoboken, NJ, 2013; and that various derivatizing agents are known to react with selected side chains or terminal residues as described for example in Fmoc Solid Phase Peptide Synthesis, A Practical Approach; Chan, W.C., White, P. D., Eds.;

Oxford University Press, 2000; Peptide Synthesis and Applications, Second Edition (Methods in Molecular Biology); Jensen, K. J., Shelton, P. T., Pedersen, S. L., Eds.; Humana Press, 2013.

Brief Description of Drawings

[55] Figure 1. Generalised synthetic scheme for preparation of amides, esters and hydrazides using the ligation and selenium chemistries disclosed herein. [56] Figure 2. General Scheme showing the solid-phase synthesis of peptide selenoesters and amino peptide ligation intermediates; additive free ligation; and selective reductive and oxidative deselenizations to afford peptides with Alanine and Serine at the ligation site. Selenocystine is the oxidised (diselenide) form of selenocysteine (Sec). Additive free ligation of selenocystine-containing dimer (H- USPGYS-NH₂)2 (1) with selenoesters 2 to 10, derived from solid phase synthesis, to give symmetrical dimers 11a to 19a and asymmetrical dimers lib to 19b. Selective reductive and oxidative deselenizations to afford peptides with Alanine (20 to 28) and Serine (29 to 33) at the ligation site are shown.

[57] Figure 3. Scheme showing additive-free ligation of a model selenoester (Ac- LYRANA-SePh, 2) and selenocystine-containing dimer (H-USPGYS-NH₂)₂ 1 in aqueous, denaturing buffer at neutral pH to afford Ac-LYRANAUSPGYS-NH₂ symmetrical dimer 11a and unsymmetrical diselenide product lib.

[58] Figure 4. (A) Model ligation Ac-LYRANS-SePh (3) + H-USPGYS-NH₂ dimer (1) in 6 M Gn-HCl, 100 mM Na₂HP0₄, pH 6.2, 2.5 mM with respect to 1; (B) Model ligation Ac-LYRANT-SePh (4) + H-USPGYS-NH₂ dimer (1) in 6 M Gn-HCl, 100 mM Na₂HP0₄, pH 6.2, 2.5 mM with respect to 1; (C) Stills of model ligation Ac- LYRANS-SePh (3) + H-USPGYS-NH₂ dimer (1) taken at t = 0 sees and t = 26 sees; (D) Stills of model ligation Ac-LYRANT-SePh (4) + H-USPGYS-NH₂ dimer (1) taken at t = 0 sees and t = 70 sees.

[59] Figure 5. Rate plot comparing the ligation between Ac-LYRANL-SePh (5) and (H-USPGYS-NH₂)₂ with the ligation of various peptides bearing an N-terminal Sec, cysteine and cystine.

[60] Figure 6. Rate plot comparison over the first 2 minutes of the ligation.

[61] Figure 7. Scheme of one-pot Ac-LYRANA-SePh + H-USPGYS-NH₂ dimer ligation followed by Sec to Ala and Sec to Ser conversion. [62] Figure 8. Synthesis of a peptide with Ser at the ligation site using a selective oxidative deselenization.

[63] Figure 9. Selenoester ligations with β-selenophenylalanine-containing peptides.

[64] Figure 10. A selenoester ligation with a 2-thiol tryptophan-containing peptide.

[65] Figure 11. Synthesis of native Chorismate Mutase (Mtb CM). [66] Figure 12. Scheme of ESAT-6 synthesis.

[67] Figure 13. Generalised synthetic scheme for the preparation of peptide on solid support using selenocystine- selenoester ligation.

[68] Figure 14. Generalised solid-phase synthesis of Ac-LYRANAUSPGYS-NH₂ (11) using selenocystine- selenoester ligation chemistry.

[69] Figure 15. Solid-phase synthesis of peptides combining ligation and Fmoc protecting group chemistries.

[70] Figure 16. One pot ligation-deselenization using a model sequence containing cysteine.

[71] Figure 17. Ligation of H-USPGYS-NH₂ dimer (1) and alkyl selenoester (51) in ligation buffer at 5 mM, pH 6.2.

[72] Figure 18. Progress plot of the ligation reaction between Ac-LYRANL-SeAlk (51) and H-USPGYS-NH₂ dimer (1) at 2.5 mM with respect to 1 over the course of 12 h. [73] Figure 19. Ligation reaction between Ac-LYRANP-SePh (52) and H- USPGYS-NH₂ dimer (1).

[74] Figure 20. Plot of the Ac-LYRANP-SePh (52) + H₂N-USPGYS-NH₂ dimer (1) ligation in 200 mM TCEP (6 M guanidine hydrochloride, 100 mM Na₂HP0₄, pH 6.2 at 2.5 mM with respect to peptide 1.

[75] Figure 21. Ligation reaction between Ac-LYRANI-SePh (6) and H- USPGYS-NH₂ dimer (1) at 2.5 mM with respect to peptide 1 in the presence of 200 mM TCEP.

[76] Figure 22. General methods for the synthesis of substituted aryl diselenides.

[77] Figure 23. Synthesis of diselenophenylacetic acid (SePhCH₂C0₂H)_2.

[78] Figure 24. Synthesis of the aryl selenoesters using Fmoc-SPPS strategy and off -resin selenoesterification.

[79] Figure 25. Synthesis of Ac -LYRANL- S ePhCH₂C 0₂H by trans- selenoesterification.

[80] Figure 26. Scheme depicting the ligation reaction of H-USPGYS-NH₂ dimer (1) with Ac-LYRANL-SePh (5) and the ligation reaction of H-USPGYS-NH₂ dimer (1) with Ac-LYRANL-SePhR (54 to 59).

[81] Figure 27. Analytical UPLC trace of HPLC purified H-USPGYS-NH₂ dimer (1); Rt 2.8 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 659.2 (100%); Mass Found (ESI+ ); 658.2 [M+2H]²⁺.

[82] Figure 28. Analytical UPLC trace of HPLC purified Ac-LYRANA-SePh (2); Rt 4.7 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+H]⁺: 889.3 (100%), [M+2H]²⁺: 445.2 (100%); Mass Found (ESf); 889.4 [M+H]⁺, 445.5 [M+2H]²⁺. [83] Figure 29. Analytical UPLC trace of HPLC purified Ac-LYRANS-SePh (3); Rt 4.4 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+H]⁺: 905.3 (100%), [M+2H]²⁺: 453.2 (100%); Mass Found (ESf); 905.5 [M+H]⁺, 453.4 [M+2H]²⁺.

[84] Figure 30. Analytical UPLC trace of HPLC purified Ac-LYRANT-SePh (4); Rt 4.4 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+H]⁺: 919.4 (100%), [M+2H]²⁺: 460.2 (100%); Mass Found (ESf); 919.5 [M+Hf, 460.4 [M+2H]²⁺.

[85] Figure 31. Analytical UPLC trace of HPLC purified Ac-LYRANL-SePh (5); Rt 5.4 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+Hf: 931.4 (100%), [M+2H]²⁺: 466.2 (100%); Mass Found (ESf); 931.5 [M+Hf, 466.3 [M+2H]²⁺.

[86] Figure 32. Analytical UPLC trace of HPLC purified Ac-LYRANI-SePh (6); Rt 5.3 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+H]²⁺: 931.4 (100%), [M+2H]²⁺: 466.2 (100%); Mass Found (ESf); 931.5 [M+Hf, 466.5 [M+2H]²⁺.

[87] Figure 33. Analytical UPLC trace of HPLC purified Ac-LYRANV-SePh (7); Rt 5.1 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+Hf: 917.4 (100%), [M+2H]²⁺: 459.2 (100%); Mass Found (ESf); 917.5 [M+Hf, 459.3 [M+2H]²⁺.

[88] Figure 34. Analytical UPLC trace of HPLC purified Ac-LYRANF-SePh (8); Rt 5.4 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+Hf: 965.4 (100%), [M+2H]²⁺: 483.2 (100%); Mass Found (ESf); 965.6 [M+Hf, 483.3 [M+2H]²⁺.

[89] Figure 35. Analytical UPLC trace of HPLC purified Ac-LYRANM-SePh (9); Rt 5.1 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+Hf: 949.3 (100%), [M+2H]²⁺: 475.7 (100%); Mass Found (ESf); 949.5 [M+H]⁺, 475.3 [M+2H]²⁺.

[90] Figure 36. Analytical UPLC trace of HPLC purified Ac-LYRANK-SePh (10); Rt 4.3 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 473.7 (100%); Mass Found (ESI⁺); 474.0 [M+2H]²⁺.

[91] Figure 37. Ligation reaction between Ac-LYRANA-SePh and H-USPGYS- NH₂ dimer to give lla-c.

[92] Figure 38. Analytical HPLC trace of the ligation reaction between Ac- LYRANA-SePh and H-USPGYS-NH₂ dimer at 60 sees; product 11a R_t 22.7 min; product lib R_t 25.6 min (0-60% B over 30 min, λ = 214 nm).

[93] Figure 39. Analytical UPLC trace of HPLC purified ligation product Ac- LYRAN AUS PG YS -NH₂ (11); Rt 4.2 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 1389.6 (100%), [M+3H]³⁺: 926.7 (100%), [M+4H]⁴⁺: 695.3 (100%); Mass Found (ESI⁺); 1390.2 [M+2H]²⁺, 926.7 [M+3H]³⁺, 695.4 [M+4H]⁴⁺.

[94] Figure 40. Preparation of 12 by ligation reaction between Ac-LYRANS-SePh + H-USPGYS-NH₂ dimer.

[95] Figure 41. Analytical HPLC trace of the ligation reaction between Ac- LYRANS-SePh and H-USPGYS-NH₂ dimer at 60 sees; product 12a R_t 23.0 min; product 12b R_t 25.3 min (0-60% B over 30 min, λ = 214 nm).

[96] Figure 42. Analytical UPLC trace of HPLC purified ligation product Ac- LYRANS US PG YS -NH₂ (11); Rt 4.1 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 1405.6 (100%), [M+3H]³⁺: 937.4 (100%), [M+4H]⁴⁺: 703.3 (100%); Mass Found (ESI⁺); 1405.8 [M+2H]²⁺, 937.5 [M+3H]³⁺. [97] Figure 43. Preparation of Ac-LYRANTUSPGYS-NH₂ (13) by ligation reaction between Ac-LYRANT-SePh + H-USPGYS-NH₂ dimer.

[98] Figure 44. Analytical HPLC trace of the ligation reaction between Ac- LYRANT-SePh and H-USPGYS-NH₂ dimer at 60 s; product 13a R_t 23.9 min; product 13b R_t 26.2 min (0-60% B over 30 min, λ = 214 nm).

[99] Figure 45. Analytical UPLC trace of HPLC purified ligation product Ac- LYRANTUSPGYS-NH₂ (13); Rt 4.1 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 1419.6 (100%), [M+3H]³⁺: 946.7 (100%), [M+4H]⁴⁺: 710.3 (100%); Mass Found (ESI⁺); 1419.1 [M+2H]²⁺, 946.7 [M+3H]³⁺.

[100] Figure 46. Preparation of 14 by ligation reaction between Ac-LYRANL-SePh + H-USPGYS-NH₂ dimer.

[101] Figure 47. Analytical HPLC trace of the ligation reaction between Ac- LYRANL-SePh and H-USPGYS-NH₂ dimer at 60 sees; product 14a R_t 24.8 min; product 14b R_t 27.3 min; product 14c R_t 25.3 min (0-60% B over 30 min, λ = 214 nm).

[102] Figure 48. Analytical UPLC trace of HPLC purified ligation product Ac- LYRANLUSPGYS-NH₂ (14); Rt 4.6 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+3H]³⁺: 954.7 (100%), [M+4H]⁴⁺: 716.3 (100%); Mass Found (ESI⁺); 954.7 [M+2H]²⁺, 716.3 [M+3H]³⁺.

[103] Figure 49. Preparation of 15 by the ligation reaction between H-USPGYS- NH₂ dimer and Ac-LYRANI-SePh.

[104] Figure 50. Analytical HPLC trace of the ligation reaction between Ac- LYRANI-SePh and H-USPGYS-NH₂ dimer at 300 s; product 15a R_t 24.7 min; product 15b R_t 27.7 min; product 15c R_t 25.2 min (0-60% B over 30 min, λ = 214 nm). [105] Figure 51. Analytical HPLC trace of the ligation reaction between Ac- LYRANI-SePh and H-USPGYS-NH₂ dimer at 600 s; product 15a R_t 24.7 min; product 15b R_t 27.9 min; product 15c R_t 25.1 min (0-60% B over 30 min, λ = 214 nm).

[106] Figure 52. Analytical UPLC trace of HPLC purified ligation product Ac- LYRANIUSPGYS-NH₂ (15); Rt 4.6 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+3H]³⁺: 954.7 (100%), [M+4H]⁴⁺: 716.3 (100%); Mass Found (ESI⁺); 954.8 [M+3H]³⁺, 716.3 [M+4H]⁴⁺.

[107] Figure 53. Ligation reaction between H-USPGYS-NH₂ dimer and Ac- LYRANV-SePh to give 16a-c.

[108] Figure 54. Analytical HPLC trace of the ligation reaction between Ac- LYRANV-SePh and H-USPGYS-NH₂ dimer at 600 s; product 16a R_t 24.2 min; product 16b R_t 27.1 min; product 16c R_t 24.5 min (0-60% B over 30 min, λ = 214 nm).

[109] Figure 55. Analytical UPLC trace of HPLC purified ligation product Ac- LYRAN VUS PG YS -NH₂ (16); Rt 4.4 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+3H]³⁺: 945.4 (100%), [M+4H]⁴⁺: 709.3 (100%); Mass Found (ESI⁺); 945.5 [M+3H]³⁺, 709.4 [M+4H]⁴⁺.

[110] Figure 56. Ligation reaction between H-USPGYS-NH₂ dimer and Ac- LYRANF-SePh to give 17a-c.

[I l l] Figure 57. Analytical HPLC trace of the ligation reaction between Ac- LYRANF-SePh and H-USPGYS-NH₂ dimer at 60 s; product 17a R_t 25.6 min; product 17b R_t 28.3 min (0-60% B over 30 min, λ = 214 nm).

[112] Figure 58. Analytical UPLC trace of HPLC purified ligation product Ac- LYRANFUSPGYS-NH₂ (17); Rt 4.6 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+3H]³⁺: 977.4 (100%), [M+4H]⁴⁺: 733.3 (100%); Mass Found (ESI⁺); 977.6 [M+3H]³⁺, 733.4 [M+4H]⁴⁺. [113] Figure 59. Ligation reaction between H-USPGYS-NH₂ dimer and Ac- LYRANM-SePh to give 18a-c. Analytical UPLC trace of HPLC purified ligation product Ac-LYRANMUSPGYS-NH₂ (18); Rt 4.4 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 1449.6 (100%), [M+3H]³⁺: 966.7 (100%), [M+4H]⁴⁺: 725.3 (100%); Mass Found (ESI⁺); 1450.1 [M+2H]²⁺, 966.4 [M+3H]³⁺, 725.0 [M+4H]⁴⁺.

[114] Figure 60. Ligation reaction between H-USPGYS-NH2 dimer and Ac- LYRANK-SePh to give 19a-c. Analytical UPLC trace of HPLC purified ligation product Ac-LYRANKUSPGYS-NH₂ (19a); Rt 17.0 min (0-50% B over 30 min, λ = 214 nm); Calculated Mass [M+3H]³⁺: 964.7 (100%), [M+4H]⁴⁺: 723.8 (100%); Mass Found (ESI⁺); 964.5 [M+3H]³⁺, 723.9 [M+4H]⁴⁺.

[115] Figure 61. Analytical UPLC trace of HPLC purified Ac-LYRANAASPGYS- NH₂ (20) following ligation and Sec to Ala conversion; Rt 3.7 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 655.8 (100%); Mass Found (ESI⁺);

656.2 [M+2H]²⁺.

[116] Figure 62. Analytical UPLC trace of HPLC purified Ac-LYRANSASPGYS- NH₂ (21) following ligation and Sec to Ala conversion; Rt 3.7 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 664.3 (100%); Mass Found (ESI⁺);

664.3 [M+2H]²⁺.

[117] Figure 63. Analytical UPLC trace of HPLC purified Ac-LYRANTASPGYS- NH₂ (22) following ligation and Sec to Ala conversion; Rt 3.7 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 670.8 (100%); Mass Found (ESI⁺);

671.3 [M+2H]²⁺.

[118] Figure 64. Analytical UPLC trace of HPLC purified Ac-LYRANIASPGYS- NH₂ (23) following ligation and Sec to Ala conversion; Rt 4.0 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 676.9 (100%), Mass Found (ESF);

677.4 [M+2H]²⁺. [119] Figure 65. Analytical UPLC trace of HPLC purified Ac-LYRANVASPGYS- NH₂ (24) following ligation and Sec to Ala conversion; Rt 3.9 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 669.8 (100%); Mass Found (ESI⁺); 670.3 [M+2H]²⁺.

[120] Figure 66. Analytical UPLC trace of HPLC purified Ac-LYRANFASPGYS- NH₂ (25) following ligation and Sec to Ala conversion; Rt 4.2 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 693.8 (100%); Mass Found (ESI⁺);

694.0 [M+2H]²⁺.

[121] Figure 67. Analytical UPLC trace of HPLC purified Ac-LYRANMASPGYS- NH₂ (26) following ligation and Sec to Ala conversion; Rt 3.9 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 685.8 (100%), Mass Found (ESF); 686.3 [M+2H]²⁺.

[122] Figure 68. Analytical UPLC trace of HPLC purified Ac-LYRANKASPGYS- NH₂ (27) following ligation and Sec to Ala conversion; Rt 3.6 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 684.4 (100%), Mass Found (ESF); 684.8 [M+2H]²⁺.

[123] Figure 69. Analytical UPLC trace of HPLC purified Ac-LYRANLASPGYS- NH₂ (28) following ligation and Sec to Ala conversion; Rt 25.4 min (0-50% B over 30 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 677.1 (100%); Mass Found (ESI⁺);

677.1 [M+2H]²⁺.

[124] Figure 70. Analytical UPLC trace of HPLC purified Ac-LYRANASSPGYS- NH₂ (29) following ligation and Sec to Ser conversion; Rt 3.7 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 663.8 (100%); Mass Found (ESI⁺); 664.3 [M+2H]²⁺.

[125] Figure 71. Analytical UPLC trace of HPLC purified Ac-LYRANTSSPGYS- NH₂ (30) following ligation and Sec to Ser conversion; Rt 3.7 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 679.2 (100%); Mass Found (ESI⁺); 679.3 [M+2H]²⁺.

[126] Figure 72. Analytical UPLC trace of HPLC purified Ac -LYRANIS S PG YS - NH₂ (31) following ligation and Sec to Ser conversion; Rt 4.0 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 684.8 (100%); Mass Found (ESI⁺); 685.3 [M+2H]²⁺.

[127] Figure 73. Analytical UPLC trace of HPLC purified Ac-LYRANVS SPGYS - NH₂ (32) following ligation and Sec to Ser conversion; Rt 3.8 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 678.3 (100%); Mass Found (ESI⁺); 678.3 [M+2H]²⁺ _.

[128] Figure 74. Analytical UPLC trace of HPLC purified Ac- LYRANM(=0)SSPGYS-NH₂ (33) following ligation and Sec to Ser conversion; Rt 3.6 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 702.3 (oxidised Met) (100%); Mass Found (ESF); 702.4 [M+2H]²⁺.

[129] Figures 75 to 78 show the ligation of Ac-LYRANT-SePh + H-USPGYS-NH₂ dimer and the conversion of the Sec residue at the ligation site to provide Alanine and Serine residues at the ligation site.

[130] Figure 75. The ligation of Ac-LYRANT-SePh + H-USPGYS-NH₂ and the conversion of the Sec residue at the ligation site to provide Alanine and Serine residues at the ligation site. Analytical UPLC chromatograms and ESI MS spectra illustrating; A) the ligation at t = 0 s; B) the crude ligation at t = 5 mins; C) the crude Sec to Ala conversion; D) the crude Sec to Ser conversion.

[131] Figure 76. Crude analytical UPLC trace of Ac-LYRANT-SePh + H- USPGYS-NH₂ dimer ligation at t = 0 s; Rt 3.1 & 4.6 min (diselenide and selenoester respectively) (0-50% B over 5 min, λ = 214 nm). [132] Figure 77. Crude analytical UPLC trace of Ac-LYRANT-SePh + H- USPGYS-NH₂ dimer ligation at t = 5 mins; Rt 4.3 (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 1419.6 (100%), [M+3H]³⁺: 946.7 (100%), [M+4H]⁴⁺: 710.3 (100%); Mass Found (ESI⁺); 1419.1 [M+2H]²⁺, 946.7 [M+3H]³⁺, 709.6 [M+4H]⁴⁺.

[133] Figure 78. Crude analytical UPLC trace of Ac-LYRANTASPGYS- NH₂ following ligation and Sec to Ala conversion; Rt 3.6 (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 670.8 (100%); Mass Found (ESI⁺); 670.0 [M+2H]²⁺.

[134] Figure 79. Direct-inject analytical HPLC trace of Ac-LYRANA-SePh + H- USPGYS-NH₂ dimer ligation at t = 60 sees. Symmetrical (11a) and asymmetrical (lib) products indicated.

[135] Figure 80. Analytical HPLC of crude reaction mixture obtained in preparing 33: (0-50% B over 5 min, λ = 230 nm).

[136] Figure 81. Analytical HPLC trace of purified Ac-LYRANM(=0)SSPGYS- NH₂ (33) following ligation and Sec to Ser conversion; Rt 22.9 min (0-50% B over 30 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 702.3 (oxidised Met) (100%); Mass Found (ESI⁺); 702.4 [M+2H]²⁺.

[137] Figure 82. Analytical HPLC of crude reaction mixture obtained in preparing Ac-LYRANGS SPGYS -NH₂ (41): (0-50% B over 30 min, λ = 230 nm).

[138] Figure 83. Analytical HPLC of 41: Rt 14.7 min (0-50% B over 30 min, λ = 280 nm); Calculated Mass [M+2H]²⁺: 656.8 (100%), 657.3 (61.6%); Mass Found (ESI⁺); 657.15 [M+2H]²⁺.

[139] Figure 84. Analytical HPLC of crude reaction mixture in synthesis of Ac- LYRAN AS S PG YS -NH₂ 29: (0-50% B over 30 min, λ = 230 nm). [140] Figure 85. Analytical HPLC of 29: Rt 15.3 min (0-50% B over 30 min, λ = 230 nm); Calculated Mass [M+H]⁺: 1326.6 (100%), [M+2H]²⁺: 663.8 (100%); Mass Found (ESI⁺); 1326.8 [M+H]⁺, 664.3 [M+2H]²⁺.

[141] Figure 86. Analytical HPLC of crude reaction mixture in synthesis of Ac- LYRANM(=0)SSPGYS-NH₂ 42 (t =1 h) after 1 dose of TCEP (50 eq.) and Oxone (50 eq.): (0-50% B over 30 min, λ = 230 nm).

[142] Figure 87. Analytical HPLC of 42: Rt 15.2 min (0-50% B over 30 min, λ = 230 nm); Calculated Mass [M+2H]²⁺: 701.8 (100%), 702.3 (64.9%); Mass Found (ESI⁺); 702.2 [M+2H]²⁺.

[143] Figure 88. Analytical HPLC of Ac-LYRANMSSPGYS-NH₂ 33: Rt 15.6 min (0-50% B over 30 min, λ = 230 nm); Calculated Mass [M+2H]²⁺: 693.8 (100%); Mass Found (ESI⁺); 693.2 [M+2H]²⁺.

[144] Figure 89. Analytical HPLC of crude reaction mixture in synthesis of Ac- LYRANFS S PG YS -NH₂ 43: (0-50% B over 30 min, λ = 230 nm).

[145] Figure 90. Analytical HPLC of 43: Rt 17.9 min (0-50% B over 40 min, λ = 230 nm); Calculated Mass [M+2H]²⁺: 701.8 (100%), 702.3 (69.2%); Mass Found (ESI⁺); 704.2 [M+2H]²⁺.

[146] Figure 91. Analytical HPLC of crude reaction mixture in synthesis of Ac- LYRANLS S PG YS -NH₂ 44 (t =1 h) after 1 dose of TCEP (50 eq.) and Oxone (50 eq.): (0-50% B over 30 min, λ = 230 nm).

[147] Figure 92. Analytical HPLC of 44: Rt 17.3 min (0-50% B over 30 min, λ = 230 nm); Calculated Mass [M+H]⁺: 1368.7 (100%), [M+2H]²⁺: 684.8 (100%), 685.4 (66.0%); Mass Found (ESF); 1368.8 [M+H]⁺, 685.3 [M+2H]²⁺. [148] Figure 93. Analytical HPLC of crude reaction mixture in synthesis of Ac- LYRANFS S PKYS -NH₂ 45: (0-50% B over 30 min, λ = 230 nm).

[149] Figure 94. Analytical HPLC of 45: Rt 19.7 min (0-40% B over 30 min, λ = 230 nm); Calculated Mass [M+2H]²⁺: 737.4 (100%), 737.9 (73.5%); Mass Found (ESI⁺); 737.8 [M+2H]²⁺.

[150] Figure 95. Analytical HPLC of crude reaction mixture in synthesis of Ac- LYRAN AS S PH YS -NH₂ 46: (0-40% B over 30 min, λ = 230 nm).

[151] Figure 96. Analytical HPLC of 46: Rt 17.0 min (0-40% B over 30 min, λ = 230 nm); Calculated Mass [M+2H]²⁺: 703.8 (100%), 704.4 (67.1%); Mass Found (ESI⁺); 704.2 [M+2H]²⁺.

[152] Figure 97. Analytical HPLC of crude reaction mixture in synthesis of Ac- LYRAN AS S PW YS -NH₂ 47: (0-50% B over 30 min, λ = 230 nm).

[153] Figure 98. Analytical HPLC (purified product 47): Rt 19.3 min (0-50% B over 30 min, λ = 230 nm); Calculated Mass [M+2H]²⁺: 728.4 (100%), 728.9 (72.5%); Mass Found (ESI⁺); 728.8 [M+2H]²⁺.

[154] Figure 99. Analytical HPLC of crude reaction mixture in synthesis of Ac- LYRAN AS S PC YS -NH₂ 48 (0-40% B over 30 min, λ = 230 nm) showing a 4: 1 ratio of peptide 48 to the corresponding Sec to Ala conversion product.

[155] Figure 100. Analytical HPLC of 48: Rt 18.8 min (0-40% B over 30 min, λ = 230 nm); Calculated Mass [M+2H]²⁺: 686.8 (100%), 687.3 (63.8%); Mass Found (ESI⁺); 687.3 [M+2H]²⁺.

[156] Figure 101. Analytical HPLC trace (0 to 50% B over 30 min, λ = 230 nm) of the treatment of Ac-LYRANACSPGYS-NH₂ with TCEP (50 eq.) and Oxone (50 eq.) according to the optimized Sec to Ser conversion protocol. Results indicate that the Cys-containing peptide is inert to the reaction conditions.

[157] Figure 102. Crude HPLC-MS trace (0 to 50% B over 30 min, λ = 280 nm) of a representative ligation between a β-selenophenylalanine-containing peptide and a peptide phenylselenoester (t = 6 h).

[158] Figure 103. ESI Mass Spectrum of the ligation product as the diselenide dimer. Calculated Mass [M+2H]²⁺: 1494.1; [M+3H]³⁺: 996.4; Mass Found (ESf); 1493.5 [M+2H]²⁺, 995.9 [M+3H]³⁺.

[159] Figure 104. ESI Mass Spectrum of the ligation product as the asymmetric phenylselenyl diselenide. Calculated Mass [M+2H]²⁺: 825.8; Mass Found (ESI⁺); 825.8 [M+2H]²⁺.

[160] Figure 105. Crude HPLC-MS trace (0 to 50% B over 30 min, λ = 280 nm) of a representative ligation between a 2-thiol tryptophan containing peptide and a peptide phenylselenoester (t = 30 min).

[161] Figure 106. ESI Mass Spectrum of a ligation product as the trans-esterified, internal thioester. Calculated Mass [M+2H]²⁺: 1171.1; [M+3H]³⁺: 781.1; Mass Found (ESI⁺); 1171.1 [M+2H]²⁺, 781.1 [M+3H]³⁺.

[162] Figure 107. HPLC trace of ligation reaction between Ac-LYRANL-SePh (5) + H-USPGYS-NH₂ dimer (1) at pH 1.6, 20 h; Rt 7.3 min (1) and 11.1 min (5) (0-50% B over 30 min, λ = 214 nm).

[163] Figure 108. HPLC trace of ligation reaction between Ac-LYRANL-SePh (5) + H-USPGYS-NH₂ dimer (1) at pH 2.3, 5 h; Rt 27.5 min (14a), 28.1 min (14c) and 30.7 min (14b) (0-50% B over 30 min, λ = 214 nm). [164] Figure 109. HPLC trace of ligation reaction between Ac-LYRANL-SePh (5) + H-USPGYS-NH₂ dimer (1) at pH 5.0, 3 min; Rt 27.4 min (14a), 27.9 min (14c) and 30.4 min (14b) (0-50% B over 30 min, λ = 214 nm).

[165] Figure 110. HPLC trace of ligation reaction between Ac-LYRANL-SePh (5) + H-USPGYS-NH₂ dimer (1) at pH 7.7, 2 min; Rt 27.1 min (14a), 27.6 min (14c) and 30.4 min (14b) (0-50% B over 30 min, λ = 214 nm).

[166] Figure 111. HPLC trace of ligation reaction between Ac-LYRANL-SePh (5) + H-USPGYS-NH₂ dimer (1) at pH 8.3; Rt 19.8 min (1) and 27.1 min (14a) (0-50% B over 30 min, λ = 214 nm).

[167] Figure 112. Analytical UPLC trace of the ligation reaction between peptides Ac-LYRANF-SePh (8) and H-USPGYS-NH₂ dimer (1) at pH 4.0 after 10 mins; products observed at 4.5 min and 4.6 min (0-50% B over 30 min, λ = 214 nm).

[168] Figure 113. HPLC trace of a ligation reaction between Ac-LYRANA-SePh (2) + H-USPGYS-NH₂ dimer (1) at 250 μΜ with respect to dimer 1 after 60 min; Rt 3.8 min (11a) and 4.7 min (lib) (0-50% B over 8 min, λ = 214 nm).

[169] Figure 114. HPLC trace of a ligation reaction between Ac-LYRANL-SePh (5) + H-USPGYS-NH₂ dimer (1) at 250 μΜ with respect to dimer 1 after 90 mins; Rt 4.4 min (14a), 4.5 min (14c) and 5.2 min (14b) (0-50% B over 8 min, λ = 214 nm).

[170] Figure 115. Analytical HPLC trace of purified C-terminal Mtb CM (35; (300 A widepore C-18 column): Rt 25.1 min (0 - 70% B over 30 min, λ = 230 nm).

[171] Figure 116. MS for purified 35 Calculated mass (ESI⁺); 601.8 [M+16H]¹⁶⁺, 641.9 [M+15H]¹⁵⁺, 687.6 [M+14H]¹⁴⁺, 740.5 [M+13H]¹³⁺, 802.1 [M+12H]¹²⁺, 874.9 [M+11H]¹¹⁺, 962.3 [M+10H]¹⁰⁺, 1069.1 [M+9H]⁹⁺ ; Observed mass (ESF); 601.9 [M+16H]¹⁶⁺, 641.9 [M+15H]¹⁵⁺, 687.5 [M+14H]¹⁴⁺, 740.4 [M+13H]¹³⁺, 802.1 [M+12H]¹²⁺, 874.8 [M+11H]¹¹⁺, 962.2 [M+10H]¹⁰⁺, 1069.1 [M+9H]⁹⁺. [172] Figure 117. Analytical HPLC trace of purified N-terminal Mtb CM (34); (300 A widepore C-18 column): Rt 23.0 min (0 - 100% B over 30 min, λ = 214 nm).

[173] Figure 118. MS for purified 34. Calculated mass (ESI⁺); 583.2 [M+8H]⁸⁺, 666.3 [M+7H]⁷⁺, 777.2 [M+6H]⁶⁺, 932.5 [M+5H]⁵⁺, 1165.3 [M+4H]⁴⁺, Observed mass (ESI⁺); 583.1 [M+8H]⁸⁺, 666.3 [M+7H]⁷⁺, 777.2 [M+6H]⁶⁺, 932.4 [M+5H]⁵⁺, 1165.1 [M+4H]⁴⁺.

[174] Figure 119. Analytical HPLC trace of the crude ligation of 34 and 35 (300 A widepore C-18 column): Rt 23.5 min (0 - 100% B over 30 min, λ = 214 nm); (ESf ) MS for ligation product of 34 and 35.

[175] Figure 120. Analytical HPLC trace of the deselenization to give 36 (300 A widepore C-18 column): Rt 23.1 min (0 - 100% B over 30 min, λ = 214 nm); (ESf) MS for crude product 36.

[176] Figure 121. Analytical HPLC trace of purified Mtb CM 36 following ligation and Sec to Ala conversion; (300 A widepore C-18 column): Rt 21.8 min (0 - 100% B over 30 min, λ = 214 nm).

[177] Figure 122. MS for purified 36. Calculated mass (ESf); 513.7 [M+18H]¹⁸⁺, 543.9 [M+17H]¹⁷⁺, 577.8 [M+16H]¹⁶⁺, 616.3 [M+15H]¹⁵⁺, 660.2 [M+14H]¹⁴⁺, 710.9 [M+13H]¹³⁺, 770.1 [M+12H]¹²⁺, 840.0 [M+11H]¹¹⁺, 923.9 [M+10H]¹⁰⁺ , 1026.4 [M+9H]⁹⁺ Observed mass (ESf); 514.9 [M+18H]¹⁸⁺, 544.2 [M+17H]¹⁷⁺, 578.1 [M+16H]¹⁶⁺, 616.5 [M+15H]¹⁵⁺, 660.4 [M+14H]¹⁴⁺, 712.3 [M+13H]¹³⁺, 770.3 [M+12H]¹²⁺, 840.2 [M+11H]¹¹⁺, 924.2 [M+10H]¹⁰⁺ , 1026.8 [M+9H]⁹⁺.

[178] Figure 123. Circular dichroism spectrum of native Mtb CM 36 (250-195) in 50 mM Tris, 0.1 M NaCl, pH 7.5. (Data interval: 1 nm, bandwidth: 1.00 nm, scanning speed: 20 nm/min, accumulations: 5).

[179] Figure 124. Rate plot for [S] = 1 mM. [180] Figure 125. Rate plot for [S] = 0.5 niM. [181] Figure 126. Rate plot for [S] = 0.25 niM. [182] Figure 127. Rate plot for [S] = 0.125 niM.

[183] Figure 128. Lineweaver-Burk plot for Mtb CM activity; Km calc. as 1.1 mM (Vmax = 0.8 μηιοΐ min^"1 mg^"1). Turnover number 2.6 sec^"1.

[184] Figure 129. Analytical UPLC trace of ESAT-6 N-terminal fragment (selenoester) 37: Rt 5.1 min (0 - 70% B over 6 min, λ = 214 nm).

[185] Figure 130. MS for ESAT-6 N-terminal fragment (selenoester) 37. Calculated mass (ESI⁺); 1437.0 [M+3H]³⁺, 1078.0 [M+4H]⁴⁺, 862.6 [M+5H]⁵⁺; Observed mass (ESr); 1437.9 [M+3H]³⁺, 1078.7 [M+4H]⁴⁺, 863.1 [M+5H]⁵⁺.

[186] Figure 131. Analytical UPLC trace of ESAT-6 mid-fragment thioester 38: Rt 5.2 min (0 - 70% B over 6 min, λ = 214 nm).

[187] Figure 132. MS for ESAT-6 mid-fragment thioester 38. Calculated mass (ESI⁺); 1787.3 [M+4H]⁴⁺, 1430.0 [M+5H]⁵⁺, 1191.8 [M+6H]⁶⁺, 1021.7 [M+7H]⁷⁺. Observed mass (ESI⁺); 1788.3 [M+4H]⁴⁺, 1431.0 [M+5H]⁵⁺, 1192.65 [M+6H]⁶⁺, 1022.3 [M+7H]⁷⁺.

[188] Figure 133. Analytical UPLC trace of ESAT-6 C-terminal fragment 39: Rt 4.0 min (0 - 70% B over 6 min, λ = 214 nm).

[189] Figure 134. MS for ESAT-6 C-terminal fragment 39. Calculated mass (ESI⁺); 1166.5 [M+2H]²⁺, 778.0 [M+3H]³⁺. Observed mass (ESf); 1167.6 [M+2H]²⁺, 778.7 [M+3H]³⁺. [190] Figure 135. Analytical HPLC trace of crude ligation product prior to desulfurization to give 40 (300 A widepore C-18 column): Rt 4.4 min (0 - 100% B over 8 min, λ = 214 nm); (ESr^1") MS for crude ligation product.

[191] Figure 136. Analytical HPLC trace of native ESAT-6 40: Rt 26.0 min (0 - 100% B over 30 min, λ = 214 nm).

[192] Figure 137. MS for native ESAT-6 40. Calculated mass (ESf); 1090.9 [M+9H]⁹⁺, 1227.1 [M+8H]⁸⁺, 1402.2 [M+7H]⁷⁺, 1635.8 [M+6H]⁶⁺, 1962.7 [M+5H]⁵⁺; Observed mass (ESI⁺); 1091 [M+9H]⁹⁺, 1228 [M+8H]⁸⁺, 1403 [M+7H]⁷⁺, 1637 [M+6H]⁶⁺, 1965 [M+5H]⁵⁺.

[193] Figure 138. Circular dichroism spectrum of native ESAT-6 40 (250-195) in 25 mM NaH₂P0₄, 0.1 M NaCl, pH 6.5. (Data interval: 1 nm, bandwidth: 1.00 nm, scanning speed: 20 nm/min, accumulations: 5).

[194] Figure 139. Synthesis of H-USPCYS-NH₂ dimer 49. Analytical HPLC: Rt 13.7 (intramolecular selenyl- sulfide, 49a), 16.5 min (diselenide dimer, 49b) (0-25% B over 30 min, λ = 230 nm); Calculated Mass (intramolecular selenyl- sulfide) [M+H]⁺: 704.2 (100%); Mass Found (ESI⁺); 704.3 [M+H]⁺; Calculated Mass (diselenide dimer) [M+H]⁺: 705.2 (100%); Mass Found (ESI⁺); 706.3 [M+H]⁺.

[195] Figure 140. Analytical UPLC trace of HPLC purified Ac- LYRANLASPCYS-NH₂ (50) following ligation and Sec to Ala conversion: Rt 26.7 min (0-50% B over 30 min, λ = 214 nm); Calculated Mass [M+2H]²⁺: 699.8 (100%); Mass Found (ESI⁺); 700.2 [M+2H]²⁺.

[196] Figure 141. Analytical UPLC trace of HPLC purified Ac-LYRANL- SeCH₂C(0)NH₂ (51); Rt 4.7 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+H]⁺: 911.4 (100%), [M+2H]²⁺: 456.7 (100%); Mass Found (ESI⁺); 912.6 [M+H]⁺, 457.1 [M+2H]²⁺. [197] Figure 142. HPLC trace of a ligation reaction between Ac-LYRANL-SeAlkyl

(51) + H-USPGYS-NH₂ dimer (1) in 6 M guanidine hydrochloride, 100 mM Na₂HP0₄, pH 6.2 after 11 h; Rt 19.2 min (1 - selenol), 20.6 min (1 - diselenide) 25.9 min (product) 26.6 min (51) (0-50% B over 30 min, λ = 214 nm). Calculated Mass product [M+3H]³⁺: 955.1 (100%), [M+4H]⁴⁺: 716.6 (100%); Mass Found product (ESI⁺); 954.7 [M+3H]³⁺, 716.9 [M+4H]⁴⁺.

[198] Figure 143. Analytical UPLC trace of HPLC purified Ac-LYRANP-SePh

(52) ; Rt 4.8 min (0-50% B over 5 min, λ = 214 nm); Calculated Mass [M+H]⁺: 915.4 (100%), [M+2H]²⁺: 458.2 (100%); Mass Found (ESF); 915.5 [M+H]⁺, 458.5 [M+2H]²⁺.

[199] Figure 144. UPLC trace of a ligation reaction between Ac-LYRANP-SePh (52) + H- USPGYS-NH₂ dimer (1) in 6 M guanidine hydrochloride, 100 mM Na₂HP0₄, pH 6.2 after 24 h; Rt 3.0 min (1), 4.9 min (52) (0-50% B over 5 min, λ = 214 nm).

[200] Figure 145. UPLC trace of a ligation reaction between Ac-LYRANI-SePh (6) + H-USPGYS-NH₂ dimer (1) in 6 M guanidine hydrochloride, 100 mM Na₂HP0₄, 200 mM TCEP, pH 6.2 after 120 mins; Rt 3.7 min (31 Sec-Ser), 3.8 min (22 Sec-Ala) and 4.4 min (15c) (0-50% B over 5 min, λ = 214 nm).

[201] Figure 146. Analytical HPLC of diselenophenylacetic acid (SePhCH₂C0₂H)₂: Rt 22.9 min (0-100% B over 30 min, λ = 230 nm).

[202] Figure 147. Synthesis of Ac-LYRANL-SeTol (54). Analytical HPLC: Rt 32.2 min (0-50% B over 30 min, λ = 230 nm).

[203] Figure 148. MS of Ac-LYRANL-SeTol (54). Calculated Mass [M+H]⁺: 945.4; [M+2H]²⁺: 473.2; Mass Found (ESI⁺); 945.4 [M+H]⁺, 473.5 [M+2H]²⁺.

[204] Figure 149. Synthesis of Ac-LYRANL-SePh(p-OMe) (55). Analytical HPLC: Rt 30.9 min (0-50% B over 30 min, λ = 230 nm). [205] Figure 150. MS of Ac-LYRANL-SePh(p-OMe) 55. Calculated Mass [M+H]⁺: 961.4; [M+2H]²⁺: 481.2; Mass Found (ESI⁺); 961.4 [M+H]⁺, 481.5 [M+2H]²⁺.

[206] Figure 151. Synthesis of Ac-LYRANL- S ePhCH₂C 0₂Me (56). Analytical HPLC: Rt 4.8 min (0-50% B over 8 min, λ = 214 nm). Calculated Mass [M+H]⁺: 1003.41; [M+2H]²⁺: 502.21; Mass Found (ESI⁺); 1003.6 [M+H]⁺, 501.7 [M+2H]²⁺.

[207] Figure 152. Synthesis of Ac-LYRANL-SePh(p-NMe₂) (57). Analytical HPLC: Rt 26.0 min (0-50% B over 30 min, λ = 230 nm).

[208] Figure 153. MS of Ac-LYRANL-SePh(p-NMe₂) 57. Calculated Mass [M+H]⁺: 974.4; [M+2H]²⁺: 487.7; Mass Found (ESI⁺); 488.0 [M+2H]²⁺.

[209] Figure 154. Synthesis of Ac-LYRANL-SePh(/?-N0₂) (58). Analytical HPLC: Rt 26.0 min (0-50% B over 30 min, λ = 230 nm).

[210] Figure 155. MS of Ac-LYRANL-SePh(p-N0₂) (58). Calculated Mass [M+H]⁺: 976.4; [M+2H]²⁺: 488.7; Mass Found (ESF); 791.6 [M+H]⁺, 396.5 [M+2H]²⁺ (hydrolyzed product observed).

[211] Figure 156. Synthesis of Ac-LYRANL-SePhCH₂C0₂H (59). Analytical HPLC: Rt 20.8 min (0-50% B over 30 min, λ = 230 nm).

[212] Figure 157. MS of Ac-LYRANL-SePhCH₂C0₂H 59. Calculated Mass [M+H]⁺: 989.4; [M+2H]²⁺: 495.2; Mass Found (ESI⁺); 989.3 [M+H]⁺, 495.4 [M+2H]²⁺.

[213] Figure 158. Analytical LCMS of on-resin ligation product Ac-LYRANGU; R_t 23.9 min (0-30% B over 30 min, λ = 230 nm); Calculated Mass [M+2H]²⁺: 884.8 (100%), [M+3H]³⁺: 590.2 (100%); Mass Found (ESI⁺); 884.7 [M+2H]²⁺, 589.8 [M+3H]³⁺.

[214] Figure 159. Analytical LC-MS of on-resin ligation product Ac- LYRANGA; R_t 12.3 min (0-50% B over 30 min, λ = 230 nm); Calculated Mass [M+H]⁺: 805.4 (100%), [M+2H]²⁺: 403.7 (100%); Mass Found (ESf); 805.6 [M+H]⁺, 403.4 [M+2H]²⁺.

[215] Figure 160. H NMR of Ac-LYRANA-SePh 2 in d₆-DMSO.

[216] Figure 161. H NMR of Ac-LYRANS-SePh 3 in d₆-DMSO.

[217] Figure 162. H NMR of Ac-LYRANL-SePh 5 in d₆-DMSO.

[218] Figure 163. H NMR of Ac-LYRANI-SePh 6 in d₆-DMSO.

[219] Figure 164. H NMR of Ac-LYRANV-SePh 7 in d₆-DMSO.

[220] Figure 165. H NMR of Ac-LYRANF-SePh 8 in d₆-DMSO.

[221] Figure 166. H NMR of Ac-LYRANM-SePh 9 in d₆-DMSO.

[222] Figure 167. H NMR of Ac-LYRANAUSPGYS-NH₂ dimer 11a in D₂0.

[223] Figure 168. H NMR of Ac-LYRANAASPGYS-NH₂ 20 in D₂0.

[224] Figure 169. H NMR of Ac-LYRANSASPGYS-NH₂ 21 in D₂0.

[225] Figure 170. H NMR of Ac-LYRANTASPGYS-NH₂ 22 in D₂0.

[226] Figure 171. H NMR of Ac-LYRANIASPGYS-NH₂ 23 in D₂0.

[227] Figure 172. H NMR of Ac-LYRANVASPGYS-NHo 24 in D₂0.

[228] Figure 173. H NMR of Ac-LYRANFASPGYS-NH₂ 25 in D₂0.

[229] Figure 174. H NMR of Ac-LYRANMASPGYS-NH₂ 26 in D₂0.

[230] Figure 175. 1H NMR of Ac-LYRANKASPGYS-NH₂ 27 in D₂0. [231] Figure 176. 1H NMR of Ac-LYRANLASPGYS-NH₂ 28 in D₂0.

[232] Figure 177. Analytical HPLC trace of purified H-( -Se)DSPGYS-NH₂ dimer (64); R_t = 18.2 min (0-60% B over 30 min, λ = 280 nm); Calculated Mass [M+H]⁺: 1405.3 (100%), [M+2H]²⁺: 703.2 (100%); Mass Found (ESI+); 1405.7 [M+H]⁺, 703.5 [M+2H]²⁺.

[233] Figure 178. Analytical HPLC trace of crude Ac-LYRANFDSPGYS-NH₂ (65) post ligation-deselenisation; R_t 24.35 min (0-60% B over 30 min, λ = 280 nm).

[234] Figure 179. Analytical UPLC trace of HPLC purified Ac-LYRANFDSPGYS- NH₂ (65); R_t 3.42 min (0-60% B over 8 min, λ = 280 nm); Calculated Mass [M+H]⁺: 1430.7 (100%), [M+2H]²⁺: 715.8 (100%); Mass Found (ESI+ ): 1431.5 [M+H]⁺, 716.4 [M+2H]²⁺.

[235] Figure 180. Analytical HPLC trace of crude Ac-LYRANMDSPGYS-NH₂ (66) post ligation-deselenisation; R_t 23.06 min (0-60% B over 30 min, λ = 280 nm).

[236] Figure 181. Analytical UPLC trace of HPLC purified Ac- LYRANMDSPGYS-NH₂ (66): R_t 3.22 min (0-60% B over 8 min, λ = 280 nm); Calculated Mass [M+H]⁺: 1414.6 (100%), [M+2H]²⁺: 707.8 (100%); Mass Found (ESI+ ): 1415.3 [M+H]⁺, 708.4 [M+2H]²⁺.

[237] Figure 182. Analytical HPLC trace of crude Ac-LYRANSDSPGYS-NH₂ (67) post ligation-deselenisation: R_t 21.83 min (0-60% B over 30 min, λ = 280 nm).

[238] Figure 183. Analytical UPLC trace of HPLC purified Ac-LYRANSDSPGYS- NH₂ (67); R_t 2.97 min (0-60% B over 8 min, λ = 280 nm); Calculated Mass [M+H]⁺: 1370.6 (100%), [M+2H]²⁺: 685.8 (100%); Mass Found (ESI+): 1371.7 [M+H]⁺, 686.4 [M+2H]²⁺. [239] Figure 184. Analytical HPLC trace of crude Ac-LYRANVDSPGYS-NH₂ (68) post ligation-deselenisation: R_t 9.14 min (0-60% B over 15 min, λ = 280 nm).

[240] Figure 185. Analytical UPLC trace of HPLC purified Ac-LYRANVDSPGYS- NH₂ (68); R_t 3.50 min (0-50% B over 8 min, λ = 280 nm); Calculated Mass [M+H]⁺: 1382.7 (100%), [M+2H]²⁺: 691.8 (100%); Mass Found (ESI+ ): 1383.5 [M+H]⁺, 692.4 [M+2H]²⁺.

[241] Figure 186. Analytical HPLC trace of crude Ac-LYRANADSPGYS-NH₂ (69) post ligation-deselenisation; R_t 21.95 min (0-60% B over 30 min, λ = 280 nm).

[242] Figure 187. Analytical UPLC trace of HPLC purified Ac-LYRANADSPGYS- NH₂ (69); Rt 3.30 min (0-60% B over 8 min, λ = 280 nm); Calculated Mass [M+2H]²⁺: 677.8 (100%); Mass Found (ESI+): 678.3 [M+2H]²⁺.

[243] Figure 188. Analytical HPLC trace of crude Ac-LYRANLDSPGYS-NH₂ (70) post ligation-deselenisation; R_t 23.87 min (0-60% B over 30 min, λ = 280 nm).

[244] Figure 189. Analytical UPLC trace of HPLC purified Ac-LYRANLDSPGYS- NH₂ (70); R_t 3.70 min (0-60% B over 8 min, λ = 280 nm); Calculated Mass [M+H]⁺: 1396.7 (100%), [M+2H]²⁺: 698.9 (100%); Mass Found (ESI+): 1397.5 [M+H]⁺, 699.4 [M+2H]²⁺.

[245] Figure 190. Analytical HPLC trace of crude Ac-LYRANYDSPGYS-NH₂ (71) post ligation-deselenisation; R_t 22.77 min (0-60% B over 30 min, λ = 280 nm).

[246] Figure 191. Analytical UPLC trace of HPLC purified Ac-LYRANYDSPGYS- NH₂ (71); R_t 3.48 min (0-60% B over 8 min, λ = 280 nm); Calculated Mass [M+H]⁺: 1446.7 (100%), [M+2H]²⁺: 723.8 (100%); Mass Found (ESI+): 1447.9 [M+H]⁺, 724.5 [M+2H]²⁺.

[247] Figure 192. Analytical HPLC trace of HPLC purified Hyalomin-3 1-25 selenoester (72); Rt 26.82 min (0-60% B over 30 min, λ = 230 nm). [248] Figure 193. Mass data for compound 72. Calculated Mass [M+2H] ⁺: 1457.1 (100%), [M+3H]³⁺: 971.7 (100%); Mass Found (ESI+): 1456.8 [M+2H]²⁺, 971.5 [M+3H]³⁺.

[249] Figure 194. Analytical HPLC trace of HPLC purified Hyalomin-3 (26-60) diselenide dimer (73); Rt 21.33 min (0-70% B over 30 min, λ = 230 nm).

[250] Figure 195. Mass data for compound 73. Calculated Mass [M+4H]⁴⁺: 1699.7 (100%), [M+5H]⁵⁺: 1360.0 (100%), [M+6H]⁶⁺: 1133.5 (100%), [M+7H]⁷⁺: 971.7 (100%), [M+8H]⁸⁺: 850.4 (100%); Mass Found (ESI+): 1700.0 [M+4H]⁴⁺, 1360.4 [M+5H]⁵⁺, 1133.6 [M+6H]⁶⁺, 971.8 [M+7H]⁷⁺, 850.4 [M+8H]⁸⁺.

[251] Figure 196. Analytical HPLC trace of crude Hyalomin-3 (74) post ligation- deselenisation; Rt 22.10 min (0-60% B over 30 min, λ = 230 nm).

[252] Figure 197. Mass data for compound 74. Calculated Mass [M+4H]⁴⁺: 1519.2 (100%), [M+5H]⁵⁺: 1215.6 (100%), [M+6H]⁶⁺: 1013.1 (100%), [M+7H]⁷⁺: 868.5 (100%), [M+8H]⁸⁺: 760.1 (100%); Mass Found (ESI+): 1519.6 [M+4H]⁴⁺, 1215.9 [M+5H]⁵⁺, 1013.3 [M+6H]⁶⁺, 868.7 [M+7H]⁷⁺, 760.1 [M+8H]⁸⁺.

[253] Figure 198. Analytical HPLC trace of HPLC purified Hyalomin-3 (74); Rt 21.87 min (0-60% B over 30 min, λ = 230 nm).

[254] Figure 199. Further mass data for compound 74. Calculated Mass [M+4H]⁴⁺: 1519.2 (100%), [M+5H]⁵⁺: 1215.6 (100%), [M+6H]⁶⁺: 1013.1 (100%), [M+7H]⁷⁺: 868.5 (100%), [M+8H]⁸⁺: 760.1 (100%); Mass Found (ESI+): 1519.7 [M+4H]⁴⁺, 1215.9 [M+5H]⁵⁺, 1013.4 [M+6H]⁶⁺, 868.3 [M+7H]⁷⁺, 760.2 [M+8H]⁸⁺.

[255] Figure 200. Analytical HPLC trace of HPLC purified Hyalomin-4 1-25 selenoester (75); Rt 18.63 min (0-100% B over 30 min, λ = 230 nm). [256] Figure 201. Mass data for compound 75. Calculated Mass [M+2H] ⁺: 1500.6 (100%), [M+3H]³⁺: 1000.7 (100%); Mass Found (ESI+): 1500.6 [M+2H]²⁺, 1000.6 [M+3H]³⁺.

[257] Figure 202. Analytical HPLC trace of HPLC purified Hyalomin-4 (26-51) diselenide dimer (76); Rt 16.44 min (0-100% B over 30 min, λ = 230 nm).

[258] Figure 203. Mass data for compound 76. Calculated Mass [M+3H]³⁺: 1774.4 (100%), [M+4H]⁴⁺: 1331.1 (100%), [M+5H]⁵⁺: 1065.1 (100%), [M+6H]⁶⁺: 887.7 (100%), [M+7H]⁷⁺: 761.0 (100%), [M+8H]⁸⁺: 666.0 (100%); Mass Found (ESI+): 1774.9 [M+3H]³⁺, 1331.0 [M+4H]⁴⁺, 1064.9 [M+5H]⁵⁺, 887.6 [M+6H]⁶⁺, 760.9 [M+7H]⁷⁺, 665.7 [M+8H]⁸⁺

[259] Figure 204. Analytical HPLC trace of crude Hyalomin-4 post ligation- deselenisation (77); Rt 22.37 min (0-60% B over 30 min, λ = 230 nm).

[260] Figure 205. Mass data for compound 77. Calculated Mass [M+3H]³⁺: 1808.5 (100%), [M+4H]⁴⁺: 1356.6 (100%), [M+5H]⁵⁺: 1085.5 (100%), [M+6H]⁶⁺: 904.7 (100%), [M+7H]⁷⁺: 775.6 (100%); Mass Found (ESI+): 1809.0 [M+3H]³⁺, 1356.8 [M+4H]⁴⁺, 1085.7 [M+5H]⁵⁺, 904.8 [M+6H]⁶⁺, 775.7 [M+7H]⁷⁺.

[261] Figure 206. Analytical HPLC trace of HPLC purified Hyalomin-4 (77); Rt 21.34 min (0-60% B over 30 min, λ = 230 nm).

[262] Figure 207. Further mass data for compound 77. Calculated Mass [M+3H]³⁺: 1808.5 (100%), [M+4H]⁴⁺: 1356.6 (100%), [M+5H]⁵⁺: 1085.5 (100%), [M+6H]⁶⁺: 904.7 (100%), [M+7H]⁷⁺: 775.6 (100%), [M+8H]⁸⁺: 678.8 (100%); Mass Found (ESI+): 1809.0 [M+3H]³⁺, 1357.0 [M+4H]⁴⁺, 1085.8 [M+5H]⁵⁺, 904.9 [M+6H]⁶⁺, 775.7 [M+7H]⁷⁺, 678.8 [M+8H]⁸⁺.

[263] Figure 208. Synthetic route towards Boc-(p-PMBSe)Asp-OH [264] Figure 209. Analytical HPLC trace of HPLC purified H-(p-Se)LSPGYS-NH₂ dimer (85); R_t = 3.68 min (0-50% B over 5 min, λ = 280 nm); Calculated Mass [M+H]⁺: 1401.44 (100%), [M+2H]²⁺: 701.22 (100%); Mass Found (ESI+); 1398.5 [M+H]⁺, 700.1 [M+2H]²⁺.

[265] Figure 210. Analytical HPLC trace of crude Ac-LYRANLLSPGYS-NH₂ (86) post ligation-deselenisation; R_t 28.63 min (0-50% B over 30 min, λ = 280 nm).

[266] Figure 211. Analytical UPLC trace of HPLC purified Ac-LYRANLLSPGYS- NH₂ (86); R_t 4.74 min (0-50% B over 5 min, λ = 280 nm); Calculated Mass [M+H]⁺: 1394.74 (100%), [M+2H]²⁺: 697.87 (100%); Mass Found (ESI+ ): 1395.65 [M+H]⁺, 698.45 [M+2H]²⁺.

[267] Figure 212. One-pot ligation-deselenisation of model peptide systems; [a] 0.5 eq. of H-(p-Se)LSPGYS-NH₂ diselenide dimer to 1.28 eq. of selenoester.

[268] Figure 213. 1H NMR of l,2-bis(4-methoxybenzyl)diselane (60) in CDC1₃.

[269] Figure 214. ¹³C NMR of l,2-bis(4-methoxybenzyl)diselane (60) in CDC1₃.

[270] Figure 215. 1H NMR of Se-(4-methoxybenzyl) benzenesulfonoselenoate (61) in CDC1₃.

[271] Figure 216. 13 C NMR of Se-(4-methoxybenzyl) benzenesulfonoselenoate (61) in CDC1₃.

[272] Figure 217. 1H NMR of 1-allyl 4-(tert-butyl) (2R)-2-((tert- butoxycarbonyl)amino)-3-((4-methoxy benzyl) selanyl) succinate (62) in CDC1₃.

[273] Figure 218. ¹³C NMR of 1-allyl 4-(tert-butyl) (2R)-2-((tert- butoxycarbonyl)amino)-3-((4-methoxy benzyl) selanyl) succinate (62) in CDC1₃. [274] Figure 219. 1H NMR of (2R)-4-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)- 3-((4-methoxybenzyl)selanyl)-4-oxobutanoic acid (Boc-(P-PMBSe)Asp-OH) (63) in CDC1₃.

[275] Figure 220. ¹³C NMR of (2R)-4-(tert-butoxy)-2-((tert- butoxycarbonyl)amino)-3-((4-methoxybenzyl)selanyl)-4-oxobutanoic acid (Βοο(β- PMBSe)Asp-OH) (63) in CDCI₃.

[276] Figure 221. 1H NMR of tert-butyl (R)-4-((R)- l-hydroxy-2-methylpropyl)-2,2- dimethyloxazolidine-3-carboxylate (78) in CDCI₃.

[277] Figure 222. ¹³C NMR of tert-butyl (R)-4-((R)-l-hydroxy-2-methylpropyl)- 2,2-dimethyloxazolidine-3-carboxylate (78) in CDCI₃.

[278] Figure 223. 1H NMR of tert-butyl ((2R,3R)-l,3-dihydroxy-4-methylpentan-2- yl)carbamate (79) in CDC1₃.

[279] Figure 224. ¹³C NMR of tert-butyl ((2R.3R)- 1 ,3-dihydroxy-4-methylpentan-2- yl)carbamate (79) in CDCI₃.

[280] Figure 225. 1H NMR of tert-butyl ((2R,3R)-l-((tert-butyldimethylsilyl)oxy)- 3-hydroxy-4-methylpentan-2-yl)carbamate (80) in CDCI₃.

[281] Figure 226. ¹³C NMR of tert-butyl ((2R,3R)-l-((tert-butyldimethylsilyl)oxy)-

3- hydroxy-4-methylpentan-2-yl)carbamate (80) in CDCI₃.

[282] Figure 227. 1H NMR of tert-butyl ((2R.3S)- l-((tert-butyldimethylsilyl)oxy)-4- methyl-3-selenocyanatopentan-2-yl)carbamate (81) in CDCI₃.

[283] Figure 228. ¹³C NMR of tert-butyl ((2R,3S)-l-((tert-butyldimethylsilyl)oxy)-

4- methyl-3-selenocyanatopentan-2-yl)carbamate (81) in CDCI₃. [284] Figure 229. 1H NMR of tert-butyl ((2R,3S)-l-hydroxy-4-methyl-3- selenocyanatopentan-2-yl)carbamate (82) in CDC1₃

[285] Figure 230. ¹³C NMR of tort-butyl ((2R,3S)-l-hydroxy-4-methyl-3- selenocyanatopentan-2-yl)carbamate (82) in CDCI₃

[286] Figure 231 1H NMR of (2R,3S)-2-((tert-butoxycarbonyl)amino)-4-methyl-3- selenocyanatopentanoic acid (83) in CDCI₃.

[287] Figure 232. ¹³C NMR of (2R,3S)-2-((tert-butoxycarbonyl)amino)-4-methyl-3- selenocyanatopentanoic acid (83) in CDCI₃.

[288] Figure 233. 1H NMR of (2R,3S)-2-((tert-butoxycarbonyl)amino)-3-((4- methoxybenzyl)selanyl)-4-methylpentanoic acid (84) in CDCI₃.

[289] Figure 234. ¹³C NMR of (2R,3S)-2-((tert-butoxycarbonyl)amino)-3-((4- methoxybenzyl)selanyl)-4-methylpentanoic acid (84) in CDCI₃.

Description of the Embodiments

[290] In an effort to develop new synthetic methodologies that allow rapid and facile access to a number of compounds the present disclosure relates to the ligation/reaction of a selenoester reagent with another reagent that contains a seleno moiety and a another nucleophilic moiety as shown in Figure 1.

[291] With regard to the synthesis of peptides, in an effort to develop a ligation that may overcome such prohibitive reaction rates at sterically encumbered C-termini, disclosed herein is a ligation method utilizing peptide fragments, one bearing a selenocystine functionality and one containing a selenoester moiety. It was reasoned that the increased nucleophilicity of a selenocysteine combined with the enhanced properties of the selenoester as an acyl donor should dramatically increase the rate of ligation. Remarkably, an NCL-based reaction between such groups followed by chemo selective deselenization chemistry has not been explored to date. To avoid the possibility of deselenization occurring due to the presence of a reducing agent (such as TCEP), the Inventors sought to develop an additive-free ligation utilizing both chemical and electrochemical reductants. In the process of these investigations it was discovered that a peptide bearing an N-terminal selenocystine (the oxidised form of Sec) and a peptide bearing a C-terminal phenylselenoester ligated within seconds in aqueous buffer at room temperature without any additive.

[292] Ligation reactions at selenocysteine are often slower than the analogous reactions at cysteine (native chemical ligation). The use of strong reductants to chemically reduce the diselenide to liberate the free selenol/selenolate in solution often leads to loss of the selenol functionality through a deselenization process.

[293] The disclosure relates to the surprising discovery that a peptide carrying an N- terminal selenocystine amino acid residue can be chemo selectively ligated to a peptide fragment bearing a C-terminal selenoester-functionalized amino acid residue. Remarkably, the reaction proceeds without any additive - no reductant is required to reduce the selenocystine to free selenocysteine, no nucleophilic thiol is required, no exogenous nucleophilic selenol is required. Furthermore, the reaction proceeds quickly using unprotected peptide fragments, in aqueous buffer with broad pH range at room temperature. Ligation with sterically encumbered C-termini has been observed to proceed with vastly enhanced reaction rates (on the minute scale) compared to conventional NCL where ligation may only be completed after 2 days. Also, without any further purification, the crude ligation product can be subjected to deselenization conditions by one-pot radical reduction or oxidation to afford Ala or Ser at the ligation junction. The strategy for the synthesis of peptides is shown in Figure 2.

The isolated yields of the intermediates and products displayed in Figure 2 are also shown below in Table 2. Reaction

Selenoester Isolated

Ester time (s) Isolated yield

Ac- Ligation yield One- yield One-pot Sec to LYRANX yield^[c] (%) pot Sec to

(%) Ala^[d] (%)

(X =) Ser (%)

Ala 47 (2) 60^[a] 72 (11) 60 (20) 80 (29) Ser 72 (3) 60^[a] 73 (12) 57 (21) ND Thr 69 (4) 60^[a] 84 (13) 71 (22) 50 (30) Leu 47 (5) 60^[a] 87 (14) 66 (28) ND He 46 (6) 600^[b] 63 (15) 63 (23) 62 (31) Val 57 (7) 300^[b] 67 (16) 79 (24) 50 (32) Phe 59 (8) 60^[a] 83 (17) 58 (25) ND Met 45 (9) 60^[a] 72 (18) 56 (26) 55 (33) Lys 52 (10) 60^[a] 79 (19) 97 (27) ND

[294] Table 2. Isolated yields of additive free selenocystine-selenoester ligation and one-pot ligation-deselenization reactions. ^[a] 0.5 eq. H-USPGYS-NH₂ dimer to 1.0 eq. selenoester. ^[b] 0.5 eq. H-USPGYS-NH₂ dimer to 1.25 eq. of selenoester ^[c] Yield calculated from combined symmetrical diselenide (XXa) and asymmetrical diselenide (XXb) products.

[295] Initial Ligation Reactions and Development

[296] A model ligation reaction between a selenoester and a selenocystine was undertaken according to the following scheme as shown in Figure 3:

[297] It was found that peptide 1, bearing an N-terminal selenocystine (the oxidised form of Sec), and a peptide bearing a C-terminal phenylselenoester (2) ligated within seconds in aqueous buffer at room temperature without additives. Due to the electrophilic nature of both the diselenide and selenoester groups this result was surprising and has no precedent. The reaction is also advantageous as the addition of a reductant can result in deselenization. As there are no additives, work-up of the crude reaction product is simplified and the crude product can be telescoped into further synthetic transformations. [298] Further investigation of the ligation using direct-inject HPLC proved the model ligation was complete within 60 seconds yielding the symmetrical diselenide product 11a and to a lesser extent the asymmetrical diselenide lib. It appeared at this stage that no exogenous reductant was required for the ligation to proceed and the ligation was extremely rapid. To verify the chemo selectivity of the ligation, 11a was independently synthesized, isolated and characterized via NMR. This confirmed the symmetrical diselenide 11a as the major ligation product.

[299] Model Ligation Reactions

[300] With a variety of selenoesters in hand a number of other model ligations were carried out. Initially each fragment was included at a molar ratio of 1: 1 in 6 M Gn»HCl, 0.1 M phosphate buffer, pH 7.0 at a concentration of 2.5 mM with respect to the diselenide and 5 mM with respect to the selenoester. Upon addition of the peptides the pH dropped to 6.2 and, for all unhindered esters, a yellow precipitate was observed within 60 seconds after addition of the selenoester, with the accompanying odour of DPDS. Attempts to quench the reaction at multiple time points by dilution and/or acidification were unsuccessful. Although slowed at 100 μΜ and/or acidification to pH 2 the reaction still proceeded towards completion. Direct-inject HPLC to monitor the ligations. All examples except for the He and Val selenoesters had reached completion within 60 seconds of peptide addition. This includes the esters of Leu and Thr, examples that usually take 24 and 48 h respectively. Figure 4 depicts stills showing the progress of two example ligations.

[301] Both symmetrical and asymmetrical ligated products were isolated in varying ratios with combined yields of between 63-87% with no detectable hydrolysis of the selenoester. At 1 molar eq. the more hindered β branched examples of He and Val did not proceed to completion. Interestingly only 1.25 molar eq. of these hindered selenoesters was required to push the ligation to completion within 10 minutes. The analogous thioester ligations are only able to reach a 50% yield over a 48 h period. [302] Investigation into the tolerance of our ligation to pH change and dilution allowed comparisons to be drawn against the more traditional Cys (and thiolated residue) ligations that slow substantially below neutral pH and under a concentration of 1 mM. Our examples were observed to be efficacious between a pH range of 2.3-7.7. Above this value the selenoester rapidly hydrolyses. For example, the ligation of H- USPGYS-NH₂ (1) and Ac-LYRANL-SePh (5) was performed in 6 M guanidine hydrochloride, 100 mM Na₂HP0₄, 2.5 mM with respect to 1, at various pH levels. The reaction was analysed at appropriate time points using UPLC (0-50% B over 30 min, λ = 214 nm) and the results are shown below in Table 3.

Final pH before Time for

injection completion

No reaction

5 h

3 min

1 min

2 min

Hydrolysed ester

Table 3. Time to completion for ligation reactions between H-USPGYS-NH₂ (1) and Ac-LYRANL-SePh (5) at pH 1.6 - 8.3.

[303] The reaction may be conducted at moderately elevated temperatures, or at room temperature or below. In general, the ligation reactions described herein are conducted at room temperature. Nonetheless, the skilled addressee would understand that the reactions can be run at a lower temperature to minimise side reactions or run at elevated temperatures to, for example, further accelerate the rate of reaction. Suitable lower temperatures are below room temperature, below 0°C down to about -100 °C; for example, -10, -20, -50 or -70°C, or about -100 to about 0°C, or about -100 to -50, -100 to -70, -50 to 0, -20 to 0 or -80 to -60°C, e.g. about -100, -90, -80, -78, -70, -60, -50, - 40, -30, -20, -10 or 0°C. Suitable elevated temperatures are above room temperature, above about 30, 40, 50, 60, 70, up to about 80 °C. About 10 to about 80°C, or about 20 to 80, 50 to 80, 70 to 80, 10 to 30, 10 to 50, 30 to 60, 30 to 40, 40 to 70 or 50 to 70°C, e.g. about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80°C.

[304] The reaction may be run in the presence of an additive where the pH of the reaction mixture ranges from about 2 to 14. It would be understood, for example, that the reaction may be run in the presence of an additive at a pH from about 2 to 14; 2 to 13; 2 to 12; 2 to 11; 2 to 10; 2 to 9; 2 to 8; 2 to 7; 2 to 6; 2 to 5; 2 to 4; 2 to 3; 3 to 14; 3 to 13; 3 to 12; 3 to 11; 3 to 10; 3 to 9; 3 to 8; 3 to 7; 3 to 6; 3 to 5; 3 to 4; 4 to 14; 4 to 13; 4 to 12; 4 to 11; 4 to 10; 4 to 9; 4 to 8; 4 to 7; 4 to 6; 4 to 5; 5 to 14; 5 to 13; 5 to 12; 5 to 11; 5 to 10; 5 to 9; 5 to 8; 5 to 7; 5 to 6; 6 to 14; 6 to 13; 6 to 12; 6 to 12; 6 to 11; 6 to 10; 6 to 9; 6 to 8; 6 to 7; 7 to 14; 7 to 13; 7 to 12; 7 to 11; 7 to 10; 7 to 9; 7 to 8; 8 to 14; 8 to 13; 8 to 12; 8 to 11; 8 to 10; 8 to 9; 9 to 14; 9 to 13; 9 to 12; 9 to 11; 9 to 10; 10 to 14; 10 to 13; 10 to 12; 10 to 11; 11 to 14; 11 to 13; 11 to 12; 12 to 14; 12 to 13; or 13 to 14. The reaction may be run in the presence of an additive for example at a pH of about 2; 3; 4; 5; 6; 7 ;8; 9; 10; 11; 12; 13 or 14.

[305] It would be understood that, for example, the reaction may be run in the absence of an additive at a pH from about 2 to 8; 2 to 7; 2 to 6; 2 to 5; 2 to 4; 2 to 3; 3 to 8; 3 to 7; 3 to 6; 3 to 5; 3 to 4; 4 to 8; 4 to 7; 4 to 6; 4 to 5; 5 to 8; 5 to 7; 5 to 6; 6 to 8; 6 to 7; or 7 to 8. The reaction may be run, for example, at a pH of about 2.0 or 2.1 or 2.2 or 2.3 or 2.4 or 2.5 or 2.6 or 2.7 or 2.8 or 2.9 or 3 or 3.1 or 3.2 or 3.3 or 3.4 or 3.5 or 3.6 or 3.7 or 3.8 or 3.9 or 4 or 4.1 or 4.2 or 4.3 or 4.4 or 4.5 or 4.6 or 4.7 or 4.8 or 4.9 or 5 or 5.1 or 5.2 or 5.3 or 5.4 or 5.5 or 5.6 or 5.7 or 5.8 or 5.9 or 6 or 6.1 or 6.2 or 6.3 or 6.4 or 6.5 or 6.6 or 6.7 or 6.8 or 6.9 or 7 or 7.1 or 7.2 or 7.3 or 7.4 or 7.5 or 7.6 or 7.7 or 7.8 or 7.9 or 8.0.

[306] The reactions may be carried out using conventional heating or microwave irradiation or with flow chemistry performed in a fluidic device e.g. a micro-fluidic reactor. The reactions may be conducted under an inert atmosphere, e.g. nitrogen, helium, argon, carbon dioxide etc. [307] In an equivalent molar ratio, most of our selenoester examples proceeded to completion at a concentration of 1 mM (notable exceptions are the Val and lie esters which necessitate 1.25 eq). At 2 molar equivalents the ligations could be effectively run at 500 μΜ. Below this concentration the reaction stalled.

[308] Comparison of Sec/Cys and seleno/thioester mediated ligations

[309] To compare the rates of Sec/Cys and seleno/thioester mediated ligations model a-Leu seleno- and thioesters were incubated with model N-terminal cysteine, cystine and selenocystine peptide fragments without the presence of a reducing agent. The Cys based ligations were quenched at appropriate time points via dilution in acidic water (0.1% TFA/H₂0) and analyzed using HPLC. (Due to the difficulties experienced in quenching the Sec ligations, these samples were analyzed at each time-point via direct inject HPLC.) The results are depicted in Figure 5.

[310] It was observed that ligation of an a-Leu thioester did not proceed with the addition of either a diselenide or a cystine. The cystine-terminated peptide did proceed to approximately 50% over 5 minutes using the a-Leu selenoester, further illustrating the interesting reactivity of this ester. The reaction stalled at this point due to the formation of unproductive thioester resulting from the reaction of the ligated product with excess ester. The standard ligation of an N-terminal cysteine and C-terminal thioester proceeded slightly further to approximately 65% over 5-10 minutes before stalling. However, the ligation of a selenocystine fragment with the hindered Leu selenoester reached 100% conversion after only one minute. The results are depicted in Figure 6.

[311] Selective Sec to Ala and Sec to Ser deselenizations

[312] The inventors have found that the ligation and selective deselenization steps described above may conveniently be conducted as a one-pot reaction. They may be conducted without isolation or purification of intermediate species. Thus, following the ligation reaction, the crude reaction mixture may be subjected, without purification of intermediates (but optionally with at least partial removal of at least one reagent or catalyst used in the ligation reaction), to suitable deselenization conditions and reagents. The resulting ligated and selectively deselenized product peptide may be obtained from the resulting reaction mixture following a suitable time for reaction.

[313] An example of a one-pot protocol for the synthesis of peptides is show in Figure 7.

[314] With reference to Figure 7, conversion of the Sec residue in the ligated sequences to Ala was successfully implemented by using a modified version of Dawson's deselenization protocol. Treating the crude reaction solution with TCEP and DTT, as per the published protocol, did not lead to the desired deselenized product. It is thought that the 1 eq. of benzeneselenol liberated upon ligation is able to quench the radical deselenization. The DPDS was therefore first extracted using hexane before the solution was degassed and 50 eq. TCEP, 5 eq. DTT added, to give the desired sequence via a one-pot reaction in 56% - 97% yield.

[315] Successful conversion to the Ser residue was also possible in a one-pot reaction by addition of 50 eq. TCEP and 50 eq. Oxone, pH 4.5 at 100 μΜ final concentration after hexane extraction of the liberated DPDS, following our previously optimized procedure (unpublished research), in 50 - 80% yield.

[316] Further, the oxidative deselenizations described herein can be extended to other peptides which are the products of conventional NCL as shown for example in Figure 8.

[317] The person skilled in the art would be aware that through judicious choice of the seleno amino peptide reagent, the amino acid at the ligation site can be modified. In this regard and as an illustration a selenoester ligation with a β-selenophenylalanine- containing peptide is depicted in Figure 9. [318] The present disclosure also contemplates the synthesis of non-natural peptides, e.g., peptides that incorporate a fluorescent dye or radioisotope. To that end, methods are disclosed for the preparation of non-natural peptides or derivatives by selenocystine-selenoester ligation chemistry.

[319] Non-limiting examples of seleno reagents that may be used to introduce proteogenic/non-proteogenic amino acids into the peptides disclosed herein are displayed below in Tables 4 to 7.

Table 4. Non-limiting examples of seleno functionalised β-substituted amino acids

[320] Table 5. Non-limiting examples of seleno functionalised γ-substituted amino acids

[321] Table 6. Non-limiting examples of seleno functionalised δ-substituted amino acids

Table 7. Non-limiting examples of seleno functionalised aromatic amino acids [322] A further effort to provide a facile synthesis of proteins through peptide ligation has focused on the ligation of disulfide peptides and peptide aryl seleno esters. For illustration, a selenoester ligation with a 2-thiol tryptophan-containing peptide is shown in Figure 10.

[323] Synthesis of the native sequence (36) of protein Chorismate Mutase (Mtb CM)

[324] To demonstrate the scope and applicability of the methods disclosed herein, the synthesis of the protein Chorismate Mutase (Mtb CM) is described. Chorismate Mutase is an enzyme isolated from Mycobacterium tuberculosis that catalyzes the conversion of chorismate to prephenate, a key intermediate in the biosynthesis of tyrosine (Tyr) and phenylalanine (Phe). The synthesis which is shown in Figure 11, involved the ligation of two fragments, Mtb CM 1-40 bearing a C-terminal methionine phenylselenoester (34) and Mtb CM 41-83 bearing an N-terminal selenocystine moiety (35).

[325] Both fragments were synthesized using Fmoc SPPS. These two fragments were dissolved in 6 M Gn»HCl, 0.1 M phosphate buffer at pH 6.2 and after 5 min the additive-free ligation had proceeded to completion to afford exclusively the symmetrical diselenide (not isolated) together with DPDS which precipitated from solution. Without purification, DPDS was extracted using hexane before treating the reaction mixture with DTT and TCEP to effect in situ deselenization and afford full length Mtb CM with excellent crude purity. Following reverse-phase HPLC, the protein was folded by dialysis into 50 mM Tris and 0.1 M NaCl to provide Mtb CM 36 in an excellent 59% overall yield. Importantly, our synthetic folded enzyme had similar structure and activity to that reported for the recombinant protein (Prakash, P.; Aruna, B.; Sardesai, A. A.; Hasnain, S. E. J. Biol. Chem. 2005, 280, 19641 and Kim, S.-K.; Reddy, S. K.; Nelson, B. C; Robinson, H.; Reddy, P. T.; Ladner, J. E. FEBS Journal 2008, 275 4824) as determined by circular dichroism (CD) spectroscopy and by a kinetic assay with chorismate. [326] Also disclosed is the synthesis of a second protein, the N-aceylated Cys-free 94 residue protein early secretory antigenic protein-6 (ESAT-6) 40, to showcase the efficiency of the additive-free ligation technology (Figure 12). ESAT-6, also from Mtb, is an important virulence factor and a potent T cell antigen (Sorensen, A. L.; Nagai, S.; Houen, G.; Andersen, P.; Andersen, A. B. Infect. Immun. 1995, 63, 1710). The protein was disconnected into three fragments, ESAT-6 1-39 (37) as an N-terminal phenylselenoester, ESAT-6 40-71 dimer (38) containing an N-terminal selenocystine moiety and C-terminal alkyl thioester and ESAT-6 72-94 (39) which we proposed to assemble via a one-pot, three-component ligation reaction using both native chemical ligation and the additive-free selenocystine-selenoester ligation methodology. Selenoester 37 and bifunctional peptide dimer 38 were first reacted in 6 M Gn»HCl, 0.1 M phosphate buffer at pH 6.2 and after 2.5 min a yellow DPDS precipitate formed and the reaction had reached completion, as judged by LCMS analysis. At this point, C- terminal fragment 39 was added, together with TCEP and the thiol additive TFET, (Thompson, R. E.; Liu, X.; Alonso-Garcia, N.; Pereira, P. J. B.; Jolliffe, K. A.; Payne, R. J. . Am. Chem. Soc. 2014, 136, 8161) before adjusting the pH to 7.5. The ligation was allowed to proceed at 37 °C for 16 h, which led to completion of the native chemical ligation reaction together with concomitant deselenization of Sec-40 to Ala due to the addition of TCEP (Metanis, N.; Keinan, E.; Dawson, P. E. Angew. Chem. Int. Ed. 2010, 49, 7049). The reaction mixture was subsequently dosed with the water- soluble radical initiator VA-044, (Wan, Q.; Danishefsky, S. J. Angew. Chem. Int. Ed.

2007, 46, 9248), glutathione (Haase, C; Rohde, H.; Seitz, O. Angew. Chem. Int. Ed.

2008, 47, 6807) and further TCEP, and the pH adjusted to 7.5 to effect desulfurization of Cys-72 to Ala, which reached completion after 16 h at 37 °C. Purification by reverse-phase HPLC then provided ESAT-6 (40) in 44% yield over the multiple-step, one-pot process.

[327] The present disclosure also contemplates the deployment of the selenium chemistries disclosed herein so that they are amenable to solid-phase synthesis of molecules, for example, peptides. [328] In practice, the majority of protein targets (with or without modifications) are >85 amino acids in length and, due to the size limitations of solid-phase peptide synthesis (SPPS), require the ligation of three or more peptide fragments for assembly. In most cases each individual ligation step requires purification by reverse-phase HPLC before proceeding to the next ligation (or deprotection) reaction, inevitably leading to the use of large quantities of solvent and significant handling losses. Moreover, each purification step entails time-consuming lyophilization procedures (ca. 24 h) to enable solvent exchange for subsequent reactions.

[329] To address these shortcomings, researchers have developed solid-phase ligation strategies in which peptides are assembled via ligation reactions on a water- swellable solid support. (L. E. Canne, P. Botti, R. J. Simon, Y. J. Chen, E. A. Dennis, S. B. H. Kent, J. Am. Chem. Soc. 1999, 121, 8720-8727; A. Brik, E. Keinan, P. E. Dawson, J. Org. Chem. 2000, 65, 3829-3835; J. A. Camarero, G. J. Cotton, A. Adeva, T. W. Muir, J. Pept. Res. 1998, 51, 303-316; E. C. B. Johnson, T. Durek, S. B. N. Kent, Angew. Chem.-Int. Edit. 2006, 45, 3283-3287.)

[330] Whilst this strategy circumvents purification of intermediates, the reaction rates of en bloc ligations are significantly reduced and the inherent size limitation associated with assembly on solid supports remains.

[331] Given the rapid rates of ligation observed in diselenide-selenoester ligation, it was anticipated that rates would still be appreciable on the solid-phase. To test this hypothesis, an experiment was conducted whereby a side chain protected, resin-bound model peptide was reacted with a phenylselenoester in aqueous buffer. Under these conditions, quantitative formation of the corresponding resin-bound ligation product in less than 30 minutes occurred as judged by HPLC-MS analysis of the peptide following acidic cleavage from the resin. The synthetic scheme is shown in Figure 13. Further strategies for the solid-phase synthesis of peptides are shown in Figure 14 and Figure 15. [332] It is disclosed that the oxidative deselenizations described herein can be extended to other peptides which are the products of conventional NCL. The syntheses of compounds 41 to 48 serve as representative examples. Further, the disclosed ligation and deselenization methodologies can also be employed in substrates containing sulphur. This is shown by work carried out with a model sequence containing Cys. Ligation of Cys containing H-USPCYS-NH₂ dimer 49 with Ac-LYRANL-SePh 5 followed by a one-pot reductive deselenization gave Ac- LYRANLASPCYS-NH₂ (50) in 63% yield (see Figure 16).

[333] Selenium ligation using an alkyl selenoester

[334] To probe the relative reactivity of an alkyl selenoester compared with an aryl selenoester a ligation reaction between Ac-LYRANL-SeAlk (5 mM, 51) and H- USPGYS-NH₂ dimer (2.5 mM, 1) was performed in 6 M guanidine hydrochloride, 100 mM Na₂HP0₄, pH 6.2 with no external additive (see Figure 17). A progress plot of the ligation is shown in Figure 18.

[335] Proline and Isoleucine ligation in the presence of a reductant

[336] To probe the course of the ligation with Proline at the ligation site the additional model selenoester Ac-LYRANP-SePh (52) was prepared. Ligation of Ac- LYRANP-SePh 52 with H-USPGYS-NH₂ dimer 1 proceeded to > 80 % completion with the addition of 200 mM TCEP to give the dimers 53a and 53b. The reaction scheme is shown in Figure 19 and a plot of the reaction course is shown in Figure 20. The reaction is also an example of a selenium ligation performed in the presence of a reductant. The effect of an external reductant on a selenoester- selenocystine ligation was explored not only with the Ac-LYRANP-SePh (52) + H-USPGYS-NH₂ dimer (1) ligation but with a ligation between Ac-LYRANI-SePh (6) + H-USPGYS-NH₂ dimer (1) in the presence of 200 mM TCEP (see Figure 21). In this latter case substantial deselenization of the main ligation product was observed over 2 h (Figure 145).

[337] Kinetic Study of Selenium Ligation with Various Substituents [338] The synthesis of appropriate selenium containing intermediates (see Figures 22 to 25) allowed the kinetics of the ligation reaction with various substituents to be studied. To assess the effect electron withdrawing and donating groups may have on the reaction rate, the ligation of H-USPGYS-NH₂ (1) with Ac-LYRANL-SePh 5 and the ligation of 1 with Ac-LYRANL-SePhR (54 to 59), was performed in 6 M guanidine hydrochloride, 100 mM Na₂HP0₄, pH 6.2, 5 mM. The synthetic scheme is shown in Figure 26. Reaction half-lives are shown in Table 8 (which is ordered according to increasing half-life).

Selenoester (R=)

54 CH₃ 12 s

5 H 24 s

55 OMe 24 s

56 CH₂COOMe 48 s

57 NMe₂ 60 s

58 NO₂ 72 s

59 CH2COO- > 1 h

Table 8. Reaction half-lives for the ligation of selenoesters 5, 54 to 59 bearing electronically variable R groups with H-USPGYS-NH₂ dimer (1).

[339] Non-limiting examples illustrating the present disclosure are presented below.

Experimental

[340] General Methods

[341] 1H NMR spectra were recorded at 300 K using a Bruker Avance DPX 400 spectrometer. Chemical shifts are reported in parts per million (ppm) and are referenced to solvent residual signals: CDC1₃ (δ 7.26 [^l H]), d₆-DMSO (δ 2.50 [^l H]), D₂0 (δ 4.79 [^l H]), MeOD (δ 3.31 [^l H]). ¹ H NMR data is reported as chemical shift (δΗ), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, ddd = doublet of doublet of doublets), relative integral, coupling constant (J Hz) and assignment where possible. Low-resolution mass spectra were recorded on a Shimadzu 2020 mass spectrometer (ESI) operating in positive mode. High resolution MALDI- FTICR mass spectra were measured on a Bruker-Daltonics Apex Ultra 7.0T Fourier transform mass spectrometer (FTICR) using a matrix of 10 mg/mL a-cyano-4- hydroxycinnamic acid in water/acetonitrile (1: 1 v/v) containing 0.1 vol.% TFA.

[342] Analytical HPLC was performed on either a Waters Acquity UPLC system equipped with PDA ek detector (λ = 210 - 400 nm), Sample Manager FAN and Quaternary Solvent Manager (H-class) modules or a Waters System 2695 separations module with an 2996 photodiode array detector. Peptides were analyzed using a Waters Sunfire 5 μιη, 2.1 x 150 mm column (C-18) or an XB ridge BEH 5 μιη, 2.1 x 150 mm wide-pore column (C-18) at a flow rate of 0.2 mL min^"1 on the HPLC system or Waters Acquity UPLC BEH 1.7 μιη 2.1 x 50 mm column (C-18) at a flow rate of 0.6 mL min^{" 1} on the UPLC system. Both instruments were ran using a mobile phase composed of 0.1% trifluoroacetic acid in H₂O (Solvent A) and 0.1% trifluoroacetic acid in acetonitrile (Solvent B). The analysis of the chromatograms was conducted using Empower 3 Pro software (2010).

[343] Direct inject analytical reverse-phase HPLC was performed on a Waters System 2695 separations module with an Alliance series column heater at 30 °C and 2996 photodiode array detector. Peptides were analyzed using a Waters Sunfire 5 μιη, 2.1 x 150 mm column (C-18) at a flow rate of 2 mL min^"1 using a mobile phase of 0.1% TFA in water (Solvent A) and 0.1% TFA in acetonitrile (Solvent B). Results were analyzed with Waters Empower software.

[344] Preparative reverse-phase HPLC was performed using a Waters 600 Multisolvent Delivery System and Waters 500 pump with 2996 photodiode array detector or Waters 490E Programmable wavelength detector operating at 230 and 254 nm. Peptides were purified on a Waters Sunfire 5 μιη (C-18) preparative column operating at a flow rate of 7 mL min^"1 or an XBridge BEH 5 μιη wide-pore (C-18) using a mobile phase of 0.1% trifluoroacetic acid in water (Solvent A) and 0.1% trifluoroacetic acid in acetonitrile (Solvent B) and a linear gradient of 0-50% B over 40 min.

[345] LC-MS was performed either on a Shimadzu LC-MS 2020 instrument consisting of a LC-M20A pump and a SPD-20A UV/Vis detector coupled to a Shimadzu 2020 mass spectrometer (ESI) operating in positive mode or a Shimadzu UPLC-MS equipped with the same modules as the LC-MS system except for a SPD- M30A diode array detector. Separations were performed on the LC-MS system either on a Waters Sunfire 5 μιη, 2.1 x 150 mm column (C-18), or wide-pore equivalent operating at a flow rate of 0.2 mL min^"1. Separations on the UPLC-MS system were performed using a Waters Acquity UPLC BEH 1.7 μιη 2.1 x 50 mm column (C-8) at a flow rate of 0.6 mL min^"1. Separations were performed using a mobile phase of 0.1% formic acid in water (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B) and a linear gradient of 0-50% B over 30 min.

[346] Materials

[347] Commercial materials were used as received unless otherwise noted. Amino acids, coupling reagents and resins were obtained from Novabiochem or GL Biochem. Reagents that were not commercially available were synthesized following literature procedures as indicated in the experimental. Dichloromethane (DCM) was distilled from calcium hydride and N,N-dimethylformamide (DMF) was obtained as peptide synthesis grade from Merck or Labscan. (Boc-Sec-OH)₂ was synthesized from commercially available (H₂N-Sec-OH)₂ (Mimotopes, VIC, Australia).

[348] The aryl diselenides described herein were prepared using modifications to established literature methods (van der Toorn, J. C. et al., Eur. J. Org. Chem. 2011, 2011, 4345 (page 4351, paragraphs 5-7); dos Santos Edos, A. et al., Bioorg. Med. Chem. Lett. 2013, 23, 4669 (supporting information page 6, paragraph 2; page 7, paragraph 4); Pinto, B. M. et al., Synth. Commun. 1986, 16, 553 (page 555, paragraph 4); Piano, D. et al, Eur. J. Med. Chem. 2011, 46, 3315 (page 3322, sections 4.1.2 and 4.1.3); Stuhr-Hansen, N. et al., Tetrahedron 2011, 67, 2633 (page 2638, sections 4.2.1 - 4.2.4); Kopylova, B. V. et al, Russ. Chem. Bull. 1973, 22, 2663 (page 2664, paragraph 6). With the exception of diselenophenylacetic acid (Se₂PAA), the aryl diselenides below are known compounds.

[349] The synthesis of aryl diselenides followed three general approaches as shown in Figure 22.

[350] Method A: Grignard approach

[351] A flame-dried flask was placed under Ar (g) and charged with Mg granules (2.0 eq.) and a magnetic stir bar. Freshly distilled THF (5 mL) and a crystal of iodine were added to the reaction flask and the solution stirred vigorously for 20 min. In a separate flask, the p- substituted bromobenzene (1.0 g, 1.0 eq.) was dissolved in dry THF (10 mL) and slowly transferred via canula to the reaction flask. The resultant solution was gradually warmed to reflux and stirred for 1-2 h to complete formation of the Grignard reagent. The pale-gray Grignard solution was transferred via canula to a clean, flame-dried flask under an atmosphere of argon. The solution was cooled to 0 °C and treated with selenium powder (1.0 eq.). The reaction mixture was warmed to room temperature and stirred for 16 h. The crude reaction mixture was poured into saturated aqueous NH₄C1 (25 mL) and extracted with EtOAc (3 x 20 mL). The combined organic extracts were dried (MgS0₄), filtered and concentrated in vacuo. The crude residue was eluted through a silica plug (0: 100 to 20:80 EtO Ac/Hex) to provide the aryl diselenide.

[352] The person skilled in the art may recognise that the Grignard approach to the preparation of aryldiselenides potentially leads to the formation of the diarylselenide (Ar-Se-Ar) byproduct, which may be difficult to separate from the corresponding diselenide. However, as the person skilled in the art would also recognise, as only the aryldiselenide is capable of reacting in the subsequent phosphine -promoted selenoester synthesis, mixtures of aryldiselenide and diarylselenide can be subjected directly to the subsequent selenoesterification reaction to afford the desired selenoester.

[353] Method B: Aryldiazonium approach [354] To a solution of /^-substituted aniline (50 mg) in 10% (v/v) aq. HCl/MeOH (0.5 mL) at 0 °C was added dropwise a solution of NaN0₂ (1.1 eq.) in H₂0 (0.5 mL). The reaction mixture was stirred at 0 °C for 30 min. The resulting diazonium salt was added to a solution of selenourea (2 eq.) and CuCl₂ (0.25 eq.) in 10% (v/v) H₂0/MeOH (0.5 mL). The resulting mixture was stirred at 35 °C for 2 h, cooled to room temperature and then extracted with CHC1₃. The aqueous layer was concentrated via lyophilization. The crude residue was resuspended in methanol (2 mL) and treated with excess 30% aqueous NH₄OH (0.5 mL). The reaction mixture was heated at 50 °C for 1 h. The crude reaction mixture was acidified with 1 M HC1 and extracted with EtOAc (3 x 20 mL). The combined organic layers were dried (MgS0₄), filtered and concentrated in vacuo. The crude product was then purified by flash column chromatography through a silica plug or via reverse-phase HPLC.

[355] Method B is a modification of the procedure published by Stuhr-Hansen and coworkers, in which an aryldiazonium species is reacted with KSeCN in the presence of NaOAc to form an arylselenocyanate. The selenocyanate may then be converted to the corresponding diselenide upon treatment with H₂S0₄ and 0₂. In the above modification, selenourea serves as the nucleophilic selenium species in place of KSeCN. Note that the addition of CuCl₂ with selenourea is based on literature precedent for the arylation of thiourea with aryldiazonium salts (Kopylova, B. V. et ah, Russ. Chem. Bull. 1973, 22, 2663 (page 2664, paragraph 6)).

[356] Method C: Aryldiazonium approach using commercially available aryldiazonium salts.

[357] Method B can be carried out with commercially available aryldiazonium salts (e.g. 4-nitrobenzenediazonium tetrafluoroborate). In this instance, a solution of commercially available diazonium salt (-100 mg) was treated directly with selenourea and CuCl₂.

[358] Diselenophenylacetic acid (SePhCH₂C0₂H)₂ [359] The title compound was prepared from the corresponding aniline (50 mg) using Method B (see Figure 23) and isolated following preparative reverse-phase HPLC (0 to 100% B over 40 min) as a pale yellow solid (12 mg, 17% over 3 steps): ¹H NMR (D₂0/CD₃CN, 300 MHz) δ 6.24 (d, 2H, J = 8.1 Hz, Ar-H), 5.90 (d, 2H, J = 8.1 Hz, Ar-H), methylene obscured by residual solvent peak; Calculated Mass [M+H]⁺: 430.93 (100%), 428.93 (95.8%); Mass Found (ESI⁺); 429.10 [M+H]⁺.

[360] Solid-Phase Peptide Synthesis

[361] Preloading Rink Amide resin

[362] Rink amide resin was initially washed with DCM (5 x 3 mL) and DMF (5 x 3 mL), followed by removal of the Fmoc group by treatment with 20% piperidine/DMF (2 x 5 min). The resin was washed with DMF (5 x 3 mL), DCM (5 x 3 mL) and DMF (5 x 3 mL). PyBOP (4 eq.) and NMM (8 eq.) were added to a solution of Fmoc-AA- OH (4 eq.) in DMF. After 5 min of pre-activation, the mixture was added to the resin. After 2 h the resin was washed with DMF (5 x 3 mL), DCM (5 x 3 mL) and DMF (5 x 3 mL), capped with acetic anhydride/pyridine (1:9 v/v) (2 x 3 min) and washed with DMF (5 x 3 mL), DCM (5 x 3 mL) and DMF (5 x 3 mL).

[363] Preloading 2-chloro-trityl chloride resin

[364] 2-Chloro-trityl chloride resin (1.22 mmol/g loading) was swollen in dry DCM for 30 min then washed with DCM (5 x 3 mL). A solution of Fmoc-AA-OH (0.5 equiv. relative to resin functionalization) and z^'P^NEt (2.0 eq. relative to resin functionalization) in DCM (final concentration 0.1 M of amino acid) was added and the resin shaken at rt for 16 h. The resin was washed with DMF (5 x 3 mL) and DCM (5 x 3mL). The resin was treated with a solution of DCM/CH30H/z^'Pr₂NEt (17:2: 1 v/v/v) for 1 h and washed with DMF (5 x 3 mL), DCM (5 x 3 mL), and DMF (5 x 3 mL). The resin was subsequently submitted to iterative peptide assembly (Fmoc-SPPS).

[365] Loading estimation of amino acid loading [366] The resin was treated with 20% piperidine/DMF (2 x 3 niL, 3 min), 50 μΐ_^ of the combined deprotection solution was diluted to 10 niL using 20% piperidine/DMF in a volumetric flask. The UV absorbance of the resulting piperidine-fulvene adduct was measured (λ = 301 nm, ε = 7800 M^"1 cm^"1) to estimate the amount of amino acid loaded onto the resin.

[367] Deprotection

[368] The resin was treated with 20% piperidine/DMF (2 x 3 mL, 3 min) and washed with DMF (5 x 3 mL), DCM (5 x 3 mL) and DMF (5 x 3 mL).

[369] General iterative peptide assembly (Fmoc-SPPS)

[370] General amino acid coupling: A solution of protected amino acid (4 eq.), PyBOP (4 eq.) and NMM (8 eq.) in DMF (final concentration 0.1 M) was added to the resin. After 1 h, the resin was washed with DMF (5 x 3 mL), DCM (5 x 3 mL) and DMF (5 x 3 mL).

[370A] Coupling conditions for (Boc-Sec-OH)2: A solution of compound (Boc-Sec- OH)₂ (1.0 eq), DIC (2.0 eq.), and HOAt (2.0 eq) in DMF was added to the resin (1.0 eq.) and shaken at rt for 6 h. The resin was then washed with DMF (5 x 3 mL), DCM (5 x 3 mL), DMF (5 x 3 mL), and DCM (10 x 3 mL).

[370B] Capping: Acetic anhydride/pyridine (1:9 v/v) was added to the resin (3 mL). After 3 min the resin was washed with DMF (5 x 3 mL), DCM (5 x 3 mL) and DMF (5 x 3 mL).

[371] Cleavage: A mixture of TFA, thioanisole, triz^'sopropylsilane (TIS) and water (90:5:2.5:2.5 v/v/v/v) was added to the resin. After 2 h, the resin was washed with TFA (3 x 2 mL).

[372] Work-up: The combined solutions were concentrated under a stream of nitrogen either to dryness or to < 5 mL. In the latter case, 30 mL of diethyl ether was added to precipitate the peptide and the suspension centrifuged. The pellet or dry residue was then dissolved in water containing 0.1% TFA, filtered and purified by preparative HPLC and analyzed by LC-MS and ESI mass spectrometry.

[373] Automated solid-phase peptide synthesis

[374] Automated Fmoc-SPPS was carried out on a Biotage Initiator+ Alstra microwave peptide synthesizer equipped with an inert gas manifold. General synthetic protocols for Fmoc deprotection and capping were carried out in accordance with the manufacturer's specifications. Standardized amino acid couplings were performed for 20 min at 50 °C under microwave irradiation in the presence of amino acid (0.3 M in DMF), Oxyma (0.3 M in DMF) and diz^'sopropylcarbodiimide (0.3 M in DMF). Peptide cleavage and work- up were carried out as described above for manual SPPS.

[375] General Procedures

[376] Selenoester synthesis (Ac-LYRANX-SePh)

[377] Model peptide selenoesters were prepared on 2-chlorotrityl chloride resin using Fmoc-SPPS as described in the general methods. Cleavage of the peptides from the resin was effected by treating with 30 vol.% HFIP in DCM for 2 h before concentrating in vacuo. The resulting residue was dissolved in anhydrous DMF and cooled to 0 °C. Diphenyl diselenide (30 eq. in DMF) was added to the solution followed by Bu₃P (30 eq.). The reaction was allowed to proceed at 0 °C for 3 h, after which time the solvent was removed in vacuo. The crude material was put on ice and the protecting groups removed via treatment with TFA:TIS:thioanisole:H₂0 (90:5:2.5:2.5 v/v/v/v). After 2 h at room temp the cleavage cocktail was removed under a stream of N₂ and the crude residue suspended in diethyl ether and cooled at -20 °C. The precipitate was pelleted by centrifugation at 4000 rpm for 5 min, the supernatant decanted and the pellet dissolved in 0.1% TFA/H₂0 with addition of 0.1% TFA/MeCN and purified via preparative HPLC. [378] Ligation procedure

[379] Peptide selenoesters (1.0-1.25 eq.) and N-terminal Sec dimer peptides (~2 mg, 0.5 eq.) were dissolved in ligation buffer (6 M guanidine hydrochloride, 100 mM Na₂HP0₄, pH 7.2) to a concentration of 10-12.5 mM and 5 mM respectively. The solutions were combined; the final pH of the solution was measured as 6.5. For preparative samples the ligation reaction was allowed to proceed for 5-10 minutes before analysis and purification. For rate studies, the ligation solution was analyzed via HPLC using direct injection at appropriate time points.

[380] As an illustration, to 100 μΐ. of H-USPGYS-NH₂ dimer (1) in 6 M guanidine hydrochloride, 100 mM Na₂HP0₄, pH 7.2 at a concentration of 5 mM, was added 100 μΐ. of Ac-LYRANX-SePh (2-10) also in 6 M guanidine hydrochloride, 100 mM Na₂HP0₄, pH 7.2 at a concentration of 10 - 12.5 mM. Each model ligation was analysed at appropriate time points (60, 90, 300 and/or 600 seconds) via direct inject HPLC with a gradient of 0-60% B over 30 mins using a Waters Sunfire 5 μιη 4.6 x 250 mm (C-18) column at a flow rate of 2 mL/min. At the sampling time, 40 μΐ_^ of the crude ligation solution was diluted up to 1 mL with 1 % TFA/H₂0 and injected into the HPLC loop.

[381] Sec to Ala conversion

[382] To the crude peptide ligation solution (5 mM), was added an equal volume of hexane, the sample agitated and the hexane removed using a pipette. The aqueous phase was then degassed using argon. A solution of TCEP (50 eq.) in water (0.25 M) was prepared and adjusted to a final pH of 7.5-7.7 with 2 M NaOH and degassed. A solution of DTT (5 eq.) in water (25 mM) was prepared via dilution of a 0.25 M solution and degassed. The peptide solution was treated with the TCEP solution and the DTT solution simultaneously to give a final concentration of 2.5 mM with respect to the peptide ligation product and a final reaction pH of 4.5-5. The solution was agitated on an orbital shaker at rt and monitored by LC-MS analysis. If necessary, additional aliquots of aqueous TCEP and DTT were added. Following completion of the reaction, the solution was purified via semi-preparative reverse-phase HPLC employing a mobile phase of 0.1% TFA in water (Solvent A) and 0.1% TFA in acetonitrile (Solvent B) with a linear gradient as specified. All peptide products were isolated as white solids following lyophilization.

[383] Sec to Ser conversion

[384] Reaction conditions A

[385] The crude peptide ligation solution (5 mM) was diluted to a concentration of 300 μΜ using distilled water. An equal volume of hexane was added, the sample agitated and the hexane removed using a pipette. A solution of TCEP (50 eq.) in water (15 mM) was prepared and adjusted to a final pH of 7.5-7.7 with 2 M NaOH. A solution of Oxone (50 eq.) in water (15 mM) was also prepared. The peptide solution was treated simultaneously with the aqueous solutions of TCEP and Oxone to give a final concentration of 100 μΜ with respect to the peptide ligation product and a final reaction pH of 4.2-4.5. The solution was agitated on an orbital shaker at rt and monitored by LC-MS analysis. If necessary, additional aliquots of aqueous TCEP (pH adjusted to 7.5-7.7) and aqueous Oxone were added. Following completion of the reaction, the solution was concentrated on a lyophilizer (16 h). The samples were reconstituted in water containing 0.1% TFA and centrifuged. The supernatant was collected and purified via semi-preparative reverse-phase HPLC employing a mobile phase of 0.1% TFA in water (Solvent A) and 0.1% TFA in acetonitrile (Solvent B) with a linear gradient as specified. All peptide products were isolated as white solids following lyophilization.

[386] Reaction conditions B

[387] The peptide ligation product (1.5-2.5 mg) was dissolved in buffer (6 M guanidine hydrochloride, 100 mM Na₂HP0₄, adjusted to pH 7.5, 5 mM with respect to the ligation product). The peptide was diluted to a concentration of 200 μΜ by the addition of distilled water. A solution of TCEP (50 eq.) in water (15 mM) was prepared and adjusted to a final pH of 7.5-7.7 with 2 M NaOH. A solution of Oxone (50 eq.) in water (30 mM) was also prepared. The peptide solution was treated simultaneously with the aqueous solutions of TCEP and Oxone to give a final concentration of 100 μΜ with respect to the peptide ligation product and a final reaction pH of 4.2-4.5. The solution was agitated on an orbital shaker at rt and monitored by LC-MS analysis. If necessary, additional aliquots of aqueous TCEP (pH adjusted to 7.5-7.7) and aqueous Oxone were added. Following completion of the reaction, crude peptide products were concentrated on a lyophilizer (16 h). The samples were reconstituted in water containing 0.1% TFA and centrifuged. The supernatant was collected and purified via semi-preparative reverse-phase HPLC employing a mobile phase of 0.1% TFA in water (Solvent A) and 0.1% TFA in acetonitrile (Solvent B) with a linear gradient as specified.

[388] In the case for peptides containing oxidatively sensitive residues, such as Trp and Cys residues, the crude reaction mixture was immediately purified (without lyophilization) by semi-preparative reverse-phase HPLC under the same conditions described above to avoid concentrating the crude peptide product with large excesses of oxidant.

[389] All peptide products were isolated as white solids following lyophilization. [390] Synthesis and analytical data for model peptides [391] Model amino diselenide peptide

[392] H-USPGYS-NH₂ dimer (1)

[393] Compound 1 was synthesized using standard Fmoc-SPPS on Rink amide resin (25 μιηοΐ) through the direct incorporation of (Boc-Sec-OH)2 as outlined in the general methods section. The crude peptide was purified by preparative reverse-phase HPLC (0 to 30% B over 40 min, 0.1% TFA) and lyophilized to afford the peptide exclusively as the diselenide dimer (11.0 mg, 60% yield). Analytical data is shown in Figure 27.

[394] Model peptide selenoesters [395] Ac-LYRANA-SePh (2)

[396] Ac-LYRANA-resin was synthesized on a Biotage automated synthesiser as outlined in the general methods section on 2-chlorotrityl 2-chloride resin (1 mmol). 43 μιηοΐ of the protected peptide was cleaved from the resin using 30 vol.% HFIP in DCM and selenoesterified as outlined in the general methods section. The crude peptide was purified by preparative reverse-phase HPLC (0 to 80% B over 30 min, 0.1% TFA) and lyophilized to afford the desired selenoester (18.1 mg, 47% yield). Analytical data is shown in Figure 28.

[397] Ac-LYRANS-SePh (3)

[398] Ac-LYRANS -resin was synthesized on a Biotage automated synthesiser as outlined in the general methods section on 2-chlorotrityl 2-chloride resin (1 mmol). 33 μιηοΐ of the protected peptide was cleaved from the resin using 30 vol.% HFIP in DCM and selenoesterified as outlined in the general methods section. The crude peptide was purified by preparative reverse-phase HPLC (0 to 80% B over 30 min, 0.1% TFA) and lyophilized to afford the desired selenoester (21.7 mg, 72% yield). Analytical data is shown in Figure 29.

[399] Ac-LYRANT-SePh (4)

[400] Ac-LYRANT -resin was synthesized on a Biotage automated synthesiser as outlined in the general methods section on 2-chlorotrityl 2-chloride resin (1 mmol). 39 μιηοΐ of the protected peptide was cleaved from the resin using 30 vol.% HFIP in DCM and selenoesterified as outlined in the general methods section. The crude peptide was purified by preparative reverse-phase HPLC (0 to 80% B over 30 min, 0.1% TFA) and lyophilized to afford the desired selenoester (25 mg, 69% yield). Analytical data is shown in Figure 30.

[401] Ac-LYRANL-SePh (5)

Ac-LYRANL-resin was synthesized on a Biotage automated synthesiser as outlined in the general methods section on 2-chlorotrityl 2-chloride resin (1 mmol). 54 μιηοΐ of the protected peptide was cleaved from the resin using 30 vol.% HFIP in DCM and selenoesterified as outlined in the general methods section. The crude peptide was purified by preparative reverse-phase HPLC (0 to 80% B over 30 min, 0.1% TFA) and lyophilized to afford the desired selenoester (23.7 mg, 47% yield). Analytical data is shown in Figure 31.

[402] Ac-LYRANI-SePh (6)

[403] Ac-LYRANI-resin was synthesized on a Biotage automated synthesiser as outlined in the general methods section on 2-chlorotrityl 2-chloride resin (1 mmol). 50 μιηοΐ of the protected peptide was cleaved from the resin using 30 vol.% HFIP in DCM and selenoesterified as outlined in the general methods section. The crude peptide was purified by preparative reverse-phase HPLC (0 to 80% B over 30 min, 0.1% TFA) and lyophilized to afford the desired selenoester (21.3 mg, 46% yield). Analytical data is shown in Figure 32.

[404] Ac-LYRANV-SePh (7)

[405] Ac-LYRANV-resin was synthesized on a Biotage automated synthesiser as outlined in the general methods section on 2-chlorotrityl 2-chloride resin (1 mmol). 36 μιηοΐ of the protected peptide was cleaved from the resin using 30 vol.% HFIP in DCM and selenoesterified as outlined in the general methods section. The crude peptide was purified by preparative reverse-phase HPLC (0 to 80% B over 30 min, 0.1% TFA) and lyophilized to afford the desired selenoester (18.9 mg, 57% yield). Analytical data is shown in Figure 33.

[406] Ac-LYRANF-SePh (8)

[407] Ac-LYRANF-resin was synthesized on a Biotage automated synthesiser as outlined in the general methods section on 2-chlorotrityl 2-chloride resin (1 mmol). 39 μιηοΐ of the protected peptide was cleaved from the resin using 30 vol.% HFIP in DCM and selenoesterified as outlined in the general methods section. The crude peptide was purified by preparative reverse-phase HPLC (0 to 80% B over 30 min, 0.1% TFA) and lyophilized to afford the desired selenoester (22.5 mg, 59% yield). Analytical data is shown in Figure 34.

[408] Ac-LYRANM-SePh (9)

[409] Ac-LYRANM-resin was synthesized on a Biotage automated synthesiser as outlined in the general methods section on 2-chlorotrityl 2-chloride resin (1 mmol). 48 μιηοΐ of the protected peptide was cleaved from the resin using 30 vol.% HFIP in DCM and selenoesterified as outlined in the general methods section. The crude peptide was purified by preparative reverse-phase HPLC (0 to 80% B over 30 min, 0.1% TFA) and lyophilized to afford the desired selenoester (20.8 mg, 45% yield). Analytical data is shown in Figure 35.

[410] Ac-LYRANK-SePh (10)

[411] Ac-LYRANK-resin was synthesized on a Biotage automated synthesiser as outlined in the general methods section on 2-chlorotrityl 2-chloride resin (1 mmol). 48 μιηοΐ of the protected peptide was cleaved from the resin using 30 vol.% HFIP in DCM and selenoesterified as outlined in the general methods section. The crude peptide was purified by preparative reverse-phase HPLC (0 to 80% B over 30 min, 0.1% TFA) and lyophilized to afford the desired selenoester (23.7 mg, 52 % yield). Analytical data is shown in Figure 36.

[412] Preparation of Ac-LYRANAUSPGYS-NH₂ (11)

[413] Ligation of H-USPGYS-NH₂ dimer (2.1 mg, 1.63 μπιοΐ) and Ac-LYRANA- SePh (2.88 mg, 3.25 μηιοΐ) was performed as outlined in the general procedures and as shown in Figure 37. Sixty second time point HPLC analysis for the ligation showed complete consumption of the starting selenoester peptide (Figure 38). Purification via preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the symmetrical diselenide ligation product (11a, 3.05 mg) and the asymmetrical diselenide ligation product (lib, 0.18 mg) as white solids (72% combined yield). Analytical data is shown in Figure 39.

[414] Ac-LYRANSUSPGYS-NH₂ (12)

[415] Ligation of H-USPGYS-NH₂ dimer (2.2 mg, 1.67 μπιοΐ) and Ac-LYRANS- SePh (3.01 mg, 3.34 μιηοΐ) was performed as outlined in the general procedures and as shown in Figure 40. Purification via preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the symmetrical diselenide ligation product (12a, 3.06 mg) and the asymmetrical diselenide ligation product (12b, 0.4 mg) as white solids (73% combined yield). Analytical data is shown in Figure 41 and Figure 42 .

[416] Preparation of Ac-LYRANTUSPGYS -NH₂ (13)

[416A] Ligation of 1 (2.2 mg, 1.65 μπιοΐ) and Ac-LYRANT-SePh (3.07 mg, 3.3 μιηοΐ) was performed as outlined in the general procedures and as shown in Figure 43. Purification via preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the symmetrical diselenide ligation product (13a, 3.37 mg) and the asymmetrical diselenide ligation product (13b, 0.69 mg) as white solids (84% combined yield). Analytical data is shown in Figure 44 and Figure 45.

[417] Preparation of Ac-LYRANLUSPGYS -NH₂ (14)

[418] Ligation of 1 (2.47 mg, 1.88 μπιοΐ) and Ac-LYRANL-SePh (3.49 mg, 3.75 μιηοΐ) was performed as outlined in the general procedures and as shown in Figure 46. Purification via preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the symmetrical diselenide ligation product (14a, 4.07 mg) and the asymmetrical diselenide ligation product (14b, 0.67 mg) as white solids (87% combined yield). Analytical data is shown in Figure 47 and Figure 48.

[419] Ac-LYRANIUSPGYS-NH₂ (15)

[420] Ligation of 1 (2.4 mg, 1.83 μπιοΐ) and Ac-LYRANI-SePh (4.24 mg, 4.56 μιηοΐ) was performed as outlined in the general procedures and as shown in Figure 49. Purification via preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the symmetrical diselenide ligation product (15a, 2.59 mg) and the asymmetrical diselenide ligation product (15b, 0.6 mg) as white solids (63% combined yield). Analytical data is shown in Figure 50, Figure 51 and Figure 52.

[421] Ac-LYRANVUSPGYS-NH₂ (16)

[422] Ligation of 1 (2.18 mg, 1.66 μπιοΐ) and Ac-LYRANV-SePh (3.79 mg, 4.14 μιηοΐ) was performed as outlined in the general procedures and as shown in Figure 53. Purification via preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the symmetrical diselenide ligation product (16a, 2.59 mg) and the asymmetrical diselenide ligation product (16b, 0.6 mg) as white solids (67% combined yield). Analytical data is shown in Figure 54 and Figure 55.

Preparation ofAc-LYRANFUSPGYS-NH₂ (17)

[424] Ligation of 1 (2.13 mg, 1.62 μπιοΐ) and Ac-LYRANF-SePh (3.12 mg, 3.24 μιηοΐ) was performed as outlined in the general procedures and as shown in Figure 56. Purification via preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the symmetrical diselenide ligation product (17a, 2.81 mg) and the asymmetrical diselenide ligation product (17b, 1.23 mg) as white solids (83% combined yield). Analytical data is shown in Figure 57 and Figure 58. [425] Ac-LYRANMUSPGYS-NH₂ (18)

[426] Ligation of 1 (2.36 mg, 1.79 μηιοΐ) and Ac-LYRANM-SePh (3.39 mg, 3.58 μηιοΐ) was performed as outlined in the general procedures. Purification via preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the symmetrical diselenide ligation product (18a, 2.84 mg) and the asymmetrical diselenide ligation product (18b, 0.91 mg) as white solids (72% combined yield). Analytical data is shown in Figure 59.

[427] Ac-LYRANKUSPGYS-NH₂ (19)

[428] The ligation of H-USPGYS-NH₂ dimer 1 (2.0 mg, 1.5 μιηοΐ) and Ac- LYRANK-SePh 10 (2.9 mg, 3.1 μιηοΐ) was performed as outlined in the general methods section. NH₂NH₂ hydrate (15 μΐ) was added to the reaction mixture before the purification via preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization to afford the symmetrical diselenide ligation product 19a (3.5 mg, 79%) as a white solid. Analytical data is shown in Figure 60.

[429] Model One-pot ligations and deselenizations [430] Model One-pot ligation - Sec to Ala conversion

[431] Ac-LYRANAASPGYS-NH₂ (20)

[432] Ac-LYRANAASPGYS-NH₂ was synthesized via ligation of the peptides Ac- LYRANA-SePh (2.94 mg, 3.31 μηιοΐ) and 1 (2.18 mg, 1.66 μηιοΐ). The crude material was subjected to conditions outlined in the general methods to convert the internal Sec residue to Ala. Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the desired peptide as a white solid (2.73 mg, 60% yield). Analytical data is shown in Figure 61.

[433] Ac-LYRANSASPGYS-NH₂ (21)

[434] Ac-LYRANSASPGYS-NH₂ was synthesized via ligation of the peptides Ac- LYRANS-SePh (2.87 mg, 3.18 μπιοΐ) and 1 (2.09 mg, 1.59 μπιοΐ). The crude material was subjected to the optimised conditions outlined in the general methods to convert the internal Sec residue to Ala. Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the desired peptide as a white solid (2.4 mg, 57% yield). [435] Ac-LYRANTASPGYS-NH₂ (22)

[436] Ac-LYRANTASPGYS-NH₂ was synthesized via ligation of the peptides Ac- LYRANT-SePh (2.58 mg, 2.81 μπιοΐ) and 1 (1.85 mg, 1.41 μπιοΐ). The crude material was subjected to the optimised conditions outlined in the general methods to convert the internal Sec residue to Ala. Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the desired peptide as a white solid (2.69 mg, 71% yield). Analytical data is shown in Figure 63.

[437] Ac-LYRANIASPGYS-NH₂ (23)

[438] Ac-LYRANIASPGYS-NH₂ was synthesized via ligation of the peptides Ac- LYRANI-SePh (3.80 mg, 4.09 μπιοΐ) and 1 (2.15 mg, 1.63 μπιοΐ). The crude material was subjected to the optimised conditions outlined in the general methods to convert the internal Sec residue to Ala. Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the desired peptide as a white solid (2.64 mg, 63% yield). Analytical data is shown in Figure 64.

[439] Ac-LYRANVASPGYS-NH₂ (24)

[440] Ac-LYRANVASPGYS-NH₂ was synthesized via ligation of the peptides Ac- LYRANV-SePh (3.48 mg, 3.80 μπιοΐ) and 1 (2.0 mg, 1.52 μπιοΐ). The crude material was subjected to the optimised conditions outlined in the general methods to convert the internal Sec residue to Ala. Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the desired peptide as a white solid (3.2 mg, 79% yield). Analytical data is shown in Figure 65.

[441] Ac-LYRANFASPGYS-NH₂ (25)

[442] Ac-LYRANFASPGYS-NH₂ was synthesized via ligation of the peptides Ac- LYRANF-SePh (2.89 mg, 3.0 μπιοΐ) and 1 (1.97 mg, 1.5 μπιοΐ). The crude material was subjected to the optimised conditions outlined in the general methods to convert the internal Sec residue to Ala. Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the desired peptide as a white solid (2.11 mg, 58% yield). Analytical data is shown in Figure 66.

[443] Ac-LYRANMASPGYS-NH₂ (26)

[444] Ac-LYRANMASPGYS-NH₂ was synthesized via ligation of the peptides Ac- LYRANM-SePh (3.1 mg, 3.27 μπιοΐ) and H-USPGYS-NH₂ dimer 1 (2.15 mg, 1.64 μιηοΐ). The crude material was subjected to the optimised conditions outlined in the general methods to convert the internal Sec residue to Ala. Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the desired peptide as a white solid (2.5 mg, 56% yield). Analytical data is shown in Figure 67.

[445] Ac-LYRANKASPGYS-NH₂ (27)

[446] Ac-LYRANKASPGYS-NH₂ was synthesized via ligation of the peptides Ac- LYRANK-SePh (2.92 mg, 3.09 μπιοΐ) and 1 (2.03 mg, 1.55 μπιοΐ). The crude material was subjected to the optimised conditions outlined in the general methods to convert the internal Sec residue to Ala. Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the desired peptide as a white solid (4.1 mg, 97% yield). Analytical data is shown in Figure 68.

[447] Ac-LYRANLASPGYS-NH₂ (28)

[448] Ac-LYRANLASPGYS-NH₂ 28 was synthesized via ligation of the peptides Ac-LYRANL-SePh 5 (3.01 mg, 3.2 μπιοΐ) and H-USPGYS-NH₂ dimer 1 (2.13 mg, 1.6 μιηοΐ). The crude material was subjected to the optimised conditions outlined in the general methods to convert the internal Sec residue to Ala. Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the desired peptide as a white solid (2.83 mg, 66% yield). Analytical data is shown in Figure 69.

[449] Model one-pot ligations - Sec to Ser conversions

[450] Ac-LYRANASSPGYS-NH₂ (29)

[451] Ac-LYRANAS SPGYS -NH₂ was synthesized via ligation of the peptides Ac- LYRANA-SePh (1.24 mg, 1.40 μιηοΐ) and 1 (0.92 mg, 0.70 μιηοΐ). The crude material was subjected to the optimised conditions outlined in the general methods to convert the internal Sec residue to Ser. Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the desired peptide as a white solid (1.49 mg, 80% yield). Analytical data is shown in Figure 70.

[452] Ac-LYRANTS SPGYS -NH₂ (30)

[453] Ac-LYRANTS SPGYS -NH₂ was synthesized via ligation of the peptides Ac- LYRANT-SePh (3.22 mg, 3.51 μπιοΐ) and 1 (2.31 mg, 1.76 μπιοΐ). The crude material was subjected to the optimised conditions outlined in the general methods to convert the internal Sec residue to Ser. Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the desired peptide as a white solid (2.38 mg, 50% yield). Analytical data is shown in Figure 71.

[454] Ac-LYRANIS SPGYS -NH₂ (31)

[455] Ac-LYRANIS SPGYS -NH₂ was synthesized via ligation of the peptides Ac- LYRANI-SePh (3.59 mg, 3.85 μπιοΐ) and H-USPGYS-NH₂ dimer (2.03 mg, 1.55 μιηοΐ). The crude material was subjected to the optimised conditions outlined in the general methods to convert the internal Sec residue to Ser. Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the desired peptide 31 as a white solid 1.4 mg, 62% yield). Analytical data is show in Figure 72.

[456] Ac-LYRANVSSPGYS-NH₂ (32)

[457] Ac-LYRANVS SPGYS -NH₂ was synthesized via ligation of the peptides Ac- LYRANV-SePh (3.92 mg, 4.27 μπιοΐ) and H-USPGYS-NH₂ dimer (2.25 mg, 1.71 μmol). The crude material was subjected to the optimised conditions outlined in the general methods to convert the internal Sec residue to Ser. Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the desired peptide 32 as a white solid (2.3 mg, 50% yield). Analytical data is shown in Figure 73.

[458] Ac-LYRANM(=0)SSPGYS-NH₂ (33)

[459] Ac-LYRANM(=0)SSPGYS-NH₂ was synthesized via ligation of the peptides Ac-LYRANM-SePh (2.95 mg, 3.12 μπιοΐ) and 1 (2.05 mg, 1.56 μπιοΐ). The crude material was subjected to the optimised conditions outlined in the general methods to convert the internal Sec residue to Ser. Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the desired peptide 33 as a white solid (2.4 mg, 55% yield). Analytical data is shown in Figure 74.

[460] Ac-LYRANGS SPGYS -NH₂ (41)

[461] Peptide 41 was synthesized from the corresponding Sec peptide ligation product (1.8 mg, 1.2 μιηοΐ) according to the optimized Sec to Ser conversion protocol outlined in the general methods (reaction condition B). Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 40 min, 0.1% TFA) followed by lyophilization afforded peptide 41 as a white solid (1.3 mg, 83% yield). Analytical data is shown in Figure 82 and Figure 83.

[462] Ac-LYRAN AS SPGYS -NH₂ (29)

[463] Peptide 29 was synthesized from selenyl-MPAA sulfide peptide ligation product (2.5 mg, 1.6 μιηοΐ) according to the optimized Sec to Ser conversion protocol outlined in the general methods (reaction condition B). Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 40 min, 0.1% TFA) followed by lyophilization afforded peptide 29 as a white solid (1.9 mg, 90% yield). Analytical data is shown in Figure 84 and Figure 85.

[464] Ac-LYRANM(=0)SSPGYS-NH₂ (42)

[465] Peptide 42 was synthesized from selenyl-MPAA sulfide peptide ligation product (1.9 mg, 1.2 μιηοΐ) according to the optimized Sec to Ser conversion protocol outlined in the general methods (reaction condition B). In this instance, complete conversion of the ligation product to 42 required an additional dose of TCEP (10 eq.) and Oxone (10 eq.) at t = 1 h. Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 40 min, 0.1% TFA) followed by lyophilization afforded peptide 42 as a white solid (1.6 mg, 97% yield). Analytical data is shown in Figure 86 and Figure 87.

[466] Ac-LYRANMS SPGYS -N¾ (33)

[467] To a solution of peptide 42 (2.5 mg, 1.8 μιηοΐ) in TFA (0.85 mL) at 0 °C was added a solution of NH₄I (5.5 mg, 38 μιηοΐ) in TFA (0.85 mL) and water (60 μί) followed by the addition of dimethyl sulfide (2.8 μί, 38 μιηοΐ). A brown precipitate formed and the solution was gently agitated at 0 °C for 30 min at which point LC-MS analysis indicated complete consumption of peptide 42. A saturated solution of ascorbic acid in water was added dropwise until the solution turned clear. The crude mixture was concentrated under a stream of nitrogen and purified by semi-preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization to afford the title compound 33 as a white solid (2.5 mg, quant, yield). Analytical data is shown in Figure 88.

[468] Ac-LYRANFS SPGYS -NH₂ (43)

[469] Peptide 43 was synthesized from the selenyl-MPAA sulfide peptide ligation product (1.5 mg, 1.0 μιηοΐ) according to the optimized Sec to Ser conversion protocol outlined in the general methods (reaction condition B). Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 40 min, 0.1% TFA) followed by lyophilization afforded peptide 43 as a white solid (1.3 mg, 93% yield). Analytical data is shown in Figure 89 and Figure 90.

[470] Ac-LYRANLS SPGYS -NH₂ (44)

[471] Peptide 44 was synthesized from the selenyl-sulfide peptide ligation product (1.0 mg, 0.64 μιηοΐ) according to the optimized Sec to Ser conversion protocol outlined in the general methods (reaction condition B). In this instance, complete conversion to 44 required an additional dose of TCEP (10 eq.) and Oxone (10 eq.) at t = 1 h. Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 40 min, 0.1 % TFA) followed by lyophilization afforded peptide 44 as a white solid (0.9 mg, quant, yield). Analytical data is shown in Figure 91 and Figure 92.

[472] Ac-LYRANFS SPKYS -NH₂ (45)

[473] Peptide 45 was synthesized from the selenyl-MPAA sulfide peptide ligation product (1.7 mg, 1.0 μιηοΐ) according to the optimized Sec to Ser conversion protocol outlined in the general methods (reaction condition B). Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 40% B over 40 min, 0.1% TFA) followed by lyophilization afforded peptide 45 as a white solid (1.3 mg, 93% yield). Analytical data is shown in Figure 93 and Figure 94.

[474] Ac-LYRAN AS SPHYS -NH₂ (46)

[475] Peptide 46 was synthesized from the selenyl-MPAA sulfide peptide ligation product (1.7 mg, 1.1 μιηοΐ) according to the optimized Sec to Ser conversion protocol outlined in the general methods (reaction condition B). Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 40% B over 40 min, 0.1% TFA) followed by lyophilization afforded peptide 46 as a white solid (1.4 mg, 95% yield). Analytical data is shown in Figure 95 and Figure 96.

[476] Ac-LYRAN AS SPWYS -NH₂ (47)

[477] Peptide 47 was synthesized from a mixture of the diselenide dimer ligation product (1.0 mg, 0.62 μιηοΐ) and the corresponding selenyl-MPAA sulfide ligation product (1.0 mg, 0.64 μιηοΐ) according to the optimized Sec to Ser conversion protocol outlined in the general methods (reaction condition B). Direct purification of the crude (unconcentrated) reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 40 min, 0.1% TFA) followed by lyophilization afforded peptide 47 as a white solid (1.5 mg, 80% yield). Analytical data is shown in Figure 97 and Figure 98.

[478] Ac-LYRAN AS SPCYS -NH₂ (48)

[479] Peptide 48 was synthesized from the selenyl-MPAA sulfide peptide ligation product (1.1 mg, 0.63 μιηοΐ) according to the optimized Sec to Ser conversion protocol outlined in the general methods (reaction condition B). Complete consumption of the ligation peptide required an additional dose of TCEP (50 eq.) and Oxone (50 eq.) at t = 1 h. Direct purification of the crude (unconcentrated) reaction mixture via semi- preparative reverse phase HPLC (0 to 40% B over 40 min, 0.1% TFA) followed by lyophilization afforded peptide 48 (0.7 mg, 80% yield) and the corresponding Sec to Ala conversion product (0.2 mg) as white solids. Analytical data is shown in Figure 99 and Figure 100.

[480] Cysteine Compatibility Assays indicating that a Cys-containing peptide is inert to the Sec to Ser reaction conditions is shown in Figure 101.

[481] Selenoester ligations with β-selenophenylalanine-containing peptides

[482] The β-selenophenylalanine-containing peptide (0.5 eq.) and the peptide phenylselenoester (1.0 - 1.5 eq.) were dissolved in ligation buffer (6 M Gn^»HCl/0.1 M Na₂HP0₄, pH = 7.2, 2.5 mM with respect to the β-selenophenylalanine-containing peptide) at room temperature. Reaction monitoring via HPLC-MS analysis at t = 6 h (0 to 50% B over 30 min) indicated consumption of the β-selenophenylalanine-containing peptide and formation of ligated peptide products as a mixture of the diselenide dimer peptide, the asymmetric phenylselenyl diselenide and the trans-esterified internal selenoester product (see Figures 102-104).

[483] Selenoester ligations with 2-thiol tryptophan disulfide-containing peptides [484] General Protocol:

[485] The 2-thiol tryptophan (Trp) disulfide-containing peptide (0.5 eq.) and the peptide phenylselenoester (1.0 - 1.5 eq.) were dissolved in ligation buffer (6 M Gn^»HCl/0.1 M Na₂HP0₄, pH = 7.2, 2.5 mM with respect to the 2-thiol-Trp disulfide containing peptide) at room temperature. Reaction monitoring via HPLC-MS analysis at t = 30 min (0 to 50% B over 30 min) indicated consumption of the 2-thiol Trp- containing peptide and formation of ligated peptide product, primarily as the trans- esterified, internal thioester adduct (see Figure 105 and Figure 106). [486] H range study

[487] The ligation of H-USPGYS-NH₂ (1) and Ac-LYRANL-SePh (5) was performed in 6 M guanidine hydrochloride, 100 mM Na₂HP0₄, 5 mM, at various pH levels. The reaction was analysed at appropriate time points using UPLC (0-50% B over 30 min, λ = 214 nm). Analytical data is show in Figures 107 to 111. The ligation reaction between peptides Ac-LYRANF-SePh (8) and H-USPGYS-NH₂ dimer (1) at pH 4.0 was also performed (Figure 112).

[488] Concentration range study (500 μΜ)

[489] The ligation of H-USPGYS-NH₂ (1) and Ac-LYRANL-SePh (5) or Ac- LYRANA-SePh (2) was performed in 6 M guanidine hydrochloride, 100 mM Na₂HP0₄, pH 6.2 at 250 μΜ with respect to peptide 1. The reaction was analysed UPLC-MS (0-50% B over 8 min, λ = 214 nm). Analytical data is show in Figure 113 and Figure 114.

[490] Synthesis of native peptides using selenoester ligation

[491] Mtb CM C-terminal fragment incorporating a diselenide moiety (35)

[492] The C-terminal 43 residues of Mtb CM were synthesized using a Biotage automated peptide synthesizer as outlined in the general methods. A (Boc-Sec-OH)₂ residue was used in place of the native N-terminal Cys. The crude peptide was purified via preparative reverse phase HPLC (0 to 80% B over 30 min, 0.1% TFA) followed by lyophilization affording the desired peptide as a white solid (42.0 mg, 11% yield). Analytical data is shown in Figure 115 and Figure 116.

[493] Mtb CM N-terminal fragment incorporating a phenyl selenoester moiety (34)

[494] The N-terminal 40 residues of Mtb CM were synthesized using a Biotage automated peptide synthesizer as outlined in the general methods. The peptide was cleaved off the resin (with protecting groups intact) and selenoesterified as outlined in the general methods. The crude peptide was purified via preparative reverse phase HPLC (0 to 100% B over 30 min, 0.1% TFA) followed by lyophilization affording the desired peptide as a white solid (19.3 mg, 7% yield). Analytical data is shown in Figure 117 and Figure 118.

[495] Mtb CM native sequence (83mer) (36)

[496] The C-terminal diselenide fragment and N-terminal selenoester fragment were ligated at a stoichiometry of 1: 1.1, and the internal Sec residue converted to Ala as outlined in the general methods (see Figures 119 and 120). The crude reaction product was purified via preparative reverse phase HPLC (0 to 80% B over 30 min, 0.1% TFA, Figure 121) followed by lyophilization affording the desired protein as a white solid (3.98 mg, 57% yield). Analytical MS data is shown in Figure 122.

[497] After HPLC purification of 36 the lyophilized TFA salt of the protein was dissolved in 6 M guanidine hydrochloride, 100 mM Na₂HP0₄, pH 7.2 to a concentration of 1.5 mM and dialyzed into 50 mM Tris, 0.1 M NaCl, pH 7.5 at 4 °C overnight. The concentration of the stock solution was confirmed by UV/Vis spectroscopy and stored at -20 °C A portion of the protein stock solution was used to interrogate the structure of the synthetic Mtb CM using circular dichroism (see Figure 123).

[498] Mtb CM (36) activity assays were carried out as described by Davidson and Hudson (Davidson, B. E., and Hudson, G. S. (1987) Methods Enzymol. 142, 440-450). Briefly, to 200 μΐ_^ of chorismic acid solution in 50 mM Tris-HCl, 10 mM mercaptoethanethiol, 0.1 mg/mL BSA, pH 7.5 (1, 0.5, 0.25 & 0.125 mM) incubated for 5 min at 37 °C was added 10 of Mtb CM (10 μΜ solution; 100 pmol). The reactions were then incubated to their desired time point (2.5, 10, 20, 30, 60, 120 or 180 min) and quenched with 200 1 M HC1. The samples were further incubated for 10 min before addition of 400 μΐ_^ 2.5 M NaOH and analysis by UV/Vis at 320 nm using a control sample minus addition of the Mtb CM enzyme as a blank. The assays are depicted in Figures 124 to 128.

[499] ESAT-6 synthesis

Ac- TEQQWNFAG IEAAASAIQG NVTSIHSLLD EGKQSLTKL AAWGGSGSEA YQGVQQKWDA TATELNNALQ NLARTISEAG QAMASTEGNV TGMFA-OH

[500] ESAT-6 N-terminal fragment (selenoester) (37)

[501] The N-terminal 39 residue fragment of ESAT-6 (37) was synthesized using a Biotage automated peptide synthesizer as outlined in the general methods section. The protected peptide was cleaved from the resin and selenoesterified as outlined in the general methods section. The crude peptide was purified via preparative reverse phase HPLC (0 to 80% B over 30 min, 0.1% TFA) followed by lyophilization affording the desired peptide as a white solid (11.1 mg, 7% yield). Analytical data is shown in Figure 129 and Figure 130.

[502] ESAT-6 mid-fragment (38)

[503] The 32 residue mid-fragment sequence of ESAT-6 (38) was synthesized using a Biotage automated peptide synthesizer as outlined in the general methods section. The protected peptide was cleaved from the resin and thioesterified as outlined in the general methods section. The crude peptide was purified via preparative reverse phase HPLC (0 to 80% B over 30 min, 0.1% TFA) followed by lyophilization affording the desired peptide as a white solid (5.35 mg, 3% yield). Analytical data is shown in Figure 131 and Figure 132.

[504] ESAT-6 C-terminal diselenide fragment (39)

[505] The C-terminal diselenide 23 residue fragment of ESAT-6 (39) was synthesized using a Biotage automated peptide synthesizer as outlined in the general methods section. The peptide was cleaved from the resin and purified via preparative reverse phase HPLC (0 to 80% B over 30 min, 0.1% TFA) followed by lyophilization affording the desired peptide as a white solid (18.03 mg, 15.5% yield). Analytical data is shown in Figure 133 and Figure 134.

[506] ESAT-6 native sequence (40)

[507] The ligation of N-terminal selenoester 37 (2.46 mg, 0.571 μιηοΐ) and mid- fragment thioester dimer 38 (1.7 mg, 0.238 μιηοΐ) was performed as outlined in the general methods section in 100 μΐ_^ ligation buffer (2.5 mM with respect to 38). An HPLC trace of the crude ligation product is shown in Figure 135. This was followed by hexane extraction and addition of 1.55 mg C-terminal fragment 39 (0.665 μιηοΐ) and TCEP (1.43 mg, 5 μιηοΐ) in an equivalent volume of buffer. The pH of the combined solution (2.5 mM) was adjusted to 7.5, the solution degassed and 4 μL· TFET added. The ligation was allowed to proceed at 37 °C overnight. After this time 29 mg TCEP (0.1 mmol) and 6.14 mg glutathione (0.02 mmol) were dissolved into 480 μΐ_^ ligation buffer and the pH adjusted to 7.5. The ligation solution was degassed, treated with the solution of TCEP and glutathione, and 3.23 mg VA-044 (0.01 mmol) were added to the combined reaction mixture as a solid. The reaction was allowed to proceed at 37 °C overnight. Purification via preparative reverse phase HPLC (0 to 80% B over 30 min, 0.1% TFA) followed by lyophilization afforded the native ESAT-6 peptide 40 (1.71 mg, 43% yield) as a white solid ( yield was corrected for loss of material during pH adjustment and analysis.). Analytical data is shown in Figure 136 and Figure 137.

[508] After HPLC purification of 40 the lyophilized TFA salt of the protein was dissolved in 25 mM NaH₂P0₄, 0.1 M NaCl, pH 6.5 at room temperature to a concentration of 1 mM (quantified by Nanodrop UV/Vis) and stored as a stock solution at -20 °C. A sample was dissolved to 10 μΜ in the folding buffer and analyzed via circular dichroism (see Figure 138).

[509] One-pot ligation-deselenization using a model sequence containing Cys

[510] Preparation of H-USPCYS-NH₂ internal selenyl-sulfide (49a) and H- USPCYS-NH₂ dimer (49b)

49a 49b

[511] H-USPCYS-NH₂ dimer was synthesized using Fmoc-strategy SPPS on Rink amide resin (25 μιηοΐ) through the direct incorporation of (Boc-Sec-OH)₂ as outlined in the general methods section. The crude peptide was purified by preparative reverse- phase HPLC (0 to 30% B over 40 min, 0.1% TFA) and lyophilized to afford the desired peptide as a mixture of the intramolecular selenyl-sulfide (49a) and diselenide dimer (49b) (9.0 mg, 51% yield). Analytical data is shown in Figure 139.

[512] Ac-LYRANLASPCYS-NH₂ (50)

[513] Ac-LYRANLASPCYS-NH₂ 50 was synthesized via ligation of the peptides Ac-LYRANL-SePh 5 (1.1 mg, 1.18 μπιοΐ) and H-USPCYS-NH₂ dimer 49 (0.73 mg, 0.52 μιηοΐ). The crude material was subjected to the optimised conditions outlined in the general methods to convert the internal Sec residue to Ala. Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded the desired peptide as a white solid (0.91 mg, 63% yield). Analytical data is shown in Figure 140.

[514] Alkyl selenoester work

[515] Ac-LYRANL-SeCH₂C(0)NH₂ (51)

[516] To selenium powder (250 mg, 3.17 mmol) in a 2-necked round-bottom flask was added water (1.5 mL) and the solution was degassed and put on ice. A solution of sodium borohydride (360 mg, 9.52 mmol) in degassed water (2.5 mL) was added dropwise and the reaction mixture was stirred at 0 °C for 10 min, rt for 20 min and then refluxed for 20 min. After cooling the solution to rt, degassed acetic acid was added dropwise until the pH of the solution reached 6-7. 0.5 mL of the resulting solution was added to the solution of Ac-LYRANL-SPh (2 mg, 2.26 μιηοΐ) in MeCN/H₂0 (1:4, v/v) and stirred overnight. This is followed by the addition of an 0.4 M solution of iodoacetamide in degassed water (0.5 mL). The resulting solution was allowed to stir at rt for 2 h. Purification via preparative reverse phase HPLC (0 to 50% B over 30 min, 0.1% TFA) followed by lyophilization afforded 29 (1.0 mg, 49%) as a white solid. Analytical data is shown in Figure 141.

[517] To probe the relative reactivity of an alkyl selenoester compared with an aryl selenoester a ligation reaction between Ac-LYRANL-SeAlk (5 mM, 51) and H- USPGYS-NH₂ dimer (2.5 mM, 1) was performed in 6 M guanidine hydrochloride, 100 mM Na₂HP0₄, pH 6.2 with no external additive. The reaction was sampled and analysed at 11 h (Figure 142).

[518] Proline and Isoleucine ligation in the presence of a reductant

[519] To probe the course of the ligation with Proline at the ligation site the additional model selenoester Ac-LYRANP-SePh (52) was prepared.

[520] Ac-LYRANP-SePh (52)

[521] Ac-LYRANP-resin was synthesized on a Biotage automated synthesiser as outlined in the general methods section on 2-chlorotrityl 2-chloride resin (1 mmol). 31 μιηοΐ of the protected peptide was cleaved from the resin using 30 vol.% HFIP in DCM and selenoesterified as outlined in the general methods section. The crude peptide was purified by preparative reverse-phase HPLC (0 to 80% B over 30 min, 0.1% TFA) and lyophilized to afford the desired selenoester (12.1 mg, 42% yield). Analytical data is shown in Figure 143.

[522] It was observed that ligation at Ac-LYRANP-SePh did not proceed without a reductant (Figure 144). Ligation of Ac-LYRANP-SePh 52 with H-USPGYS-NH₂ dimer 1 (Figure 19) proceeded to > 80 % completion with the addition of 200 mM TCEP (see Figure 20). The effect of an external reductant on a selenoester- selenocystine ligation was explored not only with the Ac-LYRANP-SePh (52) + H- USPGYS-NH₂ dimer (1) ligation but with a ligation between Ac-LYRANI-SePh (6) + H-USPGYS-NH₂ dimer (1) in the presence of 200 mM TCEP (see Figure 21). Studies of the ligation between Ac-LYRANI-SePh (6) + H-USPGYS-NH₂ dimer (1) in the presence of 200 mM showed that deselenization of the main ligation product was observed over 2 h with conversion of the internal Sec residue to both Ala and Ser occurring (Figure 145).

[523] Kinetic Study of Selenium Ligation with Various Substituents

[524] The synthesis of appropriate selenium containing intermediates allowed the kinetics of the ligation reaction to be studied. Analytical data for the intermediates is shown in Figures 146 to 157.

[525] Solid phase synthesis of peptides

[526] To a selenocystine-containing peptide dimer (1.5 μιηοΐ, 0.5 eq.) anchored to Chem Matrix Rink amide resin was added 0.6 mL of buffer (6 M guanidine hydrochloride, 0.1 M Na₂HP0₄, 2.5 mM concentration with respect to the resin-bound selenocystine peptide) containing a peptide phenylselenoester (3.0 mg, 3.4 μιηοΐ, 1.1 eq.). The resin was agitated on an orbital shaker at room temperature for 30 min, at which point the ligation solution was filtered from the resin-bound peptide. The resin was washed with buffer (6 M guanidine hydrochloride, 0.1 M Na₂HP0₄, 3 x 3 mL), followed by H₂0 (3 x 3 mL), DMF (3 x 3mL) and DCM (10 x 3 mL). The peptide was then cleaved from the resin upon treatment with a solution of TFA/z^'Pr₃SiH/H₂0 (90:5:5 v/v/v, 2 mL, 2 h) and the cleavage solution was subsequently concentrated under a stream of nitrogen. LC-MS analysis of the crude residue indicated formation of the desired ligation products as a mixture of the symmetrical diselenide dimer, the asymmetric phenylselenyl diselenide and the trans-esterified internal selenoester.

[527] Solid phase ligation methods

Ac— ( LYRANG vAN

528] Preparation of and

[529] Rink amide Chem-Matrix resin was initially washed with DCM (5 x 3 mL) and DMF (5 x 3 mL), followed by removal of the Fmoc group by treatment with 20% piperidine/DMF (2 x 5 min). The resin was then washed with DMF (5 x 3 mL), DCM (5 x 3 mL) and DMF (5 x 3 mL). PyBOP (4 eq.) and NMM (8 eq.) were added to a solution of (Fmoc-Sec-OH)₂ (2 eq.) in DMF. After 5 min of pre-activation, the mixture was added to the resin. After 2 h the resin was washed with DMF (5 x 3 mL), DCM (5 x 3 mL) and DMF (5 x 3 mL) and capped with acetic anhydride/pyridine (1:9 v/v) (2 x 3 min). The Fmoc group was then removed with 20% piperidine/DMF (2 x 5 min), the resin washed with DMF (5 x 3 mL), DCM (5 x 3 mL) and DMF (5 x 3 mL) and the loaded resin swollen in ligation buffer (6 M guanidine hydrochloride, 100 mM Na₂HP0₄, pH 7.2). The peptide selenoester was then added (1-2 eq. dissolved in ligation buffer) and the resin agitated for 30 mins. To prepare the C-terminal Ala peptide the resin was washed with ligation buffer (5 x 3 mL) followed by addition of 50 eq. TCEP and 5 eq. DTT (also in ligation buffer). After 2 h the resin was again washed with ligation buffer (5 x 3 mL) followed by DMF (10 x 3 mL) and DCM (10 x 3 mL). To prepare the C-terminal Sec peptide the resin was washed with ligation buffer followed by washes with DMF (10 x 3 niL) and DCM (10 x 3 niL). To isolate the C- terminal Ala or Sec peptide, a mixture of TFA, thioanisole, triz^'sopropylsilane (TIS) and water (90:5:2.5:2.5 v/v/v/v) was added to the resin. After 2 h, the cleavage solution was plunged into a vial and the resin washed with TFA (2 mL). The combined solutions were concentrated under a stream of nitrogen either to dryness or to < 5 mL. In the latter case, 30 mL of diethyl ether was added to precipitate the peptide and the suspension centrifuged. The pellet or dried residue was then dissolved in water containing 0.1% TFA, filtered, purified by preparative HPLC and analyzed by analytical UPLC and LC-MS. All peptide products were isolated as white solids following lyophilization. Analytical data is shown in Figure 158 and Figure 159.

[530] Synthesis of Boc-(P-PMBSe)Asp-OH

[531] l,2-bis(4-methoxybenzyl)diselane (60) {Org. Lett., 2001, 3 (9), pp 1331- 1334)

[532] To a solution of 4-methoxybenzyl chloride (1.36 mL, 10.0 mmol, 1.0 equiv.) in acetonitrile (100 mL) was added potassium selenocyanate (1.58 g, 11.0 mmol, 1.1 equiv.) portionwise and the resulting solution stirred at room temperature for 16 h. Diethyl ether (approx. 100 mL) was added to the reaction mixture, which was then filtered through celite. The filtrate was evaporated in vacuo and then re-dissolved in absolute ethanol (120 mL). Aqueous sodium hydroxide (1 M, 60 mL) was added slowly with stirring and the reaction mixture stirred 3 h at room temperature. The resulting yellow precipitate was collected by filtration, washed with 1: 1 v/v ethanohwater and dried in vacuo. This afforded the title compound (60) as fine yellow flakes (1.81 g, 4.51 mmol, 90%). [533] 1H NMR (300 MHz, CDC1₃) δ (ppm): 7.17 (4 H, d, J=7.7 Hz), 6.85 (4 H, d, J=7.7 Hz), 3.85 (4 H, s), 3.80 (6 H, s); ¹³C NMR (75 MHz, CDC1₃) S (ppm): 158.9 (C), 131.2 (C), 130.2 (CH), 114.0 (CH), 55.4 (CH₃), 32.4 (CH₂); IR v_max/cm^_1 2835, 1603, 1505, 1456, 1297, 1237, 1184, 1115, 1030; MP 75-77 °C; MS Calculated mass [M+Ag]⁺: 508.869; Found (MALDI-ToF): 508.881.

5e-(4-methoxybenzyl) benzenesulfonoselenoate (61)

[535] To diselenide 60 (60 mg, 0.15 mmol, 1.0 equiv.) and sodium benzenesulfinate (25 mg, 0.15 mmol, 1.0 equiv.) in 75% actone/water (0.6 mL) at room temperature was added a solution of silver nitrate (28 mg, 0.16 mmol, 1.1 equiv.) in water (0.1 mL) and the solution stirred vigorously at room temperature for 30 min. The black suspension was filtered through celite, then partitioned between ethyl acetate and water. The aqueous layer was extracted with ethyl acetate (2 x) and the combined organic layers washed with brine, dried over Na₂S0₄ and the solvent removed in vacuo. The residue was purified by flash column chromatography over silica (5% ethyl acetate in hexanes, then 10% ethyl acetate in hexanes) to give the title compound (61) as a yellow oil (31 mg, 60%).

[536] 1H NMR (300 MHz, CDC1₃) δ (ppm): 7.82 (2 H, d, 7=8.2 Hz), 7.66 - 7.42 (3 H, m), 7.15 (2 H, d, 7=8.4 Hz), 6.77 (2 H, d, 7=8.4 Hz), 4.49 (2 H, s), 3.77 (3 H, s). ¹³C NMR (75 MHz, CDC1₃) δ (ppm): 159.4 (C), 147.5 (C), 133.5 (CH), 130.6 (CH), 129.3 (CH), 126.6 (C), 126.5 (CH), 114.4 (CH), 55.4 (CH₃), 37.0 (CH₂); IR v_max/cm^_1 2932, 2836, 1607, 1582, 1510, 1462, 1445, 1302, 1247, 1175, 1128, 1071, 1029

[537] 1-allyl 4-(tert-butyl) (2R)-2-((tert-butoxycarbonyl)amino)-3-((4- methoxybenzyl) selanyl)succinate (62)

[538] To a solution of 1-allyl 4-(ie/ -butyl) (ieri-butoxycarbonyl)-L-aspartate (0.95 g, 2.5 mmol) in anhydrous THF (26 mL) under nitrogen atmosphere at -78 °C was added LHMDS (1 M in THF, 5.6 mL, 5.6 mmol) and the reaction mixture stirred at -78 °C for 2 h. A solution of Se-(4-methoxybenzyl) benzenesulfonoselenoate (61) (0.96 g, 2.8 mmol) in THF (12 mL) was added dropwise over several minutes and the reaction mixture was stirred a further 2 h at -78 °C. Saturated aqueous NH₄C1 (10 mL) was added to the reaction mixture at -78 °C and it was then allowed to warm to room temperature. The reaction mixture was partitioned between sat. aq. NH₄C1 and EtOAc, the organic layer separated, and the aqueous layer extracted with ethyl acetate (2 x). The combined organic layers were washed with brine, dried over Na₂S0₄ and the solvent removed in vacuo. The residue was purified by flash column chromatography over silica (5% ethyl acetate in hexanes, then 10% ethyl acetate in hexanes) to give the title compound (62) as a yellow oil (1.1 g, 81%).

[539] [a]²⁰D +109.6 (c 1.0, CHC1₃); 1H NMR (300 MHz, CDC1₃) δ (ppm): 7.27 (2 H, d, J=7.3 Hz), 6.84 (2 H, d, J=7.3 Hz), 5.99 - 5.79 (1 H, m), 5.75 (1 H, d, J=10.2 Hz), 5.39 - 5.18 (2 H, m), 4.72 - 4.50 (3 H, m), 4.06 (1 H, d, J=11.5 Hz), 3.98 (1 H, d, J=10.8 Hz), 3.79 (4 H, V), 1.50 (9 H, s), 1.46 (9 H, s); ¹³C NMR (75 MHz, CDC1₃) δ (ppm): (major diastereomer) 172.1 (C), 170.0 (C), 158.8 (C), 155.9 (C), 131.5 (CH), 130.3 (CH), 129.8 (C), 118.5 (=CH₂), 114.1 (CH), 82.5 (C), 80.1 (C), 66.2 (CH₂), 55.3 (CH₃), 55.1 (CH), 40.1 (CH), 28.6 (CH₂), 28.4 (CH₃), 28.0 (CH₃); IR v^/cm^"1 3434, 2976, 2929, 1711, 1649, 1609, 1583, 1510, 1491, 1457, 1392, 1367, 1350, 1300, 1246, 1153, 1097, 1058, 1031; MS (ESI+) 530.4 [M+H]⁺, 552.3 [M+Na]⁺; HRMS Calculated mass [M+Na]⁺: 552.14710; Found (ESI+): 552.14714.

[540] (2R)-4-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-3-((4- methoxybenzyl)selanyl)-4-oxobutanoic acid (Boc-(P-PMBSe)Asp-OH) (63)

[541] To a solution of 1-allyl 4-(ie/t-butyl) (2 ?)-2-((ieri-butoxycarbonyl)amino)-3- ((4-methoxybenzyl)selanyl)succinate (62) (1.0 g, 2.0 mmol) in CH₂CI₂ (21 mL) under nitrogen atmosphere was added phenylsilane (0.48 mL, 3.9 mmol) and Pd(PPh₃)₄ (45 mg, 0.039 mmol) and the reaction mixture stirred for 1 h at room temperature in a screw-top vial. The solvent was removed in vacuo and the residue was purified by flash column chromatography over silica (10% ethyl acetate in hexanes, then 30% ethyl acetate in hexanes containing 0.5% acetic acid) to give the title compound (63) as a yellow oil in -85: 15 mixture of diastereomers at the β position (0.82 g, 86%).

[542] [a]²⁰D +106.4 (c 1.1, CHC1₃); 1H NMR (300 MHz, CDC1₃) δ (ppm): 7.29 - 7.22 (2 H, m), 6.86 (2 H, d, 7=7.7 Hz), 5.86 (1 H, d, 7=8.6 Hz), 4.64 (1 H, dd, 7=8.8, 3.5 Hz), 4.05 (1 H, d, 7=11.3 Hz), 3.99 (1 H, d, 7=11.0 Hz), 3.81 (3 H, s), 3.73 (1 H, d, 7=3.3 Hz), 1.53 (9 H, s), 1.46 (9 H, s); ¹³C NMR (75 MHz, CDC1₃) δ (ppm): (major diastereomer) 174.0 (C), 172.9 (C), 158.8 (C), 156.0 (C), 130.2 (CH), 129.5 (C), 114.1 (CH), 83.1 (C), 80.5 (C), 55.2 (CH₃), 54.7 (CH), 39.6 (CH), 28.7 (CH₂), 28.3 (CH₃), 27.9 (CH₃); IR v cm^-1 3436, 2977, 2933, 1711, 1609, 1583, 1510, 1456, 1393, 1368, 1350, 1301, 1246, 1149, 1060, 1032; MS (ESI+) 490.3 [M+H]⁺, 512.3 [M+Na]⁺; HRMS Calculated mass [M+Na]⁺: 512.11580; Found (ESI+): 512.11563.

[543] Preparation of H-(P-Se)DSPGYS-NH₂ dimer (64)

[544] A solution of Boc-(p-PmbSe)Asp-OH (42 mg, 86 μιηοΐ), DIC (13 μΐ,, 86 μιηοΐ) and HOAt (12 mg, 86 μιηοΐ) in DMF (0.87 mL) was added to Rink amide immobilized SPGYS (0.34 g, 72 μιηοΐ, 4.7 g/mmol loading) and the reaction mixture shaken at room temperature for 16 h. The resulting peptide was cleaved by treatment with TFA/iPr₃SiH/H₂0 (80: 10: 10 v/v/v) for 2 h at room temperature and precipitated from diethyl ether, centrifuged, and the crude peptide used directly. To a solution of crude H-(p-PmbSe)DSPGYS-NH₂ (86 μηοΐ) in TFA (8.4 mL) in an ice bath was added DMSO (2.1 mL) dropwise with stirring and the resulting solution was stirred overnight at room temperature. The total reaction volume was reduced to ~4 mL under a flow of nitrogen and the peptide was precipitated from diethyl ether and centrifuged. The crude peptide was dissolved in water containing 0.1% TFA, purified by preparative HPLC (0- 30% acetonitrile in water, containing 0.1% TFA) and lyophilised to give the title compound (64) as a colourless powder (26 mg, 44% from resin loading). Analytical data is shown in Figure 177.

[545] Preparation of Ac-LYRANFDSPGYS-NH₂ (65)

[546] The title compound was prepared using the general procedure for one -pot ligation-deselenisation. Ligation of H-(P-Se)DSPGYS-NH₂ dimer (2.0 mg, 1.4 μιηοΐ) and Ac-LYRANF-SePh (3.6 mg, 3.7 μιηοΐ), followed by direct deselenisation and purification via preparative reverse phase HPLC (0 to 60% B over 30 min, 0.1% TFA) afforded the title compound (65) as a colourless solid after lyophilisation (2.1 mg, 53% yield over 2 steps). Analytical data is shown in Figure 178 and Figure 179.

[547] Preparation of Ac-LYRANMDSPGYS-NH₂ (66)

[548] The title compound was prepared using the general procedure for one -pot ligation-deselenisation. Ligation of H-(P-Se)DSPGYS-NH₂ dimer (2.0 mg, 1.4 μιηοΐ) and Ac-LYRANM-SePh (3.5 mg, 3.7 μιηοΐ) followed by direct deselenisation and purification via preparative reverse phase HPLC (0 to 60% B over 30 min, 0.1% TFA) afforded the title compound (66) as a colourless solid after lyophilisation (2.6 mg, 65% yield over 2 steps). Analytical data is shown in Figure 180 and Figure 181.

[549] Preparation of Ac-LYRANSDSPGYS-NH₂ (67)

[550] The title compound was prepared using the general procedure for one -pot ligation-deselenisation. Ligation of H-(P-Se)DSPGYS-NH₂ dimer (2.0 mg, 1.4 μιηοΐ) and Ac-LYRANS-SePh (3.3 mg, 3.7 μιηοΐ), followed by direct deselenisation and purification via preparative reverse phase HPLC (0 to 60% B over 30 min, 0.1% TFA) afforded the title compound (67) as a colourless solid after lyophilisation (2.2 mg, 57% yield over 2 steps). Analytical data is shown in Figure 182 and Figure 183.

[551] Preparation of Ac-LYRANVDSPGYS-NH₂ (68)

[552] The title compound was prepared using the general procedure for one -pot ligation-deselenisation. Ligation of H-(P-Se)DSPGYS-NH₂ dimer (2.0 mg, 1.4 μιηοΐ) and Ac-LYRANV-SePh (5.2 mg, 5.4 μιηοΐ), followed by direct deselenisation and purification via preparative reverse phase HPLC (0 to 60% B over 30 min, 0.1% TFA) afforded the title compound (68) as a colourless solid after lyophilisation (2.3 mg, 58% yield over 2 steps). Analytical data is shown in Figure 184 and Figure 185.

[553] Preparation of Ac-LYRANADSPGYS-NH₂ (69)

[554] The title compound was prepared using the general procedure for one -pot ligation-deselenisation. Ligation of H-(P-Se)DSPGYS-NH₂ dimer (2.0 mg, 1.4 μιηοΐ) and Ac-LYRANA-SePh (3.3 mg, 3.7 μιηοΐ), followed by direct deselenisation and purification via preparative reverse phase HPLC (0 to 60% B over 30 min, 0.1% TFA) afforded the title compound (69) as a colourless solid after lyophilisation (2.5 mg, 64% yield over 2 steps). Analytical data is shown in Figure 186 and Figure 187.

[555] Preparation of Ac-LYRANLDSPGYS-NH₂ (70)

[556] The title compound was prepared using the general procedure for one -pot ligation-deselenisation. Ligation of H-(P-Se)DSPGYS-NH₂ dimer (2.0 mg, 1.4 μιηοΐ) and Ac-LYRANL-SePh (3.4 mg, 3.7 μιηοΐ), followed by direct deselenisation and purification via preparative reverse phase HPLC (0 to 60% B over 30 min, 0.1% TFA) afforded the title compound (70) as a colourless solid after lyophilisation (2.9 mg, 72% yield over 2 steps). Analytical data is shown in Figure 188 and Figure 189.

[557] Preparation of Ac-LYRANYDSPGYS-NH₂ (71)

[558] The title compound was prepared using the general procedure for one -pot ligation-deselenisation. Ligation of H-(P-Se)DSPGYS-NH₂ dimer (2.0 mg, 1.4 μιηοΐ) and Ac-LYRANY-SePh (3.6 mg, 3.7 μιηοΐ), followed by direct deselenisation and purification via preparative reverse phase HPLC (0 to 60% B over 30 min, 0.1% TFA) afforded the title compound (71) as a colourless solid after lyophilisation (2.6 mg, 64% yield over 2 steps). Analytical data is shown in Figure 190 and Figure 191.

[559] Synthesis of Hyalomin 3 and 4

[560] Preparation of Hyalomin-3 N-terminal Selenoester (1-25) (72) H— (K P N L Q S R N G D G V A E T S Y E E Y P D D S

[561] The N-terminal fragment of Hyalomin 3 (residues 1-25, 75 μιηοΐ) was prepared on 2-chlorotrityl chloride resin using Fmoc-SPPS as described in the general methods. Cleavage of the peptide from the resin was effected by treating with 30 vol.% HFIP in CH₂CI₂ for 2 h before concentrating in vacuo. The resulting residue was dissolved in anhydrous DMF (1 mL per 25 μιηοΐ of resin) and cooled to 0 °C. Diphenyl diselenide (DPDS) (30 eq.) was added to the solution followed by Bu₃P (30 eq.). The reaction was allowed to proceed at 0 °C for 3 h, after which time the solvent was removed in vacuo. The crude material was put on ice and the protecting groups removed via treatment with TFA:TIS:thioanisole:H₂0 (85:5:5:5 v/v/v/v). After 2 h at room temperature the cleavage cocktail was removed under a stream of N₂ and the crude residue suspended in diethyl ether and cooled to -20 °C. The precipitate was pelleted by centrifugation at 4000 rpm for 5 min. The supernatant decanted and the pellet dissolved in 0.1% TFA/H₂0 with addition of 0.1% TFA/MeCN and purified via preparative HPLC (0-40% 0.1% TFA/MeCN in 0.1% TFA/H₂0) to afford the title compound (72) as a colourless solid after lyophilisation (8.48 mg, 3.9% yield). Analytical data is shown in Figure 192 and Figure 193.

[562] Preparation of Hyalomin 3 C-terminal Diselenide Dimer (26 - 60) (73)

[563] Fmoc-Ser-OH was loaded to 2-chlorotrityl chloride ChemMatrix resin (150 μιηοΐ) and the peptide was elongated using automated Fmoc-SPPS as outlined in the general procedures. After coupling of Boc-(P-PmbSe)Asp-OH to the N-terminus, the fully protected resin-bound peptide was cleaved and deprotected using a solution of TFA/z^'Pr₃SiH/H₂0 (89:5:5 v/v/v). The solution was agitated at room temperature for 2 h and then concentrated in vacuo. Crude peptide was precipitated from cold Et₂0, centrifuged, and used directly. A solution of N-terminal PMB-protected peptide in TFA containing 1% thioanisole was treated with 2,2'-dithiobis(5-nitropyridine) (DTNP) (3.0 eq.). The reaction mixture was stirred at room temperature for 2 h, at which point UPLC-MS analysis indicated complete consumption of starting material and the formation of a mixture of deprotected products composed of the diselenide dimer peptide and DTNP-bound selenyl sulfide peptides. The mixture was concentrated, treated with TCEP (catalytic) and immediately subjected to purification by reverse- phase HPLC followed by lyophilisation to yield the desired product (73) exclusively as the diselenide dimer peptide (6.5 mg, 1.3% yield). Analytical data is shown in Figure 194 and Figure 195.

[564] Preparation of Hyalomin-3 (1-60) (74)

[565] The ligation of Hyalomin-3 (26-60) dimer (2.67 mg, 0.4 μιηοΐ) and Hyalomin- 3 (1-25) selenoester (4.05 mg, 1.39 μιηοΐ) together with in situ deselenization was performed as outlined in the general methods section. Purification via preparative reverse phase HPLC (0 to 60% B over 30 min, 0.1% TFA) followed by lyophilization afforded the native Hyalomin-3 protein (74) (2.25 mg, 47%) as a white solid. Analytical data is shown in Figures 196 to 199.

[566] Preparation of Hyalomin-4 N-terminal Selenoester (1-25) (75)

[567] The N-terminal fragment of Hyalomin 4 (residues 1-25, 75 μιηοΐ) was prepared on 2-chlorotrityl chloride resin using Fmoc-SPPS as described in the general methods. Cleavage of the peptide from the resin was effected by treating with 30 vol.% HFIP in CH₂C1₂ for 2 h before concentrating in vacuo. The resulting residue was dissolved in anhydrous DMF (1 niL per 25 μηιοΐ of resin) and cooled to 0 °C. Diphenyl diselenide (DPDS) (30 eq.) was added to the solution followed by Bu₃P (30 eq.). The reaction was allowed to proceed at 0 °C for 3 h, after which time the solvent was removed in vacuo. The crude material was put on ice and the protecting groups removed via treatment with TFA:TIS:thioanisole:H₂0 (85:5:5:5 v/v/v/v). After 2 h at room temperature the cleavage cocktail was removed under a stream of N₂ and the crude residue suspended in diethyl ether and cooled to -20 °C. The precipitate was pelleted by centrifugation at 4000 rpm for 5 min. The supernatant decanted and the pellet dissolved in 0.1% TFA/H₂0 with addition of 0.1% TFA/MeCN and purified via preparative HPLC (0-40% 0.1% TFA/MeCN in 0.1% TFA/H₂0) to afford the title compound (75) as a colourless solid after lyophilisation (8.9 mg, 4.5 % yield). Analytical data shown in Figure 200 and Figure 201.

[568] Preparation of Hyalomin 4 C-terminal Diselenide Dimer (26 - 51) (76)

[569] Fmoc-Ser-OH was loaded to 2-chlorotrityl chloride ChemMatrix resin (150 μιηοΐ) and the peptide was elongated using automated Fmoc-SPPS as outlined in the general procedures. After coupling of Boc-(P-PmbSe)Asp-OH to the N-terminus, the fully protected resin-bound peptide was cleaved and deprotected using a solution of TFA/z^'Pr₃SiH/H₂0 (89:5:5 v/v/v). The solution was agitated at room temperature for 2 h and then concentrated in vacuo. Crude peptide was precipitated from cold Et₂0, centrifuged, and used directly. A solution of N-terminal PMB-protected peptide in TFA containing 1% thioanisole was treated with 2,2'-dithiobis(5-nitropyridine) (DTNP) (3.0 eq.). The reaction mixture was stirred at room temperature for 2 h, at which point UPLC-MS analysis indicated complete consumption of starting material and the formation of a mixture of deprotected products composed of the diselenide dimer peptide and DTNP-bound selenyl sulphide peptides. The mixture was concentrated, treated with TCEP (catalytic) and immediately subjected to purification by reverse- phase HPLC followed by lyophilisation to yield the desired product (76) exclusively as the diselenide dimer peptide (16 mg, 3.6%). Analytical data shown in Figure 202 and Figure 203.

[570] Preparation of Hyalomin-4 (1-51) (77)

[571] The ligation of Hyalomin-4 (26-51) dimer (2.47 mg, 0.46 umol) and Hyalomin-3 (1-25) selenoester (4.49 mg, 1.497 μιηοΐ) together with in situ deselenization was performed as outlined in the general methods section. Purification via preparative reverse phase HPLC (0 to 60% B over 30 min, 0.1% TFA) followed by lyophilization afforded the native Hyalomin-4 protein (77) (3.3 mg, 66%) as a white solid. Analytical data shown in Figures 204 to 207.

[572] Synthesis of (2R,3S)-2-((tert-butoxycarbonyl)amino)-3-((4- methoxybenzyl)selanyl)-4-methylpentanoic acid [Boc-(P-PmbSe)Asp-OH] (84)

[573] tert-butyl (R)-4-((/?)-l-hydroxy-2-methylpropyl)-2,2-dimethyloxazolidine- 3-carboxylate (78)

[574] Magnesium turnings (0.27 g, 11.05 mmol) were dried in a 3-necked round bottom flask in vacuo with heat and stirring for 0.5 h before the addition of dry Et₂0 (2 mL). A spatula tip of iodine was quickly added to the above mixture and the resulting brown suspension was stirred vigorously at room temperature for 30 min. Following magnesium activation, a solution of isopropyl bromide (0.52 mL, 5.53 mmol) in Et₂0 (3 mL) was added to the mixture dropwise at 0 °C and subsequently stirred at 40 °C for 1 h. A solution of Gamer' s aldehyde (0.97 g, 4.25 mmol) in Et₂0 (5 mL) was added dropwise to the Grignard reagent at 0 °C. The reaction mixture was continually stirred at 0 °C and allowed to warm up to room temperature over 5 h. The reaction was quenched with saturated aq. NH₄C1 (8 mL) at 0 °C before extraction with EtOAc (20 mL x 3). The combined organic layers were dried over MgS0₄, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography (15:85, v/v, EtOAc/Hexane, R_f = 0.32) to afford syn alcohol 78 as a white solid (0.63 g, 2.29 mmol, 54%).

[575] CH₂C1₂, ZnSe, film) 3399, 2963, 2935, 2875, 1657, 1400, 1366, 1248, 1172, 1108, 1058, 1018, 865; 1H NMR (CDC1₃, 400 MHz) δ 4.04 (br s, 1H, CHNH), 3.94-3.91 (m, 1H, CH_2a), 3.76- 3.74(d, 1H, J = 9.1 Hz, CH_2b), 3.50-3.48 (d, 1H, J = 9.0 Hz, CHOH), 1.70- 1.64 (m, 1H, CH(CH₃)₂), 1.59-1.44 (m, 15H, C(CH₃)₃, C(CH₃)₃), 1.03-1.01 (d, 3H, J=6.8 Hz, CU(CH₃)_2a), 0.90-0.89 (d, 3H, J=6.7 Hz, CH(CH^); ¹³C NMR (CDC1₃, 75 MHz) δ 155.73, 99.12, 79.41, 77.26, 65.76, 45.42, 29.85, 29.33, 28.51, 19.12, 18.69, 17.64; HRMS (ESr): mlz calcd. for Ci₄H₂₇N0₄Na [M+Na]⁺ 296.1832, found 296.1833.

[576] tert-butyl ((2R,3R)-l,3-dihydroxy-4-methylpentan-2-yl)carbamate (79)

79

[577] To a solution of syn alcohol 78 (2.54 g, 9.29 mmol) in THF (380 mL), 0.5 M aq. HC1 (20 mL) was added slowly. The mixture was stirred at room temperature for 24 h before being quenched by the addition of NaHC<¾ (1.68 g, 20 mmol), followed by co-evaporation with toluene to dryness. The crude material was diluted with EtOAc (100 mL), filtered through cotton wool and concentrated in vacuo. The crude product was purified by flash column chromatography (1 : 1, v/v, EtOAc/Hexane, R_f = 0.34) to obtain syn diol 79 as a yellow oil (2.09 g, 8.95 mmol, 96%). [578] [α]¾°= -15.1° (c 0.37, CH₂C1₂); IR (v/cm^-1, CH₂C1₂, ZnSe, film) 3377, 2961, 2927, 2874, 1685, 1503, 1392, 1366, 1258, 1166, 1064, 1012, 803; 1H NMR (CDC1₃, 400 MHz) δ 5.28 (br s, 1H, NH), 3.77-3.72 (m, 3Η, CH₂, CHNH), 3.48-3.46 (d, 1H, J = 8.3 Hz, CHOH), 3.12 (br s, 2H, OH), 1.77-1.68 (m, 1Η, CH(CH₃)₂), 1.43 (s, 9H, C(CH₃)₃), 0.99-0.98 (d, 3Η, J=6.6 Hz, CH(CH₃)_2a), 0.91-0.89 (d, 3H, J=6.7 Hz, CH(C¾j₂»); ¹³C NMR (CDC1₃, 100MHz) δ 156.6, 79.8, 78.0, 65.4, 52.4, 31.1, 28.5, 19.1, 18.9; HRMS (ESI⁺): mlz calcd. for CnH₂₃N0₄Na [M+Na]⁺ 256.1523, found 256.1524.

[579] tert-butyl ((2R,3R)-l-((tert-butyldimethylsilyl)oxy)-3-hydroxy-4- methylpentan-2-yl)carbamate (80)

80

[580] Imidazole (1.22 g, 17.9 mmol) and syn diol 79 (2.08 g, 8.95 mmol) were dissolved in dry CH₂C1₂ (8.5 mL) followed by addition of ie/ -butyldimethylsilyl chloride (1.62 g, 10.74 mmol) in one portion at 0 °C. The mixture was stirred at 0 °C and warmed to room temperature slowly over 12 h. After complete consumption of the starting material, the reaction was quenched with saturated aq. NH₄C1 (20 mL) and extracted with CH₂C1₂ (60 mL x 3). The combined organic layers were dried over MgS0₄, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography (5:95, v/v, EtOAc/Hexane, R_f = 0.28) to give TBS-protected syn diol 80 as a yellow oil (3.00 g, 8.66 mmol, 97%).

[581] [a]¾°= -26.4° (c 0.54, CH₂C1₂); IR (v/cm^"1, CH₂C1₂, ZnSe, film) 3444, 2956, 2929, 2858, 1716, 1692, 1498, 1472, 1390, 1366, 1254, 1168, 1097, 1017, 835, 776; 1H NMR (CDC1₃, 400 MHz) δ 5.20-5.18 (d, 1H, = 8.5 Hz, NH), 3.94-3.90 (dd, 1Η, = 3.4 Hz, 10.2 Hz, CH_2a 3.79-3.76 (dd, 1H, J = 2.3 Hz, 10.2 Hz, CH_2b 3.71-3.69 (d, 1H, = 8.4 Hz, CHNH), 3.52-3.50 (d, 1H, = 8.4 Hz, CHOH), 1.74-1.69 (m, 1H, CH(CH₃)₂), 1.43 (s, 9H, C(CH₃)₃), 1.00-0.98 (d, 3Η, J = 6.6 Hz, CU(CH₃)₂a), 0.91-0.87 (m, 12H, SiC(CH₃Jj, CU(CH₃)_2b), 0.07 (s, 6Η, Si(CH₃J₂); ¹³C NMR (CDC1₃, 100MHz) δ 156.0, 79.3, 67.1, 51.3, 30.9, 28.5, 26.0, 19.1, 19.0, 18.3, -5.5; HRMS (ESI⁺): mlz calcd. for Ci₇H₃₇N0₄SiNa [M+Na]⁺ 370.2384, found 370.2386.

[582] tert-butyl ((2R,35)-l-((tert-butyldimethylsilyl)oxy)-4-methyl-3- selenocyanatopentan-2-yl)carbamate (81)

81

[583] Triethylamine (1.80 mL, 12.99 mmol) and methanesulfonyl chloride (1.04 mL, 10.39 mmol) were slowly added into a solution of TBS-protected syn diol 80 (3.00 g, 8.66 mmol) in dry CH₂C1₂ (30 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 40 min before being quenched with saturated aq. NH₄C1 (40 mL) followed by extraction with CH₂C1₂ (60 mL x 3). The combined organic layers were dried over MgS0₄, filtered and concentrated in vacuo to yield crude mesylate as a pale yellow solid. The crude mesylate and potassium selenocyanate (18.71 g, 129.85 mmol) were dissolved in dry MeCN (40 mL) and stirred at 65 °C for 24 h. The reaction mixture was concentrated in vacuo, diluted with CH₂C1₂ (50 mL) and poured into water (70 mL). The organic layer was separated and the aqueous layer was extracted with DCM (70 mL x 2). The combined organic layers were dried over MgS0₄, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography (4:96, v/v, EtOAc/Hexane, R_f = 0.31) to yield selenocyanate 81 as a yellow oil (2.01 g, 4.61 mmol, 54%).

[584] -52.3° (c 0.34, CH₂C1₂); IR (v/cm^"1, CH₂C1₂, ZnSe, film) 2959, 2930, 2885, 2858, 2149 (SeCN), 1715, 1490, 1391, 1366, 1255, 1170, 1103, 1046, 836, 779; 1H NMR (CDC1₃, 400 MHz) δ 5.28-5.26 (d, 1H, = 8.6 Hz, NH), 4.08-4.07 (m, 1Η, CHNH), 3.89-3.85 (dd, 1H, J = 2.2 Hz, 10.7 Hz, CH_2a), 3.75-3.72 (dd, 1H, J = 1.92 Hz, 10.6 Hz, CH_2b), 3.51-3.47 (m, 1H, CHSeCN), 2.11-2.02 (m, 1Η, CH(CH₃)₂), 1.47 (s, 9H, C(CH₃)₃), 1.30-1.28 (m, 3Η, CH(CH₃J₂a), 1.13-1.12 (d, 3Η, J = 6.7 Hz, CH(CH₃)_2b) 0.91 (s, 9H, SiC(CH₃)₃), 0.10 (s, 3H, Si(CH₃)_2a), 0.09 (s, 3H, Si(CH₃)_2b); ¹³C NMR (CDC1₃, 100 MHz) δ 155.1, 106.3(SeCN), 80.4, 64.1, 62.7, 51.2, 33.2, 29.8, 28.5, 26.1, 21.3, 18.6, -5.2, -5.3; HRMS (ESf): mlz calcd. for Ci₈H₃₆N₂0₃SeSiNa [M+Na]⁺ 459.1552, found 459.1554.

[585] tert-butyl ((2/f,35)-l-hydroxy-4-methyl-3-selenocyanatopentan-2- yl)carbamate (82)

82

[586] A solution of tetrabutylammonium fluoride (5.02 niL, 5.02 mmol, 1 M in THF) was added dropwise to a solution of selenocyanate 81 (1.82 g, 4.18 mmol) in dry THF (32 mL) and the reaction mixture was stirred at room temperature for 30 min. CaC0₃ (1.06 g), DOWEX ^® 50W X8 (200-400 mesh, 3.11 g) and MeOH (7.53 mL) were added to the reaction flask and the reaction was stirred for 1 h. The resulting suspension was filtered through celite, washed with MeOH and concentrated in vacuo. The crude product was purified by flash column chromatography (3:7, v/v, EtOAc/Hexane, R_f = 0.32) to afford 82 as a yellow oil (0.97 g, 3.03 mmol, 72%).

[587] [a]¾°= -34.4° (c 0.28, CH₂C1₂); IR (v/cm^"1, CH₂C1₂, ZnSe, film) 3421, 2967, 2932, 2155 (SeCN), 1687, 1505, 1392, 1368, 1250, 1168, 1052, 803; 1H NMR (CDC1₃, 400 MHz) δ 5.5.34-5.32 (d, 1H, J = 7.8 Hz, NH), 4.07 (m, 1Η, CHNH), 3.99-3.95 (dd, 1H, J = 3.3 Hz, 11.2 Hz, CH_2a), 3.85-3.81 (dd, 1H, = 2.4 Hz, 11.3 Hz, CH_2b), 3.55- 3.52 (m, 1H, CHSeCN), 2.35 (br s, 1Η, ΟΗ), 2.18-2.11 (m, 1Η, CH(CH₃)₂), 1.46 (s, 9H, C(CH₃)₃), 1.21-1.20 (d, 3Η, J = 6.6 Hz, CU(CH₃)_2a), 1.15-1.13 (d, 3H, J = 6.6 Hz, CH(C¾j₂»); ¹³C NMR (CDCI₃, 100 MHz) δ 155.4, 105.2 (SeCN), 80.5, 63.2, 62.2, 52.2, 31.9, 28.5, 21.6, 21.5; HRMS (ESI⁺): mlz calcd. for Ci₂H₂₂N₂0₃SeNa [M+Na]⁺ 345.0688, found 345.0688.

[588] (2R,35)-2-((tert-butoxycarbonyl)amino)-4-methyl-3- selenocyanatopentanoic acid (83)

83

[589] The selenocyanate alcohol 82 (158 mg, 0.49 mmol) was dissolved in a mixed solvent of MeCN/H₂0 1 : 1 (v/v, 4 mL) before the addition of (diacetoxyiodo)benzene (0.7895 g, 2.451 mmol) and TEMPO (0.038g, 0.2451 mmol) in one portion. The orange solution was stirred at room temperature for 3 h and then concentrated under a stream of N₂ followed by lyophilization. The crude product was purified by flash column chromatography (96:2:2, v/v/v, DCM:MeOH:AcOH, R_f = 0.33) to provide 83 as a yellow oil (148.5 mg, 0.4429 mmol, 90%).

[590] [a]_D ²°= -42.7° (c 0.33, CH₂C1₂); IR (v/cm^"1, CH₂C1₂, ZnSe, film) 2965, 2923, 2852, 2153 (SeCN), 1705, 1503, 1457, 1393, 1369, 1253, 1160, 1057, 1019, 856, 801; 1H NMR (CDC1₃, 400 MHz) δ 5.53-5.51 (d, 1H, = 5.5 Hz, NH), 4.87-4.74 (m, 1Η, CHNH), 3.49-3.47 (m, 1H, CHSeCN), 2.30-2.22 (m, 1Η, CH(CH₃)₂), 1.47 (s, 9H, C(CH₃)₃), 1.22-1.20 (d, 6Η, = 5.9 Hz, CH(CHjj₂); ¹³C NMR (CDC1₃, 100 MHz) δ 172.5 (COOH), 155.4, 102.8 (SeCN), 81.7, 60.9, 55.2, 29.8, 28.4, 21.2; HRMS (ESI⁺): mlz calcd. for [M+Na]⁺ 359.0481, found 359.0481.

[591] (2R,3S)-2-((tert-butoxycarbonyl)amino)-3-((4-methoxybenzyl)selanyl)-4- methylpentanoic acid [Boc-(P-PmbSe)Asp-OH] (84)

84

[592] To a THF (6 mL) solution of 83 (209 mg 0.87 mmol) was added a solution of NaBH₄ (0.065g, 1.7306 mmol) in 95% EtOH (0.86 mL) at 0 °C under an argon atmosphere. Following stirring of the reaction mixture at 0 °C for 1 h, /?-methoxybenzyl chloride (0.54 g, 3.46 mmol) and aq. 2 M NaOH (degassed, 2.3 mL) were added whilst maintaining a temperature of 0 °C and the mixture was allowed to warm up to room temperature while stirring for 16 h. The reaction was quenched with AcOH (2 mL) and co-evaporated with toluene to dryness. The crude product was purified by flash column chromatography (97: 1 :2, v/v/v, CH₂Cl₂:MeOH:AcOH, R_f = 0.35) to provide β- selenoleucine 84 as a yellow oil (186 mg, 0.43 mmol, 50%).

[593] [a]¾°= -72.7° (c 0.29, CH₂C1₂); IR (v/cm^"1, CH₂C1₂, ZnSe, film) 2960, 2926, 2853, 1715, 1610, 1511, 1458, 1393, 1368, 1248, 1171, 1035, 831 ; 1H NMR (CDC1₃, 400 MHz) δ 7.20-7.117 (d, 2H, J = 8.5 Hz, Ar-H), 6.81-6.79 (d, 2Η, J = 8.0 Hz, Ar-H), 5.19-5.18 (d, 1Η, J = 7.1 Hz, NH), 4.66 (s, 1Η, CHNH), 3.77 (s, 3H, OCH₃), 3.75-3.74 (m, 2Η, CH₂), 2.61 (s, 1Η, CHSe), 2.03-2.01 (m, 1Η, CH(CH₃)₂), 1.45 (s, 9H, C(CH₃)₃), 0.970 (s, 6Η, CH(CH₃J₂); ¹³C NMR (CDC1₃, 100 MHz) δ 175.2 (COOH), 158.7, 155.5, 130.3, 114.1, 80.6, 56.2, 55.4, 51.6, 29.8, 28.9, 28.4, 21.4, 21.0; HRMS (ESI⁺): mlz calcd. for [M+Na]⁺ 454.1103, found 454.1101.

[594] Preparation of H-(P-Se)LSPGYS-NH₂ dimer (85)

ΌΗ [595] A solution of Boc-(p-PmbSe)Leu-OH (26 mg, 60 μηιοΐ), DIC (9.3 μΐ,, 60 μηιοΐ) and HO At (8.2 mg, 60 μιηοΐ) in DMF (0.6 mL) was added to Rink amide resin immobilized with SPGYS (50 μιηοΐ, 0.27 mmol/g loading) and the reaction mixture shaken at room temperature for 16 h. The resulting peptide was cleaved by treatment with TFA/iPr₃SiH/H₂0 (80: 10: 10 v/v/v) for 2 h at room temperature, precipitated from diethyl ether and centrifuged. A solution of the crude peptide in TFA (50 mL) was added dropwise into a mixture of DMSO and TFA (1:4 v/v, 10 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 16 h, the solvent was then concentrated under a stream of N₂ for 16 h, precipitated from diethyl ether and centrifuged. The crude peptide was dissolved in water containing 0.1% TFA, purified by preparative HPLC (10-20% acetonitrile in water, containing 0.1% TFA) and lyophilised to give the title compound (85) as a colourless powder (12.7 mg, 36% from resin loading). Analytical data is shown in Figure 209.

[596] Ac-LYRANLLSPGYS-NH₂ (86)

[597] The title compound was prepared using the general procedure for one -pot ligation-deselenisation. Ligation of H-(P-Se)LSPGYS-NH₂ dimer (2.03 mg, 1.45 μιηοΐ) and Ac-LYRANL-SePh (3.45 mg, 3.71 μιηοΐ), followed by direct deselenisation and purification via preparative reverse phase HPLC (15 to 45% B over 40 min, 0.1% TFA) afforded the title compound (86) as a colourless solid after lyophilisation (3.30 mg, 82% yield over 2 steps). Analytical data is shown in Figure 210 and Figure 211.

[598] It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS:

1. A method of preparing an amide containing compound comprising the step of reacting an ester with a diselenide bearing an amino group.

2. The method of claim 1, wherein the reaction proceeds in the absence of an additive.

3. The method of claim 1 or claim 2, wherein the amide containing compound is a peptide.

4. The method of claim 3, wherein the peptide is defined by formula (I):

the ester is defined by formula (II):

II and the diselenide is defined by formula (III):

(HI), wherein:

Nterm is the N-terminus of the peptide; Cterm is ^me C-terminus of the peptide; A A is an amino acid; n is an integer;

(AA)n represents a peptide comprising n number of amino acid monomers; DG is a displaceable group;

is a diselenide functionalised amino acid residue at the ligation site.

2

5. The method of claim 4, wherein w / is Sec.

6. The method of claim 4 or claim 5, wherein DG is a selenoate or a leaving group (LG).

7. The method of claim 6, wherein the selenoate is an aryl selenoate.

8. The method of claim 7, wherein the aryl selenoate is phenyl selenoate.

9. The method of claim 8, wherein the peptide is defined by formula (IV):

IV the ester is defined by formula (V):

the selenide is defined by formula (VI):

VI, wherein AAa-e, AAl-5 and X are amino acids; wherein ^< ° represents a peptide comprising the five amino acid residues AAi, AA₂, AA₃, AA₄, and AA₅;

AAa - AAb - AAc - AAd— AAe-f- . .. , . . _f. wherem ^{? s} represents a peptide comprising the five amino acid residues AAa, AAb, AAc, AAd, and AAe.

10. The method of claim 9, wherein: AA_a is L; AA_b is Y; AA_C is R; AAd is A; AAe

X is selected from the group consisting of: Ala, Ser, Thr, Leu, He, Val, Phe, Met and Lys;

AAii_g is U; AA1 is S; AA₂ is P; AA₃ is G; AA₄ is Y; AA₅ is S.

11. The method of any one of claims 1 to 10, wherein the reaction is conducted in an aqueous solution.

12. The method of claim 11, wherein the aqueous solution has a pH in the range of about 2 to 14.

13. The method of claim 12, wherein the aqueous solution is a buffer comprising a denaturing agent and an aqueous solution of Na₂HP0₄.

14. The method of claim 13, wherein the denaturing agent is 6 M guanidine hydrochloride.

15. The method of claim 14, wherein the aqueous solution of Na₂HP0₄ has a concentration of about 100 mM.

16. The method of claim 13, wherein the buffer is at a pH of about 7.2 when the reaction is run in the absence of an additive.

17. The method of claim 13, wherein the ester and the diselenide are dissolved in the buffer before the reaction step at a concentration of about 10 mM.

18. The method of claim 17, wherein the reaction is commenced by combining the solutions of the ester and the diselenide and then allowed to proceed to completion as measured by an analytical technique.

19. The method of claim 18, wherein the reaction is complete within about 60 seconds as measured by noting the consumption of the ester by HPLC excepting those reactions where X is selected from He or Val wherein the reaction is complete within about 10 minutes as measured by HPLC.

20. The method of any one of claims 1 to 19, additionally comprising the step of deselenizing the peptide.

21. The method of claim 20, wherein the deselenization comprises reacting the peptide with a mild reducing agent.

22. The method of claim 20 or claim 21, wherein the deselenization comprises reacting the peptide with a mild oxidizing agent.

23. A method of oxidatively deselenising a seleno functionalized amino acid residue in a peptide, said method comprising exposing the peptide to a mild reducing agent and a mild oxidizing agent.

24. The method of any one of claims 20 to 23, wherein the peptide comprises a cysteine residue and the deselenisation step comprises selectively deselenizing the peptide so as not to desulfurize the cysteine residue.

25. The method of any one of claims 21, 23 or 24, wherein the mild reducing agent comprises a phosphine.

26. The method of claim 25, wherein the phosphine is water soluble.

27. The method of claim 26, wherein the phosphine is tris-(2- carboxyethyl)phosphine (TCEP).

28. The method of any one of claims 21 to 27, wherein the reducing agent additionally comprises a thiol.

29. The method of claim 28, wherein the thiol is dithiothreitol.

30. The method of any one of claims 22 to 29 wherein the oxidising agent is potassium peroxy monosulfate.

31. The method of any one of claims 20 to 30, wherein the reaction step and the deselenization step are conducted in a one-pot reaction.

32. A method of preparing an ester containing compound comprising the step of reacting an ester reagent with a diselenide bearing a hydroxyl group.

33. A method of preparing a hydrazide containing compound comprising the step of reacting an ester reagent with a diselenide bearing a hydrazine group.

34. A method of preparing an amide containing compound comprising the step of reacting an ester with a dithiol bearing an amino group, wherein the reaction proceeds in the absence of an additive.

35. A method of preparing an ester containing compound comprising the step of reacting an ester with a dithiol bearing a hydroxyl group, wherein the reaction proceeds in the absence of an additive.

36. A method of preparing a hydrazide containing compound comprising the step of reacting an ester with a dithiol bearing a hydrazine group, wherein the reaction proceeds in the absence of an additive.

37. The method of any one of claims 32 to 36, wherein the ester is a selenoester.

38. A method of preparing a phenylselenoester, the method comprising the step of treating a carboxylic acid compound with diphenyldiselenide followed by Bu₃P.

39. The method of claim 31, wherein the selenoester is a peptide selenoester and the carboxylic acid is a peptide carboxylic acid.

40. A peptide of Formula (I) as defined in claim 4.

41. A peptide of Formula (IV) as defined in claim 9.