Peptides containing phosphorylcholine conjugates and methods for their synthesis

Abstract

In one aspect of the present invention, there is a compound comprising an N-protected tyrosine modified with phosphorylcholine. Further, the use of the compound for SPPS, etc. is also provided.

Description

[Technical field]

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 177,420, filed April 21, 2021, the contents of which are incorporated by reference herein in their entirety.

[0002] The present invention is directed to tyrosine-phosphorylcholine conjugates and their uses, such as for the synthesis of peptide derivatives. [Background technology]

[0003] Ocular inflammation, inflammation of any part of the eye, is one of the most common eye diseases. Ocular inflammation refers to a wide range of ocular inflammatory diseases, one of which is uveitis. These diseases are widespread across all age groups and can be associated with systemic diseases such as Crohn's disease, Behcet's disease, juvenile idiopathic arthritis and other diseases. Inflammation can also be associated with other common eye conditions such as dry eye and dry macular degeneration. Some drugs have known side effects that cause uveitis and / or dry eye. The most common treatment for ocular inflammation is steroids, especially corticosteroids. However, these treatments have some known, often severe, side effects.

[0004] Phosphorylcholine (PC) is a zwitterionic small molecule secreted by helminths that allows them to survive in the host and induces a state of immune tolerance as well as the surface of some bacteria and apoptotic cells. Tuftsin-phosphorylcholine (TRS) is a bispecific small molecule with immunomodulatory activity. TRS (Thr-Lys-Pro-Arg-Gly-Tyr-PC) is an immunomodulatory peptide derivative.

[0005] Currently, TRS is synthesized by post-synthetic modification of Thr-Lys-Pro-Arg-Gly-Tyr to couple the PC moiety to the phenolic ring of tyrosine. However, this synthetic approach results in very low yields, thus making the synthesis of TRS ineffective and costly. There is a strong need for new, simple and efficient methods to synthesize TRS. Summary of the Invention

[0006] In one aspect of the invention there is a compound or a salt thereof, the compound having formula 1: [ka] wherein R1 is hydrogen or an amine protecting group; X is hydrogen, a phenol protecting group, or [ka] where R2 is the side chain of a natural or unnatural alpha amino acid and R is hydrogen, a carboxyl protecting group, a leaving group, a solid-phase linker group or absent.

[0007] In one embodiment, the compound has formula 2: [ka] wherein R1 includes said amine protecting group.

[0008] In another embodiment, the compound of formula 3: [ka] wherein each R4 is independently selected from the group consisting of H, amino acid, peptide, polyamino acid, at least one R4 is not H, and R5 is hydrogen or a linker group attached to a solid phase, the method comprising providing a compound of the invention linked to a solid phase, (i) deprotecting the amine protecting group of the compound; (ii) coupling an N-protected amino acid moiety to said compound; (iii) contacting the solid phase with a composition comprising a cyclic amine moiety, thereby obtaining the compound of interest attached to the solid phase; Includes.

[0009] In one embodiment, the method optionally comprises performing a step (iv) of deprotecting said N-protected amino acid moiety prior to performing step (iii).

[0010] In one embodiment, the method further comprises subsequently repeating said steps (ii) and (iv) before performing said step (iii).

[0011] In one embodiment, said step (ii) comprises contacting said solid phase with a coupling composition comprising 1 to 5 molar equivalents of said N-protected amino acid moiety.

[0012] In one embodiment, the compound is: [ka] Includes.

[0013] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although methods and materials similar or equivalent to those described herein can be used to carry out or test embodiments of the present invention, exemplary methods and / or materials are described below.In case of conflict, the patent specification, including definitions, shall prevail.In addition, the materials, methods, and examples are illustrative only and are not necessarily intended to be limiting. [Brief description of the drawings]

[0014] [Figure 1A-1B]1A is a graph showing an HPLC chromatogram showing the impurity profile of TRS synthesized via SPPS post-modification (FIG. 1A) and the corresponding MS spectrum of the major impurity with retention time 30.8 min (RRT=1.27) (FIG. 1B). The peaks in the MS spectrum correspond to 422.171 M / Z[M+3H] / 3, 632.753 M / Z[M+2H] / 2, Mw=1264.576 Da. FIG. 1B shows the proposed structure of the 1.27 impurity (based on MS / MS analysis). [Figure 2A-2B] FIG. 2A is a graph showing an HPLC chromatogram (FIG. 2B) depicting the impurity profile of TRS synthesized via the methods described herein, and the corresponding MS spectrum (FIG. 2B) of the major impurity with retention time 17.044 min (RRT=0.7). The peaks in the MS spectrum correspond to 234.0 M / Z [M+2H] / 2 and 467.2 M / Z [M+1H]. FIG. 2B shows the proposed structure of the 0.7 impurity (based on MS / MS analysis). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention, in some embodiments thereof, provides a compound comprising an N-protected tyrosine modified with PPC via an azo bond. The present invention, in some embodiments thereof, is based at least in part on the surprising discovery that the compounds of the present invention having an unprotected tyrosine side chain can be successfully implemented in the SPPS synthesis of TRS. The inventors have surprisingly found that the SPPS synthesis of TRS carried out by implementing the compounds of the present invention (having an unprotected phenol group of tyrosine) results in a dramatic improvement in synthesis yield and provides the desired TRS in high purity.

[0016] In one aspect of the invention, a compound of Formula 1: [ka] wherein X is hydrogen, a phenol protecting group, or [ka] wherein each R1 is independently or includes a hydrogen or an amine protecting group, R2 is the side chain of a natural or unnatural alpha amino acid (protected or unprotected), and R is hydrogen, a carboxyl protecting group, a leaving group, a solid-phase linker group, or is absent.

[0017] In some embodiments, the salt of the compound of the present invention comprises a phosphate salt. In some embodiments, the salt of the compound of the present invention refers to a phosphate salt of any one of the compounds disclosed herein, e.g., a deprotonated phosphate group. In some embodiments, the salt of the compound of the present invention is a compound represented by Formula I: [ka] It is represented by:

[0018] In some embodiments, salts of the compounds of the invention include compounds represented by Formula 1 or I and their counterions (eg, countercations and / or counteranions).

[0019] In some embodiments, the compounds of the present invention include a phosphate salt of a compound represented by Formula 1. In some embodiments where R1 is H, the compounds of the present invention include a phosphate salt and / or an ammonium salt of a compound represented by Formula 1. In some embodiments, the compounds of the present invention include a compound represented by Formula 1 and a counterion. In some embodiments, the counterion includes a monovalent cation. In some embodiments, the counterion includes a multivalent cation (e.g., a divalent and / or trivalent cation). In some embodiments, the counterion includes a counteranion (e.g., a monovalent and / or a multivalent counteranion).

[0020] In some embodiments, R1 is hydrogen. In some embodiments, R1 is or comprises a protecting group. In some embodiments, R1 is or comprises an amine protecting group. In some embodiments, the amine protecting group is cleavable (or removable) under the conditions of solid phase peptide synthesis (SPPS). In some embodiments, the amine protecting group is cleavable (e.g., undergoes deprotection or cleavage to provide a free unprotected amine) under any of the deprotection conditions applied in SPPS. In some embodiments, the amine protecting group is selected from an acid labile amine protecting group and a base labile amine protecting group.

[0021] In some embodiments, the amine protecting group is compatible with SPPS. In some embodiments, the amine protecting group is cleavable under conditions that are compatible with SPPS. In some embodiments, the amine protecting group is cleavable under conditions suitable for Fmoc or Boc deprotection. In some embodiments, the amine protecting group is cleavable under conditions suitable for Fmoc deprotection. In some embodiments, the amine protecting group is cleavable under conditions suitable for Boc deprotection. In some embodiments, the conditions suitable for Fmoc or Boc deprotection refer to conditions applied in solid phase based synthesis (e.g., SPPS).

[0022] In some embodiments, the amine protecting group is cleavable under conditions suitable for solid-phase Fmoc deprotection. In some embodiments, the amine protecting group is cleavable under conditions suitable for solid-phase Boc deprotection. Conditions for solid-phase deprotection of Fmoc or Boc are well known in the art. For example, conditions for solid-phase deprotection of Fmoc include, inter alia, a 20% piperidine (or DBU) solution in an organic solvent.

[0023] In some embodiments, the amine protecting group comprises any one of 9-fluorenylmethyloxycarbonyl (Fmoc), Alloc, Dde, iv-Dde, benzyl, benzyloxycarbonyl, tert-butyloxycarbonyl (Boc), 2-[biphenylyl-(4)]-propyl-2-oxycarbonyl, dimethyl-3,5 dimethoxybenzyloxycarbonyl, 2-(4-nitrophenylsulfonyl)ethoxycarbonyl, 1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl, 2,7-di-tert-butyl-Fmoc, 2-fluoro-Fmoc, nitrobenzenesulfonyl, benzothiazole-2-sulfonyl, 2,2,2-trichloroethyloxycarbonyl, dithiasuccinoyl, p-nitrobenzyloxycarbonyl.

[0024] In some embodiments, the amine protecting group is Boc or Fmoc.

[0025] In some embodiments, the compounds are as described herein and R1 lacks an acetyl group. In some embodiments, the compounds are as described herein and R1 lacks an acyl group.

[0026] In some embodiments, the carboxyl protecting group comprises a protecting group that is compatible with SPPS.

[0027] In some embodiments, the carboxyl protecting group comprises any one of tert-butyl ester, methyl ester, ethyl ester, benzyl ester, silyl ester (e.g., 2-trimethylsilylethyl), (2-phenyl-2-trimethylsilyl)ethyl, 2-(trimethylsilyl)isopropyl), allyl ester, 2-chlorotrityl (2-Cl-Trt), 2,4-dimethoxybenzyl, 2-phenylisopropyl, 9-fluorenylmethyl, Dmab, carbamoylmethyl, phenacyl, p-nitrobenzyl, 4,5-dimethoxy-2-nitrobenzyl, and 1,1-dimethylallyl. Other carboxyl protecting groups are known in the art.

[0028] In some embodiments, the phenol protecting group comprises a protecting group that is compatible with SPPS.

[0029] In some embodiments, R is or includes a linker group (also referred to herein as a cleavable linker) attached to a solid phase. In some embodiments, the terms "solid phase" and "solid support" are used interchangeably herein. As used herein, the term "solid phase" refers to a polymeric resin in the form of particles (usually polymeric beads having an average diameter in the range of 1 μm to 1 mm). In some embodiments, the solid phase is or includes a solid phase that is compatible with the SPPS process (e.g., the linker covalently attached to the solid phase and the compound of the invention, or the extending peptide chain that includes it, is chemically and / or physically stable under the conditions applied during SPPS).

[0030] Non-limiting examples of solid supports include, but are not limited to, PAM, chlorotrityl, Rink amide, and Wang. Other commercially available resins for solid phase synthesis (e.g., peptide synthesis) are well known in the art.

[0031] Those skilled in the art will appreciate that a solid phase that is compatible with the SPPS process may further comprise a cleavable linker. A variety of cleavable linkers are known in the art that contain chemical moieties (e.g., MBHA linkers) that allow a compound or a polypeptide derived therefrom to be cleaved from the solid support.

[0032] In some embodiments, the compound or the polypeptide derived from the compound is covalently linked to the solid support via a cleavable linker. In some embodiments, the cleavable linker is covalently linked to the solid support and the compound of the present invention or the polypeptide derived therefrom. In some embodiments, the cleavable linker is covalently linked to the compound of the present invention via a carbonyl group or an amino group. In some embodiments, the cleavable linker is covalently linked to the compound of the present invention via R, where R represents a bond. In some embodiments, R represents a covalent bond to the solid phase.

[0033] In some embodiments, the compound or a polypeptide derived from the compound is covalently attached to the solid support via a cleavable bond. In some embodiments, the cleavable bond is configured to decompose under certain cleavage conditions. The cleavable bond is configured to be labile to a certain cleavage solution (usually an acidic solution) and release the compound or a polypeptide derived therefrom under suitable cleavage conditions, such as a cleavage solution (usually including an acidic solution such as a TFA-based solution). In some embodiments, the cleavable bond is labile to any other conditions suitable for cleavage of the cleavable bond, including exposure to thermal irradiation, UV irradiation, nucleophiles (e.g., hydroxides, alcohols, amines, thiols, hydrazines, especially under basic conditions).

[0034] In some embodiments, the compound of the invention that is covalently attached to a solid support has Formula 1A: [ka] wherein R and X are as described herein and Y represents a heteroatom; [ka] represents a solid support and / or a linker attached to the solid support (i.e., a cleavable linker). In some embodiments, Y is selected from O, NH, and S.

[0035] In some embodiments, the compound of the invention that is covalently attached to a solid support has formula 1A1: [ka] In some embodiments, the compound of the present invention is represented by formula 1A1, where R1 is as described herein. In some embodiments, the compound of the present invention is represented by formula 1A1, where R1 is an amine protecting group. In some embodiments, the compound of the present invention is represented by formula 1A1, where R1 is H, Boc, or Fmoc.

[0036] In some embodiments, the phenol protecting group comprises any one of triisopropylsilyl ether (TIPS), tert-butyldimethylsilyl ether (TBDMS), methyl ether, benzyl ether (Bn), methoxymethyl acetal (MOM), 2-(trimethylsilyl)ethoxy]methyl acetal, tert-butyl ether, 2-chlorotrityl, trityl, benzyl, benzyloxycarbonyl, and Boc.

[0037] In some embodiments, the compounds of the present invention include active esters thereof. A variety of active esters are known in the art and generally relate to stable derivatives of carboxylates that are capable of reacting with nucleophiles without any catalyst.

[0038] In some embodiments, the active ester of the compound is represented by formula 1, where R is or comprises a leaving group. In some embodiments, the leaving group comprises an active ester obtained by reacting the free carboxy group of the compound of the present invention with a coupling reagent. Various coupling reagents are well known in the art, including HATU, HOBt, PyBOP, BOP, DIC, DCC, EDAC, etc. In some embodiments, the leaving group comprises any one of halo, hydroxysuccinimide, hydroxybenzotriazole, pentafluorophenol, imidazole carbonate, and O-acylisourea.

[0039] In some embodiments, the compound of the invention further binds to an additional natural or unnatural amino acid. In some embodiments, the bond is via a peptide bond. In some embodiments, the bond is via an amino group of the compound. In some embodiments, the compound of the invention that is bound to an amino acid has formula 1B: [ka] where R, R1, and X are as described herein, and R2 is the side chain of a natural or unnatural alpha amino acid (protected or unprotected).

[0040] In some embodiments, the compounds of the invention are as described herein, and R and X are hydrogen. In some embodiments, the compounds of the invention are as described herein, and R and X are hydrogen, and R is hydrogen or an amine protecting group.

[0041] In some embodiments, the compounds of the present invention have Formula 2: [ka] or Formula II [ka] wherein R1 is as defined above.

[0042] In some embodiments, R1 is H or an amine protecting group. In some embodiments, R1 is Fmoc or Boc. In some embodiments, the compound of the present invention is represented by formula 2, where R1 is Fmoc or Boc. One non-limiting exemplary synthetic procedure of the compounds disclosed herein is provided in the Examples section.

[0043] In some embodiments, a peptide sequence or compound of interest is provided that comprises a diazotized tyrosine, synthesized by the methods of the invention, the diazotized tyrosine being represented by formula 3A: [ka] wherein R6 represents one or more substituents, and the wavy bonds represent (i) the peptide sequence of interest, and / or (ii) R1, an N-protecting group, an acyl, OR, O - , OH, and H. In some embodiments, the peptide sequence of interest is represented by Formula 2A: [ka] wherein each Z independently is or includes a peptide, an amino acid, NH, OH, or H, and at least one Z is a peptide (e.g., Z is attached to a carboxy group).

[0044] In some embodiments, the peptide sequence of interest is present in trace amounts of: [ka] In some embodiments, the peptide sequence of interest comprises: [ka] wherein R6 is as described herein and the wavy bond lacks (i) a peptide sequence of interest, and / or (ii) R1, an N-protecting group, an acyl, OR, O - , OH, and H.

[0045] In some embodiments, the peptide sequence of interest is or comprises a TRS synthesized by the methods of the present invention, wherein the TRS comprises at least one impurity of the HPLC impurity profile described in the Examples section, the impurity profile being obtained via analytical method A described herein. In some embodiments, the impurity is characterized by a relative retention time (RRT) of 0.7 and / or a MW of 466.4 Da. In some embodiments, the impurity (also referred to herein as the 0.7 impurity) is characterized by the following: [ka] and any salts thereof.

[0046] In some embodiments, the TRS synthesized by the methods of the invention is free of an impurity characterized by a relative retention time (RRT) of 1.27 and / or a MW of 1263.5 Da (also used herein as the 1.27 impurity), and the impurity profile is obtained by analytical method A described herein. In some embodiments, the TRS synthesized by the methods of the invention is free of the 1.27 impurity: [ka] is lacking.

[0047] Exemplary HPLC impurity profiles and MS spectra of the impurities are shown in Figures 1A-B and 2A-B.

[0048] In some embodiments, the peptide sequences of interest synthesized by the methods of the invention are characterized by a purity of at least 95%, at least 96%, at least 97%, at least 99%, at least 99.5%, including any range therebetween, as determined by analytical HPLC, hi some embodiments, the peptide sequences of interest synthesized by the methods of the invention are substantially pure.

[0049] In some embodiments, the terms "peptide sequence," "peptide sequence of interest," and "peptide of interest" are used interchangeably herein.

[0050] As used herein, substantially pure means that the substance is sufficiently free of readily detectable impurities as determined by standard analytical methods, such as thin layer chromatography (TLC), nuclear magnetic resonance (NMR), gel electrophoresis, high performance liquid chromatography (HPLC) and mass spectrometry (MS), gas chromatography mass spectrometry (GC-MS), and similar methods used by those skilled in the art to assess such purity, or sufficient purity such that further purification does not detectably alter the physical and chemical properties, such as the enzyme activity and biological activity, of the substance. Both traditional and modern methods for purification of compounds that produce substantially chemically pure compounds are known to those skilled in the art. However, a substantially chemically pure compound may be a mixture of stereoisomers.

[0051] In some embodiments, a pharmaceutical composition is provided that includes a peptide of interest, including any pharma- ceutically acceptable salts thereof, where the peptide of interest is synthesized by the methods of the present invention and is a pharmaceutical grade compound. In some embodiments, the pharmaceutical composition includes the peptide of interest, including any pharma- ceutically acceptable salts thereof, as a pharma- ceutical active agent. In some embodiments, the pharmaceutical composition includes a therapeutically effective amount of the peptide of interest. In some embodiments, the pharmaceutical composition consists essentially of the peptide of interest, including any pharma- ceutically acceptable salts thereof.

[0052] In some embodiments, the pharmaceutical composition is for use as a medicament (eg, for treating and / or preventing a disease and / or medical condition in a subject in need thereof).

[0053] In some embodiments, the term "amino acid" encompasses D- and / or L-amino acids, optionally containing one or more protecting groups.

[0054] The term "amino acid" as used herein refers to an organic compound that contains both a basic amino group and an acidic carboxyl group. Included in this term are naturally occurring amino acids, protected amino acids (e.g., containing one or more protecting groups on the carboxyl, amine, and / or side chain of the amino acid), unusual, non-naturally occurring amino acids, and amino acids that are known to occur biologically in free or conjugated form but do not normally occur in proteins. Included in this term are modified and unusual amino acids, such as those disclosed in Roberts and Vellaccio (1983) The Peptides. 5: 342-429.Modified, unusual, or non-naturally occurring amino acids include D-amino acids, hydroxylysine, 4-hydroxyproline, N-Cbz-protected aminovaleric acid (Nva), ornithine (O), aminooctanoic acid (Aoc), 2,4-diaminobutyric acid (Abu), homoarginine, norleucine (Nle), N-methylaminobutyric acid (MeB), 2-naphthylalanine (2Np), aminoheptanoic acid (Ahp), phenylglycine, β-phenylproline, tert-leucine, 4-aminocyclohexylalanine (Cha), N-methyl-norleucine, 3,4-dehydroproline, N,N-dimethylaminoglycine, N-methylaminoglycine, 4-aminopipetdine-4-carboxylic acid, acid), 6-aminocaproic acid, trans-4-(aminomethyl)-cyclohexanecarboxylic acid, 2-, 3-, and 4-(aminomethyl)-benzoic acid, 1-aminocyclopentanecarboxylic acid, 1-aminocyclopropanecarboxylic acid, cyanopropionic acid, 2-benzyl-5-aminopentanoic acid, norvaline (Nva), 4-O-methyl-threonine (TMe), 5-O-methyl-homoserine (hSM), tert-butylalanine (tBu), cyclopentylalanine (Cpa), 2-aminoisobutyric acid (Aib), N-methylglycine (MeG), N-methylalanine (MeA), N-methylphenylalanine (MeF), 2-thienylalanine (2Th), 3-thienylalanine (3Th), O-methyltyrosine (YMe), 3-benzothienylalanine (Bzt), and D-alanine (DAl).

[0055] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a polymer of amino acid residues.

[0056] The terms "peptide," "polypeptide," and "protein," as used herein, encompass natural peptides, peptide derivatives such as beta-peptides, peptidomimetics (which typically contain non-peptide bonds or other synthetic modifications), and peptide analogs peptoids and semipeptoids, or any combination thereof. In another embodiment, the terms "peptide," "polypeptide," and "protein" apply to amino acid polymers in which at least one amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0057] The term "derivative" or "chemical derivative" includes any chemical derivative of a polypeptide having one or more residues chemically derivatized by reaction on a side chain or any functional group within the peptide. Such derivatized molecules include, for example, peptides having one or more protecting groups (e.g., side chain protecting group(s) and / or N-terminal protecting group) and / or peptides in which free amino groups have been derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, acetyl groups, or formyl groups. Free carboxyl groups may be derivatized to form amides, salts, methyl and ethyl esters, or other types of esters, or hydrazides thereof. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are peptides containing one or more naturally occurring amino acid derivatives of the twenty standard amino acid residues. For example, 4-hydroxyproline may be substituted for proline, 5-hydroxylysine may be substituted for lysine, 3-methylhistidine may be substituted for histidine, homoserine may be substituted for serine, and Dab, Daa, and / or ornithine (O) may be substituted for lysine.

[0058] Additionally, peptide derivatives may differ from the native sequence of the peptides of the invention by chemical modifications including, but not limited to, terminal NH2 acylation, acetylation, or thioglycolic acid amidation, as well as by amidation of terminal and / or side chain carboxy groups, e.g., with ammonia, methylamine, etc. Peptides may be either linear, cyclic, branched, or the like, with any conformation that may be achieved using methods known in the art.

[0059] method In another aspect, there is a method of synthesizing a compound of interest and / or any salt thereof, the compound of interest having formula 3: [ka] wherein each R4 is independently selected from the group consisting of H, an amino acid, a peptide, a polyamino acid, and at least one R4 is not H and R5 is hydrogen or a linker group attached to a solid phase, the method comprising providing a compound of the invention: (i) deprotecting the amine protecting group of a compound of the invention; (ii) coupling an N-protected amino acid moiety to a compound of the invention, thereby inducing elongation of a solid phase-bound peptide chain; (iii) contacting the growing peptide chain with an amine base, thereby obtaining a solid phase-bound compound of interest; The compounds of the present invention include those of formula 1: [ka] wherein X is hydrogen or a phenol protecting group, R1 comprises an amine protecting group, and R represents a linker group attached to a solid phase. In some embodiments, the terms "solid phase" and "solid support" are used interchangeably herein. In some embodiments, the compound of interest comprises a peptide, and the sequence of the peptide comprises a diazotized tyrosine (e.g., Tyr-PPC) as described herein. In some embodiments, the diazotized tyrosine is located at the N-terminus, C-terminus, and / or within the peptide sequence.

[0060] In some embodiments, the method of the invention comprises synthesizing a compound of interest by providing a solid phase-bound compound of the invention followed by performing steps i-iii, where the solid phase-bound compound of the invention is represented by formula 1A, where R1 is or includes an amine protecting group (e.g., Fmoc), X is hydrogen, and Y is as described herein. In some embodiments, the solid phase-bound compound of the invention is represented by formula 1A, where R1 is or includes an amine protecting group (e.g., Fmoc), X is hydrogen, and Y is O. In some embodiments, steps i-iii are performed in order.

[0061] In another aspect, the method of the invention comprises providing a compound of the invention bound to a solid phase, followed by: - coupling an N-protected amino acid moiety to the N-terminus of the compound of the invention, thereby inducing the elongation of a solid phase-bound peptide chain; and - contacting the growing peptide chain (and / or its bound solid phase) with a sufficient amount of an amine base, thereby obtaining a solid phase-bound compound of interest. The compound of the present invention comprises: [ka] wherein X is hydrogen or a phenol protecting group, R is H, and R represents a linker group attached to the solid phase. In some embodiments, X is H.

[0062] In another aspect, a method is provided for synthesizing a peptide sequence of interest represented by the formula AXB-NH2, where A represents a first amino acid or a first amino acid sequence, B represents a second amino acid or a second amino acid sequence, and X is one of the following: [ka] wherein the wavy bonds represent attachment points to the peptide sequence, and the method comprises: - providing a first amino acid or a first amino acid sequence bound to a solid phase, - carrying out a deprotection step to obtain a deprotected first amino acid or a deprotected first amino acid sequence (e.g. comprising a deprotected N-terminal amine); - coupling a deprotected first amino acid or a deprotected first amino acid sequence to a compound of any one of formulas II or 2, including any salt thereof, wherein R1 comprises an amine protecting group; - deprotecting the amine protecting group to obtain a deprotected N-terminal amine; - coupling a second amino acid or performing SPPS of a second amino acid sequence; - followed by step iii, thereby obtaining the peptide sequence of interest bound to the solid phase; Includes.

[0063] In some embodiments, the method further comprises cleaving the peptide sequence to obtain the peptide sequence of interest.

[0064] In some embodiments, each of the steps of the method (e.g., steps i-iii) is carried out by applying a solution of the corresponding reagent. One skilled in the art will appreciate that the solid-phase reaction is carried out by applying a solution containing the reagent to a solid support in contact with the growing chain or compound to induce a reaction between the reagent and the growing chain or compound. Furthermore, it should be clear that the reaction on the solid support requires a solvent employed to provide sufficient swelling to the resin, thereby facilitating the reaction or improving the reaction yield of each step. Furthermore, the solvent must be compatible with the resin, e.g., the solvent must be inert to the resin without inducing physical or chemical degradation of the resin. Various solvents such as DMF, DCM, NMP, etc. can be implemented in the solid-phase reaction. Other solvents suitable or compatible for solid-phase reactions are known in the art. The exact solvent depends on the chemical composition of the resin, resin swelling, the ability of the solvent to dissolve any of the reagents, etc., and preferably, a dry solvent is used for any one of the steps of the method of the present invention. In particular, it is desirable to use a dry solvent (water content less than 1%) for step ii of the method.

[0065] In some embodiments, each of the method steps (e.g., steps i-iii) is carried out under conditions sufficient to induce significant conversion, e.g., a reaction yield of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, including any range therebetween. In some embodiments, each of the method steps (e.g., steps i-iii) is carried out under air at a temperature ranging from 10 to 50° C., including any range therebetween. In some embodiments, each of the method steps (e.g., steps i-iii) is carried out at a temperature below the boiling point of the solvent.

[0066] In some embodiments, each of the steps of the method (e.g., steps i-iii) is performed once. In some embodiments, any one of the steps of the method (e.g., any one of steps i-iii) is repeated. In some embodiments, any one of the steps of the method is performed multiple times (e.g., 2-20 times, 2-4 times, 4-6 times, 6-10 times, 10-20 times (including any range in between)). In some embodiments, any one of the steps of the method is performed once or multiple times until the reaction is complete. One of ordinary skill in the art would be able to determine when the reaction is complete. For example, the conversion efficiency of the coupling (step ii) can be determined by performing a ninhydrin test (also called the Kaiser test). The efficiency of the deprotection step i can be determined by measuring the UV absorbance of the deprotection solution and thus determining the concentration of the cleaved amine protecting group (e.g., of Fmoc deprotection). Furthermore, the duration of any of the steps of the present invention can be adjusted to obtain the highest efficiency by monitoring the yield of each step as described herein.

[0067] In some embodiments, step i of the methods of the invention comprises applying a deprotection solution to the solid support-bound compound or growing chain for a period of at least 1 second, at least 1 minute (m), at least 5 m, at least 10 m, at least 20 m (including any range in between). In some embodiments, step i results in deprotection of the amine or cleavage of the amine protecting group.

[0068] In some embodiments, the deprotection solution comprises a suitable amount of a deprotecting agent (e.g., acid or base) in an amount sufficient to deprotect at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, at least 99.9%, including any range therebetween, of the amine protecting groups. A variety of deprotection solutions are well known in the art (such as a 20% piperidine solution for Fmoc deprotection, or a 50% TFA solution for Boc deprotection).

[0069] In some embodiments, step ii of the method of the invention comprises applying a coupling solution to the solid support-bound compound or growing chain for a period of at least 1 minute (m), at least 5 m, at least 10 m, at least 20 m, at least 30 m, at least 1 hour, at least 10 h, or more, including any range therebetween, where the coupling solution comprises a sufficient amount of an amino acid moiety. In some embodiments, step ii of the method of the invention results in the formation of a peptide bond between the amino group and the next amino acid (or results in the coupling of the next amino acid). In some embodiments, step ii of the method of the invention comprises coupling an amino acid moiety (e.g., an N-protected amino acid moiety) to the amino group of the compound of the invention, thereby inducing chain growth on the solid phase. In some embodiments, step ii of the method of the invention is for growing a peptide chain. In some embodiments, step ii of the method of the invention induces growth of a growing peptide chain bound to a solid phase.

[0070] In some embodiments, the amino acid moiety comprises an N-protected amino acid moiety. In some embodiments, the amino acid moiety comprises an active ester of an amino acid. In some embodiments, the amino acid moiety comprises an N-protected active ester of an amino acid. The active ester is as described above. In some embodiments, the amino acid moiety comprises an amino acid.

[0071] In some embodiments, the coupling solution comprises a sufficient amount of an N-protected amino acid moiety, and optionally, a sufficient amount of an organic base (such as DIPEA or collidine). In some embodiments, the coupling solution comprises a sufficient amount of an N-protected amino acid moiety, and optionally, a sufficient amount of a coupling agent.

[0072] In some embodiments, the coupling solution comprises a sufficient amount of N-protected amino acid and coupling agent. Coupling agents are well known in the art, and exemplary coupling agents are described above. In some embodiments, the coupling solution comprises 1-2 molar equivalents (relative to the amine of the compound or growing chain) of N-protected amino acid and coupling agent. In some embodiments, the coupling solution comprises 1-2 molar equivalents of N-protected amino acid and coupling agent, and lacks a base. In some embodiments, the coupling solution comprises 1-5 molar equivalents (including any range in between) of N-protected amino acid and coupling agent, and lacks a base.

[0073] In some embodiments, the coupling solution comprises 1-5 molar equivalents (including any range in between) of an N-protected amino acid moiety and lacks a coupling agent and a base. In some embodiments, the coupling solution comprises 1-5, 1-2, 2-4 molar equivalents (including any range in between) of an N-protected active ester of an amino acid (e.g., an NHS ester) and lacks a coupling agent and a base. In some embodiments, the coupling solution comprises less than 4 molar equivalents, less than 3 molar equivalents, less than 2 molar equivalents, less than 1.6 molar equivalents of a base (e.g., an organic amine base such as a tertiary amine), including any range in between.

[0074] In some embodiments, the coupling solution comprises 1-6, 1-4, 1-2, 2-4 molar equivalents of the N-protected amino acid moiety. In some embodiments, the coupling solution comprises the N-protected amino acid moiety in an amount sufficient for a coupling yield of at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, at least 99.9%, including any range therebetween. In some embodiments, the coupling solution comprises the N-protected amino acid moiety in an amount sufficient to provide selective coupling to the free amine groups of the growing chain. As used herein, the term coupling selectivity refers to the ratio of coupling to amino groups versus coupling to the hydroxy group of the phenolic ring of tyrosine.

[0075] As depicted in Scheme 1, coupling can occur at both the amino group (at the N-terminus of the growing chain) and at the OH group of the tyrosine side chain. It is desirable to maximize coupling selectivity in order to minimize OH-coupling by-products. [ka]

[0076] One of skill in the art will appreciate that the exact composition of the coupling solution will depend on the amino acid moiety and, optionally, on the particular coupling agent. Furthermore, the exact composition of the coupling solution can be adjusted based on the selectivity and yield of the coupling step. Non-limiting exemplary coupling solutions are as described in the Examples section.

[0077] In some embodiments, the method steps (eg, steps i-iii) are performed in order.

[0078] In some embodiments, the method of the present invention comprises carrying out a step of loading the compound of the present invention onto a solid phase, thereby obtaining a compound of the present invention bound to the solid phase. The loading is carried out according to well-known procedures.

[0079] In some embodiments, the method of the present invention includes performing step iii, thereby removing by-products including the growing chain coupled to the hydroxy group (e.g., of the phenol ring) of tyrosine, as shown in Scheme 1 above. As described in the Examples section, the inventors have performed a novel approach to SPPS of peptides (e.g., TRS) containing diazotized tyrosine. By implementing the SPPS route described herein, as illustrated in Scheme 1, the inventors observed significant by-product formation due to side reactions occurring at the unprotected phenol ring of diazotized tyrosine (e.g., compound 13, see Scheme 2 below). Therefore, the by-products must be cleaved to obtain the desired product in sufficient yield.

[0080] To this end, the present invention, in some embodiments thereof, provides an efficient and simple procedure for the removal of by-products (step iii of the invention described herein), thus facilitating the SPPS synthesis of peptides containing diazotized tyrosines, such as TRS.

[0081] In some embodiments, step iii comprises contacting the growing peptide chain with an amine base or a solution or composition comprising a sufficient amount of an amine base. In some embodiments, step iii comprises contacting the growing peptide chain with a cleavage solution comprising a sufficient amount of an amine base under suitable conditions, the sufficient amount being an amount that induces substantial (at least 90%, at least 95%, at least 99%) cleavage or removal of by-products. In some embodiments, the sufficient amount comprises at least 1, at least 10, at least 50, at least 100, at least 200, at least 500, 10-500, 10-100, 50-500, 50-100, 10-50, 1-100, 1-10, 1-500 molar equivalents of amine base, including any range therebetween. In some embodiments, suitable conditions include contact times ranging from 1 second to 1 hour, and / or operational temperatures ranging from 5-90, or 15-40, or 15-30° C., including any range therebetween.

[0082] In some embodiments, the amine base is an Fmoc deprotecting agent. In some embodiments, the amine base can fully deprotect Fmoc (e.g., result in at least 80%, at least 90%, at least 95%, at least 99% Fmoc deprotection on the solid support). In some embodiments, the amine base is selected from primary amines, secondary amines, guanidine-based compounds, and amidine-based compounds, including any combination thereof. In some embodiments, the amine base is a linear amine or a cyclic amine.

[0083] In some embodiments, the amine base comprises any one of piperidine, DBU, cyclohexylamine, ethanolamine, pyrrolidine, morpholine, 4-methylpiperidine, tetramethylguanidine, and DBN, or any combination thereof.

[0084] In some embodiments, the amine base is substantially devoid of tertiary amines, hi some embodiments, the amine base is substantially devoid of DIPEA, TEA, or both.

[0085] In some embodiments, the methods of the invention optionally comprise performing a step (iv) of deprotecting the N-protected amino acid moiety prior to performing step (iii). In some embodiments, step iv comprises applying a deprotection solution to the resin-bound growing chain, thereby removing the amine protecting group, wherein the deprotection solution is as described herein.

[0086] In some embodiments, the method of the present invention further comprises subsequently repeating steps (ii) and (iv) before performing step (iii). In some embodiments, steps (ii) and (iv) are performed and subsequently repeated, thereby extending the peptide chain. In some embodiments, steps (ii) and (iv) are repeated to synthesize a predetermined sequence (e.g., a predetermined amino acid sequence). In some embodiments, steps (ii) and (iv) are repeated multiple times to synthesize a predetermined sequence, the multiple times being as described herein.

[0087] In some embodiments, the methods of the invention further comprise cleaving the synthesized compound (eg, peptide) from the solid support.

[0088] In some embodiments, the methods of the present invention comprise a process comprising the step of reacting a compound represented by formula 4: [ka] wherein the method comprises steps i-iii and further comprises repeating steps (ii) and (iv) before performing step (iii) to synthesize a peptide sequence represented by formula 4.

[0089] In some embodiments, SPPS is performed to form a peptide chain on a solid support: [ka] where PG is a protecting group (e.g., Fmoc) and the peptide chain further comprises one or more protecting groups at the side chain of the amino acid(s); and (ii) subsequently cleaving the peptide chain (under conditions sufficient to preserve the PG and lysine protecting groups), thereby producing a protected peptide chain: [ka] wherein the side chains of lysine, arginine, threonine, or both are optionally bound to a protecting group; and (iii) combining the protected peptide chain with the compound: [ka] There are methods of synthesizing TRS, comprising coupling with: [ka] This is done by adding to a solution of the active ester.

[0090] In some embodiments, the compound: [ka] is synthesized by providing a compound of formula 2, where R1 is Boc (e.g., the compound described in Example 1), and deprotecting the Boc as described herein.

[0091] In some embodiments, the protected peptide chain is: [ka] and providing a protected peptide chain and a compound: [ka] There are methods of synthesizing TRS that include coupling with, thereby obtaining TRS.

[0092] In some embodiments, the method includes providing a diazotized tyrosine bound to a solid support, deprotecting the amine protecting group, thereby obtaining a free amine group, and coupling the free amine group to a next amino acid or polypeptide. In some embodiments, the method further includes repeating the deprotection and coupling steps (e.g., multiple times as described herein) to synthesize a predetermined sequence (e.g., a predetermined amino acid sequence).

[0093] In another aspect, there is a method for synthesizing a peptide sequence of interest that comprises a diazotized tyrosine (including any derivative thereof), the diazotized tyrosine being linked to an azo bond. [ka] In some embodiments, the diazotized tyrosine comprises a substituent attached to the phenolic ring of tyrosine via Formula 3A: [ka] wherein R6 comprises one or more substituents, the wavy bonds represent points of attachment to a peptide sequence of interest, and at least one of the wavy bonds represents a point of attachment to the peptide sequence. In some embodiments, one of the wavy bonds represents a point of attachment to the peptide sequence, and another wavy bond represents a point of attachment to the peptide sequence, and the other ... - , OH, and H; (ii) representing the point of attachment to the peptide sequence of interest.

[0094] In some embodiments, the method comprises administering to a subject a compound: [ka] to the growing peptide chain on the solid support, and performing step iii, thereby obtaining the desired peptide sequence bound to the solid support, wherein R1 comprises an amine protecting group and R6 is as described herein.

[0095] In some embodiments, the method further comprises coupling the next N-protected amino acid to the N-terminus and deprotecting the amine protecting group to obtain a deprotected N-terminal amine, the coupling and deprotection steps being performed before performing step iii. In some embodiments, the method further comprises repeating the coupling and deprotection steps, thereby obtaining a solid support-bound peptide sequence of interest. In some embodiments, the method further comprises cleaving the peptide sequence to obtain a peptide sequence of interest.

[0096] In some embodiments, the peptide sequence of interest is represented by the formula AXB-NH2, where A represents a first amino acid sequence, B represents a second amino acid sequence, and X represents diazotized tyrosine, and the method comprises: - providing a first amino acid sequence bound to a solid phase; - carrying out a deprotection step to obtain a deprotected first amino acid sequence (e.g., comprising a deprotected N-terminal amine); - reacting the deprotected first amino acid sequence with the compound: [ka] wherein R1 comprises an amine protecting group and R6 is as described herein; - deprotecting the amine protecting group to obtain a deprotected N-terminal amine; - performing SPPS of a second amino acid sequence, thereby extending the peptide chain; followed by step iii, thereby obtaining the peptide sequence of interest bound to the solid phase; Includes.

[0097] In some embodiments, the method further comprises cleaving the peptide sequence to obtain the peptide sequence of interest.

[0098] In some embodiments, R6 is phosphorylcholine, (C0-C6)alkyl-aryl, (C0-C6)alkyl-heteroaryl, (C0-C6)alkyl-(C3-C8)cycloalkyl, optionally substituted C3-C8 heterocyclyl, halogen, -NO2, -CN, -OH, -CONH2, -CONR2, -CNNR2, -CSNR2, -CONH-OH, -CONH-NH2, -NHCOR, -NHCSR, -NHCNR, -NC(=O)OR, -NC(=O)NR, -NC(=S)OR, -NC(=S)NR, -S02R, -SOR, -SR, -S02OR, -SON(R)2, -NHNR2, -NNR, C1-C6 haloalkyl, optionally substituted The substituents include C1-C6 alkyl, -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy(C1-C6 alkyl), hydroxy(C1-C6 alkoxy), alkoxy(C1-C6 alkyl), alkoxy(C1-C6 alkoxy), C1-C6 alkyl-NR2, C1-C6 alkyl-SR, -CONH(C1-C6 alkyl), -CON(C1-C6 alkyl)2, -CO2H, -CO2R, -OCOR, -OCOR, -OC(=O)OR, -OC(=O)NR, -OC(=S)OR, -OC(=S)NR, polyamino acids, peptides, or combinations thereof. Further substituents are well known in the art.

[0099] In some embodiments, the method includes performing SPPS, thereby synthesizing a peptide chain, and coupling a diazotized tyrosine to the peptide chain (e.g., to the N-terminus of the growing peptide chain). In some embodiments, the diazotized tyrosine is represented by Formula 3B: [ka] wherein R6 and R4 are as described herein, and R5 is H, an active ester, or absent. In some embodiments, at least one R4 is or includes an amine protecting group (e.g., Fmoc).

[0100] In some embodiments, the method comprises (i) performing SPPS, thereby synthesizing a peptide chain, (ii) subsequently cleaving the peptide chain (under conditions sufficient to preserve protecting groups at either the N-terminus or C-terminus and / or side chains), thereby obtaining a peptide chain comprising a deprotected N-terminus or a deprotected C-terminus, and (iii) coupling the deprotected N-terminus or the deprotected C-terminus with a diazotized tyrosine described herein (e.g., diazotized tyrosine of formula 3B), where step (iii) is performed in solution (e.g., by utilizing an organic solvent that is compatible with the reactants).

[0101] In some embodiments, the diazotized tyrosine bound to the solid support has formula 3C: [ka] wherein R6 is as described herein and at least one R4 includes an amine protecting group.

[0102] In some embodiments, the methods of the invention further comprise removing the by-products by performing step iii, as described herein, thereby obtaining a predetermined sequence that is substantially devoid of by-products.

[0103] In some embodiments, the methods of the invention result in the formation of less than 10 mol%, less than 8 mol%, less than 5 mol%, less than 1 mol%, less than 0.5 mol%, less than 0.1 mol%, including any range therebetween. In some embodiments, the amount of by-product relates to the molar percentage of product relative to the total amount of peptide sequence (e.g., bound to the solid support or after cleavage).

[0104] definition As used herein, the term "alkyl" refers to an aliphatic hydrocarbon, including straight-chain and branched-chain groups. Preferably, the alkyl group has 21-100 carbon atoms, more preferably 21-50 carbon atoms. Whenever a numerical range, for example "21-100", is stated herein, it is implied that the group, in this case the alkyl group, may contain up to 100 carbon atoms, such as 21 carbon atoms, 22 carbon atoms, 23 carbon atoms, etc. In the context of the present invention, a "long-chain alkyl" is an alkyl having at least 20 carbon atoms in its backbone (the longest path of consecutive covalently bonded atoms). Thus, a short-chain alkyl has 20 or fewer backbone carbons. An alkyl, as defined herein, may be substituted or unsubstituted.

[0105] The term "alkyl," as used herein, also includes saturated or unsaturated hydrocarbons, and thus, this term further includes alkenyls and alkynyls.

[0106] The term "alkenyl," as defined herein, refers to an unsaturated alkyl having at least two carbon atoms and at least one carbon-carbon double bond. Alkenyl can be unsubstituted or substituted with one or more substituents, as described above.

[0107] The term "alkynyl," as defined herein, is an unsaturated alkyl having at least two carbon atoms and at least one carbon-carbon triple bond. Alkynyl can be unsubstituted or substituted with one or more substituents, as described above.

[0108] The term "cycloalkyl" refers to an all-carbon monocyclic or fused ring (i.e., rings which share adjacent pairs of carbon atoms) group in which one or more of the rings does not have a completely conjugated pi-electron system. Cycloalkyl groups can be substituted or unsubstituted as indicated herein.

[0109] The term "aryl" refers to an all-carbon monocyclic or fused polycyclic (i.e., rings which share adjacent pairs of carbon atoms) group having a completely conjugated pi-electron system. Aryl groups can be substituted or unsubstituted as indicated herein.

[0110] The term "alkoxy" refers to both an --O-alkyl and an --O-cycloalkyl group, as defined herein.

[0111] The term "aryloxy" refers to -O-aryl as defined herein.

[0112] Each of the alkyl, cycloalkyl, and aryl groups in the general formulas herein can be substituted with one or more substituents, each of which can be, independently, for example, halide, alkyl, alkoxy, cycloalkyl, nitro, amino, hydroxyl, thiol, thioalkoxy, carboxy, amido, aryl, and aryloxy, depending on the group substituted and its position in the molecule. Additional substituents are also contemplated.

[0113] The terms "halide," "halogen," or "halo" refer to fluorine, chlorine, bromine, or iodine.

[0114] The term "haloalkyl" refers to an alkyl group, as defined herein, further substituted with one or more halide.

[0115] The term "haloalkoxy" refers to an alkoxy group, as defined herein, further substituted with one or more halide.

[0116] The terms "hydroxyl" or "hydroxy" refer to an --OH group.

[0117] The term "mercapto" or "thiol" refers to a -SH group.

[0118] The term "thioalkoxy" refers to both an --S-alkyl group, and an --S-cycloalkyl group, as defined herein.

[0119] The term "thioaryloxy" refers to both an --S-aryl and an --S-heteroaryl group, as defined herein.

[0120] The term "amino" refers to a -NR'R'' group, with R' and R'' as described herein.

[0121] The term "heterocyclyl" refers to a monocyclic or fused ring group having one or more atoms such as nitrogen, oxygen, and sulfur in the ring(s). The ring may also have one or more double bonds. However, the ring does not have a completely conjugated pi-electron system. Representative examples are piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholino, etc.

[0122] The term "carboxy" or "carboxylate" refers to the group -C(O)OR', where R' is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl (bonded through a ring carbon), or heterocyclyl (bonded through a ring carbon), as defined herein.

[0123] The term "carbonyl" refers to the group --C(O)R', where R' is as defined above.

[0124] The above terms also include thio derivatives thereof (thiocarboxy and thiocarbonyl).

[0125] The term "thiocarbonyl" refers to a -C(S)R' group, where R' is as defined above.

[0126] A "thiocarboxy" group refers to a -C(S)OR' group, where R' is as defined herein.

[0127] A "sulfinyl" group refers to a -S(O)R' group, where R' is as defined herein.

[0128] A "sulfonyl" or "sulfonate" group refers to a -S(O)2R' group, where R' is as defined herein.

[0129] A "carbamyl" or "carbamate" group refers to an --OC(O)NR'R'' group, where R' is as defined herein and R'' is as defined for R'.

[0130] A "nitro" group refers to a -NO2 group.

[0131] The term "amide" as used herein includes C-amide and N-amide.

[0132] The term "C-amide" refers to a -C(O)NR'R'' terminal group or a -C(O)NR'- linking group, as these terms are defined above, where R' and R'' are as defined herein.

[0133] The term "N-amido" refers to an -NR"C(O)R' terminal group or an -NR'C(O)- linking group, as these terms are defined above, where R' and R" are as defined herein.

[0134] The term "carboxylic acid derivatives" as used herein includes carboxy, amide, carbonyl, anhydride, carbonate, and carbamate.

[0135] A "cyano" or "nitrile" group refers to a --CN group.

[0136] The terms "azo" or "diazo" refer to an --N.dbd.NR' terminal group or an --N.dbd.N- linking group, as these terms are defined above, with R' defined above.

[0137] The term "guanidine" refers to the -R'NC(N)NR''R''' terminal group or the -R'NC(N)NR''- linking group, as these terms are defined above, where R', R'', and R''' are as defined herein.

[0138] As used herein, the term "azido" refers to the group --N3.

[0139] The term "sulfonamide" refers to a -S(O)2NR'R'' group, with R' and R'' as defined herein.

[0140] The term "phosphonyl" or "phosphonate" refers to the group -OP(O)-(OR')2, with R' as defined above.

[0141] The term "phosphinyl" refers to a -PR'R'' group, with R' and R'' defined above.

[0142] The term "alkylaryl" refers to an alkyl, as defined herein, substituted with an aryl, as described herein. An exemplary alkylaryl is benzyl.

[0143] The term "heteroaryl" refers to a monocyclic (e.g., C5-C6 heteroaryl ring) or fused ring (i.e., rings sharing adjacent pairs of atoms) group having one or more atoms, such as nitrogen, oxygen, and sulfur, in the ring(s) and further having a fully conjugated pi-electron system. In some embodiments, the terms "heteroaryl" and "C5-C6 heteroaryl" are used interchangeably herein. Non-limiting examples of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, and purine. Heteroaryl groups can be substituted or unsubstituted by one or more substituents, as described above. Representative examples are thiadiazole, pyridine, pyrrole, oxazole, indole, purine, and the like.

[0144] As used herein, the terms "halo" and "halide," which are considered interchangeable herein, refer to an atom of the halogens, i.e., fluorine, chlorine, bromine, or iodine, also referred to herein as fluoride, chloride, bromide, and iodide.

[0145] The term "haloalkyl" refers to an alkyl group, as defined above, further substituted with one or more halide.

[0146] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated above and as claimed in the claims section below finds experimental support in the following examples.

[0147] It is understood that certain features of the invention that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described in the context of a single embodiment for brevity may also be provided separately or in any suitable subcombination, or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0148] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0149] All publications, patents, and patent applications mentioned herein are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. Furthermore, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent section headings are used, they should not be construed as necessarily limiting.

[0150] Example Example 1 Conjugation of phosphorylcholine to BOC-TYR 1) Preparation of diazonium salts 4-Aminophenyl(2-(trimethylammonio)ethyl)phosphate (50 mg, 0.18 mmol) was dissolved in 1 M aqueous HCl (1 mL), cooled in an ice-water bath, and sodium nitrite (12.6 mg, 0.18 mmol) was added in a single batch. The resulting solution was stirred at 0° C. for 30 min.

[0151] 2) Azo coupling A new mixture was prepared containing BOC-L-tyrosine (107 mg, 0.38 mmol) in NaHCO3 (1 M) + NaOH buffer (pH 10) (3.3 mL) + acetonitrile (1.2 mL). The mixture was cooled in an ice-water bath. The diazonium salt mixture was added dropwise. A red solution was formed. Mixing was continued at 0° C. for 6 min. The reaction mixture was acidified to pH=~3 with 1N aqueous HCl.

[0152] The resulting solution was lyophilized overnight and subsequently purified (e.g., by preparative MPLC) to give the compound: [ka] where R is Boc.

[0153] Example 2 Preparation of exemplary compounds of the invention Preparation of diazonium salts: [ka] 4-Aminophenyl(2-(trimethylammonio)ethyl)phosphate (250 mg, 0.912 mmol) was dissolved in 1M aqueous HCl (5 mL) and cooled in an ice-water bath, and sodium nitrite (62.9 mg, 0.912 mmol) was added in a single batch. The resulting solution was stirred at 0 °C for 30 min. Azo coupling, a new mixture was prepared containing Fmoc-Tyr-OH (739 mg, 1.832 mmol) in saturated NaHCO3 (17 mL) + acetonitrile (12.5 mL). The resulting suspension / solution was cooled in an ice-water bath. The diazonium salt mixture was added dropwise. Mixed at 0 °C. The reaction mixture gradually became yellow. After 5.5 h, LCMS showed complete conversion. The reaction mixture was acidified to pH ~6 with 1N HCl, and the yellowish suspension changed to a clear orange solution, which was lyophilized. This provided 2.10 g. It was dissolved in a mixture of DMSO / HO / MeCN (~1:1:1) and purified by acidic preparative MPLC in five runs. The fractions were combined and lyophilized overnight to give the desired product (compound 10).

[0154] Example 3 SPPS synthesis of TRS Facing difficulties in protecting the hydroxy group of compound 10, the inventors explored a novel strategy for the SPPS synthesis of TRS.

[0155] The inventors, The SPPS synthesis was initiated by implementing N-protected (Fmoc) phosphorylcholine modified tyrosine (e.g., compound 10), and 200 mg of compound 10 was loaded onto the CTC resin. Briefly, 2-chlorotrityl chloride resin (1.0-1.2 mmol / g, 200-400 mesh) (450 mg, 1.441 mmol) was swollen by rocking in dichloromethane (12 mL) for 30 min. The solvent was removed and a solution of (S,E)-4-((5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-carboxyethyl)-2-hydroxyphenyl)diazenyl)phenyl(2-(trimethylammonio)-ethyl)phosphate (200 mg, 0.290 mmol) in dichloromethane (12 mL) containing DIPEA (0.177 mL, 1.016 mmol) was added (substrate was not soluble in DCM, solution was obtained after addition of DIPEA).

[0156] After 17 h, the solution was removed and the resin was washed with dichloromethane (3 x 10 mL, each wash step >2 min). Capping solution (CH2Cl2:MeOH:DIPEA 9:1:0.5) was added (10.5 mL) and the resin was rocked for 1 h. The resin was then washed with dichloromethane (3 x 10 mL) and dried in vacuum.

[0157] This resin was then split equally in order to explore several conditions of subsequent chemistry in parallel, aimed at preventing the formation of the previously discovered tyrosine O-acylation, as seen in the isolation of compound 13 (see Scheme 2). The different reaction conditions are outlined in Table 1 (see below). [ka] [Table 1]

[0158] Various coupling conditions were tested, as shown in Table 1. Entries a-c led to the formation of substantial amounts of the by-product (13). An improvement was obtained by using Fmoc-Gly-OSu in DMF (entry d). In this case, the formation of the by-product (13) was reduced to only 3% relative to the desired compound 12. Nevertheless, none of these methods could completely suppress the formation of 13, which still represents a risk for further peptide synthesis, since it could lead to the accumulation of the by-product (compound 13).

[0159] Surprisingly, the inventors have found that the by-product (or the phenolic ester by-product represented by compound 13 in Scheme 3) can be cleaved under standard Fmoc deprotection conditions with piperidine or DBU in DMF to neatly provide compound 15, as shown below. [ka]

[0160] The inventors have further successfully implemented additional amine bases (other than piperidine and DBU) for the cleavage of the by-product. A number of amine bases, including primary and secondary amines, guanidine-based compounds, and amidine-based compounds, have been successfully implemented for the cleavage of the by-product. Furthermore, the inventors have observed that tertiary amines can also be implemented for the cleavage of the by-product, but that tertiary amines often require longer reaction times and / or subsequent repetition of the by-product cleavage step. To this end, the inventors have found that primary and secondary amines, guanidine-based compounds, and amidine-based compounds are characterized by improved reactivity and thus result in more efficient cleavage of the by-product (e.g., resulting in improved yield and / or purity of the compound of interest) compared to tertiary amines under similar reaction conditions.

[0161] The amine bases that the inventors have successfully tested (cleavage of at least about 95% of the by-products) are as follows: piperidine, DBU, cyclohexylamine, ethanolamine, pyrrolidine, morpholine, 4-methylpiperidine, tetramethylguanidine, and DBN. The amine bases are implemented in the form of 5-20% solutions in DMF (incubation time of about 30 minutes). The inventors have further observed that pyrrolidine, piperidine, DBU, cyclohexylamine, and ethanolamine have excellent efficiency (nearly 100% yield) for cleavage of the by-products. Moreover, it was found that primary and secondary amines, guanidine-based compounds, as well as amidine-based compounds (especially pyrrolidine, piperidine, DBU, cyclohexylamine, and ethanolamine) resulted in almost complete Fmoc deprotection, resulting in compound 15 being obtained in 99-100% yield.

[0162] After loading compound 10 onto the resin to obtain compound 11 (see Scheme 2), peptide coupling with Fmoc-Gly-OH was repeated on the solid phase under standard conditions, followed by Fmoc deprotection. Then, another peptide coupling step with Boc-Arg(Pbf)-OH was performed (Scheme 4, steps 1-4). The resin was then split into two parts. The first part was directly treated with HFIP for resin cleavage (step 5a), which provided a mixture of compounds 16 and 17. The second batch was first treated with piperidine in DMF to cleave the phenolic ester (step 5b). Then, a resin cleavage step was performed (step 6), which provided the desired tripeptide 16 in very high purity without traces of the di-coupled compound 17. [ka]

[0163] These results pave the way for the solid-phase synthesis of longer peptides under standard SPPS conditions, and the problem of O-acylation of the free tyrosine OH is efficiently overcome by cleavage of the phenolic ester by-product during the Fmoc deprotection step with piperidine in DMF.

[0164] A standard, non-limiting procedure for the SPPS synthesis of TRS is described below: [ka]

[0165] Compound 10 (Scheme 2) was loaded onto the solid support as described above, which resulted in the formation of compound 11.

[0166] Fmoc deprotection: The resin coupled to compound 10 (compound 11) was rocked in piperidine 20% (w / w) in peptide grade DMF (2 mL, 4.05 mmol) for 30 min. The piperidine solution was removed and the resin was washed with DMF (5 mL, peptide grade). This procedure was repeated twice.

[0167] For Boc-based SPPS synthesis, Boc-protected alternatives of compound 10 can be used. SPPS synthesis is almost identical to Fmoc-based SPPS synthesis, but Boc cleavage is usually performed under acidic conditions, including 50% TFA in DCM. The exact conditions for Boc cleavage are well known in the art. It is understood that Boc-based SPPS requires a solid support that is compatible with it. Solid supports for Boc-based SPPS are well known in the art.

[0168] The resin-bound Fmoc-tyrosine-PPC (11) was then used in a peptide solid-phase synthesis procedure to TRS (Scheme 10). Each amino acid was coupled under standard conditions using HBTU and HOBt with DIPEA in DMF. In some embodiments, each coupling step was performed multiple times (e.g., twice).

[0169] Removal of phenolic ester by-products and peptide cleavage: The resin was rocked in 20% (w / w) piperidine in peptide grade DMF (2 mL, 4.05 mmol) for 30 min and then washed with DMF (2 x 3 mL). This procedure was repeated twice, resulting in almost complete removal of the by-products (confirmed by analytical studies). Alternatively, a solution of DBU in DMF was successfully implemented for the removal of the by-products.

[0170] Finally, the peptide was cleaved from the resin using TFA / TIS / H2O (18:1:1) while removing all acid-labile protecting groups. The crude peptide was dissolved in water, and then the Pbf residues could be easily removed by extraction with EtOAc and Et2O. After lyophilization, crude TRS (TFA salt) was obtained in 63% yield and 88% purity. Purification by preparative HPLC provided 240 mg (10 to 51%) of the desired compound in high purity. Alternatively, the crude peptide (e.g., TRS and any one of the additional peptides disclosed below) was purified by preparative reversed-phase MPLC to obtain the desired peptide in high purity.

[0171] In some embodiments, removal of the by-products and deprotection of the N-terminal protecting group are performed simultaneously (e.g., by applying an amine base cleavage solution to the solid support-bound peptide chain to simultaneously remove both the N-terminal protecting group and the by-products).

[0172] Moreover, the TRS synthesized as described herein was further analyzed by HPLC and LC / MS to obtain its impurity profile. The impurity profile was further compared to that of TRS synthesized by post-SPPS modification (diazotization) of Thr-Lys-Pro-Arg-Gly-Tyr to couple the PC moiety to the phenolic ring of tyrosine. Surprisingly, the inventors confirmed that the TRS synthesized as described herein is characterized by a different impurity profile as shown in Figures 1A and 2A. The inventors observed that the TRS synthesized by post-SPPS modification is characterized by the 1.27 impurity, while the TRS synthesized as described herein is devoid of such impurity. Based on the proposed structure of the 1.27 impurity, it should be clear that such impurity is unique to post-SPPS modification (diazotization).

[0173] Additionally, the TRS synthesized as described herein was characterized by a process-specific impurity (the 0.7 impurity disclosed above) that corresponds to the compound of the present invention lacking the protecting group(s). Thus, it is hypothesized that the TRS synthesized as described herein (or any peptide having a diazotized tyrosine moiety) can be differentiated based on the process-specific impurity(s), such as the 0.7 impurity described above. Thus, the diazotized tyrosine moiety (e.g., [ka] The presence of impurities including 1,2-dichlorophenyl ether (DHA) and / or derivatives thereof is indicative of a peptide synthesized by the methods of the present invention.

[0174] To this end, the inventors have successfully synthesized various peptides having Tyrosine-PPC by utilizing the compound of the present invention (Fmoc-Tyrosine-PPC) based on the method described herein. Exemplary peptides are as follows: H-Leu-Phe-Orn-Gly-TyrPPC-OH; H-Leu-Phe-TyrPPC-Orn-Gly-OH; and H-TyrPPC-Leu-Phe-Orn-Gly-OH.

[0175] One of skill in the art will appreciate that any peptide sequence having a diazotized tyrosine moiety can be synthesized according to any one of the methods disclosed herein.

[0176] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will occur to those skilled in the art. It is therefore to be understood that the appended claims are intended to cover all such modifications and changes that fall within the true spirit of the invention.

Claims

1. Formula 1: 【Chemistry 1】 or a salt thereof, wherein X is hydrogen, a phenol protecting group, or 【Chemistry 2】 wherein R2 is a protected or unprotected side chain of a natural or non-natural alpha amino acid, each R1 is independently hydrogen or an amine protecting group, and R is hydrogen, a carboxyl protecting group, a leaving group, a solid-phase linker group, or absent, or a salt thereof.

2. 2. The compound of claim 1, wherein the amine protecting group comprises any one of 9-fluorenylmethyloxycarbonyl (Fmoc), Alloc, Dde, iv-Dde, benzyl, benzyloxycarbonyl, tert-butyloxycarbonyl (Boc), 2-[biphenylyl-(4)]-propyl-2-oxycarbonyl, dimethyl-3,5 dimethoxybenzyloxycarbonyl, 2-(4-nitrophenylsulfonyl)ethoxycarbonyl, 1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl, 2,7-di-tert-butyl-Fmoc, 2-fluoro-Fmoc, nitrobenzenesulfonyl, benzothiazole-2-sulfonyl, 2,2,2-trichloroethyloxycarbonyl, dithiasuccinoyl, p-nitrobenzyloxycarbonyl.

3. 2. The compound of claim 1, wherein the carboxyl protecting group comprises any one of tert-butyl ester, methyl ester, ethyl ester, benzyl ester, silyl ester, 2-(trimethylsilylethyl), (2-phenyl-2-trimethylsilyl)ethyl, 2-(trimethylsilyl)isopropyl), allyl ester, 2-chlorotrityl (2-Cl-Trt), 2,4-dimethoxybenzyl, 2-phenylisopropyl, 9-fluorenylmethyl, Dmab, carbamoylmethyl, phenacyl, p-nitrobenzyl, 4,5-dimethoxy-2-nitrobenzyl, and 1,1-dimethylallyl.

4. 2. The compound of claim 1, wherein the phenol protecting group comprises one of triisopropylsilyl ether (TIPS), tert-butyldimethylsilyl ether (TBDMS), methyl ether, benzyl ether (Bn), methoxymethyl acetal (MOM), 2-(trimethylsilyl)ethoxy]methyl acetal, tert-butyl ether, 2-chlorotrityl, trityl, benzyl, benzyloxycarbonyl, or Boc.

5. 2. The compound of claim 1, wherein the leaving group comprises one of a halo, a hydroxysuccinimide, a hydroxybenzotriazole, a pentafluorophenol, an imidazole carbonate, an O-acylisourea.

6. Formula 2: 【Chemistry 3】 2. The compound of claim 1, represented by:

7. The compound of claim 6, wherein R1 is Fmoc or Boc.

8. Formula 1: 【Chemistry 4】 wherein: X is hydrogen or a phenol protecting group; R1 comprises an amine protecting group; R is a linker group attached to a solid phase; A compound or a salt thereof.

9. 9. The compound of claim 8, wherein the amine protecting group is or comprises Fmoc.

10. 9. The compound of claim 8, wherein X is H.

11. A method for synthesizing a peptide sequence of interest, including any salt thereof, comprising: 【Chemistry 5】 wherein each R4 is independently selected from the group consisting of H, amino acid, peptide, polyamino acid, and at least one R4 is not H and R5 is hydrogen or a linker group attached to a solid phase, the method comprising providing a compound according to any one of claims 8 to 10 linked to a solid phase; (i) deprotecting the amine protecting group of the compound; (ii) coupling an N-protected amino acid moiety to said compound; (iii) contacting said solid phase with an amine base, thereby obtaining said peptide sequence of interest attached to said solid phase; A method comprising:

12. 12. The method of claim 11 , wherein the amine base is an Fmoc deprotecting agent.

13. 12. The method of claim 11, wherein the amine base is selected from primary amines, secondary amines, guanidine-based compounds, and amidine-based compounds, including any combination thereof.

14. 12. The method of claim 11, optionally comprising performing a step (iv) of deprotecting the N-protected amino acid moiety prior to performing step (iii).

15. 15. The method of claim 14, further comprising subsequently repeating steps (ii) and (iv) before performing step (iii).

16. The method of claim 11, further comprising cleaving the peptide sequence from the solid support, thereby obtaining the desired peptide sequence.

17. 12. The method of claim 11, wherein said step (ii) comprises contacting said solid phase with a coupling composition comprising 1 to 5 molar equivalents of said N-protected amino acid moiety.

18. The method of claim 11 , wherein the N-protected amino acid moiety optionally comprises an active ester of the N-protected amino acid moiety.

19. 20. The method of claim 18, wherein the coupling composition is substantially devoid of a base.

20. 12. The method of claim 11, wherein said step (ii) comprises contacting said solid phase with a coupling composition comprising 1 to 5 molar equivalents of said N-protected amino acid moiety.

21. The peptide sequence of interest is 【Chemistry 6】 The method of claim 11 , comprising:

22. 1. A peptide synthesized by the method of the present invention, comprising: 【Chemistry 7】 and further comprising a trace impurity characterized by: (i) a MW of 466.4 Da; (ii) a relative retention time (RRT) of about 0.7; or both (i) and (ii).

23. The impurities are selected from the group consisting of: 【Chemistry 8】 23. The peptide of claim 22, including any salts thereof.

24. 23. The peptide of claim 22, which is devoid of impurities characterized by: (i) a MW of 1263.5 Da; (ii) a relative retention time (RRT) of about 1.27; or both (i) and (ii).

25. 23. The peptide of claim 22, wherein the RRT of the impurity is determined via analytical method A.

26. Formula 3A: 【Chemistry 9】 wherein R6 represents one or more substituents, at least one of the wavy bonds represents a point of attachment to the peptide sequence of interest, and another wavy bond represents (i) OH, O － (ii) a point of attachment to said peptide sequence of interest, said method comprising: (i) Compound: 【Chemistry 10】 to a growing peptide chain, wherein R1 comprises an amine protecting group and R5 is OH, O － , hydrogen, an active ester, or a linker group attached to a solid phase, wherein (a) the growing peptide chain or (b) the compound is attached to the solid phase; and (ii) contacting said solid phase with an amine base, thereby obtaining said peptide sequence of interest attached to said solid phase; A method comprising:

27. 27. The method of claim 26, wherein the amine base is an Fmoc deprotecting agent.

28. 27. The method of claim 26, wherein the amine base is selected from primary amines, secondary amines, tertiary amines, guanidine-based compounds, and amidine-based compounds, including any combination thereof.

29. 27. The method of claim 26, further comprising: (iii) deprotecting the amine protecting group to obtain a deprotected N-terminal amine; and (iv) coupling a next N-protected amino acid to the deprotected N-terminal amine, wherein steps (iii) and (iv) are performed before performing step (ii).

30. 30. The method of claim 29, wherein steps (iii) and (iv) are subsequently performed, optionally repeated one or more times.

31. 27. The method of claim 26, further comprising cleaving the solid phase-linked peptide sequence of interest to obtain the peptide sequence of interest.

32. R6 is the following: 【Chemistry 11】 The method of claim 26, wherein the compound is or comprises:

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