WO2016205798A1

WO2016205798A1 - Selective arylation of dichalcogenides in biomolecules

Info

Publication number: WO2016205798A1
Application number: PCT/US2016/038372
Authority: WO
Inventors: Stephen L. Buchwald; Bradley L. PENTELUTE; Daniel T. COHEN; Chi Zhang
Original assignee: Massachusetts Institute of Technology
Current assignee: Massachusetts Institute of Technology
Priority date: 2015-06-19
Filing date: 2016-06-20
Publication date: 2016-12-22
Anticipated expiration: 2017-12-19
Also published as: US10117948B2; US20160367693A1

Abstract

Disclosed are chemical transformations for the conjugation of unprotected peptide biomolecules. The processes feature several significant advantages over existing methods of peptide modification, including specificity towards selenocysteine over other nucleophiles (e.g., amines, hydroxyls), excellent functional group tolerance, and mild reaction conditions.

Description

Selective Arylation of Dichalcogenides in Biomolecules

RELATED APPLICATIONS

This application claims the benefit of priority to United States Provisional Patent Application serial number 62/182, 146, filed June 19, 2015, the contents of which are hereby incorporated by reference.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant Nos. GM046059 and GM1 10535 awarded by the National Institutes of Health. The government has rights in this invention.

BACKGROUND

Peptide-based technologies have facilitated the elucidation of disease mechanisms and serve as novel and effective therapeutics. An emerging theme in biotechnology is to use peptide variants to disrupt protein-protein interactions because compared to small molecules they have a larger surface area for binding, can recognize targets with higher specificity/affinity, and can be generated in weeks by phage display. For this and other reasons, for many years researchers in the field of bioconjugate chemistry have needed well-defined ligation strategies that can be used for the at-will modification of biomolecules. Efficient bioconjugation strategies generally involve high levels of functional group tolerance, compatibility with water and other solvents, and efficient conversions (e.g., fast reaction times and high yields).

Selenocysteine (Sec or U), discovered by Stadtman in 1974, is the 21^st proteinogenic amino acid. Selenocysteine is a structural analogue to cysteine with a selenol in place of the thiol. There are several other notable differences between cysteine and selenocysteine. The greater acidity of selenocysteine (p a = 5.47) versus cysteine (pK_a = 8.14) causes it to be deprotonated under physiological pH and its lower reduction potential makes it an integral part of antioxidant proteins. Selenocysteine is essential for enzymatic activity in enzymes including glutathione peroxidases, iodothyronine deiodinases, formate dehydrogenases, and methionine-R-sulfoxide reductase. Mutation of the catalytic selenocysteine to cysteine in the aforementioned enzymes results in a decrease in activity of > 100 fold. The inherent nucleophilicity of selenols makes selenocysteine an appealing handle for chemoselective bioconjugation in peptides and proteins. Nevertheless, reports on bioconjugation with this amino acid have been sparse due to several challenges associated with its functionalization. Namely, a selenol is easily oxidized to the diselenide or seleninic acid. In addition, because selenium is highly polarizable, it can be eliminated to generate dehydroalanine. Reports of selenocysteine functionalization have paralleled methods to modify cysteine and relied upon alkylation and maleimide conjugate addition with the selenol group (Figure 1, Panel (1)). In contrast to common cysteine conjugation methods, a reducing agent [i.e., /ra(2-carboxyethyl)phosphine (TCEP)] and exclusion of oxygen are needed to generate the selenol in situ prior to reaction with the electrophile.

There exists a need for development of a direct, robust, and selective method for the functionalization of selenocysteine that overcomes these limitations and allow for site- selective modification of unprotected peptides.

SUMMARY

In certain embodiments, the invention relates to a compound comprising substructure I:

I wherein, independently for each occurrence,

A¹ is H, an amine protecting group, a natural or unnatural amino acid, a peptide, an oligopeptide, a polypeptide, or a protein;

A⁵ is OH, -0(carboxylate protecting group), a natural or unnatural amino acid, a peptide, an oligopeptide, a polypeptide, or a protein;

is a substituted or unsubstituted aryl or heteroaryl radical; x is 0, 1, 2, 3, 4, 5, or 6; and

R is H or alkyl. In certain embodiments, the invention relates to a compound comprising substructure II:

II wherein, independently for each occurrence,

is a substituted or unsubstituted aryl or heteroaryl radical, provided is not a perfluoroaryl radical; x is 0, 1, 2, 3, 4, 5, or 6; and

R is H or alkyl.

In certain embodiments, the invention relates to a compound comprising substructure III:

III wherein, independently for each occurrence,

A², A³, and A⁴ are selected from the group consisting of a natural amino acid, an unnatural amino acid, and a plurality of natural amino acids or unnatural amino acids; A⁵ is OH, -0(carboxylate protecting group), a natural or unnatural amino acid, a peptide, an oligopeptide, a polypeptide, or a protein; x is 0, 1, 2, 3, 4, 5, or 6;

is a substituted or unsubstituted aryl or heteroaryl diradical; and R is H or alkyl.

In certain embodiments, the invention relates to a compound comprising substructure IV:

IV

wherein, independently for each occurrence,

A², A³, and A⁴ are selected from the group consisting of a natural amino acid, an unnatural amino acid, and a plurality of natural amino acids or unnatural amino acids;

A⁵ is OH, -0(carboxylate protecting group), a natural or unnatural amino acid, a peptide, an oligopeptide, a polypeptide, or a protein; x is 0, 1, 2, 3, 4, 5, or 6;

is a substituted or unsubstituted aryl or heteroaryl diradical, provided is not a perfluoroaryl diradical; and

R is H or alkyl. In certain embodiments, the invention relates to a compound comprising substructure V or substructure VII:

V

VII wherein, independently for each occurrence,

In certain embodiments, the invention relates to a compound comprising substructure VI or substructure VIII:

VI

VIII wherein, independently for each occurrence,

R is H or alkyl.

In certain embodiments, the invention relates to a hybrid composition, wherein the hybrid composition comprises a linker, any one of the aforementioned compounds, and a detectable moiety; and the linker links the compound to the detectable moiety.

In certain embodiments, the invention relates to a hybrid composition, wherein the hybrid composition comprises a linker, any one of the aforementioned compounds, and a biomolecule; and the linker links the compound to the biomolecule.

In certain embodiments, the invention relates to a peptide, an oligopeptide, a polypeptide, or a protein, wherein the peptide, oligopeptides, polypeptide, or protein comprises substructure I, substructure II, substructure III, substructure IV, substructure V, substructure VI, substructure VII, or substructure VIII.

In certain embodiments, the invention relates to an affibody comprising substructure I, substructure II, substructure III, substructure IV, substructure V, substructure VI, substructure VII, or substructure VIII. In certain embodiments, the invention relates to a method according to Scheme 1:

Scheme la

Scheme lb

Scheme lc

Scheme Id wherein, independently for each occurrence,

A¹ is H, an amine protecting group, a natural or unnatural amino acid, a peptide, an oligopeptide, a polypeptide, or a protein; A², A³, and A⁴ are selected from the group consisting of a natural amino acid, an unnatural amino acid, and a plurality of natural amino acids or unnatural amino acids;

is a substituted or unsubstituted aryl or heteroaryl radical;

is a substituted or unsubstituted aryl or heteroaryl diradical

is a heteroaromatic moiety substituted with an electron withdrawing group; x is 0, 1, 2, 3, 4, 5, or 6; and R is H or alkyl.

In certain embodiments, the invention relates to a method according to Scheme 2:

Scheme 2a

Scheme 2b

Scheme 2d wherein, independently for each occurrence,

is not a perfluoroaryl radical;

is a substituted or unsubstituted aryl or heteroaryl diradical, provided is not a perfluoroaryl radical;

In certain embodiments, the invention relates to a method according to Scheme 3:

Scheme 3a

third temperature

Scheme 3b

Scheme 3c

Scheme 3d

wherein, independently for each occurrence, A¹ is H, an amine protecting group, a natural or unnatural amino acid, a peptide, an oligopeptide, a polypeptide, or a protein;

is an aryl or heteroaryl radical substituted with at least one electron donating group;

is a substituted aryl or heteroaryl diradical substituted with at least one electron donating group;

In certain embodiments, the invention relates to a method according to Scheme 4:

Scheme 4a

Scheme 4b

Scheme 4c

Scheme 4d wherein, independently for each occurrence,

A⁵ is OH, -0(carboxylate protecting group), a natural or unnatural amino acid, a peptide, an oligopeptide, a polypeptide, or a protein; x is 0, 1, 2, 3, 4, 5, or 6; _B2B

is an aryl or heteroaryl radical substituted with at least one electron donating provided (?) is not a perfluoroaryl radical;

is an aryl or heteroaryl diradical substituted with at least one electron donating group, provided

a perfluoroaryl radical;

is a heteroaromatic moiety substituted with an electron withdrawing group; x is 0, 1, 2, 3, 4, 5, or 6; and

R is H or alkyl.

In certain embodiments, the invention relates to a method according to Scheme 5:

Scheme 5a

Scheme 5b

Scheme 5d wherein, independently for each occurrence,

is not a perfluoroaryl radical; is a substituted or unsubstituted aryl or heteroaryl diradical, provided is not a perfluoroaryl radical;

In certain embodiments, the invention relates to a method according to Scheme 6:

Scheme 6a

Scheme 6b

Scheme 6c

Scheme 6d

A⁵ is OH, -0(carboxylate protecting group), a natural or unnatural amino acid, a peptide, an oligopeptide, a polypeptide, or a protein; x is 0, 1, 2, 3, 4, 5, or 6; l or heteroaryl radical substituted with at least one electron donating group, p

rovided is not a perfluoroaryl radical;

a perfluoroaryl radical;

In certain embodiments, the invention relates to a method according to Scheme 7:

Scheme 7a

Scheme 7b

Scheme 7c

Scheme 7d

wherein, independently for each occurrence,

A¹ is H, an amine protecting group, a natural or unnatural amino acid, a peptide, oligopeptide, a polypeptide, or a protein;

A², A³, and A⁴ are selected from the group consisting of a natural amino acid, unnatural amino acid, and a plurality of natural amino acids or unnatural amino acids;

A⁵ is OH, -0(carboxylate protecting group), a natural or unnatural amino acid peptide, an oligopeptide, a polypeptide, or a protein;

x is 0, 1, 2, 3, 4, 5, or 6;

R is H or alkyl.

In certain embodiments, the invention relates to a method according to Scheme 8:

Scheme 8a

Scheme 8b

Scheme 8c

Scheme 8d wherein, independently for each occurrence,

A⁵ is OH, -0(carboxylate protecting group), a natural or unnatural amino acid, a peptide, an oligopeptide, a polypeptide, or a protein; x is 0, 1, 2, 3, 4, 5, or 6, l or heteroaryl radical substituted with at least one electron donating group, p

rovided is not a perfluoroaryl radical;

a perfluoroaryl radical;

In certain embodiments, the invention relates to a method of killing or inhibiting the growth or proliferation of a bacterium, a fungus, a virus, or a parasite, comprising the step of: contacting with the bacterium, fungus, virus, or parasite an effective amount of any one of the aforementioned compounds, thereby treating killing or inhibiting the growth or proliferation of the bacterium, fungus, virus, or parasite.

In certain embodiments, the invention relates to a method of treating a disease in a subject in need thereof comprising the step of: administering to the subject an effective amount of any one of the aforementioned compounds, thereby treating the disease.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts three panels ((l)-(3)) showing reaction schemes comparing the bioconjugation of cysteine (panel (1)) and selenocysteine (panel (2)). The proposed reaction pathway of the reaction shown in panel (2) is shown in panel (3).

Figure 2 depicts a table summarizing reaction conditions and % yield for various arylation reactions. ^α Amino acids are shown in a one-letter code. R = no reaction. ^Yields determined by integration of total ion currents (TIC) from LC-MS analysis of the unpurified reaction mixture. ^c Elimination and diselenide were the only observable products. ^rf0.5 mM CuS0₄, 0.5 mM L2, and 0.5 mM boronic acid were used. ^e0.25 mM CuS0₄, 0.25 mM L2, and 0.25 mM boronic acid were used.^•'Selenocysteine-T P residue replaced with a serine. ¾elenocysteine-2-thiol-5-nitropyridine residue replaced with a cysteine. ^ASelenocysteine-2-thiol-5-nitropyridine residue replaced with a cysteine-2-thiol-5- nitropyridine. 'Selenocysteine-2-thiol-5-nitropyridine residue replaced with a methionine.

Figure 3 depicts five panels (a-e) summarizing reaction substrates, boronic acids, and yields for various arylation reactions. ^α Amino acids are shown in a one-letter code. ^Yields determined by integration of total ion currents (TIC) from LC-MS analysis of the unpurified reaction mixture (Average of two runs). ^h2 h reaction time. ^c1.5 h reaction time. ^d2 mM CuS0₄, 2 mM L2, and 2 mM boronic acid were used. ^e2 mM CuS0₄, 2 mM L2, 2 mM boronic acid, and H₂0:DMF (90: 10) for 3 h. Panel a shows the yield of peptides 4a-41 using aryl boronic acids. Panel b shows the yield of peptides 5a-5j using heteroaryl boronic acids. Panel c shows the yields of peptides 6a-6e using various biorelevant aryl boronic acids. Panel d shows a reaction scheme and an LCMS trace of arylated peptide 8 in the product mixture. Panel e shows a reaction scheme and an LCMS trace of starting material 9 in the product mixture.

Figure 4 depicts data from a stability study of functionalized selenocysteine in peptides. % yields determined by integration of total ion currents (TIC) from LC-MS analysis of the unpurified reaction mixture.

Figure 5A depicts the yield of Dha elimination product (%) at pH 8.0 for various selenocysteine peptides at 37 °C (left bar) and at room temperature (about 23 °C) (right bar).

Figure 5B depicts the yield of Dha elimination product (%) at pH 10.0 for various selenocysteine peptides at 37 °C (left bar) and at room temperature (about 23 °C) (right bar).

Figure 6 depicts a reaction scheme showing a competition experiment between a functionalized selenocysteine-containing peptide (top starting material) and a functionalized cysteine-containing peptide (bottom starting material). The mole ratio of product peptides is 90: 10 Se:S.

Figure 7 depicts two applications of the various Cu-catalyzed reactions of the invention: peptide stapling with a bis-boronic acid (top) and macrocyclization (bottom).

Figure 8 depicts an application of the various reactions of the invention: tagging of a biologically active peptide with a pharmaceutical or natural product.

Figure 9 depicts various pharmaceuticals and biologically active natural products containing electron-rich aromatic rings suitable for conjugation to a selenocysteine- containing peptide.

Figure 10 depicts various pharmaceuticals and biologically active natural products containing electron-rich aromatic rings suitable for conjugation to a selenocysteine- containing peptide.

Figure 11 depicts various pharmaceuticals and biologically active natural products containing electron-rich aromatic rings suitable for conjugation to a selenocysteine- containing peptide. Figure 12 depicts a reaction scheme and % yield for various arylation reactions using substituted aromatic groups or substituted or unsubstituted indoles in the presence of CuSC catalyst (top yields), and in the absence of a metal catalyst and ligand (bottom yields).

Figure 13 depicts a reaction scheme and % yield for various arylation reactions using substituted or unsubstituted heteroaromatic groups, natural products, or pharmaceuticals in the presence of CuSCvcatalyst (top yields), and in the absence of a metal catalyst and ligand (bottom yields).

Figure 14 depicts a reaction scheme and % yield for various arylation reactions using natural products or pharmaceuticals in the presence of CuSCvcatalyst (top yields), and in the absence of a metal catalyst and ligand (bottom yields).

Figure 15 depicts (top) a reaction scheme showing the Cu-catalyzed arylation of a peptide containing a protected cysteine residue near its N-terminus, and (bottom) a reaction scheme showing the Rh-catalyzed arylation of a peptide containing a protected cysteine residue in the middle of its sequence.

Figure 16 depicts (top) a reaction scheme showing the Cu-catalyzed arylation of symmetrically Se-Se-coupled peptides containing selenocysteine residues near their N- termini, and (bottom) a reaction scheme showing the Rh-catalyzed arylation of symmetrically Se-Se-coupled peptides containing selenocysteine residues in the middle of their sequences.

Figure 17 depicts (top) a reaction scheme showing the Cu-catalyzed arylation of symmetrically S-S-coupled peptides containing cysteine residues near their N-termini, and (bottom) a reaction scheme showing the Rh-catalyzed arylation of symmetrically S-S- coupled peptides containing cysteine residues in the middle of their sequences.

Figure 18 depicts (top) a reaction scheme showing the metal-catalyzed arylation of symmetrically S-S-coupled peptides containing cysteine residues, and (bottom) the yields of these reactions in the presence of various Ir or Ru metal-catalysts.

Figure 19 depicts (top) a reaction scheme showing that glutathione (GSH, a tripeptide), which is found in 5 mM concentration in animal cells, can promote dissociation of the native drug or natural product by reduction of the C-Se bond; (bottom) shows a bar graph depicting the amount of starting material remaining after the exposure to GSH at room temperature or 37 °C for 3 h.

Figure 20 depicts (top) a reaction scheme showing the conditions for conjugating vancomycin to a model peptide, and (bottom) the reaction conditions, structure of vancomycin, and reaction yield by LC-MS.

Figure 21 depicts (top) a reaction scheme showing the conditions for conjugating vancomycin to an antibacterial peptide, and (bottom) a table of various peptide sequences (note, the first two sequences are random sequences with no reported antibacterial activity) that have been conjugated to vancomycin and screened against the gram positive and gram negative bacteria in Table 3.

Figure 22A depicts various antibacterial sequences conjugated to vancomycin, and the results of screening the conjugates against gram-positive strains.

Figure 22B depicts various antibacterial sequences conjugated to vancomycin, and the results of screening the conjugates against gram-negative strains.

Figure 23 depicts (top) a reaction scheme showing the conditions for conjugating small molecules to a polyglycine-containing peptide, and (bottom) various Ar-H moieties to be conjugated.

Figure 24 depicts a schematic representation of the process of sortase-tagging an antibody with a peptide-drug conjugate.

Figure 25 depicts a schematic representation of making a peptide-drug conjugate and then sortase-tagging an antibody with the peptide-drug conjugate.

Figure 26 depicts a reaction scheme showing the conditions for cyclizing a model peptide.

Figure 27 depicts a reaction scheme showing the conditions for cyclizing an HIV-1 capsid with selenocysteine.

Figure 28 a reaction scheme showing the conditions for attempting to cyclize a peptide lacking tryptophan.

Figure 29 depicts a reaction scheme showing the conditions for cyclizing an HIV-1 capsid with cysteine. Figure 30 depicts a reaction scheme showing the formation of disulfide starting material (e.g., the disulfide shown in Figure 29) on a resin. Cleavage with 95:5 TFA:H₂0 at 60 °C for 45 min provides completely cyclized material in 60% yield after FfPLC purification.

Figure 31A depicts the structure of the phallotoxins. Figure 31B depicts the structure of the amatoxins.

Figure 32A depicts a reaction scheme and reactants for copper-mediated arylation of disulfide-containing peptides.

Figure 32B depicts a reaction scheme and reactants for rhodium-mediated arylation of disulfide-containing peptides.

Figure 33 depicts a reaction scheme and concentrations of reactants for rhodium- catalyzed arylation of a disulfide-containing peptide.

Figure 34 depicts a reaction scheme showing the arylation of a cysteine disulfide. The ability to affect functionalization with only one equivalent of rhodium has important implications for functionalization of antibodies without prior reduction.

Figure 35 depicts (top) a reaction scheme showing the metal-free arylation of mixed disulfides, and (bottom) various reagents and yields.

Figure 36 depicts (top) a reaction scheme showing the copper-catalyzed arylation of a disulfide, and (bottom) the results of this reaction with disulfides having varying sequences.

Figure 37 depicts the results of the reaction depicted in Figure 36 when the position of the disulfide is varied within the peptide.

Figure 38 depicts (top) a reaction scheme showing the copper-catalyzed arylation of a disulfide, and (bottom) various boronic acid arylating agents.

Figure 39 depicts a reaction scheme and % yield for various arylation reactions using substituted aromatic or heteroaromatic groups in the presence of CuSC catalyst (top yields), and in the absence of a metal catalyst and ligand (bottom yields). DETAILED DESCRIPTION

Overview

In certain embodiments, the invention relates to the discovery and subsequent development of a new chemical transformation for the conjugation of unprotected peptide biomolecules. This process features several significant advantages over existing methods of peptide modifications such as specificity towards selenocysteine over other nucleophiles (e.g., amines, hydroxyls), excellent functional group tolerance, and mild reaction conditions. While the reaction times normally vary depending on the concentration and chemical identity of the peptide, the reaction conditions usually can be optimized where full conversion is accomplished within 1-6 hours.

In certain embodiments, this method is employed for synthesis of functionalized peptides, "stapled" peptides, macrocyclized peptides, or heterocoupled peptide systems. In certain embodiments, this methodology is useful for producing hybrid biomaterials featuring other biomolecules such as DNA, RNA, PNA, proteins, oligosaccharides, and a combination of thereof. In certain embodiments, the hybrid biomaterials are useful for cell penetration and intracellular targeting. In certain embodiments, the invention relates to organic polymer-peptide conjugates, peptides decorated with functional small-molecules (such as fluorescent dye labels), natural products or small-molecule drugs (such as antibacterial agents, antifungal agents, antiviral agents, anticonvulsant agents, and antipsychotic agents), inorganic MRI, radio-contrast agents, and also various functionalized metal-based nanoparticle scaffolds.

In certain embodiments, the invention employs an Umpolung approach that utilizes the electrophilic character of the oxidized selenocysteine together with a copper, ligand, and nucleophilic boronic acid combination to provide the arylated selenocysteine in unprotected peptides (Figure 1, Panel (2)). While not wishing to be bound by any particular theory, the proposed reaction pathway involves an initial oxidative addition of copper to the Se-S bond (Figure 1, Panel (3)). A subsequent transmetalation with an arylboronic acid and reductive elimination furnishes the arylated selenocysteine. This method does not require oxygen-free conditions or a reducing agent and takes place in water rich media (e.g., 0.1 M Tris buffer (pH = 8.0), 95:5 H₂0:EtOH). In certain embodiments, reaction yields are excellent for a wide range of substrates. In certain embodiments, this approach is selective for selenocysteine over cysteine. In certain embodiments, the invention relates to the synthesis of arylated selenocysteine, which begins by combining unprotected peptide 1, /?-tolylboronic acid, CuSC"4, and a ligand in buffered aqueous conditions [0.1 M Tris (pH = 8.0), 95:5 H₂0:EtOH] at 37 °C. In certain other embodiments, 2-thiol-5-nitropyridine protected selenocysteine (1) is used as a starting material, as it is the reaction product from the deprotection of /?-methoxybenzyl protecting group in solid-phase peptide synthesis. In certain embodiments, different bipyridine and phenanthroline ligands (Figure 2, entries 1-4) may be used. The formation of the diselenide, seleninic acid, and elimination to dehydroalanine (Dha) were the observed side products. In the absence of ligand, only 30% conversion of 1 and 5% arylation to 4a was observed (Figure 2, entry 5). Other sources of copper provided similar yields (Figure 2, entries 6-8). In the absence of copper, no arylation was observed (Figure 2, entry 9). Lastly, decreasing the number of equivalents of copper, ligand, or boronic acid resulted in lower levels of conversion and increased side reactions (Figure 2, entries 10-11). No product was observed in a control reaction with selenocysteine-to-serine variant (Figure 2, entry 12), confirming that arylation occurs exclusively at selenium. To probe the reactivity of selenocysteine versus cysteine, two peptides in which the selenocysteine was replaced with cysteine (free thiol) or cysteine linked with a 2-thiol-5-nitropyridine as the masking agent (Figure 2, entries 13-14) were examined. For both peptides we observed low conversion to the arylated cysteine, rather the starting material (1) was converted to the respective disulfide or sulfinic acid. This result demonstrates the enhanced reactivity of selenocysteine over cysteine toward copper- mediated arylation. See also Figure 6. It also indicates cysteine is not compatible under these reaction conditions. Lastly, no oxidation was detected for the selenocysteine-to- methionine variant (Figure 2, entry 15), which proves that this method could be applied to methionine containing peptides.

In certain embodiments, the invention relates to a reaction methodology applicable to a wide variety of substrates. See Figure 3. Aryl boronic acids with electron-donating functional groups were examined (Figure 3, Panel a). Methoxy (4b), ortho-phenol (4c), dioxolane (4d), -CH₂N(H)Boc (4e), and /?ara-dimethylamine (4f) groups were readily tolerated. Under these reaction conditions, Boc deprotection was not observed (4e), thus demonstrating the mild conditions needed to achieve the arylation.

In certain embodiments, the invention relates to a method of functionalizing a selenocysteine residue using an aryl boronic acid having an electron-withdrawing functional group (Figure 3, Panel a). Despite the decreased nucleophilicity of these electron-poor boronic acids, the corresponding selenium conjugates were formed in good to excellent yield. In some cases, the number of equivalents of the Cu/L and boronic acid needed to be increased to provide full conversion to product while suppressing side reactions. Ester (4g), amide (4h), nitro (4i), nitrile (4j), and halogen (4k and 41) functional groups were compatible with these conditions. Peptide 41 was formed quantitatively despite 2,6-difluorophenyl boronic acid having the tendency to undergo protodeboronation under basic conditions.

In certain embodiments, the invention relates to a method of functionalizing a selenocysteine residue using a heteroaryl boronic acid (Figure 3, Panel b). Known bioactivities comprising heterocycles makes heterocycles an important class of molecules for conjugation to selenocysteine. The arylation of five-membered heterocycles such as indole (5a and 5b), benzofuran (5c), pyrazole (5d), 2, 5 -di substituted isoxazole (5e), and thiophene (5f and 5g) proceeded with excellent yields. Six-membered heterocycles, such as dibenzofuran (5h), phenoxathiine (5i), and pyrimidine (5j) likewise furnished the corresponding conjugate in greater than 90% yield.

In certain embodiments, the invention relates to the functionalization of selenocysteine-containing peptides with biorelevant molecules (Figure 3, Panel c). The linking of protected phenylalanine and coumarin proceeded in good yield (6a and 6b respectively). The boronic acids of anti-inflammatory drugs tolfenamic ester and paracetamol furnished the corresponding conjugates in moderate to good yields (6c and 6d respectively). Lastly, estrone was coupled to selenium in moderate yield (6e).

In certain embodiments, the invention relates to a robust, selective reaction. As an example, peptide 7 was prepared; peptide 7 contains most of the key functional groups found in polyamides (e.g., histidine, arginine, aspartic acid, tyrosine, and asparagine) excluding sulfur-based amino acids (Figure 3, Panel d). Exposure of 7 to 2,6-difluorophenyl boronic acid under reaction conditions provided the corresponding arylated product (8) in quantitative yield based on LC-MS analysis. No other peptidic species were observed (see LC-MS trace, Figure 3, Panel d). The arylation occurred exclusively on the selenocysteine as a selenocysteine-to-serine control variant (9) provided no product under identical reaction conditions (Figure 3, Panel e). In certain embodiments, the invention relates to peptides comprising aryl- functionalized selenocysteine. The stability of these aryl conjugates relative to the corresponding benzylated variant under basic conditions was investigated. In a pH 8.0 buffered solution at room temperature or 37 °C, neither the arylated nor alkylated selenocysteine peptide underwent elimination to dehydroalanine (la, Dha) even after 11 hours. Figure 5A. However, in a buffered solution at pH 10.0, appreciable amounts of elimination were observed for both the alkylated and arylated derivatives depending on the substituent on the selenium. Figure 5B.

In certain embodiments, the invention relates to peptides comprising aryl- functionalized selenocysteine that are relatively stable under oxidative conditions. Exposure of these peptide derivatives to a large excess of H₂0₂ (10 mM) at room temperature lead to the formation of varying amounts of dehydroalanine (la) depending on the R-group on the selenocysteine (Figure 4). In general, the arylated selenocysteine derivatives were less prone to oxidation/elimination than the benzylated selenocysteine (4n). This result is in agreement with previous reports that alkylated or arylated selenocysteine can be readily converted to dehydroalanine (la) even under mildly oxidative conditions. With regards to the different arenes, the more electron deficient the aryl ring on the selenium, the slower formation of Dha, la. So, in certain embodiments, the invention relates to the ability to stabilize the arylated selenocysteine by modulating the electronic nature of the arene substituent.

Exemplary Functionalized Compounds

I wherein, independently for each occurrence,

A¹ is H, an amine protecting group, a natural or unnatural amino acid, a peptide, an oligopeptide, a polypeptide, or a protein; A⁵ is OH, -0(carboxylate protecting group), a natural or unnatural amino acid, a peptide, an oligopeptide, a polypeptide, or a protein;

is a substituted or unsubstituted aryl or heteroaryl radical; x is 0, 1, 2, 3, 4, 5, or 6; and R is H or alkyl.

In certain embodiments, the invention relates to a compound comprising substructure II:

II wherein, independently for each occurrence,

i_{s a} substituted or unsubstituted aryl or heteroaryl radical, provided [_s not a perfluoroaryl radical; x is 0, 1, 2, 3, 4, 5, or 6; and

R is H or alkyl.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound is depicted in the Figures.

In certain embodiments, the invention relates to any one of the aforementioned

substituted aryl radical. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein

is a substituted phenyl radical.

In certain embodiments, the invention relates to any one of the aforementioned compound

is an unsubstituted aryl radical.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein a substituted heteroaryl radical. In certain embodiments, the invention relates to

any one of the aforementioned compounds, wherein is a substituted indole, pyrazolo, thiophene, pyrimidine, benzofuran, or isoxazole radical.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein

an unsubstituted heteroaryl radical. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein

is an unsubstituted indole, pyrazolo, thiophene, pyrimidine, benzofuran, or isoxazole radical.

In certain embodiments, the invention relates to any one of the aforementioned

compounds, wherein

is

-32-

stereoisomer thereof. In certain embodiments,

is any of the aforementioned moieties, wherein the moiety is further substituted.

In certain embodiments, the invention relates to any one of the aforementioned

stereoisomer thereof. In certain embodiments,

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein a substituted aryl radical. In certain embodiments, the invention relates to a

ny one of the aforementioned compounds, wherein is a substituted phenyl radical. In certain embodiments, the invention relates to any one of the aforementioned compound

is an unsubstituted aryl radical.

In certain embodiments, the invention relates to any one of the aforementioned

In certain embodiments, the invention relates to any one of the aforementioned

stereoisomer thereof. In certain embodiments, is any of the aforementioned moieties, wherein the moiety is further substituted.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound is a compound comprising substructure I; and none of A¹ and A⁵ comprises selenocysteine.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound is a compound comprising substructure I; and one or more of A¹ and A⁵ comprises arginine, cysteine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound is a compound comprising substructure II; and none of A¹ and A⁵ comprises cysteine. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound is a compound comprising substructure II; and one or more of A¹ and A⁵ comprises arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein A¹ is an amine protecting group selected from the group consisting of an N,0-acetal, allyloxycarbonyl (Aloe), benzyl (Bn), benzyloxycarbonyl (Cbz), benzyloxymethyl (BOM), t-butoxycarbonyl (Boc), t-butyldimethylsilyl (TBDMS), t- butyldiphenylsilyl (TBDPS), diphenylmethyl, diphenylmethylene, ethoxycarbonyl, 9- fluorenylmethoxycarbonyl (Fmoc), /?-methoxybenzyl (PMB), methoxycarbonyl, methoxymethyl (MOM), /?-methoxyphenyl (PMP), /?-nitrocinnamyloxycarbonyl (Noc), tosyl (Ts), 2-tosylethoxycarbonyl (Tsoc), 2,2,2-trichloroethoxycarbonyl (Troc), trifluoroacetyl, triisopropylsilyl (TIPS), trimethylsilyl (TMS), 2- (trimethylsilyl)ethoxycarbonyl (Teoc), 2-(trimethylsilyl)ethoxymethyl (SEM), or trityl (Tr).

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein A⁵ is -0(carboxylate protecting group), wherein the carboxylate protecting group is selected from the group consisting of allyl, benzyl, benzyloxymethyl (BOM), t-Bu, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), diphenylmethyl, 9-fluorenylmethyl (Fm), 2-methoxyethoxymethyl (MEM), methoxymethyl (MOM), /?-nitrobenzyl (PNB), an ester, a 1,3-oxazoline, pi valoyloxy methyl (Pom), 2- tosylethyl (TSE), 2,2,2-trichloroethyl (TCE), triethylsilyl (TES), trimethylsilyl (TMS), 2- (trimethylsilyl)ethoxymethyl (SEM), or 2-(trimethylsilyl)ethyl (TMSE).

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein A¹ or A⁵ is an antimicrobial peptide, such as a compound comprising magainin/pexiganan (a 22-amino acid linear peptide that may be isolated from the skin of the African clawed frog {Xenopus laevis)), omiganan (a cationic peptide that may be derived from indolicidin), OP- 145 (a 24-mer peptide that may be derived from LL-37 that may bind to lipopolysaccharides or lipoteichoic acid), novexatin (a cyclic cationic peptide of 1093 Da), LTX-109 (Lytixar, a membrane-degrading peptide), NVB302 (a class B lantibiotic), MU1140 (a lantibiotic), arenicin (a 21-mer that is rich in arginine and hydrophobic amino acids), avidocin (a R-type bacteriocin that may be derived from Pseudomanas aeruginosa), purocin (a R-type bacteriocin that may be derived from Pseudomanas aeruginosa), IMX924 (a 5-mer with defense regulation capabilities),

GIGKFLHSAKKFGKAFVGEIMNS, or GIKKFLKK AGKF GK AF . In certain embodiments, A¹ or A⁵ is an antimicrobial peptide described in Fox, J.L. Nature Biotechnology 2013, 31, 5, 379 or Arnusch, C.J. et al. PLoS ONE 2012, 7(6), e39768.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein R is H.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein x is 1, 2, or 3. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein x is 1.

In certain embodiments, the invention relates to any one of the compounds described herein. Exemplary Stapled or Macrocyclized Compounds

III wherein, independently for each occurrence,

IV wherein, independently for each occurrence,

R is H or alkyl.

In certain embodiments, the invention relates to a compound compri substructure V or substructure VII:

V

VII wherein, independently for each occurrence,

VI

VIII

wherein, independently for each occurrence,

R is H or alkyl.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound is depicted in the Figures. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein A², A³, and A⁴ are natural or unnatural amino acids.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein A² represents one natural or unnatural amino acid. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein A³ represents one natural or unnatural amino acid. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein A⁴ represents one natural or unnatural amino acid.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein A² represents two natural or unnatural amino acids. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein A³ represents two natural or unnatural amino acids. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein A⁴ represents two natural or unnatural amino acids.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein A² represents three natural or unnatural amino acids. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein A³ represents three natural or unnatural amino acids. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein A⁴ represents three natural or unnatural amino acids.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound is a compound comprising substructure III, V, or VII; and none of A¹, A², A³, A⁴, and A⁵ comprises selenocysteine.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound is a compound comprising substructure III, V, or VII; and one or more of A¹, A², A³, A⁴, and A⁵ comprises arginine, cysteine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound is a compound comprising substructure IV, VI, or VIII; and none of A¹, A², A³, A⁴, and A⁵ comprises cysteine. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein the compound is a compound comprising substructure IV, VI, or VIII; and one or more of A¹, A², A³, A⁴, and A⁵ comprises arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein A⁵ is -0(carboxylate protecting group); and the carboxylate protecting group is selected from the group consisting of allyl, benzyl, benzyloxymethyl (BOM), t-Bu, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), diphenylmethyl, 9- fluorenylmethyl (Fm), 2-methoxyethoxymethyl (MEM), methoxymethyl (MOM), p- nitrobenzyl (P B), an ester, a 1,3-oxazoline, pi valoyloxy methyl (Pom), 2-tosylethyl (TSE), 2,2,2-trichloroethyl (TCE), triethylsilyl (TES), trimethylsilyl (TMS), 2- (trimethylsilyl)ethoxymethyl (SEM), or 2-(trimethylsilyl)ethyl (TMSE).

is a substituted aryl diradical. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein

is a substituted phenyl diradical. In certain embodiments, the invention relates to any one of the aforementioned compound

is an unsubstituted aryl diradical.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein is a substituted heteroaryl diradical. In ents, the invention relates

to any one of the aforementioned compounds, is a substituted indole, pyrazolo, thiophene, pyrimidine, benzofuran, or isoxazole diradical.

is an unsubstituted heteroaryl diradical. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein

( ) is an unsubstituted indole, pyrazolo, thiophene, pyrimidine, benzofuran, or isoxazole diradical.

In certain embodiments, the invention relates to any one of the aforementioned compounds, wher is a substituted aryl diradical. In certain embodiments, the invention relates

to any one of the aforementioned compounds, wherein is a substituted phenyl diradical.

is 1,4-disubstituted phenylene, l,4-disubstituted-2,5- dimethy lpheny 1 ene, 1 , 3 -di sub stituted- 5 -fluorophenyl ene, 1,4-di sub stituted-2, 5 - difluorophenylene, l,4-disubstituted-2,5-bis(trifluoromethyl)phenylene, 4,4'-disubstituted- Ι, -biphenylene, 4,4'-disubstituted-oxy(l,l '-bisphenylene), or 4,4'-disubstituted-oxy(l,l '- bis(3-methylphenylene)).

In ents, the invention relates to any one of the aforementioned compound

is an unsubstituted aryl diradical. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherein

is a substituted heteroaryl diradical. In certain embodiments, the invention relates to any one of the aforementioned compounds, wherei

is a substituted indole, pyrazolo, thiophene, pyrimidine, benzofuran, or isoxazole diradical.

an unsubstituted indole, pyrazolo, thiophene, pyrimidine, benzofuran, or isoxazole diradical.

In certain embodiments, the invention relates to any one of the compounds described herein.

Exemplary Conjugated Compounds

In certain embodiments, the invention relates to any one of the aforementioned hybrid compositions, wherein the detectable moiety is a fluorescent moiety, a dye moiety, a radionuclide, or an MRI contrast agent.

In certain embodiments, the invention relates to a hybrid composition, wherein the hybrid composition comprises a linker, any one of the aforementioned compounds, and a biomolecule; and the linker links the compound to the biomolecule. In certain embodiments, the invention relates to any one of the aforementioned hybrid compositions, wherein the biomolecule is a protein.

In certain embodiments, the invention relates to any one of the aforementioned hybrid compositions, wherein the biomolecule is DNA or RNA.

In certain embodiments, the invention relates to any one of the aforementioned hybrid compositions, wherein the biomolecule is siRNA.

In certain embodiments, the invention relates to any one of the hybrid compositions described herein.

Exemplary peptides, oligopeptides, polypeptides, and proteins

In certain embodiments, the invention relates to a peptide, an oligopeptide, a polypeptide, or a protein, wherein the peptide, oligopeptides, polypeptide, or protein comprises a plurality of substructures selected from the group consisting of substructure I, substructure II, substructure III, substructure IV, substructure V, substructure VI, substructure VII, or substructure VIII.

In certain embodiments, the invention relates to any one of the peptides, oligopeptides, polypeptides, or proteins described herein.

Exemplary Affibodies

In certain embodiments, the invention relates to an affibody comprising substructure I, substructure II, substructure III, substructure IV, substructure V, substructure VI, substructure VII, or substructure VIII.

In certain embodiments, the invention relates to an affibody comprising a plurality of substructures selected from the group consisting of substructure I, substructure II, substructure III, substructure IV, substructure V, substructure VI, substructure VII, or substructure VIII.

As used herein the term "affibody" refers to a small protein composed of alpha helices. In certain embodiments, the affibody is engineered to bind a target protein or target peptide with high affinity. In certain embodiments, affibodies are antibody mimetics. In certain embodiments, affibodies lack disulfide bridges. In certain embodiments, the affibody comprises a three-helix bundle. In certain embodiments, the affibody has a molar mass of less than about 8 kDa. In certain embodiments, the affibody has a molar mass of about 6 kDa.

In certain embodiments, the invention relates to any one of the affibodies described herein.

Exemplary Methods

In certain embodiments, the invention relates to a method according to Scheme 1:

Scheme la

Scheme lb

Scheme lc

Scheme Id wherein, independently for each occurrence,

is a substituted or unsubstituted aryl or heteroaryl radical;

is a substituted or unsubstituted aryl or heteroaryl diradical

In certain embodiments, the invention relates to a method according to Scheme 2

Scheme 2a

Scheme 2b

Scheme 2c

Scheme 2d wherein, independently for each occurrence,

is a substituted or unsubstituted aryl or heteroaryl radical, provided not a perfluoroaryl radical;

Scheme 3a

Scheme 3b

Scheme 3c

Scheme 3d wherein, independently for each occurrence,

R is H or alkyl.

Scheme 4a

Scheme 4b

Scheme 4c

Scheme 4d wherein, independently for each occurrence,

rovided is not a perfluoroaryl radical;

a perfluoroaryl radical;

is a heteroaromatic moiety substituted with an electron withdrawing group; xisO, 1,2,3,4, 5, or 6; and

R is H or alkyl.

In certain embodiments, the invention relates to a method accordin

Scheme 5a

Scheme 5b

Scheme 5c

Scheme 5d

Scheme 6a

Scheme 6b

Scheme 6c

Scheme 6d wherein, independently for each occurrence,

a perfluoroaryl radical;

R is H or alkyl.

Scheme 7a

Scheme 7b

Scheme 7c

Scheme 7d wherein, independently for each occurrence,

Scheme 8a

Scheme 8b

Scheme 8c

Scheme 8d

rovided is not a perfluoroaryl radical;

a perfluoroaryl radical;

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the product is a hybrid biopolymer.

In certain embodiments, the invention relates to any one of the aforementioned

methods, wherein is a substituted aryl radical. In certain embodiments, the invention relates to any one of the aforementioned methods, whe

is a substituted phenyl radical.

In certain embodiments, the invention relates to any one of the aforementioned

methods, wherein is an unsubstituted aryl radical. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein

is a substituted heteroaryl radical. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein

a substituted indole, pyrazolo, thiophene, pyrimidine, benzofuran, or isoxazole radical.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein

an unsubstituted heteroaryl radical. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein

In certain embodiments, the invention relates to any one of the aforementioned

-66-

In certain embodiments, the invention relates to any one of the aforementioned

embodiments,

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein a substituted aryl radical. In certain embodiments, the invention relates to any one

of the aforementioned methods, whe is a substituted phenyl radical.

an unsubstituted aryl radical. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein

In certain embodiments, the invention relates to any one of the aforementioned

-71-

In certain embodiments, the invention relates to any one of the aforementioned

embodiments,

of the aforementioned methods, whe is a substituted phenyl radical.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein is a substituted heteroaryl radical. In certain embodiments, the invention relates to

any one of the aforementioned methods, wherein a substituted indole, pyrazolo, thiophene, pyrimidine, benzofuran, or isoxazole radical. In certain embodiments, the invention relates to any one of the aforementioned

ertain embodiments, is any of the aforementioned moieties, wherein the moiety is further substituted.

a substituted aryl radical. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein

is a substituted phenyl radical.

any one of the aforementioned methods, wherein a substituted indole, pyrazolo, thiophene, pyrimidine, benzofuran, or isoxazole radical.

In certain embodiments, the invention relates to any one of the aforementioned

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein A², A³, and A⁴ are natural or unnatural amino acids.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein A² represents one natural or unnatural amino acid. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein A³ represents one natural or unnatural amino acid. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein A⁴ represents one natural or unnatural amino acid.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein A² represents two natural or unnatural amino acids. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein A³ represents two natural or unnatural amino acids. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein A⁴ represents two natural or unnatural amino acids.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein A² represents three natural or unnatural amino acids. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein A³ represents three natural or unnatural amino acids. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein A⁴ represents three natural or unnatural amino acids.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first solvent, the second solvent, the third solvent, the fourth solvent, the fifth solvent, the sixth solvent, the seventh solvent, or the eighth solvent is water, DMF, CH₃CN, CH₃CH₂OH, CH₃OH, DMSO, tris(2-carboxyethyl)phosphine (TCEP), dibutyl ether, tetrahydrofuran (THF), 1,4-dioxane, DME, dichloromethane, dichloroethane, acetone, diethyl ether, hexanes, or a mixture thereof.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first buffer, the second buffer, the third buffer, the fourth buffer, the fifth buffer, the sixth buffer, the seventh buffer, or the eighth buffer comprises Na₃P0₄, MES, or tris(hydroxymethyl)aminomethane (TRIS).

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first time, the second time, the third time, the fourth time, the fifth time, the sixth time, the seventh time, or the eighth time is from about 30 min to about 24 h. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first time, the second time, the third time, the fourth time, the fifth time, the sixth time, the seventh time, or the eighth time is about 30 min, about 40 min, about 50 min, about 1 h, about 2 h, about 3 h, about 4 h, about 5 h, about 6 h, about 7 h, about 8 h, about 9 h, about 10 h, about 11 h, about 12 h, about 13 h, about 14 h, or about 15 h.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein A¹ is an amine protecting group selected from the group consisting of an N,0-acetal, allyloxycarbonyl (Aloe), benzyl (Bn), benzyloxycarbonyl (Cbz), benzyloxymethyl (BOM), t-butoxycarbonyl (Boc), t-butyldimethylsilyl (TBDMS), t- butyldiphenylsilyl (TBDPS), diphenylmethyl, diphenylmethylene, ethoxycarbonyl, 9- fluorenylmethoxycarbonyl (Fmoc), /?-methoxybenzyl (PMB), methoxycarbonyl, methoxymethyl (MOM), /?-methoxyphenyl (PMP), /?-nitrocinnamyloxycarbonyl (Noc), tosyl (Ts), 2-tosylethoxycarbonyl (Tsoc), 2,2,2-trichloroethoxycarbonyl (Troc), trifluoroacetyl, triisopropylsilyl (TIPS), trimethylsilyl (TMS), 2- (trimethylsilyl)ethoxycarbonyl (Teoc), 2-(trimethylsilyl)ethoxymethyl (SEM), or trityl (Tr).

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein A⁵ is -0(carboxylate protecting group); and the carboxylate protecting group is selected from the group consisting of allyl, benzyl, benzyloxymethyl (BOM), t-Bu, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), diphenylmethyl, 9- fluorenylmethyl (Fm), 2-methoxyethoxymethyl (MEM), methoxymethyl (MOM), p- nitrobenzyl (P B), an ester, a 1,3-oxazoline, pi valoyloxy methyl (Pom), 2-tosylethyl (TSE), 2,2,2-trichloroethyl (TCE), triethylsilyl (TES), trimethylsilyl (TMS), 2- (trimethylsilyl)ethoxymethyl (SEM), or 2-(trimethylsilyl)ethyl (TMSE).

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein R is H.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein x is 1, 2, or 3. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein x is 1.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first, second, third, fourth, fifth, sixth, seventh, or eighth temperature is between about 10°C and about 50 °C. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first, second, third, fourth, fifth, sixth, seventh, or eighth temperature is about 10 °C, about 11 °C, about 12 °C, about 13 °C, about 14 °C, about 15 °C, about 16 °C, about 17 °C, about 18 °C, about 19 °C, about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, about 40 °C, about 41 °C, about 42 °C, about 43 °C, about 44 °C, about 45 °C, about 46 °C, about 47 °C, about 48 °C, about 49 °C, or about 50 °C.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the Cu/ligand comprises CuS0₄. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the Cu/ligand comprises a substituted or unsubstituted bipyridine ligand or a substituted or unsubstituted phenanthroline ligand. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the Cu/ligand comprises 4,4'-di-tert-butyl-2,2'-bipy.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the Rh/ligand comprises [RhCp*Cl2]2-

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the catalytic metal is not Cu or Rh or absent, but comprises Fe, Co, Ni, Au, Pd, Pt, Ru, or Ir. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the catalytic metal is not Cu or Rh or absent, but is selected from the group consisting of Fe, Co, Ni, Au, Pd, Pt, Ru, and Ir.

The reactions typically proceed at mild temperatures and pressures to give high yields of the product. Thus, yields of desired products greater than 45%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95% may be obtained from reactions at mild temperatures according to the invention.

In certain embodiments, the reactions take place under an inert atmosphere of a gas such as nitrogen or argon.

The reaction processes of the invention can be conducted in continuous, semi- continuous or batch fashion and may involve a liquid recycle operation as desired. The processes of this invention are preferably conducted in batch fashion. Likewise, the manner or order of addition of the reaction ingredients, base, and solvent are not generally critical to the success of the reaction, and may be accomplished in any conventional fashion. The reaction can be conducted in a single reaction zone or in a plurality of reaction zones, in series or in parallel or it may be conducted batchwise or continuously in an elongated tubular zone or series of such zones. The materials of construction employed should be inert to the starting materials during the reaction and the fabrication of the equipment should be able to withstand the reaction temperatures and pressures. Means to introduce and/or adjust the quantity of starting materials or ingredients introduced batchwise or continuously into the reaction zone during the course of the reaction can be conveniently utilized in the processes especially to maintain the desired molar ratio of the starting materials. The reaction steps may be effected by the incremental addition of one of the starting materials to the other. When complete conversion is not desired or not obtainable, the starting materials can be separated from the product and then recycled back into the reaction zone.

The processes may be conducted in glass lined, stainless steel, fluoropolymer coated (e.g., Teflon coated) or similar type reaction equipment. The reaction zone may be fitted with one or more internal and/or external heat exchanger(s) in order to control undue temperature fluctuations, or to prevent any possible "runaway" reaction temperatures.

Furthermore, one or more of the reactants can be immobilized on or incorporated into a polymer or other insoluble matrix by, for example, derivatization with one or more of the substituents of the aryl group or an amino acid residue.

The ability to provide synthesis schemes for the compounds of the invention that can be carried out under mild conditions has broad application.

In addition, the subject methods can be used as part of combinatorial synthesis schemes to yield libraries of compounds. Accordingly, another aspect of the invention relates to use of the subject method to generate variegated libraries of compounds, and to the libraries themselves. The libraries can be soluble or linked to insoluble supports, e.g., through a substituent of a reactant (prior to carrying out a reaction of the invention).

Further, the methods of the invention can be used to produce synthetic intermediates that, after being subjected to additional methods known in the art, are transformed to desired end products. Exemplary Therapeutic Methods

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the disease is an infection, for example, a bacterial infection, a fungal infection, a viral infection, or a parasitic infection.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the disease is cancer. In one group of embodiments, the invention relates to any one of the aforementioned methods, wherein the disease is selected from the group consisting of breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's disease, non- Hodgkin's lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma. In some embodiments, the cancer being treated is metastatic. In other embodiments, the cancer being treated is resistant to anticancer agents.

In certain embodiments, the invention relates to any one of the aforementioned methods, further comprising the step of: monitoring the cell or the subject by ¹⁹F MR.

A subject in need thereof (or a mammal in need thereof) may include, for example, a subject who has been diagnosed with any one of the aforementioned diseases, or a subject who has been treated for any one of the aforementioned diseases, including subjects that have been refractory to the previous treatment.

In some embodiments, the subject pharmaceutical compositions of the present invention will incorporate the substance or substances to be delivered in an amount sufficient to deliver to a patient a therapeutically effective amount of an incorporated therapeutic agent or other material as part of a prophylactic or therapeutic treatment. The desired concentration of the active agent will depend on absorption, inactivation, and excretion rates of the drug as well as the delivery rate of the compound. It is to be noted that dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Typically, dosing will be determined using techniques known to one skilled in the art.

The dosage of the subject agent may be determined by reference to the plasma concentrations of the agent. For example, the maximum plasma concentration (Cma_X) and the area under the plasma concentration-time curve from time 0 to infinity (AUC (0-4)) may be used. Dosages for the present invention include those that produce the above values for Cmax and AUC (0-4) and other dosages resulting in larger or smaller values for those parameters.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could prescribe and/or administer doses of the agents of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of an agent of the invention will be that amount of the agent which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

If desired, the effective daily dose of the agent may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

The precise time of administration and amount of any particular agent that will yield the most effective treatment in a given patient will depend upon the activity, pharmacokinetics, and bioavailability of a particular agent, physiological condition of the patient (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage and type of medication), route of administration, and the like. The guidelines presented herein may be used to optimize the treatment, e.g., determining the optimum time and/or amount of administration, which will require no more than routine experimentation consisting of monitoring the subject and adjusting the dosage and/or timing.

While the subject is being treated, the health of the subject may be monitored by measuring one or more of the relevant indices at predetermined times during a 24-hour period. All aspects of the treatment, including supplements, amounts, times of administration and formulation, may be optimized according to the results of such monitoring. The patient may be periodically reevaluated to determine the extent of improvement by measuring the same parameters, the first such reevaluation typically occurring at the end of four weeks from the onset of therapy, and subsequent reevaluations occurring every four to eight weeks during therapy and then every three months thereafter. Therapy may continue for several months or even years, with a minimum of one month being a typical length of therapy for humans. Adjustments, for example, to the amount(s) of agent administered and to the time of administration may be made based on these reevaluations.

Treatment may be initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage may be increased by small increments until the optimum therapeutic effect is attained. In addition, the combined use an agent that modulates an autotrophy-associated gene product and a second agent, e.g., another agent useful for the treatment of the autophagy-related disease, may reduce the required dosage for any individual agent because the onset and duration of effect of the different compounds and/or agents may be complimentary.

In one aspect of the invention, a compound of the invention, or a pharmaceutically acceptable salt thereof, can be used alone or in combination with another therapeutic agent to treat diseases such cancer. It should be understood that the compounds of the invention can be used alone or in combination with an additional agent, e.g., a therapeutic agent, said additional agent being selected by the skilled artisan for its intended purpose. For example, the additional agent can be a therapeutic agent that is art-recognized as being useful to treat the disease or condition being treated by the compound of the present invention. The additional agent also can be an agent that imparts a beneficial attribute to the therapeutic composition e.g., an agent that affects the viscosity of the composition.

The combination therapy contemplated by the invention includes, for example, administration of a compound of the invention, or a pharmaceutically acceptable salt thereof, and additional agent(s) in a single pharmaceutical formulation as well as administration of a compound of the invention, or a pharmaceutically acceptable salt thereof, and additional agent(s) in separate pharmaceutical formulations. In other words, coadministration shall mean the administration of at least two agents to a subject so as to provide the beneficial effects of the combination of both agents. For example, the agents may be administered simultaneously or sequentially over a period of time.

It should further be understood that the combinations included within the invention are those combinations useful for their intended purpose. The agents set forth below are illustrative for purposes and not intended to be limited. The combinations, which are part of this invention, can be the compounds of the present invention and at least one additional agent selected from the lists below. The combination can also include more than one additional agent, e.g., two or three additional agents if the combination is such that the formed composition can perform its intended function.

In certain embodiments, the co-administration of two or more therapeutic agents (e.g., a pharmaceutical and a peptide) achieves a synergistic effect, i.e., a therapeutic effect that is greater than the sum of the therapeutic effects of the individual components of the combination. The term "synergistic" refers to a combination which is more effective than the additive effects of any two or more single agents. A synergistic effect permits the effective treatment of a disease using lower amounts (doses) of individual therapy. The lower doses result in lower toxicity without reduced efficacy. In addition, a synergistic effect can result in improved efficacy. Finally, synergy may result in an improved avoidance or reduction of disease as compared to any single therapy. Combination therapy can allow for the product of lower doses of the first therapeutic or the second therapeutic agent (referred to as "apparent one-way synergy" herein), or lower doses of both therapeutic agents (referred to as "two-way synergy" herein) than would normally be required when either drug is used alone.

Definitions

For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The term "heteroatom" is art-recognized and refers to an atom of any element other than carbon or hydrogen. Illustrative heteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur and selenium.

The term "alkenyl" as used herein, means a straight or branched chain hydrocarbon containing from 2 to 10 carbons and containing at least one carbon-carbon double bond formed by the removal of two hydrogens. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5- hexenyl, 2-heptenyl, 2-methyl-l-heptenyl, and 3-decenyl. The term "alkoxy" means an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy.

The term "alkoxycarbonyl" means an alkoxy group, as defined herein, appended to the parent molecular moiety through a carbonyl group, represented by -C(=0)-, as defined herein. Representative examples of alkoxycarbonyl include, but are not limited to, methoxy carbonyl, ethoxy carbonyl, and tert-butoxy carbonyl.

The term " alkoxy sulfonyl" as used herein, means an alkoxy group, as defined herein, appended to the parent molecular moiety through a sulfonyl group, as defined herein. Representative examples of alkoxysulfonyl include, but are not limited to, methoxysulfonyl, ethoxysulfonyl and propoxysulfonyl.

The term "arylalkoxy" and "heteroalkoxy" as used herein, means an aryl group or heteroaryl group, as defined herein, appended to the parent molecular moiety through an alkoxy group, as defined herein.

The term "alkyl" means a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, and n-hexyl.

The term "alkylcarbonyl" as used herein, means an alkyl group, as defined herein, appended to the parent molecular moiety through a carbonyl group, as defined herein. Representative examples of alkylcarbonyl include, but are not limited to, acetyl, 1- oxopropyl, 2,2-dimethyl-l-oxopropyl, 1-oxobutyl, and 1-oxopentyl.

The term "alkylcarbonyloxy" and "arylcarbonyloxy" as used herein, means an alkylcarbonyl or arylcarbonyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkylcarbonyloxy include, but are not limited to, acetyloxy, ethylcarbonyloxy, and tert-butylcarbonyloxy. Representative examples of arylcarbonyloxy include, but are not limited to phenyl carbonyloxy.

The term "alkyl sulfonyl" as used herein, means an alkyl group, as defined herein, appended to the parent molecular moiety through a sulfonyl group, as defined herein. Representative examples of alkylsulfonyl include, but are not limited to, methyl sulfonyl and ethyl sulfonyl.

The term "alkylthio" as used herein, means an alkyl group, as defined herein, appended to the parent molecular moiety through a sulfur atom. Representative examples of alkylthio include, but are not limited, methylthio, ethylthio, tert-butylthio, and hexylthio. The terms "arylthio," "alkenylthio" and "arylakylthio," for example, are likewise defined.

The term "alkynyl" as used herein, means a straight or branched chain hydrocarbon group containing from 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond. Representative examples of alkynyl include, but are not limited, to acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, and 1-butynyl.

The term "amido" as used herein, means - HC(=0)-, wherein the amido group is bound to the parent molecular moiety through the nitrogen. Examples of amido include alkylamido such as CH₃C(=0)N(H)- and CH₃CH₂C(=0)N(H)-.

The term "amino" as used herein, refers to radicals of both unsubstituted and substituted amines appended to the parent molecular moiety through a nitrogen atom. The two groups are each independently hydrogen, alkyl, alkylcarbonyl, alkylsulfonyl, arylcarbonyl, or formyl. Representative examples include, but are not limited to methylamino, acetylamino, and acetylmethylamino.

The term "aromatic" refers to a planar or poly cyclic structure characterized by a cyclically conjugated molecular moiety containing 4n+2 electrons, wherein n is the absolute value of an integer. Aromatic molecules containing fused, or joined, rings also are referred to as bicyclic aromatic rings. For example, bicyclic aromatic rings containing heteroatoms in a hydrocarbon ring structure are referred to as bicyclic heteroaryl rings.

The term "aryl," as used herein, means a phenyl group or a naphthyl group. The aryl groups of the invention can be optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from the group consisting of alkenyl, alkoxy, alkoxycarbonyl, alkoxysulfonyl, alkyl, alkylcarbonyl, alkylcarbonyloxy, alkylsulfonyl, alkylthio, alkynyl, amido, amino, carboxy, cyano, formyl, halo, haloalkoxy, haloalkyl, hydroxyl, hydroxyalkyl, mercapto, nitro, phosphinyl, silyl and silyloxy.

The term "arylene," is art-recognized, and as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms of an aryl ring, as defined above. The term "arylalkyl" or "aralkyl" as used herein, means an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, and 2-naphth-2-ylethyl.

The term "arylalkoxy" or "arylalkyloxy" as used herein, means an arylalkyl group, as defined herein, appended to the parent molecular moiety through an oxygen. The term "heteroarylalkoxy" as used herein, means an heteroarylalkyl group, as defined herein, appended to the parent molecular moiety through an oxygen.

The term "arylalkylthio" as used herein, means an arylalkyl group, as defined herein, appended to the parent molecular moiety through an sulfur. The term "heteroaryl alky lthio" as used herein, means an heteroarylalkyl group, as defined herein, appended to the parent molecular moiety through an sulfur.

The term "arylalkenyl" as used herein, means an aryl group, as defined herein, appended to the parent molecular moiety through an alkenyl group. A representative example is phenyl ethyl enyl.

The term "arylalkynyl" as used herein, means an aryl group, as defined herein, appended to the parent molecular moiety through an alkynyl group. A representative example is phenylethynyl.

The term "arylcarbonyl" as used herein, means an aryl group, as defined herein, appended to the parent molecular moiety through a carbonyl group, as defined herein. Representative examples of arylcarbonyl include, but are not limited to, benzoyl and naphthoyl.

The term "arylcarbonylalkyl" as used herein, means an arylcarbonyl group, as defined herein, bound to the parent molecule through an alkyl group, as defined herein.

The term "arylcarbonylalkoxy" as used herein, means an arylcarbonylalkyl group, as defined herein, bound to the parent molecule through an oxygen.

The term "aryloxy" as used herein, means an aryl group, as defined herein, appended to the parent molecular moiety through an oxygen. The term "heteroaryloxy" as used herein, means a heteroaryl group, as defined herein, appended to the parent molecular moiety through an oxygen.

The term "carbonyl" as used herein, means a -C(=0)- group. The term "carboxy" as used herein, means a -C0₂H group.

The term "cycloalkyl" as used herein, means monocyclic or multicyclic (e.g., bicyclic, tricyclic) hydrocarbons containing from 3 to 12 carbon atoms that is completely saturated or has one or more unsaturated bonds but does not amount to an aromatic group. Examples of a cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl.

The term "cycloalkoxy" as used herein, means a cycloalkyl group, as defined herein, appended to the parent molecular moiety through an oxygen.

The term "cyano" as used herein, means a -CN group.

The term "formyl" as used herein, means a -C(=0)H group.

The term "halo" or "halogen" means -CI, -Br, -I or -F.

The term "haloalkoxy" as used herein, means at least one halogen, as defined herein, appended to the parent molecular moiety through an alkoxy group, as defined herein. Representative examples of haloalkoxy include, but are not limited to, chloromethoxy, 2- fluoroethoxy, trifluoromethoxy, and pentafluoroethoxy.

The term "haloalkyl" means at least one halogen, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, and 2-chloro-3-fluoropentyl.

The term "heterocyclyl", as used herein, include non-aromatic, ring systems, including, but not limited to, monocyclic, bicyclic and tricyclic rings, which can be completely saturated or which can contain one or more units of unsaturation, for the avoidance of doubt, the degree of unsaturation does not result in an aromatic ring system) and have 3 to 12 atoms including at least one heteroatom, such as nitrogen, oxygen, or sulfur. For purposes of exemplification, which should not be construed as limiting the scope of this invention, the following are examples of heterocyclic rings: azepines, azetidinyl, morpholinyl, oxopiperidinyl, oxopyrrolidinyl, piperazinyl, piperidinyl, pyrrolidinyl, quinicludinyl, thiomorpholinyl, tetrahydropyranyl and tetrahydrofuranyl. The heterocyclyl groups of the invention are substituted with 0, 1, 2, 3, 4 or 5 substituents independently selected from alkenyl, alkoxy, alkoxycarbonyl, alkoxysulfonyl, alkyl, alkylcarbonyl, alkylcarbonyloxy, alkylsulfonyl, alkylthio, alkynyl, amido, amino, carboxy, cyano, formyl, halo, haloalkoxy, haloalkyl, hydroxyl, hydroxyalkyl, mercapto, nitro, phosphinyl, silyl and silyloxy.

The term "heteroaryl" as used herein, include aromatic ring systems, including, but not limited to, monocyclic, bicyclic and tricyclic rings, and have 3 to 12 atoms including at least one heteroatom, such as nitrogen, oxygen, or sulfur. For purposes of exemplification, which should not be construed as limiting the scope of this invention: azaindolyl, benzo(b)thienyl, benzimidazolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzoxadiazolyl, furanyl, imidazolyl, imidazopyridinyl, indolyl, indolinyl, indazolyl, isoindolinyl, isoxazolyl, isothiazolyl, isoquinolinyl, oxadiazolyl, oxazolyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrrolyl, pyrrolo[2,3-d]pyrimidinyl, pyrazolo[3,4-d]pyrimidinyl, quinolinyl, quinazolinyl, triazolyl, thiazolyl, thiophenyl, tetrahydroindolyl, tetrazolyl, thiadiazolyl, thienyl, thiomorpholinyl, triazolyl or tropanyl. The heteroaryl groups of the invention are substituted with 0, 1, 2, 3, 4 or 5 substituents independently selected from alkenyl, alkoxy, alkoxycarbonyl, alkoxysulfonyl, alkyl, alkylcarbonyl, alkylcarbonyloxy, alkylsulfonyl, alkylthio, alkynyl, amido, amino, carboxy, cyano, formyl, halo, haloalkoxy, haloalkyl, hydroxyl, hydroxyalkyl, mercapto, nitro, phosphinyl, silyl and silyloxy.

The term "heteroarylene," is art-recognized, and as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms of a heteroaryl ring, as defined above.

The term "heteroarylalkyl" or "heteroaralkyl" as used herein, means a heteroaryl, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of heteroarylalkyl include, but are not limited to, pyridin- 3-ylmethyl and 2-(thien-2-yl)ethyl.

The term "hydroxy" as used herein, means an -OH group.

The term "hydroxyalkyl" as used herein, means at least one hydroxy group, as defined herein, is appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of hydroxyalkyl include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypentyl, and 2-ethyl-4- hydroxyheptyl.

The term "mercapto" as used herein, means a -SH group. The term "nitro" as used herein, means a -N0₂ group.

The term "phosphinyl" as used herein includes derivatives of the H₃P- group, wherein the hydrogens are independently replaced with alkyl, adamantyl, fluoroalkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, aryloxy, or heteroaryloxy groups.

The term "silyl" as used herein includes hydrocarbyl derivatives of the silyl (H₃Si-) group (i.e., (hydrocarbyl)₃Si-), wherein a hydrocarbyl groups are univalent groups formed by removing a hydrogen atom from a hydrocarbon, e.g., ethyl, phenyl. The hydrocarbyl groups can be combinations of differing groups which can be varied in order to provide a number of silyl groups, such as trimethyl silyl (TMS), tert-butyldiphenylsilyl (TBDPS), tert- butyldimethylsilyl (TBS/TBDMS), triisopropyl silyl (TIPS), and [2- (trimethylsilyl)ethoxy]methyl (SEM).

The term "silyloxy" as used herein means a silyl group, as defined herein, is appended to the parent molecule through an oxygen atom.

The definition of each expression, e.g., alkyl, m, n, and the like, when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, /?-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively. The terms triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, /?-toluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, /?-toluenesulfonyl and methanesulfonyl, respectively. A more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; this list is typically presented in a table entitled Standard List of Abbreviations.

Certain compounds contained in compositions of the invention may exist in particular geometric or stereoisomeric forms. In addition, polymers of the invention may also be optically active. The invention contemplates all such compounds, including cis- and trans-isomers, R- and ^-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of compound of the invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.

It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.

The term "substituted" is also contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents may be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.

The phrase "protecting group" as used herein means temporary substituents which protect a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively. The field of protecting group chemistry has been reviewed (Greene, T.W.; Wuts, P.G.M. Protective Groups in Organic Synthesis, 2ⁿ ed.; Wiley: New York, 1991). Protected forms of the inventive compounds are included within the scope of this invention.

A "polar solvent" means a solvent which has a dielectric constant (ε) of 2.9 or greater, such as DMF, THF, ethylene glycol dimethyl ether (DME), DMSO, acetone, acetonitrile, methanol, ethanol, isopropanol, n-propanol, t-butanol or 2-methoxyethyl ether. Preferred polar solvents are DMF, DME, NMP, and acetonitrile.

An "aprotic solvent" means a non-nucleophilic solvent having a boiling point range above ambient temperature, preferably from about 25 °C to about 190 °C, more preferably from about 80 °C to about 160 °C, most preferably from about 80 °C to 150 °C, at atmospheric pressure. Examples of such solvents are acetonitrile, toluene, DMF, diglyme, THF or DMSO.

A "polar, aprotic solvent" means a polar solvent as defined above which has no available hydrogens to exchange with the compounds of this invention during reaction, for example DMF, acetonitrile, diglyme, DMSO, or TFIF.

A "hydroxylic solvent" means a solvent that comprises a hydroxyl moiety; for example, water, methanol, ethanol, tert-butanol, and ethylene glycol are hydroxylic solvents.

For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

EXEMPLIFICATION

The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the invention, and are not intended to limit the invention.

Example 1: Copper-Catalyzed Arylation of Selenocysteine in Unprotected Peptides

Materials

1. Chemicals

Jm(2-carboxyethyl)phosphine hydrochloride (TCEP-HC1) was purchased from Hampton Research (Aliso Viejo, CA). l-[Bis(dimethylamino)methylene]-lH-l,2,3- triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), Fmoc-L-Gly-OH, Fmoc-L- Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L- Asn(Trt)-OH, Fmoc-L-Asp(tBu)-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Phe-OH, Fmoc-L- Ser(tBu)-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-Ser-OH, and Fmoc-L-His(Trt)-OH were purchased from Chem-Impex International (Wood Dale, IL). Fmoc-L-4-methoxybenzyl- selenocysteine (Fmoc-L-Sec(Pmb)-OH ) was prepared from Fmoc-Ser-OH using standard literature procedure. Schroll, A. L.; Hondal, R. J.; Flemer, S. J. Pept. Sci. 2012, 18, 155. Peptide synthesis-grade N, N-dimethylformamide (DMF), dichloromethane (DCM), diethyl ether, HPLC-grade acetonitrile, and guanidine hydrochloride were obtained from VWR International (Philadelphia, PA). All reactions were set up on the bench top open to air. Water was deionized and used as is. Ethanol, copper, ligands were purchased from commercial sources and used as received. Boronic acids were purchase from commercial sources or prepared according to standard literature procedure. Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem., 1995, 60, 7508.

2. Reaction Vessels a) 0.6 mL Axygen Tubes (For 100 μΜ reaction) - Axygen Cat. No. MCT-060- L-C b) Scintillation Vials (For 1 mM scale-up reactions) - VWR Cat. No.

VW74510-20

Methods for LC-MS Analysis

LC-MS chromatograms and associated mass spectra were acquired using Agilent 6520 ESI-Q-TOF mass spectrometer unless noted. Mobile phases are: 0.1% formic acid in water (solvent C) and 0.1% formic acid in acetonitrile (solvent D) Following LC-MS methods were used:

Method A - LC conditions: Zorbax SB C3 column: 2.1 x 150 mm, 5 μιτι, column temperature: 40 °C, gradient: 0-2 minutes 1% D, 2-1 1 minutes 1-61% D, 1 1-12 minutes 61%) D, flow rate: 0.8 mL/min. MS conditions: positive electrospray ionization (ESI) extended dynamic mode in mass range 300 - 3000 m/z, temperature of drying gas = 350 °C, flow rate of drying gas = 1 1 L/min, pressure of nebulizer gas = 60 psi, the capillary, fragmentor, and octupole if voltages were set at 4000, 175, and 750, respectively. Method B - LC conditions: Zorbax SB C3 column: 2.1 x 150 mm, 5 μιτι, column temperature: 40 °C, gradient: 0-1 minutes 5-25% D, 1-5 minutes 25-75%) D, flow rate: 0.8 mL/min. 5-6 minutes 75-95% D, flow rate: 1.5 mL/min. MS conditions are same as Method A.

Method C - LC conditions: Zorbax SB C3 column: 2.1 x 150 mm, 5 μιτι, column temperature: 40 °C, gradient: 0-2 minutes 1% D, 2-23 minutes 1-61% D, 23-24 minutes 61% D, flow rate: 0.8 mL/min. MS conditions are same as Method A.

Method D - LC conditions: Zorbax SB C18 column: 2.1 x 150 mm, 5 μιτι, column temperature: 40 °C, gradient: 0-2 minutes 1%> D, 2-11 minutes 1-61% D, 11-12 minutes 61% D, flow rate: 0.8 mL/min. MS conditions are same as Method A.

All reactions for peptide stability studies were analyzed by Agilent 6550 ESI-Q- TOF mass spectrometer. Mobile phases are: 0.1%> formic acid in water (solvent C) and 0.1%) formic acid in acetonitrile (solvent D). Following LC-MS method was used:

Method E - LC conditions: EclipsePlus C18 column: 2.1 x 50 mm, RRHD 1.8 μιη, column temperature: 40 °C, gradient: 0-1 minutes 5%> D, 1-6 minutes 5-50%> D, 6-8 minutes 50-95%) D, 8-10 minutes 95%> D, flow rate: 0.5 mL/min. MS conditions: positive electrospray ionization (ESI) extended dynamic mode in mass range 300 - 3000 m/z, temperature of drying gas = 200 °C, flow rate of drying gas = 17 L/min, pressure of nebulizer gas = 35 psi, the capillary, fragmentor, and nozzle voltages were set at 3500, 380, and 500, respectively.

All data were processed using Agilent MassHunter software package. Y-axis in all chromatograms shown represents total ion current (TIC) unless noted; mass spectrum corresponds to the integration of the TIC peak unless noted.

All yields reported were determined by integrating TIC spectra. First, using Agilent MassHunter software package, the peak areas for all relevant peptidic species on the chromatogram were integrated. Then the yield was calculated as following: %>yield = S_p/Saii where S_p is the peak area of the desired product, and S_aii is sum of the peak areas of all peptidic species.

General Method for Preparation of Peptides 1) Fast-flow peptide synthesis All peptide sequences C-terminal to selenocysteine were synthesized on a 0.2-mmol scale using manual Fmoc-SPPS (Solid phase peptide synthesis) chemistry under flow using a 3-minute cycle for each amino acid. Simon, M. D.; et al., ChemBioChem 2014, 15, 713. Specifically, all reagents and solvents are delivered to a stainless steel reactor containing resins at a constant flow rate using an FIPLC pump; temperature of the reactor was maintained at 60 °C during the synthesis using a water bath. The procedure for each amino acid coupling cycle included a 30 second coupling with 1 mmol Fmoc-protected amino acid, 1 mmol HATU, and 500 μΐ_^ of diisopropyl ethyl amine (DIEA) in 2.5 mL of DMF at a flow rate of 6 mL/min (note that for the coupling of cysteine and histidine, 190 μΕ of DIEA was used to prevent racemization); 1 min wash with DMF at a flow rate of 20 mL/min; 20 second deprotection with 20% (v/v) piperidine in DMF at a flow rate of 20 mL/min; and 1 minute wash with DMF at a flow rate of 20 mL/min. After completion of the fast-flow synthesis, the resins are washed with DCM (3X) and dried under vacuum. The dried resins are used in batch synthesis for coupling of selenocysteine and the rest of the peptide sequence.

Table 1. Peptide Sequences. Amino acids were incorporated through fast-flow SPPS except

for those underlined, which were synthesized through batch SPPS.

Peptide Sequence

1 NH₇-Leu-Phe-Glv-Glv-Sec(TNP)-Glv-Leu-Leu-Lvs-Asn-Lvs-C0NH₇

1-Ser5 NH₂-Leu-Phe-Gly-Gly-Ser-Gly-Leu-Leu-Lys-Asn-Lys-CONH₂

1-Cys5 NH₂-Leu-Phe-Gly-Gly-Cys-Gly-Leu-Leu-Lys-Asn-Lys-CONH₂

1-Cys5-TNP NH₂-Leu-Phe-Gly-Gly-Cys(TNP)-Gly-Leu-Leu-Lys-Asn-Lys-CONH₂

1-Met5 NH₂-Leu-Phe-Gly-Gly-Met-Gly-Leu-Leu-Lys-Asn-Lys-CONH₂

7 NH₂-Glv-SecrTNP)-Ala-Asn-Ser-Leu-Arg-Phe-Tvr-His-AsD-Lvs-CONH₂

9 NH₂-Gly-Ser-Ala-Asn-Ser-Leu-Arg-Phe-Tyr-His-Asp-Lys-CONH₂

2) Solid-phase peptide synthesis (SPPS) in batch

Selenocysteine and amino acids N-terminal to selenocysteine were coupled to the resin under batch SPPS conditions on a 0.2-mmol scale. Each amino acid was incorporated into the peptide sequence through a cycle of coupling, washing, deprotection, and washing steps. Procedure for the coupling of selenocysteine included a 20 min coupling with 0.4 mmol Fmoc-L-Sec(Pmb)-OH, 0.4 mmol HATU, and 38 of DIEA in 2 mL of DMF. For other amino acids, coupling was performed for 10 min with 1 mmol Fmoc-protected amino acids, 1 mmol HATU, 500 μΕ of DIEA in 2.5 mL of DMF. After coupling, the resin was washed with DMF (3X). Piperidine (20% (v/v) in DMF) was added to the resin for 2 X 5 min each. The resin was washed with DMF (3X) and then subjected to coupling of the next amino acid.

Peptide sequences synthesized using batch SPPS are underlined in Table 1.

3) Peptide cleavage and deprotection

Peptides containing selenocysteine were cleaved from the resin and the side-chain was simultaneously deprotected by treatment with 5% (v/v) water, 95% (v/v) trifluoroacetic acid (TFA), 0.4 M 2,2' -dithiobis(5-nitropyri dine) (DTNP) for 7 min at 60 °C. 5 mL of cleavage cocktail was used for 0.2 mmol of peptide. The resulting solution was triturated and washed with cold diethyl ether (pre-chilled in -80 °C freezer) this was repeated a total of three times. The obtained solids were dissolved in 50% H₂0: 50% acetonitrile containing 0.1% TFA and lyophilized. These same solvent compositions were used in the majority of experiments and will be referred to as A: 0.1 % TFA in H₂0 and B: 0.1% TFA in acetonitrile.

Peptides without selenocysteine were cleaved from the resin and the side-chain was simultaneously deprotected by treatment with 2.5% (v/v) water, 2.5% (v/v) 1,2- ethanedithiol (EDT), 1% (v/v) triisopropylsilane (TIPS) in neat TFA for 7 min at 60 °C, 5 mL of cleavage cocktail was used for 0.2 mmol of peptide. The resulting solution was triturated and washed with cold ether (pre-chilled in -80 °C freezer). The trituration was repeated a total of three times. The obtained solids were dissolved in 50% A and 50% B and lyophilized.

Peptide containing methionine was cleaved from the resin and the side-chain was simultaneously deprotected by treatment with 1% (v/v) triisopropylsilane (TIPS), 2.5% (v/v) water, 2.5% (v/v) 1,2-ethanedithiol (EDT), 2% (v/v) methyl disulfide, 92% (v/v) trifluoroacetic acid (TFA), and saturated ammonium iodide for 8 min at 60 °C. 5 mL of this cleavage cocktail was used for 0.2 mmol of peptide. The resulting solution was triturated and washed with cold diethyl ether (pre-chilled in -80 °C freezer) this was repeated a total of three times. The obtained solids were dissolved in 50% H₂0: 50% acetonitrile containing 0.1% TFA and lyophilized.

4) RP-HPLC purification of peptides

The crude peptide was dissolved in 95% A: 5% B with 6 M guanidinium hydrochloride and purified by semi-preparative RP-HPLC (Agilent Zorbax SB C18 column: 21.2 x 250 mm, 7 μπι, linear gradient: 5-50% B over 90 min, flow rate: 5 mL/min). 1 μΐ. of each HPLC fraction was mixed with 1 μΐ_^ of a-cyano-4-hydroxycinnamic acid (CHCA) matrix in 75% A: 25% B, spotted with MALDI, and checked for fractions with desired molecular mass. The purity of fractions was confirmed by analytical RP-HPLC (Agilent Zorbax SB C3 column: 2.1 x 150 mm, 5 μπι, gradient: 0-2 minutes 5% B, 2-11 minutes 5-65% B, 11-12 minutes 65% B, flow rate: 0.8 mL/min). HPLC fractions containing only product materials were confirmed by LC-MS analysis, combined, and then lyophilized. Peptides purified by RP-HPLC are listed in Table 1.

Peptide 1: LCMS Analysis Method A. HRMS (ESI) Mass. calcd. for Cs_TH^NnO^SSe [M+H]⁺, 1350.58. Found [M+H]⁺, 1350.58.

Peptide 1-Ser5: LCMS Analysis Method A. HRMS (ESI) Mass. calcd. for C52H90N15O13 [M+H]⁺, 1132.68. Found [M+H]⁺, 1132.67.

Peptide 1-Cys5: LCMS Analysis Method A. HRMS (ESI) Mass. calcd. for C52H90N15O12S [M+H]⁺, 1148.66. Found [M+H]⁺, 1148.67.

Peptide 1-Cys5-TNP: Peptide was synthesized using fast-flow peptide synthesis procedure. The TNP protecting group was installed using cleavage cocktail: 5% (v/v) H₂0, 95% (v/v) TFA, 0.4 M DTNP, at 60 °C for 5 minutes. LCMS Analysis Method A. HRMS (ESI) Mass. calcd. for C57H91N17O14S2 [M+H]⁺, 1302.64. Found [M+H]⁺, 1302.64.

Peptide 1-Met5: LCMS Analysis Me thodA. HRMS (ESI) Mass. calcd. for C54H94N15O12S [M+H]⁺, 1176.69. Found [M+H]⁺, 1176.73.

Peptide 7: LCMS Analysis Method A. HRMS (ESI) Mass. calcd. for C₆₆H₉₅N₂₂0i₉SSe [M+ ⁺, 1611.60. Found [M+H]⁺, 1611.60.

Peptide 9: LCMS Analysis Method A. HRMS (ESI) Mass. calcd. for C61H93N20O18 [M+H]⁺, 1393.69. Found [M+H]⁺, 1393.72. General Procedure (A) for the Synthesis of Arylated Selenocysteine Α^Γ

A 0.6 mL Eppendorf tube was charged with 75 μΕ of deionized H₂0, 10 μΕ of 1.0 M Tris Buffer (pH = 8.0), 10 [iL of peptide (1 mM stock solution in H₂0). A separate 1.7 mL Eppendorf tube was charged with copper (20 μιηοΐ), ligand (20 μιηοΐ), arylboronic acid (20 μπιοΐ), and 1 mL or 0.5 mL of 200 proof EtOH (making a 20 or 40 mM stock solution, respectively). The heterogeneous solution was subjected to sonication for 1 min, vortexed for 30 sec, and 5 μΕ of the resulting solution was added to the peptide solution in the 0.6 mL Eppendorf tube. The resulting reaction mixture was capped, vortexed for 30 seconds, and placed in a 37 °C water bath for the indicated time (1-3 h). The reaction mixture was quenched with 5 μί of EDTA (200 mM in H₂0) and 100 μL· of 50% A: 50% B. The quenched reaction mixture was subjected to LC-MS analysis.

Optimization Table

Selenopeptide Arylation Optimization

Table 2

•;v«> S a: l a*

iir

-;;-:·;; £¾ VSS SB rs 2

so :■

&:BO ; - :;o o ;

;oo : i ^■ 2 s ΐ-

^Conversion and yields were determined by measuring the total ion currents (TIC) of LC- MS using Method A. ^Reaction run without any ligand. ^cReaction run without any copper. ^rf0.5 mM CuS0₄, 0.5 mM L2, and 0.5 mM boronic acid were used. ^e0.25 mM CuS0₄, 0.25 mM L2, and 0.25 mM boronic acid were used.

Serine Control Reaction

no reaction

peptide 1-Ser5

Cysteine Control Reaction

Cysteine- TNP Control Reaction

peptide 1- ye5-TNP

Methionine Control Reaction

no reaction

peptide 1^« et5

LC-MS Analysis of Arylation Reactions for Substrate Scope

(4a): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuS0₄, L2, and /?-tolylboronic acid stock solution (1 mM) at 37 °C for 2 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 4a: HRMS

(ESI

(4b): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuS0₄, L2, and (4-methoxyphenyl)boronic acid stock solution (1 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 4b: HRMS (ESI) Mass. calcd. for

[M+2H]²⁺, 651.83. Found [M+2H]²⁺, 651.84.

(4c): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuS0₄, L2, and (2-hydroxyphenyl)boronic acid stock solution (1 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 4c: HRMS (ESI) Mass. calcd. for CsuHwNisOisSe [M+H]⁺, 1288.63. Found [M+H]⁺, 1288.61.

(4d): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuS0₄, L2, and benzo[<i][l,3]dioxol-5-ylboronic acid stock solution (1 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 4d: HRMS (ESI) Mass. calcd. for

[M+2H]²⁺, 658.82. Found [M+2H]²⁺, 658.81.

(4e): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuS0₄, L2, and (4-(((tert-butoxycarbonyl)amino)methyl)phenyl)boronic acid stock solution (1 mM) at 37 °C for 1.5 h. The quenched reaction mixture was analyzed using LC- MS Method A. Analytical data for 4e: HRMS (ESI) Mass. calcd. for C₆₄Hio₅Ni₆Oi₄Se [M+H]⁺, 1401.72. Found [M+H]⁺, 1401.70.

(4f): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuS0₄, L2, and (4-(dimethylamino)phenyl)boronic acid stock solution (1 mM) at 37 °C for 2 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 4f: HRMS (ESI) Mass. calcd. for CeoHggNieOuSe [M+H]⁺, 1315.68. Found [M+H]⁺, 1315.69.

(4g): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuS0₄, L2, and (4-(methoxycarbonyl)phenyl)boronic acid stock solution (2 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 4g: HRMS (ESI) Mass. calcd. for CeiHiooNieOisSe [M+2H]²⁺, 665.82. Found [M+2H]²⁺, 665.82.

(4h): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuS0₄, L2, and (4-(dimethylcarbamoyl)phenyl)boronic acid stock solution (2 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 4h: HRMS (ESI) Mass. calcd. for CeiHiooNieOisSe [M+2H]²⁺, 672.34. Found [M+2H]²⁺, 672.35.

(4i): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuS0₄, L2, and (2-nitrophenyl)boronic acid stock solution (1 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 4i: HRMS (ESI) Mass. calcd. for

[M+2H]²⁺, 659.31. Found [M+2H]²⁺, 659.32.

NH;.

(4j): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuS0₄, L2, and (3-cyanophenyl)boronic acid stock solution (2 mM) at 37 °C for 2 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 4j: HRMS (ESI) Mass. calcd. for

[M+2H]²⁺, 649.32. Found [M+2H]²⁺, 649.33.

(4k): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuS0₄, L2, and (2-chlorophenyl)boronic acid stock solution (1 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 4k: HRMS (ESI) Mass. calcd. for

[M+H]⁺, 1306.59. Found [M+H]⁺, 1306.58.

(41): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuS0₄, L2, and (2,6-difluorophenyl)boronic acid stock solution (1 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 41: HRMS (ESI) Mass. calcd. for

[M+2H]²⁺, 654.81. Found [M+2H]²⁺, 654.82.

(5a): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuS0₄, L2, and (lH-indol-5-yl)boronic acid stock solution (1 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 5a: HRMS (ESI) Mass. calcd. for CeoHgeNieOuSe [M+2H]²⁺, 656.32. Found [M+2H]²⁺, 656.32.

(5b): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuS0₄, L2, and (l-(phenylsulfonyl)-lH-indol-2-yl)boronic acid acid stock solution (1 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 5b: HRMS (ESI) Mass. calcd. for CeeHiooNieOnSSe [M+2H]²⁺, 726.32.

Found [M+2H]²⁺, 726.33

(5c): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuS0₄, L2, and benzofuran-2-ylboronic acid stock solution (1 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 5c: HRMS (ESI) Mass. calcd. for CeoHgsNisOisSe [M+2H]²⁺, 656.81. Found [M+2H]²⁺, 656.82.

(5d): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuSC"4, L2, and (1 -methyl- lH-pyrazol-4-yl)boronic acid stock solution (1 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 5d: HRMS (ESI) Mass. calcd. for Csei^NnO^Se [M+H]⁺, 1276.64. Found [M+H]⁺, 1276.63.

(5e): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuSC"4, L2, and (3,5-dimethylisoxazol-4-yl)boronic acid stock solution (1 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 5e: HRMS (ESI) Mass. calcd. for CsvHgeNieOisSe [M+2H]²⁺, 646.32. Found [M+2H]²⁺, 646.32.

(5f): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuSC>4, L2, and thiophen-3-ylboronic acid stock solution (1 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 5f: HRMS (ESI) Mass. calcd. for

[M+2H]²⁺, 639.79. Found [M+2H]²⁺, 639.79.

(5g): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuSC"4, L2, and (2-formylthiophen-3-yl)boronic acid stock solution (1 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 5g: HRMS (ESI) Mass. calcd. for Cs_THgsNisOnSSe [M+2H]²⁺, 653.80. Found [M+2H]²⁺, 653.81.

(5h): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuSC"4, L2, and dibenzo[£,<i]furan-4-ylboronic acid stock solution (1 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 5h: HRMS (ESI) Mass. calcd. for C₆₄H₉₇Ni₅Oi₃Se [M+2H]²⁺, 681.83. Found [M+2H]²⁺, 681.83.

(5i): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CUSO4, L2, and phenoxathiin-4-ylboronic acid stock solution (1 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method C. Analytical data for 5i: HRMS (ESI) Mass. calcd. for C₆4H₉₇Ni₅0i₃SSe [M+2H]²⁺, 697.81. Found [M+2H]²⁺, 697.82.

(5j): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuS0₄, L2, and (2-(piperidin-l-yl)pyrimidin-5-yl)boronic acid stock solution (1 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 5j: HRMS (ESI) Mass. calcd. for C₆iHio₂Ni₈Oi₂Se [M+2H]²⁺, 679.35. Found [M+2H]²⁺, 679.36.

(6a): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuS0₄, L2, and (,S)-(4-(2-((tert-butoxycarbonyl)amino)-3-methoxy-3- oxopropyl)phenyl)boronic acid stock solution (2 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 6a: HRMS (ESI) Mass. calcd. for CevHiosNieOieSe [M+H]⁺, 1473.73. Found [M+H]⁺, 1473.71.

(6b): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuS0₄, L2, and (2-oxo-2H-chromen-6-yl)boronic acid stock solution (2 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 6b: HRMS (ESI) Mass. calcd. for

[M+2H] , 670.82. Found [M+2H] , 670.83.

(6c): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuSC"4, L2, and (2-((2-(methoxycarbonyl)phenyl)amino)-6-methylphenyl)boronic acid stock solution (1 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method D. Analytical data for 6c: HRMS (ESI) Mass. calcd. for

[M+2H]²⁺, 718.35. Found [M+2H]²⁺, 718.36.

(6d): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuSC"4, L2, and (4-acetamidophenyl)boronic acid stock solution (2 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 6d: HRMS (ESI) Mass. calcd. for

[M+2H]²⁺, 665.33. Found [M+2H]²⁺, 665.34.

(6e): Prepared according to the general procedure (A) using peptide 1 (100 μΜ) and CuS0₄, L2, and ((8R,9^13^, 145)-13-methyl-17-oxo-7,8,9,l 1, 12,13, 14, 15,16, 17- decahydro-6H-cyclopenta[a]phenanthren-3-yl)boronic acid solution (2 mM) at 37 °C for 3 h. The only exception is 10% DMF was used instead of 5% EtOH. The quenched reaction mixture was analyzed using LC-MS Method B. Analytical data for 6e: HRMS (ESI) Mass. calcd. for C₇oHiiiNi₅Oi₃Se [M+2H]²⁺, 724.88. Found [M+2H]²⁺, 724.89.

(8): Prepared according to the general procedure (A) using peptide 7 (100 μΜ) and CuS0₄, L2, and (2,6-difluorophenyl)boronic acid stock solution (1 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 8: HRMS (ESI) Mass. calcd. for C₆₇H₉₆F₂N₂oOi₇Se [M+H]⁺, 785.32. Found [M+H]⁺, 785.32.

Procedure for 1 mM Reactions

General Procedure (B) for arylation reactions with arylboronic acids

A 20-mL scintillation vial equipped with a stir bar was charged with 3 mL of deionized H₂0, 400 of 1.0 M Tris Buffer (pH = 8.0), 400 μΐ, of peptide 1 (10 mM stock solution). A separate 1.7 mL Eppendorf tube was charged with copper (200 μτηοΐ), ligand (200 μτηοΐ), arylboronic acid (200 μτηοΐ), and 1 mL of 200 proof EtOH. The heterogeneous solution was subjected to sonication for 1 min, vortexed for 30 sec, and 200 μΕ of the resulting solution was added to the peptide solution in the 20 mL scintillation vial. The resulting reaction mixture was capped, and stirred at 800 rpm in 37 °C water bath for the indicated time (10-60 min). The reaction mixture was quenched with 4 mL of 100 mM aqueous ETDA. The resulting mixture was centrifuged at 4,000 rpm for 10 min. The supernatant was filtered through a 0.22 μπι nylon filter and was subjected to purification by HPLC. The ligands were completely removed after filtering through nylon filter. General Procedure for alkylation of selenocysteine

A 20 mL scintillation vial was charged with 3 mL of deionized H₂0, 400 μΐ_^ of 1.0 M Tris Buffer with 200 mM TCEP (pH = 8.0), 400 of peptide 1 (10 mM stock solution). A separate 1.7 mL Eppendorf tube was charged with benzylbromide (200 μπιοΐ) and 1 mL of 200 proof EtOH. 200 μΕ of the resulting solution was added to the peptide solution in the 20 mL scintillation vial. The resulting reaction mixture was capped, and stirred at 800 rpm in 37 °C water bath for 30 min. The reaction mixture was diluted with 4 mL of deionized water. The resulting solution was filtered through a 0.22 μπι nylon filter and was subjected to purification by HPLC.

(4b): Prepared according to the general procedure (B) using peptide 1 (1 mM) and CuS0₄, L2, and (4-methoxyphenyl)boronic acid (10 mM) at 37 °C for 10 minutes. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 4b: HRMS (ESI) Mass. calcd. for

[M+H]⁺, 1302.64. Found [M+H]⁺, 1302.64. 4b was obtained as white power (3.55 mg, 68%) after HPLC purification and lyophlization.

Ofcte

(4g): Prepared according to the general procedure (B) using peptide 1 (1 mM) and CuS0₄, L2, and (4-(methoxycarbonyl)phenyl)boronic acid (10 mM) at 37 °C for 1 h. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 4g: HRMS (ESI) Mass. calcd. for CeiHuoNieOisSe [M+2H]²⁺, 665.82. Found [M+2H]²⁺, 665.82. 4g was obtained as white power (3.91 mg, 74%) after HPLC purification and lyophlization.

(4m): Prepared according to the general procedure (B) using peptide 1 (1 mM) and CuS0₄, L2, and phenylboronic acid (10 mM) at 37 °C for 30 minutes. The quenched reaction mixture was analyzed using LC-MS Method A. Analytical data for 4m: HRMS (ESI) Mass. calcd. for

[M+H]⁺, 1272.63. Found [M+H]⁺, 1272.63. 4m was obtained as white power (3.54 mg, 70%) after HPLC purification and lyophlization.

(4n): Prepared according to the alkylation procedure using peptide 1 (1 mM) and benzylbromide (10 mM) at 37 °C for 30 minutes. The reaction mixture was analyzed using LC-MS Method A. Analytical data for 4n: HRMS (ESI) Mass. calcd. for CssHgsNisOuSe [M+H]⁺, 1286.65. Found [M+H] ⁺, 1286.65. 4n was obtained as white power (3.63 mg, 71%)) after HPLC purification and lyophlization. Stability Studies of Functionalized Selenocysteine Peptides

1) Stability in pH = 8.0 buffer *" ¾: peptide

100 u h urs a (Dha)

A 0.6 mL tube was charged with 178 μΐ. of deionized H₂0, 20 μΐ. of 1.0 M Tris Buffer (pH 8.0, 2 μΐ. of peptide (10 mM stock solution). The resulting reaction mixture was capped and incubated in 37 °C water bath or was left at room temperature for 1 1 hours. 5 μΐ. of the crude reaction mixture was quenched by addition of 200 μΐ. of 50% A: 50% B and was subjected to LC-MS analysis Me thodE. Figure 5 A.

2) Stability in pH = 10.0 buffer peptide

1 a (Dha}

A 0.6 mL tube was charged with 178 μΐ, of deionized H₂0, 20 uL of 1.0 M CAPS Buffer (pH 10.0), 2 μΐ_^ of peptide (10 mM stock solution). The resulting reaction mixture was capped and incubated in 37 °C water bath or was left at room temperature for 1 1 hours. 5 μΐ. of the crude reaction was quenched by addition of 200 μΐ. of 50% A: 50% B and was subjected to LC-MS analysis Me thodE. Figure 5B.

3) Stability under oxidative conditions *^* pe tide

10 μ rt, 1 1 hours 1 a (Dha)

A 300-μί LC-MS vial was charged with 158 μΐ, of deionized H₂0, 20 μΐ, of 1.0 M Tris Buffer (pH = 8.0), 2 μΐ, of peptide (1 mM stock solution), 20 μΐ, of H₂0₂ (100 mM stock solution in water). The resulting reaction mixture was capped and was monitored by LC- MS (Method E).

Example 2 - Drug Release from Functionalized Selenocysteines

As important as new bioconjugation methods are, it is also important for release of the drug molecule once the bioconjugate is delivered to the treatment site.

Figure 19 shows that glutathione (GSH, a tripeptide), which is found in 5 mM concentration in animal cells, can promote dissociation of the native drug or natural product. There is no apparent degradation of the small molecule (LCMS). This is an important breakthrough for the field of drug delivery.

So, selenocysteine peptide conjugates can be delivered to the treatment site and the glutathione found in vivo can promote slow release of the native pharmaceutical.

Example 3 - Conjugation of Vancomycin to Peptides

General Procedure

Vancomycin was conjugated to a variety of peptides. As an example: A 20-mL scintillation vial equipped with a stir bar was charged with a peptide (10 mg, 0.00621 mmol), vancomycin hydrochloride (46 mg, 0.031 mmol), DI H₂0 (5.6 mL), and 620 [iL 1.0 M Tris Buffer (pH = 8.0). The reaction mixture was capped, and stirred at 600 rpm in 37 °C water bath for 3 h. Upon consumption of the peptide (monitored by LCMS) the reaction mixture was quenched with 2 mL of 0.1% TFA in 95:5 H20: MeCN and centrifuged at 4,000 rpm for 4 min. The supernatant was filtered through a 0.22-μπι nylon filter and was subjected to purification by HPLC. Analyzed by LC-MS Method D. Analytical data for 111: HRMS (ESI) Mass. calcd. For Ci₂₇Hi₆₇Cl₂N₂90₄iSe [M+2H]²⁺, 1452.52. Found [M+2H]²⁺, 1453.05. The following peptide conjugate (111) was obtained as white power (7.7 mg, 43%) after FIPLC purification and lyophlization.

Ill With Copper and Ligand

A 0.6 mL Eppendorf tube was charged with 32.5 μΐ. of deionized H₂0, 5 μΐ. of 1.0 M Tris Buffer (pH = 8.0), 10 μΐ. of peptide (1 mM stock solution in H₂0). A separate 0.6 mL Eppendorf tube was charged with copper (0.80 mg, 5 μιηοΐ), ligand (1.4 mg, 5 μιηοΐ), vancomycin (15 mg, 10 μιηοΐ), and 0.25 mL of 200 proof EtOH (making a 40 or 80 mM stock solution, respectively). The heterogeneous solution was subjected to sonication for 1 min, vortexed for 30 sec, and 2.5 μΕ of the resulting solution was added to the peptide solution in the 0.6 mL Eppendorf tube. The resulting reaction mixture was capped, vortexed for 30 seconds, and placed in a 37 °C water bath for the indicated time (3 h). The reaction mixture was quenched with 5 μΐ, of EDTA (200 mM in H₂0), 145 μΐ, of 0.1% TFA in 1 : 1 H₂0:MeCN. The quenched reaction mixture was subjected to immediate LC-MS analysis using LC-MS Method D (injection: 1 uL). Analytical data for 111: HRMS (ESI) Mass. calcd. for Ci₂₇Hi₆₈Cl₂N₂₉0₄iSe [M+3H]²⁺, 968.68. Found [M+3H]³⁺, 969.04.

Without Copper and Ligand

A 0.6 mL Eppendorf tube was charged with 32.5 μΕ of deionized H₂0, 5 μΕ of 1.0 M Tris Buffer (pH = 8.0), 10 μΕ of peptide (1 mM stock solution in H₂0). A separate 0.6 mL Eppendorf tube was charged with vancomycin (15 mg, 10 μιηοΐ), and 0.25 mL of DI H₂0 (making a 80 mM stock solution). The heterogeneous solution was subjected to sonication for 1 min, vortexed for 30 sec, and 2.5 μΕ of the resulting solution was added to the peptide solution in the 0.6 mL Eppendorf tube. The resulting reaction mixture was capped, vortexed for 30 seconds, and placed in a 37 °C water bath for the indicated time (3 h). The reaction mixture was quenched with 150 μΕ of 0.1% TFA in 1 : 1 H₂0:MeCN. The quenched reaction mixture was subjected to immediate LC-MS analysis using LC-MS Method D (injection: 1 uL). Analytical data for 111: HRMS (ESI) Mass. calcd. for Ci₂₇Hi₆₈Cl₂N₂₉0₄iSe [M+3H]²⁺, 968.68. Found [M+3H]³⁺, 969.03.

Example 4 - Functionalized Selenocysteines as Antibacterial Agents

Many bacteria have developed a resistance to previously used antibacterial pharmaceuticals.

Vancomycin has been shown to be active against a few strains of gram-positive bacteria by inhibiting the peptidoglycan synthesis. However, vancomycin is not active against gram-negative bacteria due to the bacterium's outer cell membrane. Additionally several strains of gram-positive bacteria have developed resistance towards vancomycin. Vancomycin was conjugated to a variety of antibacterial peptides and screened against (a) resistant strains of gram-positive bacteria and b) gram-negative bacteria. See Figure 20, Figure 21, Figure 22, and Table 3.

While there was some modest increase in activity for a few examples it is also important to point out that the MIC values are based on mass and the mass of the peptide conjugates are doubled (sometimes more) relative to vancomycin. This means that the conjugates of the invention are an order of magnitude more active than vancomycin.

Table 3. MIC of vancomycin alone as compared to vancomycin/peptide conjugates.

Example 5 - Selenocysteine Antibody-Drug Conjugates

Polyglycine peptides with a conjugated small molecule can be effectively sortased onto a mutant trastuzumab antibody. See Figure 23, Figure 24, and Figure 25.

An antibody-drug conjugate (ADC) of genistein was screened against BT-474 cells (mammary gland cancer cells). These results show that selenocysteine is not toxic to cells and can be used as a handle in bioconjugation.

Example 6 - Selenocysteine or Cysteine Macrocyclization

Phallotoxins and amatoxins are classes of highly cytotoxic macrocyclic natural products that have a cysteine/tryptophan linkage. These classes of natural products have been isolated from the poisonous mushrooms 1937-1941. Previous strategies to synthesize these natural products required two steps (Savige-Fontana reaction) or monomer synthesis to introduce the cysteine/tryptophan linkage.

However, the selenocysteine or cysteine conjugates described herein may be cyclized to form the required linkage. See Figures 26-3 IB.

Example 7 - Cysteine Arylation

Various disulfides, mixed or symmetrical, were arylated with a variety of arylating agents. Variations in the peptide sequence were also investigated to determine their influence on the success of the arylation reaction. Copper-catalyzed, rhodium-catalyzed, or metal-free conditions were used. See Figures 32A-38.

Example 8 - Copper-free Synthesis of Arylated Selenocysteine

A 0.6 mL Eppendorf tube was charged with 32.5 iL of deionized H₂0, 5 iL of 1.0 M Tris Buffer (pH = 8.0), 10 iL of peptide (1 mM stock solution in H₂0). A separate 0.6 mL Eppendorf tube was charged with (hetero)aryl nucleophile (40 or 80 μπιοΐ) and 0.5 mL or 0.25 mL of 200 proof EtOH (making a 40 or 80 mM stock solution, respectively). The heterogeneous solution was subjected to sonication for 1 min, vortexed for 30 sec, and 2.5 μΕ of the resulting solution was added to the peptide solution in the 0.6 mL Eppendorf tube. The resulting reaction mixture was capped, vortexed for 30 seconds, and placed in a 37 °C water bath for the indicated time (3 h). The reaction mixture was quenched with 50 [iL of H₂0 and 100 [iL of 0.2% TFA in MeCN. The quenched reaction mixture was subjected to immediate LC-MS analysis.

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. published patent applications cited herein are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

1. A compound comprising substructure I:

wherein, independently for each occurrence,

2. The compound of claim 1, wherein substituted aryl radical.

3. The compound of claim 1, wherein substituted phenyl radical.

4. The compound of claim 1, wherein unsubstituted aryl radical.

5. The compound of claim 1, wherein substituted heteroaryl radical.

6. The compound of claim 1, wher

ein is a substituted indole, pyrazolo, thiophene, pyrimidine, benzofuran, or isoxazole radical.

7. The compound of claim 1, wherein an unsubstituted heteroaryl radical

8. The compound of claim 1, wherei

n is an unsubstituted indole, pyrazolo, thiophene, pyrimidine, benzofuran, or isoxazole radical.

-127-

selenocysteine.

11. The compound of any one of claims 1-9, wherein one or more of A¹ and A⁵ comprises arginine, cysteine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

12. A compound comprising substructure II:

II wherein, independently for each occurrence,

is a substituted or unsubstituted aryl or heteroaryl radical, provided not a perfluoroaryl radical; x is 0, 1, 2, 3, 4, 5, or 6; and

R is H or alkyl.

13. The compound of claim 12, wherein is a substituted aryl radical.

14. The compound of claim 12, wherein is a substituted phenyl radical.

15. The compound of claim 12, wherein unsubstituted aryl radical.

16. The compound of claim 12, wherein

is a substituted heteroaryl radical. B

17. The compound of claim 12, wherein ^ is a substituted indole, pyrazolo, thiophene, pyrimidine, benzofuran, or isoxazole radical.

18. The compound of claim 12, wherein an unsubstituted heteroaryl radical

19. The compound of claim 12, wherein

cysteine.

22. The compound of any one of claims 12-20, wherein one or more of A¹ and A⁵ comprises arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

23. The compound of any one of claims 1-22, wherein A¹ is an amine protecting group selected from the group consisting of an N,0-acetal, allyloxycarbonyl (Aloe), benzyl (Bn), benzyloxycarbonyl (Cbz), benzyloxymethyl (BOM), t-butoxycarbonyl (Boc), t- butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), diphenylmethyl, diphenylmethylene, ethoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc), p- methoxybenzyl (PMB), methoxycarbonyl, methoxymethyl (MOM), /?-methoxyphenyl (PMP), /?-nitrocinnamyloxycarbonyl (Noc), tosyl (Ts), 2-tosylethoxycarbonyl (Tsoc), 2,2,2- trichloroethoxycarbonyl (Troc), trifluoroacetyl, triisopropylsilyl (TIPS), trimethylsilyl (TMS), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), 2-(trimethylsilyl)ethoxymethyl (SEM), or trityl (Tr).

24. The compound of any one of claims 1-23, wherein A⁵ is -0(carboxylate protecting group); and the carboxylate protecting group is selected from the group consisting of allyl, benzyl, benzyloxymethyl (BOM), t-Bu, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), diphenylmethyl, 9-fluorenylmethyl (Fm), 2-methoxyethoxymethyl (MEM), methoxymethyl (MOM), /?-nitrobenzyl (P B), an ester, a 1,3-oxazoline, pivaloyloxymethyl (Pom), 2-tosylethyl (TSE), 2,2,2-trichloroethyl (TCE), triethylsilyl (TES), trimethylsilyl (TMS), 2-(trimethylsilyl)ethoxymethyl (SEM), or 2-(trimethylsilyl)ethyl (TMSE).

25. The compound of any one of claims 1-22, wherein A¹ or A⁵ is an antimicrobial peptide, such as magainin/pexiganan (a 22-amino acid linear peptide that may be isolated from the skin of the African clawed frog (Xenopus laevis)), omiganan (a cationic peptide that may be derived from indolicidin), OP- 145 (a 24-mer peptide that may be derived from LL-37 that may bind to lipopoly saccharides or lipoteichoic acid), novexatin (a cyclic cationic peptide of 1093 Da), LTX-109 (Lytixar, a membrane-degrading peptide), NVB302 (a class B lantibiotic), MU1140 (a lantibiotic), arenicin (a 21-mer that is rich in arginine and hydrophobic amino acids), avidocin (a R-type bacteriocin that may be derived from Pseudomanas aeruginosa), purocin (a R-type bacteriocin that may be derived from Pseudomanas aeruginosa), IMX924 (a 5-mer with defense regulation capabilities),

GIGKFLHSAKKFGKAFVGEIMNS, or GIKKFLKKAGKFGKAF.

26. The compound of any one of claims 1-25, wherein R is H.

27. The compound of any one of claims 1-26, wherein x is 1, 2, or 3.

28. The compound of any one of claims 1-26, wherein x is 1.

29. A compound comprising substructure III:

III

is a substituted or unsubstituted aryl or heteroaryl diradical; and

R is H or alkyl.

A compound comprising substructure V or substructure VII:

V

VII wherein, independently for each occurrence,

A⁵ is OH, -0(carboxylate protecting group), a natural or unnatural amino acid, a peptide, an oligopeptide, a polypeptide, or a protein; x is 0, 1, 2, 3, 4, 5, or 6; _B1A

is a substituted or unsubstituted aryl or heteroaryl diradical; and

R is H or

31. TThhee ccoormi pound of claim 29 or 30, wherein none of A¹, A², A³, A⁴, and A⁵ comprises selenocysteine.

32. The compound of claim 29 or 30, wherein one or more of A¹, A², A³, A⁴, and A⁵ comprises arginine, cysteine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

33. The compound of any one of claims 29-32, wherein

diradical. B'

34. The compound of any one of claims 29-32, wherein

diradical.

The compound of any one of claims 29-32, wherein

is an unsubstituted aryl

36. The compound of any one of claims 29-32, wherein

is a substituted heteroaryl diradical.

37. The compound of any one of claims 29-32, wherein

38. The compound of any one of claims 29-32, wherein ^ is an unsubstituted heteroaryl diradical.

39. The compound of any one of claims 29-32, wherein

is an unsubstituted indole, pyrazolo, thiophene, pyrimidine, benzofuran, or isoxazole diradical. A compound comprising substructure IV:

IV wherein, independently for each occurrence,

is a substituted or unsubstituted aryl or heteroaryl diradical, provided is not a perfluoroaryl diradical; and R is H or alkyl.

41. A compound comprising substructure VI or substructure VIII:

VIII wherein, independently for each occurrence, A¹ is H, an amine protecting group, a natural or unnatural amino acid, a peptide, an oligopeptide, a polypeptide, or a protein;

R is H or alkyl.

42. The compound of claim 40 or 41, wherein none of A¹, A², A³, A⁴, and A⁵ comprises cysteine.

43. The compound of claim 40 or 41, wherein one or more of A¹, A², A³, A⁴, and A⁵ comprises arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.

44. The compound of any one of claims 40-43, wherein

is a substituted aryl diradical.

45. The compound of any one of claims 40-43, wherein

is a substituted phenyl diradical.

46. The compound of any one of claims 40-43, wherein

is an unsubstituted aryl diradical.

47. The compound of any one of claims 40-43, wherein

is a substituted heteroaryl diradical. _B1 B

48. The compound of any one of claims 40-43, wherein ^ is a substituted indole, pyrazolo, thiophene, pyrimidine, benzofuran, or isoxazole diradical.

49. The compound of any one of claims 40-43, wherein

is an unsubstituted heteroaryl diradical.

50. The compound of any one of claims 40-43, wherein

is an unsubstituted indole, pyrazolo, thiophene, pyrimidine, benzofuran, or isoxazole diradical.

51. The compound of any one of claims 29-50, wherein A², A³, and A⁴ are natural or unnatural amino acids.

52. The compound of any one of claims 29-51, wherein A is an amine protecting group selected from the group consisting of an N,0-acetal, allyloxycarbonyl (Aloe), benzyl (Bn), benzyloxycarbonyl (Cbz), benzyloxymethyl (BOM), t-butoxycarbonyl (Boc), t- butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), diphenylmethyl, diphenylmethylene, ethoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc), p- methoxybenzyl (PMB), methoxycarbonyl, methoxymethyl (MOM), /?-methoxyphenyl (PMP), /?-nitrocinnamyloxycarbonyl (Noc), tosyl (Ts), 2-tosylethoxycarbonyl (Tsoc), 2,2,2- trichloroethoxycarbonyl (Troc), trifluoroacetyl, triisopropylsilyl (TIPS), trimethylsilyl (TMS), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), 2-(trimethylsilyl)ethoxymethyl (SEM), or trityl (Tr).

53. The compound of any one of claims 29-52, wherein A⁵ is -0(carboxylate protecting group); and the carboxylate protecting group is selected from the group consisting of allyl, benzyl, benzyloxymethyl (BOM), t-Bu, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), diphenylmethyl, 9-fluorenylmethyl (Fm), 2-methoxyethoxymethyl (MEM), methoxymethyl (MOM), /?-nitrobenzyl (P B), an ester, a 1,3-oxazoline, pivaloyloxymethyl (Pom), 2-tosylethyl (TSE), 2,2,2-trichloroethyl (TCE), triethylsilyl (TES), trimethylsilyl (TMS), 2-(trimethylsilyl)ethoxymethyl (SEM), or 2-(trimethylsilyl)ethyl (TMSE).

54. The compound of any one of claims 29-51, wherein A¹ or A⁵ is an antimicrobial peptide, such as magainin/pexiganan (a 22-amino acid linear peptide that may be isolated from the skin of the African clawed frog (Xenopus laevis)), omiganan (a cationic peptide that may be derived from indolicidin), OP- 145 (a 24-mer peptide that may be derived from LL-37 that may bind to lipopoly saccharides or lipoteichoic acid), novexatin (a cyclic cationic peptide of 1093 Da), LTX-109 (Lytixar, a membrane-degrading peptide), NVB302 (a class B lantibiotic), MU1140 (a lantibiotic), arenicin (a 21-mer that is rich in arginine and hydrophobic amino acids), avidocin (a R-type bactenocin that may be derived from Pseudomanas aeruginosa), purocin (a R-type bactenocin that may be derived from Pseudomanas aeruginosa), IMX924 (a 5-mer with defense regulation capabilities),

GIGKFLHSAKKFGKAFVGEIMNS, or GIKKFLKKAGKFGKAF.

55. The compound of any one of claims 29-54, wherein R is H.

56. The compound of any one of claims 29-55, wherein x is 1, 2, or 3.

57. The compound of any one of claims 29-55, wherein x is 1. A method according to Scheme 1

Scheme la

Scheme lb

Scheme lc

Scheme Id wherein, independently for each occurrence,

A¹ is H, an amine protecting group, a natural or unnatural amino acid, a peptide, oligopeptide, a polypeptide, or a protein; A², A³, and A⁴ are selected from the group consisting of a natural amino acid, an unnatural amino acid, and a plurality of natural amino acids or unnatural amino acids;

A⁵ is OH, -0(carboxylate protecting group), a natural or unnatural amino acid, a peptide, an oligopeptide, a polypeptide, or a protein; x is 0, 1, 2, 3, 4, 5, or 6,

is a substituted or unsubstituted aryl or heteroaryl radical;

is a substituted or unsubstituted aryl or heteroaryl diradical

is a heteroaromatic moiety substituted with an electron withdrawing x is 0, 1, 2, 3, 4, 5, or 6; and R is H or alkyl.

The method of claim 58, wherein bstituted aryl radical.

61. The method of claim 58, wherein bstituted phenyl radical.

62. The method of claim 58, wherein unsubstituted aryl radical.

63. The method of claim 58, wherein bstituted heteroaryl radical.

64. The method of claim 58, whe

rein is a substituted indole, pyrazolo, thiophene, pyrimidine, benzofuran, or isoxazole radical.

65. The method of claim 58, wherein n unsubstituted heteroaryl radical

66. The method of claim 58, where

in is an unsubstituted indole, pyrazolo, thiophene, pyrimidine, benzofuran, or isoxazole radical.

-146-

-147-

or or a regioisomer or stereoisomer thereof.

A method according to Scheme 3:

Scheme 3a

Scheme 3b

Scheme 3c

Scheme 3d wherein, independently for each occurrence,

69. The method of claim 68, wherein bstituted aryl radical

70. The method of claim 68, wherein bstituted phenyl radical

The method of claim 68, wherein bstituted heteroaryl radical

72. The method of claim 68, whe

The method of claim 68, wherein

IS

-151- WO 2016/205798

or a regioisomer or stereoisomer thereof.

74. The method of any one of claims 58-73, wherein the Cu/ligand comprises CuS0₄.

75. The method of any one of claims 58-74, wherein the Cu/ligand comprises a substituted or unsubstituted bipyridine ligand or a substituted or unsubstituted phenanthroline ligand.

76. The method of any one of claims 58-74, wherein the Cu/ligand comprises 4,4'-di fert-butyl-2,2'-bipy.

77. A method according to Scheme 7:

Scheme 7a

Scheme 7b

Scheme 7c

Scheme 7d

The method of claim 77, wherein bstituted aryl radical.

79. The method of claim 77, wherein bstituted phenyl radical.

80. The method of claim 77, wherein bstituted heteroaryl radical.

81. The method of claim 77, whe

-156-

-157-

or a regioisomer or stereoisomer thereof. A method according to Scheme 2:

Scheme 2a

Scheme 2b

Scheme 2c

Scheme 2d wherein, independently for each occurrence,

84. The method of claim 83, wherein bstituted aryl radical.

85. The method of claim 83, wherein bstituted phenyl radical.

86. The method of claim 83, wherein unsubstituted aryl radical.

87. The method of claim 83, wherein bstituted heteroaryl radical.

88. The method of claim 83, whe

89. The method of claim 83, wherein n unsubstituted heteroaryl radical.

90. The method of claim 83, where

-162-

-163-

-164-

or or a regioisomer or stereoisomer thereof.

A method according to Scheme 4:

Scheme 4a

Scheme 4b

Scheme 4c

Scheme 4d wherein, independently for each occurrence,

rovided is not a perfluoroaryl radical;

a perfluoroaryl radical;

The method of claim 92, wherein bstituted aryl radical

The method of claim 92, wherein bstituted phenyl radical

The method of claim 92, wherein bstituted heteroaryl radical

96. The method of claim 92, whe

The method of claim 92, wherein

IS

-169-

or a regioisomer or stereoisomer thereof.

The method of any one of claims 83-97, wherein the Cu/ligand comprises CuS0₄

99. The method of any one of claims 83-98, wherein the Cu/ligand comprises a substituted or unsubstituted bipyridine ligand or a substituted or unsubstituted phenanthroline ligand.

100. The method of any one of claims 83-98, wherein the Cu/ligand comprises 4,4'-di- fer/-butyl-2,2'-bipy.

101. A method according to Scheme 5:

Scheme 5a

Scheme 5b

Scheme 5c A

Scheme 5d wherein, independently for each occurrence,

The method of claim 101, wherein is a substituted aryl radical.

The method of claim 101, wherein

is a substituted phenyl radical.

104. The method of claim 101, wherein unsubstituted aryl radical

The method of claim 101, wherein ubstituted heteroaryl radical

106. The method of claim 101, wher

107. The method of claim 101, wherein unsubstituted heteroaryl radical

108. The method of claim 101, whe

rein is an unsubstituted indole, pyrazolo, thiophene, pyrimidine, benzofuran, or isoxazole radical.

109. The method of claim 101, wherein

is

-174-

stereoisomer thereof. A method according to Scheme 6:

Scheme 6a

Scheme 6b

Scheme 6c

Scheme 6d wherein, independently for each occurrence,

is an aryl or heteroaryl radical substituted with at least one electron donating group, provided

is not a perfluoroaryl radical;

a perfluoroaryl radical;

111. The method of claim 110, wherein is a substituted aryl radical.

112. The method of claim 110, wherein is a substituted phenyl radical.

113. The method of claim 110, wherein

is a substituted heteroaryl radical.

114. The method of claim 110, wherein ^ is a substituted indole, pyrazolo, thiophene, pyrimidine, benzofuran, or isoxazole radical.

-179-

-180-

or a regioisomer or stereoisomer thereof.

116. The method of any one of claim 101-115, wherein the Rh/ligand comprises [RhCp*Cl₂]₂.

117. A method according to Scheme 8:

Scheme 8b

Scheme 8c

Scheme 8d wherein, independently for each occurrence,

rovided is not a perfluoroaryl radical;

a perfluoroaryl radical;

118. The method of claim 117, wherein is a substituted aryl radical

119. The method of claim 117, wherein

is a substituted phenyl radical

( B* )

120. The method of claim 117, wherein is a substituted heteroaryl radical.

(?)

121. The method of claim 117, wherein is a substituted indole, pyrazolo, thiophene, pyrimidine, benzofuran, or isoxazole radical.

122. The method of claim 117, wherein

IS

-185- WO 2016/205798

or a regioisomer or stereoisomer thereof.

123. The method of any one of claims 58-122, wherein A², A³, and A⁴ are natural or unnatural amino acids.

124. The method of any one of claims 58-123, wherein the first solvent, the second solvent, the third solvent, the fourth solvent, the fifth solvent, the sixth solvent, the seventh solvent, or the eighth solvent is water, DMF, CH₃CN, CH₃CH₂OH, CH₃OH, DMSO, tris(2- carboxyethyl)phosphine (TCEP), dibutyl ether, tetrahydrofuran (THF), 1,4-dioxane, DME, dichloromethane, dichloroethane, acetone, diethyl ether, hexanes, or a mixture thereof.

125. The method of any one of claims 58-124, wherein the first buffer, the second buffer, the third buffer, the fourth buffer, the fifth buffer, the sixth buffer, the seventh buffer, or the eighth buffer comprises Na₃P0₄, MES, or tris(hydroxymethyl)aminomethane (TRIS).

126. The method of any one of claims 58-125, wherein the first time, the second time, the third time, the fourth time, the fifth time, the sixth time, the seventh time, or the eighth time is from about 30 min to about 24 h.

127. The method of any one of claims 58-126, wherein A¹ is an amine protecting group selected from the group consisting of an N,0-acetal, allyloxycarbonyl (Aloe), benzyl (Bn), benzyloxycarbonyl (Cbz), benzyloxymethyl (BOM), t-butoxycarbonyl (Boc), t- butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), diphenylmethyl, diphenylmethylene, ethoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc), p- methoxybenzyl (PMB), methoxycarbonyl, methoxymethyl (MOM), /?-methoxyphenyl (PMP), /?-nitrocinnamyloxycarbonyl (Noc), tosyl (Ts), 2-tosylethoxycarbonyl (Tsoc), 2,2,2- trichloroethoxycarbonyl (Troc), trifluoroacetyl, triisopropylsilyl (TIPS), trimethylsilyl (TMS), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), 2-(trimethylsilyl)ethoxymethyl (SEM), or trityl (Tr).

128. The method of any one of claims 58-127, wherein A⁵ is -0(carboxylate protecting group); and the carboxylate protecting group is selected from the group consisting of allyl, benzyl, benzyloxy methyl (BOM), t-Bu, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), diphenylmethyl, 9-fluorenylmethyl (Fm), 2-methoxyethoxymethyl (MEM), methoxymethyl (MOM), /?-nitrobenzyl (P B), an ester, a 1,3-oxazoline, pivaloyloxymethyl (Pom), 2-tosylethyl (TSE), 2,2,2-trichloroethyl (TCE), triethylsilyl (TES), trimethylsilyl (TMS), 2-(trimethylsilyl)ethoxymethyl (SEM), or 2-(trimethylsilyl)ethyl (TMSE).

129. The method of any one of claims 58-128, wherein R is H.

130. The method of any one of claims 58-129, wherein x is 1, 2, or 3.

131. The method of any one of claims 58-129, wherein x is 1.

132. The method of any one of claims 58-131, wherein the first, second, third, fourth, fifth, sixth, seventh, or eighth temperature is between about 10°C and about 50 °C.

133. The method of any one of claims 58-132, wherein the catalytic metal is not Cu or Rh or absent, but is selected from the group consisting of Fe, Co, Ni, Au, Pd, Pt, Ru, and Ir.

134. A method of killing or inhibiting the growth or proliferation of a bacterium, a fungus, a virus, or a parasite, comprising the step of: contacting with the bacterium, fungus, virus, or parasite an effective amount of a compound of any one of claims 1-57, thereby treating killing or inhibiting the growth or proliferation of the bacterium, fungus, virus, or parasite.

135. A method of treating a disease in a subject in need thereof comprising the step of: administering to the subject an effective amount of a compound of any one of claims 1-57, thereby treating the disease.

136. The method of claim 135, wherein the disease is an infection, for example, a bacterial infection, a fungal infection, a viral infection, or a parasitic infection.

137. The method of claim 135, wherein the disease is cancer.