Peptide therapeutics including amidines

Abstract

A compound having the same chemical structure as a peptide therapeutic free of amidine moieties other than having one or more amide moieties of the peptide therapeutic free of amidine moieties replaced by one or more amidine moieties. A method of forming a product compound including one or more amidine moieties includes reacting a thioimidatc-containing compound including a thioimidate with a nitrogen-containing compound to form the product compound including an amidine in place of the thioimidate in the thioimidate-containing compound. The nitrogen-containing compound includes a primary amine, a secondary amine, a salt thereof, or a combination thereof. The nitrogen-containing compound is an amino acid, an amino acid derivative, an aliphatic amine, an aromatic amine, or a peptide. The thioimidate includes -S-R1, wherein R1 is chosen from -OH, -H, substituted or unsubstituted (C1-C20)hydrocarbyl, substituted or unsubstituted (C1-C20)alkyl, substituted or unsubstituted (C6-C20)aryl, (C6-C20)heteroaryl, acyl, carbonyl, carbonate, and carbamate.

Description

900.344W01, ISURF 05723PEPTIDE THERAPEUTICS INCLUDING AMIDINESCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 756,666 filed Feb. 10, 2025, the disclosure of which is incorporated herein in its entirety by reference.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under R35 GM142883 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Peptide therapeutics have emerged as a significant class of drugs due to their high specificity, potency, and ability to modulate complex biological processes and drug targets. However, a major challenge in the development of peptide-based drugs is their susceptibility to rapid degradation by proteolytic enzymes in vivo, which limits their therapeutic efficacy and pharmacokinetic profiles. Proteases, which catalyze the hydrolysis of peptide bonds, pose a significant barrier to the stability and bioavailability of peptide therapeutics.

[0004] Traditional strategies to enhance the metabolic stability of peptides include the incorporation of non-natural amino acids, backbone modifications, cyclization, and conjugation to macromolecular carriers. These approaches aim to disrupt the recognition of proteolytic enzymes without compromising the biological activity of the peptides. Despite these efforts, the need for more effective methods to improve peptide stability remains.SUMMARY OF THE INVENTION

[0005] Various aspects of the present disclosure provide a compound having the same chemical structure as a peptide therapeutic free of amidine moieties other than having one or more amide moieties of the peptide therapeutic free of amidine moieties replaced by one or more amidine moieties.

[0006] Various aspects of the present disclosure provide a pharmaceutical composition including a compound having the same chemical structure as a peptide900.344W01, ISURF 05723therapeutic free of amidine moieties other than having one or more amide moieties of the peptide therapeutic free of amidine moieties replaced by one or more amidine moieties.

[0007] Various aspects of the present disclosure provide a method of treating a condition using a compound having the same chemical structure as a peptide therapeutic free of amidine moieties other than having one or more amide moieties of the peptide therapeutic free of amidine moieties replaced by one or more amidine moieties. The method includes administering the compound to a patient to treat the condition.

[0008] Various aspects of the present disclosure provide a method of forming a product compound including one or more amidine moieties. The method includes reacting a thioimidate-containing compound including a thioimidate with a nitrogen-containing compound to form the product compound including an amidine in place of the thioimidate in the thioimidate-containing compound. The nitrogen-containing compound includes a primary amine, a secondary amine, a salt thereof, or a combination thereof. The nitrogencontaining compound is an amino acid, an amino acid derivative, an aliphatic amine, an aromatic amine, or a peptide. The thioimidate includes -S-R1, wherein R1is chosen from -OH, -H, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (Ce-C2o)aryl, (Ce-C2o)heteroaryl, acyl, carbonyl, carbonate, and carbamate.

[0009] Various aspects of the present disclosure provide a method of forming a peptide including one or more amidine moieties. The method includes reacting a thioimidate-containing compound including a thioimidate, the thioimidate-containing compound bound to a solid support, with a nitrogen-containing compound to form the peptide including an amidine in place of the thioimidate in the thioimidate-containing compound. The nitrogencontaining compound includes a primary amine or a secondary amine. The nitrogencontaining compound is an amino acid, an amino acid derivative, or a peptide. The thioimidate-containing compound is an amino acid, an amino acid derivative, a peptide, or a peptidomimetic. The thioimidate includes -C(=NHR2+)-S-R1, wherein R1and R2are independently chosen from -OH, -H, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (Ce-C2o)aryl, (Ce-C2o)heteroaryl, acyl, carbonyl, carbonate, and carbamate.

[0010] Various compounds of the present disclosure have advantages over other peptides. Various aspects of the method of the present disclosure for forming compounds such as peptides including at least one amidine bond have advantages over other methods of peptide synthesis. For example, various compounds of the present disclosure have metabolic900.344W01, ISURF 05723stability advantages over other peptides, with the one or more amidine moieties conferring enhanced resistance to proteolytic degradation, while the amidine moieties mimic the hydrogen-bonding patterns of native peptide bonds. Various compounds of the present disclosure provide an improved pharmacokinetic profile compared to the native peptide therapeutic free of amidine moieties due to increased stability against enzymatic hydrolysis enabled by the one or more amidine moieties. Various aspects of the method of the present disclosure of forming peptides provides convenient access to various compounds of the present disclosure.

[0011] Various compounds of the present disclosure advantageously preserve the biological activity of the native peptide therapeutic free of amidine moieties by maintaining the structural integrity of the peptide backbone. Various compounds of the present disclosure advantageously provide the potential to modulate receptor selectivity and binding affinity, and can improve metabolic stability.

[0012] Various aspects of the method of the present disclosure for forming peptides provide advantageous synthetic flexibility, with the ability to introduce amidines at various positions within the peptide sequence using conventional solid-phase peptide synthesis techniques as well as solution-phase peptide synthesis. Various aspects of the method of the present disclosure provide advantageous compatibility with a wide range of peptide-drug discovery programs, allowing for the modification of existing therapeutics.

[0013] Various aspects of the method of the present disclosure for forming peptides and various compounds of the present disclosure provide advantageous therapeutic potential, with various compounds including one or more amidines providing therapeutic applicability to a broad spectrum of conditions, such as chronic pain, inflammation, and cancer pain. Various aspects of the method of the present disclosure for forming peptides provide the advantageous potential to rescue promising peptide drug candidates that were previously limited by poor metabolic stability.

[0014] Various aspects of the method of the present disclosure for forming peptides and the compounds of the present disclosure advantageously establish amidines as a platform technology for enhancing peptide stability, which can be applied to various peptide-based drugs. Various aspects of the method of the present disclosure for forming peptides advantageously provide the opportunity to develop a new class of peptide therapeutics with improved stability and efficacy profiles while retaining or improving on the biological activity of existing peptide therapeutics. Various aspects of the aspects of the method of the900.344WG1, ISURF 05723present disclosure for forming peptides advantageously provide access to a new class of peptides that are useful in various industries such as the cosmetics and food industries.BRIEF DESCRIPTION OF THE FIGURES

[0015] The drawings illustrate generally, by way of example, but not by way of limitation, various aspects of the present invention.

[0016] FIG. 1 A illustrates proteolytic cleavage of a peptide with an enzyme, in accordance with various aspects of the present disclosure.

[0017] FIG. IB illustrates various techniques for protecting peptide bonds from proteolysis.

[0018] FIG. 1C illustrates stabilization of peptide bonds from proteolysis using an amidine, in accordance with various aspects of the present disclosure.

[0019] FIG. 2A illustrates one-pot palladium catalyzed allyl deprotection and preactivation with PyBOP / HOBt to enable access to thioimidates which can undergo elongation and final amidine formation on-resin, in accordance with various aspects of the present disclosure.

[0020] FIG. 2B illustrates primary thioimidate glycine hydroiodide 2, which effected efficient amidinylation on-resin in the presence of imidazole, in accordance with various aspects of the present disclosure.

[0021] FIG. 2C illustrates three amidinopeptide derivatives of Leu-enkephalin synthesized herein, in accordance with various aspects of the present disclosure.

[0022] FIG. 3 illustrates Leu-enkaphalin (left) and amidinopeptide G1 (right), along with enzymatic proteolysis half-life of cleavage of the Tyri-Gly2 site by metallopeptidase AP-N, in accordance with various aspects of the present disclosure.

[0023] FIG. 4 illustrates half-life of cleavage of Leu-Enkephalin and G1 and G3 derivatives with aminopeptidase-N, in accordance with various aspects of the present disclosure.900.344W01, ISURF 05723DETAILED DESCRIPTION OF THE INVENTION

[0024] Reference will now be made in detail to certain aspects of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0025] Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of “about 0.1% to about 5%’’ or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0026] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as “A, B, or A and B.” In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section.

[0027] In the methods described herein, the acts can be carried out in a specific order as recited herein. Alternatively, in any aspect(s) disclosed herein, specific acts may be carried out in any order without departing from the principles of the invention, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately or the plain meaning of the claims would require it. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0028] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range. The term “substantially”900.344W01, ISURF 05723as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term “substantially free of” as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that about 0 wt% to about 5 wt% of the composition is the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than or equal to about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.

[0029] The term “substituted” as used herein in conjunction with a molecule or an organic group as defined herein refers to the state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The term “functional group” or “substituent” as used herein refers to a group that can be or is substituted onto a molecule or onto an organic group. Examples of substituents or functional groups include, but arc not limited to, a halogen (c.g., F, Cl, Br, and I); an oxygen atom in groups such as hydroxy groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups. Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, Cl, Br, I, OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azido, CF3, OCF3, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2)O-2N(R)C(O)R, (CH2)O-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, and C(=NOR)R, wherein R can be hydrogen or a carbon-based moiety; for example, R can be hydrogen, (Ci-Cioojhydrocarbyl, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl; or wherein two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl.

[0030] The term “organic group” as used herein refers to any carbon-containing functional group. Examples can include an oxygen-containing group such as an alkoxy group, aryloxy group, aralkyloxy group, oxo(carbonyl) group; a carboxyl group including a900.344W01, ISURF 05723carboxylic acid, carboxylate, and a carboxylate ester; a sulfur-containing group such as an alkyl and aryl sulfide group; and other heteroatom-containing groups. Non-limiting examples of organic groups include OR, OOR, OC(O)N(R)2, CN, CF3, OCF3, R, C(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2)O-2N(R)C(0)R, (CH2)O-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, C(=NOR)R, and substituted or unsubstituted (Ci-Cioo)hydrocarbyl, wherein R can be hydrogen (in examples that include other carbon atoms) or a carbon-based moiety, and wherein the carbon-based moiety can be substituted or unsubstituted.

[0031] The term “alkyl” as used herein refers to straight chain and branched alkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbons or, in some aspects, from 1 to 8 carbon atoms. Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. As used herein, the term “alkyl” encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl.Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

[0032] The term “hydrocarbon” or “hydrocarbyl” as used herein refers to a molecule or functional group that includes carbon and hydrogen atoms. The term can also refer to a molecule or functional group that normally includes both carbon and hydrogen atoms but wherein all the hydrogen atoms are substituted with other functional groups. The term “hydrocarbyl” refers to a functional group derived from a straight chain, branched, or cyclic hydrocarbon, and can be alkyl, alkenyl, alkynyl, aryl, cycloalkyl, acyl, or any combination thereof. Hydrocarbyl groups can be shown as (Ca-Cb)hydrocarbyl, wherein a and b are integers and mean having any of a to b number of carbon atoms. For example, (Ci-C4)hydrocarbyl means the hydrocarbyl group can be methyl (Ci), ethyl (C2), propyl (C3), or butyl (C4), and (Co-Cb)hydrocarbyl means in certain aspects there is no hydrocarbyl group. A hydrocarbylene group is a diradical hydrocarbon, e.g., a hydrocarbon that is bonded at two locations.900.344W01, ISURF 05723Compound, compositions including the compound, and methods of using the compound.

[0033] Various aspects of the present disclosure provide a compound having the same chemical structure as a peptide therapeutic free of amidine moieties other than having one or more amide moieties of the peptide therapeutic free of amidine moieties replaced by one or more amidine moieties. Each one of the replaced amide moieties are replaced by a single amidine moiety.

[0034] The compound can be a peptide therapeutic. The compound can have the same, about the same, or enhanced biological activity compared to the peptide therapeutic free of amidine moieties.

[0035] As compared to the peptide therapeutic free of amidine moieties, the compound can have any number of amide moieties replaced by amidine moieties. For example, the compound can have one and not more than one amide moiety of the peptide therapeutic free of amidine moieties replaced by an amidine moiety. The compound can have two and not more than two amide moieties of the peptide therapeutic free of amidine moieties replaced by amidine moieties. The compound can have three and not more than three amide moieties of the peptide therapeutic free of amidine moieties replaced by amidine moieties. The compound can have 1-100 amide moieties of the peptide therapeutic free of amidine moieties replaced by amidine moieties, or 1-10, or 1-5, or less than or equal to 100 and greater than or equal to 1 and less than, equal to, or greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 amide moieties of the peptide therapeutic free of amidine moieties replaced by amidine moieties.

[0036] At least one or all of the one or more moieties of the peptide therapeutic free of amidine moieties that is replaced by an amidine moiety can be an amide that is at an internal location of the peptide therapeutic and that is directly bound to two amino acid residues of the peptide therapeutic.

[0037] The peptide therapeutic free of amidine moieties can be one of the compounds listed in Table 1 of the present disclosure. The peptide therapeutic can be Leu-enkephalin, having the amino acid sequence Tyr-Gly-Gly-Phe-Leu.

[0038] The compound can have the structure:900.344W01, ISURF 05723wherein X1is NH, X2is O, and X3is O.

[0039] The compound can have the structure:wherein X1is O, X2is NH, and X3is O.

[0040] The compound can have the structure:wherein X1is O, X2is O, and X3is NH.

[0041] Various aspects of the present disclosure provide a pharmaceutical composition including the compound of the present disclosure. The pharmaceutical composition can optionally include one or more excipients such as a filler, a solubilizer, a stabilizer, a buffer, a tonicity modifier, a bulking agent, a viscosity enhancer or reducer, a chelating agent, an adjuvant, a disintegrant, a glidant, a lubricant, a binder, or a surfactant.

[0042] Various aspects of the present disclosure provide a method of treating a condition. The method can include administering the compound of the present disclosure, or a pharmaceutical composition including the same, to a patient to treat the condition. The condition can be one or more of the conditions listed in the same row of Table 1 as the900.344W01, ISURF 05723peptide therapeutic that is free of amidine moieties. Although aspects of the method of treating a condition of the present disclosure are not restricted to any particular drug target and / or mechanism of action, various aspects of the method can include treating the condition via the drag target and / or mechanism of action listed in the same row of Table 1 as the peptide therapeutic free of amidine moieties.

[0043] Table 1. Peptide therapeutics that are free of amidine moieties.900.344W01, ISURF 05723900.344W01, ISURF 05723900.344W01, ISURF 05723900.344W01, ISURF 05723900.344W01, ISURF 05723900.344W01, ISURF 05723900.344W01, ISURF 05723

[0044] The peptide therapeutic free of amidine moieties can be one of the compounds listed in Table 2 of the present disclosure. The peptide therapeutic free of amidine moieties can be Feleucin-K3, having the amino acid sequence FLKLLKKLL. The peptide therapeutic free of amidine moieties can be Temporin-L, having the amino acid sequence FVQWFSKFLGRIL. The peptide therapeutic free of amidine moieties can be XT-7, having the amino acid sequence GLLGPLLKIAAKVGSNLL. The peptide therapeutic free of amidine moieties can be Ga-W2, having the amino acid sequence FLGWLFKWASK. The peptide therapeutic free of amidine moieties can be Aurein 1.2, having the amino acid sequence GLFDIIKKIAESF. The peptide therapeutic free of amidine moieties can be LL-37 (Ropocamptidc), having the amino acid sequenceLLGDFFRKS KEKIGKEFKRI VQRIKDFLRNLVPRTES .

[0045] Various aspects of the present disclosure provide a method of treating a condition. The method can include administering the compound of the present disclosure, or a pharmaceutical composition including the same, to a patient to treat the condition. The condition can be one or more of the conditions listed in the same row of Table 2 as the peptide therapeutic that is free of amidine moieties.900.344W01, ISURF 05723

[0046] Table 2. Short helical anti-microbial peptides (AMPs) that are free of amidine moieties.

[0047] Various aspects of the present disclosure provide a method of treating a condition. The method can include administering the compound of the present disclosure, or a pharmaceutical composition including the same, to a patient to treat the condition. The condition can be one or more of the conditions listed in the same row of Table 2 as the peptide therapeutic that is free of amidine moieties. Although aspects of the method of treating a condition of the present disclosure are not restricted to any particular drug target and / or mechanism of action, various aspects of the method can include treating the condition via the drug target and / or mechanism of action listed in the same row of Table 2 as the peptide therapeutic free of amidine moieties.Method of forming a product compound including one or more amidine moieties.900.344W01, ISURF 05723

[0048] Various aspects of the present disclosure provide a method of forming a product compound including one or more amidine moieties. The method can include reacting a thioimidate-containing compound including a thioimidate with a nitrogen-containing compound to form the product compound including an amidine in place of the thioimidate in the thioimidate-containing compound. The nitrogen-containing compound can include a primary amine, a secondary amine, a salt thereof (e.g., an ammonium salt, or another amine salt), or a combination thereof. The nitrogen-containing compound is an amino acid, an amino acid derivative, an aliphatic amine, or an aromatic amine. The thioimidate includes -S-R1, wherein R1is chosen from -OH, -H, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (C&-C2o)aryl, (C&-C2o)heteroaryl, acyl, carbonyl, carbonate, and carbamate. R1can be substituted or unsubstituted (Ci-C2o)hydrocarbyl or -H. The variable R1can be (Ci-C2o)alkyl. The variable R1can be methyl. The variable R1can be -H.

[0049] The thioimidate can be at any suitable position in the thioimidate-containing compound. In various aspects, the thioimidate can be at an internal location in the thioimidate-containing compound. In various aspects, the thioimidate can be at a terminal end of the thioimidate-containing compound.

[0050] The amidine can include -C(=NH)NH- or -C(=NR2)NH-, wherein R2is chosen from -OH, -H, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (C6-C2o)aryl, (C6-C2o)heteroaryl, acyl, carbonyl, carbonate, and carbamate. R2can be chosen from -OH, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (Ce-C2o)aryl, and (C6-C2o)heteroaryl. The variable R2can be substituted or unsubstituted (Ci-C2o)hydrocarbyl. The variable R2can be (Ci-C2o)alkyl. The variable R2can be methyl.

[0051] In various aspects, the reaction of the thioimidate-containing compound with the nitrogen-containing compound is performed in the absence of added solvent, e.g., less than 2 wt% added solvent, or less than 1 wt%, less than 0.5 wt%, or less than 0.1 wt%, or 0 wt%. In various aspects, the reaction of the thioimidate-containing compound with the nitrogen-containing compound is performed in an added solvent. The added solvent can be any suitable solvent. The added solvent can include an organic solvent. The added solvent can include a polar aprotic solvent. The added solvent can include a halogenated solvent, imidazole, chloroform, methylene chloride, tetrahydrofuran, dimethylformamide, 1,2-dimethyoxyethane, 1,3 -dioxolane, dimethylsulfoxide, dimethyl acetamide, a fluoroalcohol, or a combination thereof. The added solvent can include imidazole, dimethylformamide, 2,2,2-900.344W01, ISURF 05723trifluoroethanol, or a combination thereof. The added solvent can include imidazole. The added solvent can be substantially free of water. A reaction milieu including the thioimidate-containing compound and the nitrogen-containing compound can be substantially free of water. For example, the reaction milieu can be 0 wt% to 2 wt% water, or 0 wt% to 0.1 wt% water, or less than or equal to 2 wt% and greater than or equal to 0 wt% and less than, equal to, or greater than 0.001 wt%, 0.005, 0.01, 0.05, 0.1, 0.5, 1, or 1.5 wt%.

[0052] The thioimidate can be a protonated thioimidate and can have the structure -C(=NH2+)-S-R1or -C(=NHR2+)-S-R1, wherein R2is chosen from -OH, -H, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (C6-C2o)aryl, (C6-C2o)heteroaryl, acyl, carbonyl, carbonate, and carbamate. R2can be chosen from -OH, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (C6-C2o)aryl, and (Ce-C2o)heteroaryl. The variable R2can be substituted or unsubstituted (Ci-C2o)hydrocarbyl. The variable R2can be (Ci-C2o)alkyl. The variable R2can be methyl. The thioimidate can be a thioimidate hydrohalide (e.g., a thioimidate that is protonated and having a halide counterion). The thioimidate can be a thioimidate hydroiodide (e.g., a thioimidate that is protonated and having an iodide counterion).

[0053] In various aspects a reaction milieu including the thioimidate-containing compound and the nitrogen-containing compound further includes an acid. In various aspects, the acid can protonate the thioimidate (having an original structure of -C(=NH)-S-R1or -C(=NR2)-S-R1) to form the protonated structure -C(=NH2+)-S-R1or -C(=NHR2+)-S-R1. The acid can include any suitable acid, such as a carboxylic acid, a mineral acid, an organic acid, formic acid, a sulfonic acid, or a combination thereof. The acid can include acetic acid. Any suitable amount of acid can be present. For example, when compared to the amount of thioimidate-containing compound present in the reaction milieu, 0.1 to 5 equivalents of the acid can be present, or 0.5 to 2 equivalents, or less than or equal to 5 and greater than or equal to 0.1 and less than, equal to, or greater than 4.5 equivalents, 4, 3.5, 3, 2.5, 2, 1.8, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2 equivalents of the acid can be present. In various aspects the reaction milieu is substantially free of acids; for example, acids can be 0 wt% to 2 wt% of the reaction milieu, or 0 wt% to 1 wt%, or less than or equal to 2 wt% and greater than or equal to 0 wt% and less than, equal to, or greater than 0.001 wt%, 0.005, 0.01, 0.05, 0.1, 0.5, 1, or 1.5 wt%.

[0054] The reacting of the thioimidate-containing compound and the nitrogencontaining compound can be performed at any suitable temperature such that the product900.344W01, ISURF 05723compound is formed. The reacting of the thioimidate-containing compound and the nitrogencontaining compound can be performed at about room temperature. The reacting of the thioimidate-containing compound and the nitrogen-containing compound can be performed at a temperature of 10 °C to 100 °C, or 20 °C to 50 °C, or 30 °C to 45 °C, or less than or equal to 100 °C and greater than or equal to 0 °C and less than, equal to, or greater than 12 °C, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 65, 70, 75, 80, 85, 90, or 95 °C. The reacting of the thioimidate-containing compound and the nitrogen-containing compound can be performed at any suitable pressure such that the product compound is formed. The reacting of the thioimidate-containing compound and the nitrogen-containing compound can be performed at ambient pressure. The reacting of the thioimidate-containing compound and the nitrogen-containing compound can be performed at a pressure of 20 kPa to 150 kPa, or 80 kPa to 120 kPa, or less than or equal to 150 kPa and greater than or equal to 20 kPa and less than, equal to, or greater than 25 kPa, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, or 145 kPa. The reacting of the thioimidate-containing compound and the nitrogen-containing compound can be performed for any suitable duration such that the product compound is formed. The reacting of the thioimidate-containing compound and the nitrogen-containing compound can be performed for a duration of 1 min to 72 h, or 1 h to 4 h, or less than or equal to 72 h and greater than or equal to 1 min and less than, equal to, or greater than 2 min, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 min, 1 h, 1.5, 2, 2.5. 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 36, 48, or 60 h.

[0055] The thioimidate-containing compound can be any suitable thioimidate-containing compound that includes a thioimidate. The thioimidate-containing compound can be an amino acid, an amino acid derivative, a peptide, or a peptidomimetic. The product compound can be a peptide. The thioimidate group of the thioimidate-containing compound is a modified carboxylic acid group of the amino acid. For example, the thioimidate-containing compound can be or include Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, He, Feu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Typ, or Vai, or a derivative thereof. The thioimidate-containing compound can be an amino acid and the product compound can be a dipeptide. The thioimidate-containing compound can be an amino acid or a dipeptide and the product compound can be a tripeptide. The product compound be a peptide that includes more than three amino acid residues. The product compound can be a polypeptide.

[0056] The nitrogen-containing compound is a separate compound from the thioimidate-containing compound, and the reaction between the nitrogen-containing900.344W01, ISURF 05723compound and the thioimidate-containing compound to generate the product compound is an intermolecular reaction. Compared to the amount of nitrogen-containing compound present in the reaction milieu that includes the thioimidate-containing compound and the nitrogencontaining compound, any suitable number of equivalents of the thioimidate-containing compound can be present, such as 1 to 20 equivalents, 1 to 10 equivalents, or less than or equal to 20 equivalents and greater than or equal to 1 equivalent and less than, equal to, or greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 equivalents of the thioimidate-containing compound.

[0057] The nitrogen-containing compound can be an amino acid, an amino acid derivative, an aliphatic amine, an aromatic amine, or a peptide including an amino acid, amino acid derivative, aliphatic amine, or aromatic amine. The nitrogen-containing compound can be an amino acid, an amino acid derivative, or a peptide including an amino acid or amino acid derivative. The reactive nitrogen atom of the nitrogen-containing compound that forms the amidine is a nitrogen atom of an amino acid or amino acid derivative that may be part of a peptide. For example, the nitrogen-containing compound can be or include Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Typ, or Vai, or a derivative thereof. A derivative of an amino acid can an amino acid with one or more modified functional groups (e.g., modified to include on or more protecting groups), a peptide including the amino acid, or a combination thereof. The nitrogen-containing compound can include the primary amine, e.g., the nitrogen-containing compound can include Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, lie, Leu, Lys, Met, Phe, Ser, Thr, Trp, Typ, or Vai, or a derivative thereof. The nitrogen-containing compound can include a secondary amine, e.g., the nitrogen-containing compound can include Pro or a derivative thereof. The nitrogen-containing compound can include an ammonium salt (e.g., which is a primary or secondary amine).

[0058] In various aspects, a non-thioimidate amine end of the thioimidate-containing compound is protected prior to formation of the amidine. When the thioimidate-containing compound is an amino acid, an amino acid derivative, or a peptide, the non-thioimidate amine end is the end opposite the thioimidate end of the thioimidate-containing compound. The non-thioimidate amine end of the thioimidate-containing compound can be protected with any suitable protecting group, such as Fmoc, Boc, Cbz, acetyl, trifluoroacetyl, carbonyl, carbamate, carbonate, sulfonamide, or any suitable alkyl or acyl group. The non-thioimidate amine end of the thioimidate-containing compound can be protected via an Fmoc group.900.344W01, ISURF 05723

[0059] Following formation of the product compound, the method can further include adding one or more additional amino acids to the product compound via formation of peptide bonds, such as via reaction with the non-thioimidate amine end of the thioimidate-containing compound residue in the product compound. The thioimidate-containing compound that is reacted with the nitrogcn-containing compound can be protected prior to the reaction to form the product compound by a protecting group at the non-thioimidate amine end of the thioimidate-containing compound. The method can further include deprotecting the non-thioimidate amine end of the thioimidate-containing compound in the product compound prior to the adding of the one or more additional amino acids to the product compound via peptide bond formation.

[0060] The method can further include protecting the amidine group of the product compound. The method can include protecting the amidine group prior to adding one or more additional amino acids to the product compound via peptide bond formation. The protected amidine group can have the structure -(C=NH)-NP1-, -(C=NR2)-NP1-, -(C=NP1)-NP1-, or -(C=NP1)-NR2-, wherein P1is the protecting group and R2is chosen from -OH, -H, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (Ce-C2o)aryl, (C6-C2o)heteroaryl, acyl, carbonyl, carbonate, and carbamate. R2can be chosen from -OH, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (C6-C2o)aryl, and (Ce-C2o)heteroaryl. The protected amidine group can have the structure -(C=NH)-NP1-. The variable P1can be Fmoc, Boc, Boc, Cbz, acetyl, trifluoroacetyl, carbonyl, carbamate, carbonate, sulfonamide, or any suitable alkyl or acyl group. The variable P1can be Boc.

[0061] The method includes performing the reacting of the thioimidate-containing compound and the nitrogen-containing compound while the nitrogen-containing compound is bonded to a solid support (e.g., on-resin). The method can include adding one or more additional amino acids to the product compound while the product compound is still bonded to the solid support. The method can further include bonding the nitrogen-containing compound, or a precursor compound thereof, to the solid support. The method can include performing one or more synthetic transformations of the precursor compound while the precursor compound is bonded to the solid support to form the nitrogen-containing compound bonded to the solid support. The method can further include cleaving the product compound from the solid support.900.344WG1, ISURF 05723

[0062] The method can further include isolating and / or increasing the purity of the product compound. For example, the method can further include subjecting the product compound to chromatography to increase the purity thereof.

[0063] The method can further include reacting a precursor compound including a thioamidc to form the thioimidatc-containing compound including the thioimidatc in place of the thioamide in the precursor compound. The precursor compound can be a peptide, an amino acid, or an amino acid derivative. The precursor compound can include or can be Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Ti , Typ, or Vai, or a derivative thereof. The thioimidate-containing compound can be identical to the precursor compound other than the transformation of the thioamide to the thioimidate.Reacting the precursor compound to form the thioimidate-containing compound can include reacting the precursor compound with an alkyl iodide in the presence of a base.

[0064] The reaction of the thioimidate-containing compound with the nitrogencontaining compound to form the product compound can have any suitable yield of the product compound. For example, a yield of the product compound from the nitrogencontaining compound can be 0.1% to 100%, or 40% to 99%, or less than or equal to 100% and greater than or equal to 0.1% and less than, equal to, or greater than 0.5%, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 82, 84, 86, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.9, or 99.99%.

[0065] A reaction milieu including the thioimidate-containing compound and the nitrogen-containing compound can be substantially free of silver or silver-containing compounds. For example, silver or silver-containing compound can be 0 wt% to 2 wt% of the reaction milieu, or 0 wt% to 0.1 wt%, or less than, equal to, or greater than 0.001 wt%, 0.005, 0.01, 0.05, 0.1, 0.5, 1, or 1.5 wt% of the reaction milieu.Examples

[0066] Various aspects of the present invention can be better understood by reference to the following Examples which are offered by way of illustration. The present invention is not limited to the Examples given herein.Example 1. Amidine as a Single-Atom Substitution Isostere: A Novel Strategy for Metabolic Stabilization of Peptide Amide Bonds.

[0067] Abstract. The therapeutic potential of peptides is severely limited by rapid metabolism mediated by protease enzymes in the human body. Proteases catalyze the900.344W01, ISURF 05723hydrolysis of amide bonds in peptides and proteins, posing a major challenge for peptide-based therapeutics due to their susceptibility to proteolysis and poor pharmacokinetics. Traditional metabolic stabilization strategies, including multi-atom bioisosteric substitutions and the incorporation of unnatural amino acids, often undermine pharmacological profile by altering hydrogcn-bonding patterns, conformational integrity, and physicochemical properties.

[0068] To overcome these challenges, we have developed a single-atom substitution of the amide bond (O to N) to stabilize metabolically labile sites while preserving molecular and conformational integrity. This strategy has broad potential applications in drug design and discovery, enabling the metabolic stabilization of therapeutic peptides without compromising their activity, and potentially rescuing promising candidates that could not be pursued due to metabolic instability.

[0069] Introduction. Peptides are an increasingly attractive target for the translation of natural products into therapeutics. One of the most critical challenges in the field of bioactive peptides is developing proteolytically stable versions that can enact their mode of action before enzymatic breaking down in serum, plasma, or gastrointestinal tract. FIG. 1A illustrates proteolytic cleavage of a peptide with an enzyme. Proteolytic stability is therefore a fundamental component of peptide drug discovery and arguably of comparable importance to efficacy and toxicity data. Strategies to attenuate proteolysis typically include unnatural amino acid substitution at or near the scissile bond (D-, - «,«-dialkyl, peptoids and N-methylation), or promotion of secondary structure via cyclization or stapling, as shown in FIG. IB.

[0070] All of these strategies leave the carbonyl oxygen intact. Because all proteases (serine / threonine / cysteine, aspartyl, metallo) operate by activating the carbonyl oxygen of the peptide bond towards hydrolysis, the opportunity for proteolysis remains. An alternative strategy would be to alter the properties of the carbonyl oxygen itself, disrupting the finely tuned acid-base interactions within the active site and preventing the protease from effecting its mechanism of hydrolysis. Thioamides are an excellent example of this approach, as shown in FIG. IB, but their lipophilicity typically leads to loss of target recognition and activity. As illustrated in FIG. 1C, we have found that amidines are well tolerated in folded peptide structure and are better suited than thioamides to replicating the shape and hydrogen bonding patterns of native amides. Thus, amidines have the potential to (1) confer protease resistance with (2) minimal impact on interactions that are critical for drug action.900.344W01, ISURF 05723

[0071] Design of single-atom substitution isostere of peptide bonds as amidines.As a minimally perturbing substitution, single atom isosteres of the amide bond are valuable non-natural substitutions in peptides. This is exemplified in the thioamide, shown to be a valuable tool to increase proteolysis resistance and in the design of cell-permeable peptides. While thioamidcs arc tolerated in some secondary structures, activity tends to be significantly lower due to the large size of the C=O to C=S bond. A C=X amide isostere more favorable for increasing stability without degrading activity would retain favorable proteolytic stability while keeping a similar size and hydrogen-bonding character to the native amide bond. Many backbone modifications significantly alter hydrogen-bonding: esters and N-methyl amides have similar geometry to native amides but no hydrogen bond donors, while thioamides have significantly altered hydrogen bonding angles. To that end, amidines are an underexplored moiety with surprising flexibility. Recent exploration of an amidino-vancomycin by Boger and coworkers found favorable H-bonding capabilities which were critical to retain activity against vancomycin-resistant bacteria. Our group has recently shown that the amidine is the only isostere of the amide that is able to adopt both hydrogenbond donating and accepting interactions necessary to stabilize peptide folds.

[0072] Proteases, also referred to as peptidases or proteolytic enzymes, play a critical role in the hydrolysis of amide bonds in peptides and proteins. Protease activity is vital to numerous physiological and pathological processes. Based on mechanism, various proteases are classified into six distinct groups based on their catalytic mechanisms: serine proteases, cysteine proteases, threonine proteases, metalloproteases, aspartyl proteases, and proteases of unspecified mechanism. Each enzyme is characterized by a unique catalytic strategy, substrate specificity, and functional roles in biological systems. These proteases play a significant role in peptide-based therapeutics, contributing to rapid proteolysis and the metabolic instability of peptides in the human body.

[0073] Synthesis of Peptides. Synthesis of diverse amidinopeptides was enabled by utilization of our previously reported thioimidate dipeptide methodology, as described in Byerly-Duke, J. et al., Complementary Strategies for Installation of Thioimidates into Peptide Backbones, J. Org. Chem., 2024, 89, 14755-14761, and U.S. patent publication no.2024 / 0300888, both of which are hereby incorporated by reference in their entirety.Thioimidate dipeptides can be synthesized from commercially available starting materials and coupled onto solid phase. These thioimidates, which are robust in the course of elongation while also protecting a-stereochemistry, can be carried through peptide synthesis and converted to amidines on-resin. FIG. 2A illustrates one-pot palladium catalyzed allyl900.344W01, ISURF 05723deprotection and preactivation with PyBOP / HOBt enables access to thioimidates which can undergo elongation and final amidine formation on-resin. While this dipeptide strategy is very powerful for synthesizing diverse targets, we found a previously unknown limitation with dipeptide thioimidates containing glycine as the N-terminal residue (i.e., when Xaa= Gly). Such dipcptidcs exhibited poor stability in our hands, likely due to the unhindered thioimidate accelerating hydrolysis. This limitation required a separate approach, simplified by the lack of any a-stereocenter in glycine. Primary thioimidate glycine hydroiodide 2, in the presence of imidazole, effected efficient amidinlyation on-resin (FIG. 2B) which was used to furnish G2 and G3 herein. Boc protection of the resulting amidine with Boc-ON and DIEA was sufficient to mask the nucleophilic amidine and continue elongation without observation of any off-target N-acylation. With a robust set of tools for synthesis of amidinopeptides, we synthesized three amidinopeptide derivatives of Leu-enkephalin to examine their stability to enzymatic degradation and biological activity (FIG. 2C).

[0074] Metabolic Stability. Neuropeptide Lcu-cnkcphalin (Tyr-Gly-Gly-Phc-Lcu) acts as an agonist of the 5-opioid receptor (DOR) with 1-5 -fold binding affinity over the p receptor and > 1000-fold over the K-receptor. This peptide has a pharmacological profile that is useful for treating chronic pain, inflammation, and cancer pain while avoiding respiratory depression and addiction. Despite the medicinally relevant profile, in-vivo administration of Leu-enkephalin is limited by a poor pharmacokinetic profile due to rapid proteolysis of Tyn-Gly? by aminopeptidase in human plasma, and of Gly3-Phe4 by angiotensin-converting enzyme at the blood-brain barrier (BBB). Therefore, the metabolic instability of amide bonds of Leu-enkephalin limits its penetration into the central nervous system (CNS) and utility for developing drug-like compounds. Leu-enkephalin has known and predominant metabolic cleavage at the Tyri-Gly? bond by AP-N and at the Gly3-Phe4 bond by angiotensin-converting enzyme, with a plasma half-life (tl / 2) of less than 10 minutes. Among these, AP-N is the primary enzyme responsible for its metabolism.

[0075] To address this metabolic instability, we employed a novel and systematic single-atom substitution strategy aimed at metabolically stabilizing the amide bond cleavage sites without altering the conformational integrity of the peptide. Specifically, single amidine substitutions were utilized to block proteolysis at the predominate AP-N cleavage site Tyn-Gly2. Adjacent amide linkages were also substituted to interrogate effects of a proximal amidine. Amidines being non-natural. but retaining much of the native structure of an amide bond, thereby may improve the pharmacological profile and metabolic instability of Leu-enkephalin.900.344W01, ISURF 05723

[0076] While we found the half-life of the native amide incubated with AP-N to be 16.5 minutes (FIG. 3), the amidinopeptide G1 was essentially stable over the course of three hours (FIG. 3). These results indicate that the single-atom substitution of O to N completely stabilizes the predominant metabolic site against AP-N. Interestingly, even G3 of Gly -Phe4 site with substitution two residues away from the cleavage site afforded some protection from proteolysis with a half-life of 43.6 minutes. Unsubstituted amidines tend to be more susceptible to hydrolysis than similar esters and amides, yet in this case amidines showed remarkable resistance to enzymatic degradation. Hydrolysis of amidines in water is slowed significantly by both protonation and participation in a hydrogen-bonding network — both of which can be expected in an enzyme active site. FIG. 4 illustrates half-life of cleavage of Leu-Enkephalin and G1 and G3 derivatives with aminopeptidase-N (porcine kidney, supplied by Calbiochem) at 37 °C in pH 7.4 DPBS. Intact peptide determined by UPLC absorbance at 280 nm (area intact) I (sum intact + proteolyzed fraction). Time points collected in triplicate, displayed as an average. Error bars represent one standard deviation.

[0077] A general experimental procedure for determining the half-life of the peptides is as follows. The peptide was dissolved in 100 microliters of pH 7.4 gibco DPBS to a concentration of 2.5 mM, verified by nanodrop. The peptide was incubated at 37 °C for 30 minutes. Then, 0.25 micrograms of aminopeptidase M was added from a stock solution. To terminate the reaction at a given time point, 10% v / v 0.1 M HC1 (10 microliters) was added to the reaction. Protein was removed by spin down cartridge, prewashed with 200 microliters DPBS buffer. Samples were stored at -80 °C for later analysis by HPLC. Reactions were run in triplicate per time point. Oxoamide time points were 0, 5, 10, 30, 60, and 120 minutes. Amidine time points were 0, 5, 10, 60, 120, and 240 minutes.

[0078] Overall, the enzymatic stability results clearly establish amidine as an effective bioisosteric replacement capable of site- selectively mitigating and blocking metabolism by a specific class of proteases. The complete inhibition of the Tyri-Gly2 primary cleavage site by the AP-N enzyme highlights the effectiveness and utility of this approach, demonstrating its potential for enhancing peptide stability against enzymatic and proteolytic degradation.

[0079] Further experimentation should be conducted to determine if amidinopeptides can act as competitive inhibitors of AP-N to clarify the mechanism of this stability.Conserved geometry, hydrogen bonding, and secondary structure of amidinopeptides suggest that binding to the peptidase may be conserved. On the other hand, enhanced stability of G3 indicates that amidine modification may inhibit binding to the AP-N active site.900.344W01, ISURF 05723

[0080] Conclusions. In summary, we have developed a novel single-atom substitution (O to N) approach to the metabolic stabilization of proteolytically susceptible sites in peptides. The developed amidine substitution approach demonstrates significant potential for addressing the metabolic instability of therapeutic peptides such as Leu-cnkcphalin. Through systematic single-atom modifications, we successfully stabilized the predominant cleavage site (Tyn-Gly2) against AP-N-mediated metabolism, extended the peptide’s half-life and improved its enzymatic stability.

[0081] The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the aspects of the present invention. Thus, it should be understood that although the present invention has been specifically disclosed by specific aspects and optional features, modification and variation of the concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be within the scope of aspects of the present invention.Exemplary Aspects.

[0082] The following exemplary aspects are provided, the numbering of which is not to be construed as designating levels of importance:

[0083] Aspect 1 provides a compound having the same chemical structure as a peptide therapeutic free of amidine moieties other than having one or more amide moieties of the peptide therapeutic free of amidine moieties replaced by one or more amidine moieties.

[0084] Aspect 2 provides the compound of Aspect 1, wherein the compound is a peptide therapeutic.

[0085] Aspect 3 provides the compound of any one of Aspects 1-2, wherein the compound has the same or similar biological activity as the peptide therapeutic free of amidine moieties.

[0086] Aspect 4 provides the compound of any one of Aspects 1-3, wherein the compound has one and not more than one amide moiety of the peptide therapeutic free of amidine moieties replaced by an amidine moiety.

[0087] Aspect 5 provides the compound of any one of Aspects 1-3, wherein the compound has two and not more than two amide moieties of the peptide therapeutic free of amidine moieties replaced by amidine moieties.900.344W01, ISURF 05723

[0088] Aspect 6 provides the compound of any one of Aspects 1-3, wherein the compound has three and not more than three amide moieties of the peptide therapeutic free of amidine moieties replaced by amidine moieties.

[0089] Aspect 7 provides the compound of any one of Aspects 1-6, wherein the compound has 1-100 amide moieties of the peptide therapeutic free of amidine moieties replaced by amidine moieties.

[0090] Aspect 8 provides the compound of any one of Aspects 1-7, wherein one of the one or more moieties of the peptide therapeutic free of amidine moieties that is replaced by an amidine moiety is an amide that is at an internal location of the peptide therapeutic and that is directly bound to two amino acid residues of the peptide therapeutic.

[0091] Aspect 9 provides the compound of any one of Aspects 1-8, wherein one of the one or more moieties of the peptide therapeutic free of amidine moieties that is replaced by an amidine moiety is an amide that is at a terminus of the peptide therapeutic.

[0092] Aspect 10 provides the compound of any one of Aspects 1-9, wherein the peptide therapeutic free of amidine moieties is one of the compounds listed in Table 1 of the present disclosure, or wherein the peptide therapeutic free of amidine moieties is one of the compounds listed in Table 2 of the present disclosure.

[0093] Aspect 11 provides the compound of any one of Aspects 1-10, wherein the peptide therapeutic is Leu-enkephalin.

[0094] Aspect 12 provides the compound of Aspect 11, wherein the peptide therapeutic has the amino acid sequence Tyr-Gly-Gly-Phe-Leu.

[0095] Aspect 13 provides the compound of any one of Aspects 1-10, wherein the peptide therapeutic is Feleucin-K3 having the amino acid sequence FLKLLKKLL, Temporin-L having the amino acid sequence FVQWFSKFLGRIL, XT-7 having the amino acid sequence GLLGPLLKIAAKVGSNLL, Ga-W2 having the amino acid sequence FLGWLFKWASK, Aurein 1.2 having the amino acid sequence GLFDIIKKIAESF, or LL-37 (Ropocamptide) having the amino acid sequenceLLGDFFRKS KEKIGKEFKRI VQRIKDFLRNLVPRTES .

[0096] Aspect 14 provides the compound of Aspect 1, wherein the compound has the structure:900.344W01, ISURF 05723wherein X1is NH, X2is O, and X3is O.

[0097] Aspect 15 provides the compound of Aspect 1, wherein the compound has the structure:wherein X1is O, X2is NH, and X3is O.

[0098] Aspect 16 provides the compound of Aspect 1, wherein the compound has the structure:wherein X1is O, X2is O, and X3is NH.

[0099] Aspect 17 provides a pharmaceutical composition comprising the compound of any one of Aspects 1-16.

[0100] Aspect 18 provides a method of treating a condition using the compound of any one of Aspects 1-16, the method comprising:administering the compound of any one of Aspects 1-16 to a patient to treat the condition.900.344W01, ISURF 05723

[0101] Aspect 19 provides the method of Aspect 18, wherein the condition is one or more of the conditions listed in the same row of Table 1 or Table 2 as the peptide therapeutic free of amidine moieties.

[0102] Aspect 20 provides the method of Aspect 18, wherein the method comprises treating the condition via the drug target and / or mechanism of action listed in the same row of Table 1 as the peptide therapeutic free of amidine moieties.

[0103] Aspect 21 provides a method of forming a product compound comprising one or more amidine moieties, the method comprising:reacting a thioimidate-containing compound comprising a thioimidate with a nitrogen-containing compound to form the product compound comprising an amidine in place of the thioimidate in the thioimidate-containing compound;whereinthe nitrogen-containing compound comprises a primary amine, a secondary amine, a salt thereof (c.g., an ammonium salt), or a combination thereof,the nitrogen-containing compound is an amino acid, an amino acid derivative, an aliphatic amine, an aromatic amine, or a peptide, andthe thioimidate comprises -S-R1, wherein R1is chosen from -OH, -H, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (C6-C2o)aryl, (C6-C2o)heteroaryl, acyl, carbonyl, carbonate, and carbamate. R1can be substituted or unsubstituted (Ci-C2o)hydrocarbyl or -H.

[0104] Aspect 22 provides the method of Aspect 21, wherein R1is (Ci-C2o)alkyl.

[0105] Aspect 23 provides the method of any one of Aspects 21-22, wherein R1is methyl.

[0106] Aspect 24 provides the method of any one of Aspects 21-23, wherein the thioimidate is at a terminal end of the thioimidate-containing compound.

[0107] Aspect 25 provides the method of any one of Aspects 21-24, wherein the amidine comprises -C(=NH)NH- or -C(=NR2)NH-, wherein R2is chosen from -OH, -H, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (Ce-C2o)aryl, (C6-C2o)heteroaryl, acyl, carbonyl, carbonate, and carbamate. R2can be chosen from -OH, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (Ce-C2o)aryl, and (Ce-C2o)heteroaryl.900.344W01, ISURF 05723

[0108] Aspect 26 provides the method of any one of Aspects 21-25, wherein the reaction of the thioimidate-containing compound with the nitrogen-containing compound is performed in the absence of added solvent.

[0109] Aspect 27 provides the method of any one of Aspects 21-26, wherein the reaction of the thioimidate-containing compound with the nitrogcn-containing compound is performed in an added solvent.

[0110] Aspect 28 provides the method of Aspect 27, wherein the added solvent comprises an organic solvent.

[0111] Aspect 29 provides the method of any one of Aspects 27-28, wherein the added solvent comprises a polar aprotic solvent.

[0112] Aspect 30 provides the method of any one of Aspects 27-29, wherein the added solvent comprises a halogenated solvent, imidazole, chloroform, methylene chloride, tetrahydrofuran, dimethylformamide, 1,2-dimethyoxyethane, 1,3-dioxolane, dimcthylsulfoxidc, dimethyl acetamide, a fluoroalcohol, or a combination thereof.

[0113] Aspect 31 provides the method of any one of Aspects 27-30, wherein the added solvent comprises imidazole, dimethylformamide, 2,2,2-trifluoroethanol, or a combination thereof.

[0114] Aspect 32 provides the method of any one of Aspects 27-31, wherein the added solvent comprises imidazole.

[0115] Aspect 33 provides the method of any one of Aspects 27-32, wherein the added solvent is substantially free of water.

[0116] Aspect 34 provides the method of any one of Aspects 21-33, wherein a reaction milieu comprising the thioimidate-containing compound and the nitrogen-containing compound is substantially free of water.

[0117] Aspect 35 provides the method of Aspect 34, wherein the reaction milieu is 0 wt% to 2 wt% water.

[0118] Aspect 36 provides the method of any one of Aspects 34-35, wherein the reaction milieu is 0 wt% to 0.1 wt% water.

[0119] Aspect 37 provides the method of any one of Aspects 21-36, wherein the thioimidate is protonated and has the structure -C(=NH2+)-S-R1or -C(=NHR2+)-S-R1, wherein R2is chosen from -OH, -H, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (C&-C2o)aryl, (C&-C2o)heteroaryl, acyl, carbonyl, carbonate, and carbamate. R2can be chosen from -OH,900.344W01, ISURF 05723substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (C6-C2o)aryl, and (C6-C2o)heteroaryl.

[0120] Aspect 38 provides the method of any one of Aspects 21-37, wherein the thioimidate is a thioimidate hydrohalide.

[0121] Aspect 39 provides the method of any one of Aspects 21-38, wherein the thioimidate is a thioimidate hydroiodide.

[0122] Aspect 40 provides the method of any one of Aspects 21-39, wherein a reaction milieu comprising the thioimidate-containing compound and the nitrogen-containing compound further comprises an acid.

[0123] Aspect 41 provides the method of Aspect 40, wherein the acid protonates the thioimidate to form the structure -C(=NH2+)-S-R1or -C(=NHR2+)-S-R1, wherein R2is chosen from -OH, -H, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (Ce-C2o)aryl, (C6-C2o)heteroaryl, acyl, carbonyl, carbonate, and carbamate. R2can be chosen from -OH, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (Ce-C2o)aryl, and (C6-C2o)heteroaryl.

[0124] Aspect 42 provides the method of any one of Aspects 40-41, wherein the acid comprises a carboxylic acid, a mineral acid, an organic acid, formic acid, a sulfonic acid, or a combination thereof.

[0125] Aspect 43 provides the method of any one of Aspects 40-42, wherein the acid comprises acetic acid.

[0126] Aspect 44 provides the method of any one of Aspects 40-43, wherein compared to the amount of thioimidate-containing compound present in the reaction milieu, 0.1 to 5 equivalents of the acid are present.

[0127] Aspect 45 provides the method of any one of Aspects 40-44, wherein compared to the amount of thioimidate-containing compound present in the reaction milieu, 0.5 to 2 equivalents of the acid are present.

[0128] Aspect 46 provides the method of any one of Aspects 21-45, wherein the reacting of the thioimidate-containing compound and the nitrogen-containing compound is performed at room temperature.

[0129] Aspect 47 provides the method of any one of Aspects 21-46, wherein the reacting of the thioimidate-containing compound and the nitrogen-containing compound is performed at a temperature of 10 °C to 100 °C.900.344WG1, ISURF 05723

[0130] Aspect 48 provides the method of any one of Aspects 21-47, wherein the reacting of the thioimidate-containing compound and the nitrogen-containing compound is performed at a temperature of 20 °C to 50 °C.

[0131] Aspect 49 provides the method of any one of Aspects 21-48, wherein the reacting of the thioimidate-containing compound and the nitrogen-containing compound is performed at ambient pressure.

[0132] Aspect 50 provides the method of any one of Aspects 21-49, wherein the reacting of the thioimidate-containing compound and the nitrogen-containing compound is performed at a pressure of 20 kPa to 150 kPa.

[0133] Aspect 51 provides the method of any one of Aspects 21-50, wherein the reacting of the thioimidate-containing compound and the nitrogen-containing compound is performed at a pressure of 80 kPa to 120 kPa.

[0134] Aspect 52 provides the method of any one of Aspects 21-51, wherein the reacting of the thioimidate-containing compound and the nitrogen-containing compound is performed for a duration of 1 min to 72 h.

[0135] Aspect 53 provides the method of any one of Aspects 51-52, wherein the reacting of the thioimidate-containing compound and the nitrogen-containing compound is performed for a duration of 1 h to 4 h.

[0136] Aspect 54 provides the method of any one of Aspects 51-53, wherein the thioimidate-containing compound is a peptide, a peptidomimetic, an amino acid, or an amino acid derivative and the product compound is a peptide, and wherein the thioimidate-containing compound comprises Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Typ, or Vai, or a derivative thereof.

[0137] Aspect 55 provides the method of Aspect 54, wherein the product compound is a dipeptide.

[0138] Aspect 56 provides the method of Aspect 54, wherein the product compound is a tripeptide.

[0139] Aspect 57 provides the method of Aspect 54, wherein the product compound includes more than three amino acid residues.

[0140] Aspect 58 provides the method of any one of Aspects 54-57, wherein the product compound is a polypeptide.

[0141] Aspect 59 provides the method of any one of Aspects 21-58, wherein the nitrogen-containing compound is a separate compound from the thioimidate-containing compound.900.344W01, ISURF 05723

[0142] Aspect 60 provides the method of Aspect 59, wherein compared to the amount of nitrogen-containing compound present in the reaction milieu, 1 to 20 equivalents of the thioimidate-containing compound are present.

[0143] Aspect 61 provides the method of any one of Aspects 59-60, wherein compared to the amount of nitrogen-containing compound present in the reaction milieu, 1 to 10 equivalents of the thioimidate-containing compound are present.

[0144] Aspect 62 provides the method of any one of Aspects 59-61, wherein the nitrogen-containing compound comprises the primary amine.

[0145] Aspect 63 provides the method of any one of Aspects 59-62, wherein the nitrogen-containing compound comprises the secondary amine.

[0146] Aspect 64 provides the method of any one of Aspects 59-63, wherein the nitrogen-containing compound comprises the salt of the primary or secondary amine (e.g., an ammonium salt).

[0147] Aspect 65 provides the method of Aspect 64, wherein a reaction milieu comprising the thioimidate-containing compound and the nitrogen-containing compound is substantially free of acids.

[0148] Aspect 66 provides the method of any one of Aspects 21-65, wherein the nitrogen-containing compound comprises or is Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Typ, or Vai, or a derivative thereof.

[0149] Aspect 67 provides the method of any one of Aspects 21-66, wherein a non-thioimidate amine end of the thioimidate-containing compound is protected prior to formation of the amidine.

[0150] Aspect 68 provides the method of Aspect 67, wherein the non-thioimidate amine end of the thioimidate-containing compound is protected via Fmoc, Boc, Cbz, acetyl, trifluoroacetyl, carbonyl, carbamate, carbonate, sulfonamide, or any suitable alkyl or acyl group.

[0151] Aspect 69 provides the method of any one of Aspects 67-68, wherein the non-thioimidate amine end of the thioimidate-containing compound is protected via an Fmoc group.

[0152] Aspect 70 provides the method of any one of Aspects 21-70, further comprising adding one or more additional amino acids to the product compound via formation of peptide bonds.

[0153] Aspect 71 provides the method of Aspect 70, wherein a non-thioimidate amine end of the thioimidate-containing compound is protected, further comprising deprotecting the900.344W01, ISURF 05723non-thioimidate amine end of the thioimidate-containing compound in the product compound prior to the adding of the one or more additional amino acids to the product compound via peptide bond formation.

[0154] Aspect 72 provides the method of any one of Aspects 70-71, further comprising protecting the amidine group prior to the adding of the one or more additional amino acids to the product compound.

[0155] Aspect 73 provides the method of Aspect 72, wherein the protected amidine group has the structure -(C=NH)-NP1-, -(C=NR2)-NP1-, -(C=NP1)-NP1-, or -(C=NP1)-NR2-, wherein P1is the protecting group and R2is chosen from -OH, -H, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (Ce-C2o)aryl, (Ce-C2o)heteroaryl, acyl, carbonyl, carbonate, and carbamate. R2can be chosen from -OH, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (Ce-C2o)aryl, and (C6-C2o)heteroaryl.

[0156] Aspect 74 provides the method of Aspect 73, wherein the protected amidine group has the structure -(C=NH)-NP1-.

[0157] Aspect 75 provides the method of any one of Aspects 73-74, wherein P1is Boc, Fmoc, Boc, Boc, Cbz, acetyl, trifluoroacetyl, carbonyl, carbamate, carbonate, sulfonamide, or any suitable alkyl or acyl group.

[0158] Aspect 76 provides the method of any one of Aspects 73-75, wherein P1is Boc.

[0159] Aspect 77 provides the method of any one of Aspects 21-76, wherein the method comprises performing the reacting of the thioimidate-containing compound and the nitrogen-containing compound while the nitrogen-containing compound is bonded to a solid support.

[0160] Aspect 78 provides the method of Aspect 77, wherein the method further comprises bonding the nitrogen-containing compound, or a precursor compound thereof, to the solid support.

[0161] Aspect 79 provides the method of any one of Aspects 77-78, further comprising performing one or more synthetic transformations of the precursor compound while the precursor compound is bonded to the solid support to form the nitrogen-containing compound bonded to the solid support.

[0162] Aspect 80 provides the method of any one of Aspects 77-79, further comprising cleaving the product compound from the solid support.900.344W01, ISURF 05723

[0163] Aspect 81 provides the method of any one of Aspects 21-80, further comprising isolating and / or increasing the purity of the product compound.

[0164] Aspect 82 provides the method of any one of Aspects 21-81, further comprising subjecting the product compound to chromatography to increase the purity thereof.

[0165] Aspect 83 provides the method of any one of Aspects 21-82, further comprising reacting a precursor compound comprising a thioamide to form the thioimidate-containing compound comprising the thioimidate in place of the thioamide in the precursor compound.

[0166] Aspect 84 provides the method of Aspect 83, wherein the thioimidate-containing compound is identical to the precursor compound other than the transformation of the thioamide to the thioimidate.

[0167] Aspect 85 provides the method of any one of Aspects 83-84, wherein reacting the precursor compound to form the thioimidatc-containing compound comprises reacting the precursor compound with an alkyl iodide in the presence of a base.

[0168] Aspect 86 provides the method of any one of Aspects 21-85, wherein a yield of the product compound from the nitrogen-containing compound is 0.1% to 100%.

[0169] Aspect 87 provides the method of any one of Aspects 21-86, wherein a yield of the product compound from the nitrogen-containing compound is 40% to 99%.

[0170] Aspect 88 provides the method of any one of Aspects 21-87, wherein a reaction milieu comprising the thioimidate-containing compound and the nitrogen-containing compound is substantially free of silver or silver-containing compounds.

[0171] Aspect 89 provides a method of forming a peptide comprising one or more amidine moieties, the method comprising:reacting a thioimidate-containing compound comprising a thioimidate with a nitrogen-containing compound that is bound to a resin to form the peptide comprising an amidine in place of the thioimidate in the thioimidate-containing compound;whereinthe nitrogen-containing compound comprises a primary amine or a secondary amine,the nitrogen-containing compound is an amino acid, an amino acid derivative, or a peptide,the thioimidate-containing compound is an amino acid, an amino acid derivative, a peptide, or a peptidomimetic, and900.344W01, ISURF 05723the thioimidate comprises -C(=NHR2+)-S-R1, wherein R1and R2are independently chosen from -OH, -H, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (C6-C2o)aryl, (Cs-C2o)heteroaryl, acyl, carbonyl, carbonate, and carbamate. The thioimidate can include -(C=NH2+)-S-MC.

[0172] Aspect 90 provides the compound, composition, or method of any one or any combination of Aspects 1-89 optionally configured such that all elements or options recited are available to use or select from.

Claims

900. 344W01, ISURF 05723CLAIMSWhat is claimed is:

1. A compound having the same chemical structure as a peptide therapeutic free of amidine moieties other than having one or more amide moieties of the peptide therapeutic free of amidine moieties replaced by one or more amidine moieties.

2. The compound of claim 1, wherein the peptide therapeutic free of amidine moieties is one of the compounds listed in Table 1 or Table 2 of the present disclosure.

3. The compound of claim 1, wherein the peptide therapeutic is Feleucin-K3 having the amino acid sequence FLKLLKKLL, Temporin-L having the amino acid sequence FVQWFSKFLGRIL, XT-7 having the amino acid sequence GLLGPLLKIAAKVGSNLL, Ga-W2 having the amino acid sequence FLGWLFKWASK, Aurein 1.2 having the amino acid sequence GLFDIIKKIAESF, or LL-37 (Ropocamptidc) having the amino acid sequence LLGDFFRKS KEKIGKEFKRI VQRIKDFLRNLVPRTES .

4. The compound of claim 1, wherein the peptide therapeutic is Leu -enkephalin.

5. The compound of claim 1, wherein the compound has the structure:whereinX1is NH, X2is O, and X3is O, orX1is O, X2is NH, and X3is O, orX1is O, X2is O, and X3is NH.

6. A pharmaceutical composition comprising the compound of claim 1.900.344W01, ISURF 057237. A method of treating a condition using the compound of claim 1, the method comprising:administering the compound of claim 1 to a patient to treat the condition.

8. The method of claim 6, wherein the condition is one or more of the conditions listed in the same row of Table 1 or Table 2 of the present disclosure as the peptide therapeutic free of amidine moieties.

9. A method of forming a product compound comprising one or more amidine moieties, the method comprising:reacting a thioimidate-containing compound comprising a thioimidate with a nitrogen-containing compound to form the product compound comprising an amidine in place of the thioimidate in the thioimidate-containing compound;whereinthe nitrogen-containing compound comprises a primary amine, a secondary amine, a salt thereof, or a combination thereof,the nitrogen-containing compound is an amino acid, an amino acid derivative, an aliphatic amine, an aromatic amine, or a peptide, andthe thioimidate comprises -S-R1, wherein R1is chosen from -OH, -H, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (Ce-C2o)aryl, (C6-C2o)heteroaryl, acyl, carbonyl, carbonate, and carbamate.

10. The method of claim 9, wherein the amidine comprises -C(=NH)NH- or -C(=NR2)NH-, wherein R2is chosen from -OH, -H, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (Ce-C2o)aryl, (C6-C2o)heteroaryl, acyl, carbonyl, carbonate, and carbamate.

11. The method of claim 9, wherein the reaction of the thioimidate-containing compound with the nitrogen-containing compound is performed in an added solvent comprising a polar aprotic solvent.

12. The method of claim 11, wherein the added solvent comprises imidazole.900.344W01, ISURF 0572313. The method of claim 9, wherein the thioimidate is protonated and has the structure -C(=NH2+)-S-R1or -C(=NHR2+)-S-R1, wherein R2is chosen from -OH, -H, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (C6-C2o)aryl, (C6-C2o)hctcroaryl, acyl, carbonyl, carbonate, and carbamate.

14. The method of claim 9, wherein the reacting of the thioimi date-containing compound and the nitrogen-containing compound is performed at a temperature of 10 °C to 100 °C, at a pressure of 20 kPa to 150 kPa, and for a duration of 1 min to 72 h.

15. The method of claim 9, wherein the thioimi date-containing compound is a peptide, a peptidomimetic, or amino acid and the product compound is a peptide, and wherein the thioimidate-containing compound comprises Ala, Arg, Asn, Asp, Cys, Glu, Cdn, Gly, His, He, Leu, Lys, Met, Phc, Pro, Ser, Thr, Trp, Typ, or Vai, or a derivative thereof.

16. The method of claim 9, wherein the nitrogen-containing compound comprises or is Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Typ, or Vai, or a derivative thereof.

17. The method of claim 9, wherein a non-thioimidate amine end of the thioimidate-containing compound is protected prior to formation of the amidine.

18. The method of claim 9, further comprising adding one or more additional amino acids to the product compound via formation of peptide bonds.1 . The method of claim 9, wherein the method comprises performing the reacting of the thioimidate-containing compound and the nitrogen-containing compound while the nitrogencontaining compound is bonded to a solid support.

20. A method of forming a peptide comprising one or more amidine moieties, the method comprising:reacting a thioimidate-containing compound comprising a thioimidate with a nitrogen-containing compound bound to a solid support to form the peptide comprising an amidine in place of the thioimidate in the thioimidate-containing compound;900.344W01, ISURF 05723whereinthe nitrogen-containing compound comprises a primary amine or a secondary amine,the nitrogen-containing compound is an amino acid, an amino acid derivative, or a peptide,the thioimidate-containing compound is an amino acid, an amino acid derivative, a peptide, or a peptidomimetic, andthe thioimidate comprises -C(=NHR2+)-S-R1, wherein R1and R2are independently chosen from -OH, -H, substituted or unsubstituted (Ci-C2o)hydrocarbyl, substituted or unsubstituted (Ci-C2o)alkyl, substituted or unsubstituted (C&-C2o)aryl, (C&-C2o)heteroaryl, acyl, carbonyl, carbonate, and carbamate.

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