Building block for difficult peptide synthesis and preparation method thereof

Abstract

A building block structure containing N, O / S-benzylidene acetal dipeptide (NBD) is described. The unique core structure of the NBD can efficiently convert those difficult peptide / protein sequences into simple peptide / protein sequences, thereby allowing for smooth synthesis of those challenging targets. Furthermore, the production of NBD is highly robust and can be easily amplified, which can facilitate the research and development of future customized peptides / proteins.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 486,748, filed on February 24, 2023, the entire contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] The present invention is generally in the field of protein / peptide synthesis. Background Art

[0004] Chemical protein synthesis plays an important role in basic biochemical research and the development of macromolecule-based therapeutics. It allows precise modifications at the atomic level, such as mirror-image proteins. The advent of solid-phase peptide synthesis (SPPS) allows peptides or small molecule proteins to be synthesized chemically, allowing site-specific installation of chemical probes or post-translational modification of peptides to achieve their biological functions. After SPPS, various coupling reagents, orthogonal protecting groups and Fmoc-based SPPS have also been fully developed, ensuring that peptide synthesis has become a convenient and commonly used technique. SPPS remains the gold standard method and often faces challenges in synthesizing difficult peptides / proteins that tend to aggregate on the resin. There are still many peptide sequences that cannot be synthesized by direct SPPS because they have a tendency to aggregate during peptide synthesis, which classifies them as difficult peptides. Current strategies to address this problem are less cost-effective and their application in industrial production is limited. To overcome the problem of difficult peptide synthesis, three methods have been developed to inhibit hydrogen bond interchain association, including pseudoproline dipeptide, HMB (2-hydroxy-4-methoxybenzyl) and isoacyl dipeptide as backbone protecting groups, reported in 1992 (Haack & Mutter, Tetrahedron Lett. 33, 1589–1592 (1992)), 1995 (Johnson, T. et al., J. Chem. Soc. Chem. Commun. 369–372 (1993)) and 2004 (Sohma, Y. et al., Chem. Commun. 124-125 (2004)). These compounds can disrupt peptide aggregation during SPPS, but because the synthesis of these building blocks is not very cost-effective, they are not widely used in industry.

[0005] New and improved methods for synthesizing challenging peptides / proteins at low cost are needed for both basic chemical biology research and therapeutic research and development. Summary of the Invention

[0006] This article describes the building block structure of an N,O / S-benzylidene acetal dipeptide (NBD). The unique core structure of the NBD (also referred to herein as the "compound") can effectively convert difficult peptide / protein sequences into simpler ones, thereby facilitating the synthesis of challenging targets. Furthermore, the production of the NBD is highly robust and can be easily scaled up, which could facilitate the research and development of tailored peptides / proteins.

[0007] In some forms, the compound may have the structure of Formula I:

[0008] ,

[0009] Where: (i) R 1 can be a protected or unprotected side chain of an amino acid; (ii) R 2 can be an amine protecting group; (iii) R 3 ~R 6 R may independently be hydrogen, hydroxy, halogen, -CN, haloalkyl (such as -CF3), -NO2, unsubstituted C1~C6 alkyl, unsubstituted C1~C6 alkenyl, unsubstituted aryl, unsubstituted heteroalkyl, unsubstituted cyclic group, unsubstituted heterocycle, unsubstituted aralkyl, alkoxy, amino, amide, carbonyl or thiol; (iv) R 7 and R 8 R can independently be hydrogen, methyl or other protected / unprotected side chains of amino acids; (v) 9 (vi) X may be hydrogen or a functional group suitable for protecting and / or activating a carboxylic acid group, such as benzyl, allyl, unsubstituted C1-C4 alkyl, and the like, and an activating group, for example, 1-hydroxy-7-azabenzotriazole (HOAt), 1-hydroxybenzotriazole (HOBt), ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma), N-hydroxysuccinimide (NHS), pentafluorophenol (Pfp), and the like; and (vi) X may be O or S.

[0010] When R 1 In the case of a protected side chain of an amino acid, the protecting group can be a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, a carbonyl group (e.g., a carboxylic acid or a carboxylate), an amide, an amino group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, or a substituted or unsubstituted heterocycle.

[0011] In some forms, the compound may have the structure of Formula I':

[0012] ,

[0013] Where: R 1 ~R 9 and X may be as defined above, and R 10 It may be hydrogen or a functional group suitable for hindering the reactivity of the phenyl group, such as a C1-C4 acyl group (e.g., formyl, acetyl, propionyl), a carbonyl group (e.g., alloc), or a carbamate group (e.g., tert-butyl methyl(2-(methylamino)ethyl)carbamate).

[0014] The method for making the compounds disclosed herein is described. Generally, the method comprises two reaction steps. In brief, an L- or D-amino acid with suitable protection on an amine and optionally its side chain functional group can be first coupled with salicylic aldehyde or its single / multiple substituted form. Then, the obtained salicylic aldehyde ester is reacted with serine / threonine / cysteine / penicillamine (Ser / Thr / Cys / Pen) or its ester in a suitable solvent. The obtained ester can be further derivatized to a related acid with or without phenol protection. Using the method disclosed herein, NBD can be obtained in high yield (at least 40%).

[0015] Also disclosed are methods for synthesizing difficult peptides / proteins using NBD as a preformed dipeptide building block for peptide / protein synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shown is a comparison of the synthesis of PD-L1 (121-123) using the disclosed method and conventional SPPS.

[0017] Figure 2 Shown is a comparison of the synthesis of IL-2(125-133) using the disclosed method and conventional SPPS.

[0018] Figure 3 A comparison of the synthesis of amylin using the disclosed method and conventional SPPS is shown.

[0019] Figure 4 A comparison of the synthesis of Rantes using the disclosed method and conventional SPPS is shown. DETAILED DESCRIPTION

[0020] Difficult peptides / proteins often tend to aggregate on the resin through hydrophobic interactions of amino acid side chains and hydrogen bonding of amide bonds, which can lead to incomplete coupling or deprotection during the SPPS process. Without being bound by any theory, it is believed that the disclosed N,O / S-benzylidene acetal dipeptide (NBD) can act as a conformationally twisted dipeptide building block, forming a pseudo-proline turn to disrupt peptide-peptide interactions. As a result, aggregation can be successfully prevented, allowing for smooth synthesis of the target sequence.

[0021] I. Definitions

[0022] The term "alkyl" refers to a branched or straight-chain monovalent saturated aliphatic hydrocarbon radical having from one to eight carbon atoms. This term is further exemplified by radicals such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl, n-hexyl, and the like.

[0023] The term "alkenyl" refers to a branched or straight chain hydrocarbon group having from two to eight carbon atoms and a structural formula containing at least one carbon-carbon double bond.

[0024] The term "alkynyl" refers to a branched or straight chain hydrocarbon group having from two to eight carbon atoms and a structural formula containing at least one carbon-carbon triple bond.

[0025] The term "aryl" refers to a C5~C 20 Examples of aromatic groups are benzene, naphthalene, anthracene, phenanthrene, chrysene, pyrene, coronene, and the like.

[0026] The term "cyclic group" refers to a saturated or unsaturated monocyclic or polycyclic ring (such as those formed by a monocyclic or fused ring system), such as a cycloalkyl, cycloalkenyl or cycloalkynyl group, which may have from three to ten carbon atoms when geometric constraints allow.

[0027] The term "heteroalkyl" refers to a linear or branched carbon-containing alkyl group containing at least one heteroatom in the carbon backbone. Suitable heteroatoms include, but are not limited to, O, N, Si, P, and S, wherein the nitrogen, phosphorus, and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized.

[0028] The term "heteroaryl" refers to a C5~C 20A monoatomic aromatic ring or fused aromatic ring system wherein one or more carbon atoms on one or more aromatic ring structures are replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, oxygen, sulfur, and nitrogen. Examples of heteroaryl groups include, but are not limited to, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine. Examples of heteroaryl rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, hexazinyl, decahydroquinolinyl, 2H,6H-1, 5,2-dithiazinyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, naphthyridinyl, octahydroisoquinolinyl, 1,2, 3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiol, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridoxazole, pyridoimidazole, pyridothiazole, pyridinyl , pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydroisoquinolyl, tetrahydroquinolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl. One or more of these rings may be substituted as defined below for "substituted heteroaryl."

[0029] The term "heterocyclic group" refers to a cyclic group as defined above wherein at least one of the ring carbon atoms is replaced by a heteroatom such as nitrogen, oxygen, sulfur or phosphorus.

[0030] The term "aralkyl" refers to an aryl group or heteroaryl group having an alkyl, alkynyl or alkenyl group as defined above attached to an aromatic group such as aryl, heteroaryl, polyaryl or polyheteroaryl, etc. An example of an aralkyl group is a benzyl group.

[0031] The term "alkoxy" refers to a group of the formula -OR v A compound represented by v Including but not limited to alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic radical, cycloalkenyl group, aryl, heteroaryl, aralkyl, assorted alkyl etc.Exemplary alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy etc. " low alkoxy " group is an alkoxy group containing one to six carbon atoms. " ether " is two functional groups covalently linked by oxygen as defined below. Therefore, the alkyl substituent that makes alkyl become ether is or is similar to alkoxy, such as can be represented by one of-O-alkyl,-O-alkenyl,-O-alkynyl,-O-aralkyl,-O-aryl,-O-heteroaryl,-O-cyclic radical,-O-heterocyclic radical etc.

[0032] As used herein, the term "amino" includes the following groups: (primary amino group), (Secondary amino group), (tertiary amino group), and (quaternary amino), wherein E is absent or E is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, or substituted or unsubstituted heterocyclyl, wherein, independently of E, R x 、R xi and R xii Each independently represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, a substituted or unsubstituted carbonyl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted aralkyl group (e.g., a substituted or unsubstituted alkylaryl group, a substituted or unsubstituted aralkyl group), a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted polyaryl group, a substituted or unsubstituted polyheteroaryl group, a substituted or unsubstituted heterocyclic group, a hydroxyl group, an alkoxy group, a phosphonium group, a phosphanyl group, a phosphino group, a sulfinyl group, a silyl group, a mercapto group, an amide group, an amino group, or -(CH2) m -R'''; R''' represents a hydroxyl group, a substituted or unsubstituted carbonyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted cycloalkenyl group, a substituted or unsubstituted heterocyclyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted polyaryl group, a substituted or unsubstituted polyheteroaryl group, an alkoxy group, a phosphonium group, a phosphatidyl group, an amide group or an amino group; and m is zero or an integer from 1 to 8. The term "quaternary amino group" also includes groups wherein nitrogen, R x 、R xi and R xii The N +A group forming a heterocyclyl or heteroaryl group having 3 to 14 atoms in the ring structure. It is understood by those skilled in the art that the E groups listed above are divalent (e.g., methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylene, cyclohexane-1,2-diyl).

[0033] The terms "amide" or "amido" are used interchangeably to refer to both "unsubstituted amide" and "substituted amide" and are represented by the following general formula:

[0034] ,

[0035] wherein E is absent or E is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, or substituted or unsubstituted heterocyclyl, wherein R and R′, independent of E, each independently represent hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted aralkyl), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclyl, hydroxy, alkoxy, phosphonium, phosphanyl, phosphino, sulfinyl, silyl, thiol, amide, amino, or —(CH 2 ) m -R''', or R and R' together with the N atom to which they are attached form a heterocyclic ring having 3 to 14 atoms in the ring structure; R''' represents hydroxy, substituted or unsubstituted carbonyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, alkoxy, phosphonium, phosphanyl, amide or amino; and m is zero or an integer ranging from 1 to 8. In some forms, when E is oxygen, a carbamate is formed. It is understood by those skilled in the art that the E groups listed above are divalent (e.g., methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylene, cyclohexane-1,2-diyl).

[0036] As used herein, "carbonyl" is art-recognized and includes moieties such as those represented by the following formula:

[0037] ,

[0038] wherein X is a bond or represents oxygen or sulfur, and R represents hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted aralkyl), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclyl, hydroxy, alkoxy, phosphonium, phosphanyl, amide, amino, or -(CH2) m -R", or a pharmaceutically acceptable salt; E" is absent, or E" is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclyl; R' represents hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted aralkyl), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclyl, hydroxy, alkoxy, phosphonium, phosphanyl, amide, amino, or -(CH2) m-R"; R" represents hydroxy, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, alkoxy, phosphonium, phosphanyl, amide or amino; and m is zero or an integer ranging from 1 to 8. Such substituents can be any of the substituents described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (such as thioester, thioacetate or thioformate), alkoxy, phosphonium, phosphanyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (such as quaternized amino), amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, and combinations thereof. It will be understood by those skilled in the art that the E'' groups listed above are divalent (e.g., methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylene, cyclohexane-1,2-diyl). When X is oxygen and R is as defined above, this moiety is also referred to as a carboxyl group. When X is oxygen and R is hydrogen, the formula represents a "carboxylic acid." When X is oxygen and R' is hydrogen, the formula represents a "formate." When X is oxygen and R or R' is not hydrogen, the formula represents an "ester." Generally speaking, when the oxygen atom in the above formula is replaced by a sulfur atom, the formula represents a "thiocarbonyl" group. When X is sulfur and R or R' is not hydrogen, the formula represents a "thioester." When X is sulfur and R is hydrogen, the formula represents a "thioformic acid." When X is sulfur and R' is hydrogen, the formula represents a "thioformate." When X is a bond and R is not hydrogen, the above formula represents a "ketone." When X is a bond and R is hydrogen, the above formula represents an "aldehyde."

[0039] The term "thiol" is used interchangeably with and is represented by -SR, where R can be hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted aralkyl, etc.), substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted carbonyl, phosphonium, phosphanyl, amido, amino, alkoxy, oxo, phosphono, sulfinyl, or silyl, as described above. Such substituents can be any of the substituents described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl, etc.), silyl, ether, ester, thiocarbonyl (such as thioester, thioacetate or thioformate, etc.), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino (such as quaternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, polyaryl, polyheteroaryl, and combinations thereof.

[0040] The use of the term "about" is intended to describe values ​​that are within approximately + / - 10% above or below the stated value that the term "about" modifies. When the term "about" is used before a numerical range (i.e., about 1 to 5) or before a series of numbers (i.e., about 1, 2, 3, 4, etc.), unless otherwise indicated, it is intended to modify both ends of the numerical range and / or each number listed in the entire series.

[0041] As used herein, "substituted" refers to all permissible substituents of the compounds or functional groups described herein. In the broadest sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Illustrative substituents include, but are not limited to, halogen, hydroxyl, or any other organic group containing any number of carbon atoms (preferably 1 to 14 carbon atoms), and optionally containing one or more heteroatoms, such as oxygen, sulfur, or nitrogen groups, in a linear, branched, or cyclic structure format. Representative substituents include substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted phenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted aralkyl, halogen, hydroxy, alkoxy, phenoxy, aryloxy, silyl, mercapto, alkylthio, substituted alkylthio, phenylthio, arylthio, cyano, isocyano, nitro, substituted or unsubstituted carbonyl, carboxyl, amino, amido, oxo, sulfinyl, sulfonyl, sulfonic acid, phosphonium, phosphanyl, phosphoryl, phosphono, and amino acid. Such substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted phenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted aralkyl, halogen, hydroxy, alkoxy, phenoxy, aryloxy, silyl, mercapto, alkylthio, substituted alkylthio, phenylthio, arylthio, cyano, isocyano, nitro, substituted or unsubstituted carbonyl, carboxyl, amino, amide, oxo, sulfinyl, sulfonyl, sulfonic acid, phosphonium, phosphanyl, phosphoryl, phosphono and amino acid may be further substituted.

[0042] The heteroatoms (such as nitrogen) may have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valences of the heteroatoms. It is understood that "substitution" or "substituted" includes the implicit proviso that such substitution is made according to the permissible valences of the substituted atom and the substituent, and that such substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformations (e.g., rearrangement, cyclization, elimination, etc.).

[0043] The following table provides the full names of some abbreviations used in this article:

[0044]

[0045] II. Composition

[0046] The disclosed compositions comprise building block structures containing N,O / S-benzylidene acetal dipeptides (NBDs). The disclosed NBDs (also referred to herein as "compounds") are simple and effective building blocks for the synthesis of difficult peptides / proteins. Without being bound by any theory, it is believed that the NBDs disclosed herein can act as conformationally twisted dipeptide building blocks, forming pseudo-proline turns to disrupt interactions between peptides, thereby disrupting aggregation of difficult peptides / proteins on the resin during the SPPS process (typically due to hydrophobic interactions of amino acid side chains and hydrogen bonding of amide bonds).

[0047] In some forms, the disclosed compounds may have the structure of Formula I:

[0048] ,

[0049] Where: (i) R 1 can be a protected or unprotected side chain of an amino acid; (ii) R 2 It may be an amino protecting group; (iii) R 3 ~R 6 R may independently be hydrogen, hydroxy, halogen, -CN, haloalkyl (such as -CF3), -NO2, unsubstituted C1~C6 alkyl, unsubstituted C1~C6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, unsubstituted cyclic group, unsubstituted heterocycle, unsubstituted aralkyl, alkoxy, amino, amide, carbonyl or thiol; (iv) R 7 and R 8 R can independently be hydrogen, methyl or other protected / unprotected side chains of amino acids; (v) 9 (vi) X may be hydrogen or a functional group suitable for protecting and / or activating a carboxylic acid group, such as benzyl, allyl, unsubstituted C1-C4 alkyl, and an activating group, for example, 1-hydroxy-7-azabenzotriazole (HOAt), 1-hydroxybenzotriazole (HOBt), ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma), N-hydroxysuccinimide (NHS), pentafluorophenol (Pfp), and the like; and (vi) X may be O or S.

[0050] When R 1 When it is a protected side chain of an amino acid, the protecting group can be a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a carbonyl (e.g., a carboxylic acid or a carboxylic ester), an amide, an amino group, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a heterocycle. In some forms, R 1It is a protected side chain of an amino acid, and the protecting group can be an unsubstituted alkyl group (e.g., an unsubstituted C1~C6 or C1~C4 alkyl group), an unsubstituted alkenyl group (e.g., an unsubstituted C1~C6 or C1~C4 alkenyl group), an unsubstituted alkynyl group (e.g., an unsubstituted C1~C6 or C1~C4 alkynyl group), a carbonyl group (e.g., a carboxylic acid or a carboxylate), an amide, an amino group, an unsubstituted aryl group, an unsubstituted heteroaryl group, or an unsubstituted heterocycle.

[0051] The “ " represents the carbon stereogenic center (to which the wavy bond is attached), which may be in S- or R-configuration. For example, to -C(O)OR 9 The acetyl carbon of can be in S- or R- configuration. It is also understood that R 1 The side chain may be that of any suitable amino acid (natural or synthetic), and the amino acid may be in its L- or D-configuration, or may be various mixtures of their isomers.

[0052] R 1 It can be any suitable α-amino acid (natural or synthetic) protected or unprotected side chain. 1 When the side chain of an amino acid with a hydroxyl group, the hydroxyl group is optionally protected by a suitable hydroxyl protecting group known in the art. For side chains with additional amino groups, the amino group is optionally protected by a suitable amino protecting group known in the art. In some forms, when R 1 When it is a protected side chain of an amino acid, the protecting group can be t Bu, Boc, Thz, Acm, Trt, Cbz, Alloc, azido, Bn, Bz, Ac or Pbf, or a combination thereof.

[0053] In some forms, R 1 It can be a protected or unprotected side chain of glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, cysteine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, serine or threonine. Any of these amino acids can be in its L configuration or D configuration. In these forms, when R 1 When it is a protected side chain, the protecting group can be t Bu, Boc, Thz, Acm, Trt, Cbz, Alloc, azido, Bn, Bz, Ac or Pbf, or a combination thereof.

[0054] R 2It can be any substituent conventionally used to hinder the reactivity of amino groups. For example, suitable amino protecting groups are described in the following literature: Green T., "Protective Groups in Organic Synthesis", Chapter 7, John Wiley and Sons, Inc., 1991, 309-385. In some forms, R 2 It can be Fmoc, Cbz, Moz, Boc, Troc, Teoc, Alloc or Voc.

[0055] R 3 ~R 6 R may independently be hydrogen, hydroxy, halogen, -CN, haloalkyl (such as -CF3, etc.), -NO2, unsubstituted alkyl (such as unsubstituted C1~C8 alkyl, etc.), unsubstituted alkenyl (for example, unsubstituted C1~C8 alkenyl), unsubstituted alkynyl (such as unsubstituted C1~C8 alkynyl, etc.), unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, unsubstituted cyclic group, unsubstituted heterocycle, unsubstituted aralkyl, alkoxy, amino, amide, carbonyl or thiol. In some forms, R 3 ~R 6 can independently be hydrogen, halogen, -CN, -CF3, -NO2, unsubstituted C1~C6 or C1~C4 alkyl, unsubstituted C1~C6 or C1~C4 alkenyl, unsubstituted C1~C6 or C1~C4 alkynyl, alkoxy and unsubstituted aryl. In some forms, R 3 ~R 6 At least one of, optionally R 3 ~R 6 Two or more of are not hydrogen. For example, R 3 ~R 6 One of the 3 ~R 6 Two of them, R 3 ~R 6 Three or R 3 ~R 6 All of the ions are not hydrogen.

[0056] In some forms, R 7 and R 8 can independently be hydrogen or methyl. In some forms, R 7 and R 8 Can independently be protected / unprotected side chains of amino acids.

[0057] In some forms, R 9 It can be hydrogen, benzyl, allyl or unsubstituted C1~C4 alkyl. In some forms, R 9can be hydrogen. In some forms, R 9 It can be any substituent conventionally used to protect carboxylic acid groups. For example, suitable amino protecting groups are described in Green T., "Protective Groups in Organic Synthesis", Chapter 5, John Wiley and Sons, Inc., 2006, 533-646. In some forms, R 9 It may also be any substituent conventionally used to activate a carboxylic acid group. For example, suitable amino protecting groups are described in the following literature: Albericio F. "Peptide Coupling Reagents, More than a Letter Soup", Chem. Rev. 2011, 111, 6557-6602. In some forms, R 9 It can be benzyl, allyl or unsubstituted C1~C4 alkyl. In some forms, R 9 It can be an activating group such as 1-hydroxy-7-azabenzotriazole (HOAt), 1-hydroxybenzotriazole (HOBt), 2-cyano-2-(hydroxyimino)ethyl acetate (Oxyma), N-hydroxysuccinimide (NHS), pentafluorophenol (Pfp), etc. In these forms, It is an activated ester group, such as 1-hydroxy-7-azabenzotriazole (HOAt) ester, 1-hydroxybenzotriazole (HOBt) ester, ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) ester, N-hydroxysuccinimide (NHS) ester, pentafluorophenol (Pfp) ester, etc.

[0058] In some forms, the disclosed compounds may have the structure of Formula I':

[0059] ,

[0060] where R 1 ~R 9 and X may be any form as defined above, and R 10 It may be hydrogen or a functional group suitable for hindering the reactivity of the phenyl group, such as a C1-C4 acyl group (e.g., formyl, acetyl, propionyl), a carbonyl group (e.g., alloc), or a carbamate group (e.g., tert-butyl methyl(2-(methylamino)ethyl)carbamate).

[0061] In some forms, R 10 can be hydrogen. In some forms, R 10It can be any substituent conventionally used to hinder the reactivity of the phenol group. For example, suitable amino protecting groups are described in Green T., "Protective Groups in Organic Synthesis", Chapter 3, John Wiley and Sons, Inc., 2006, 367-430. In some forms, R 10 It may be a C1-C4 acyl group (eg, formyl, acetyl, propionyl), a carbonyl group (eg, alloc), or a carbamate group (eg, tert-butyl methyl(2-(methylamino)ethyl)carbamate).

[0062] In some forms, R 9 may be hydrogen, and R 10 In some forms, R 9 It can be benzyl, allyl, unsubstituted C1~C4 alkyl or activated group, such as 1-hydroxy-7-azabenzotriazole (HOAt), 1-hydroxybenzotriazole (HOBt), 2-cyano-2-(hydroxyimino)acetic acid ethyl ester (Oxyma), N-hydroxysuccinimide (NHS), pentafluorophenol (Pfp), etc., and R 10 In some forms, R 9 may be hydrogen, and R 10 It can be a C1-C4 acyl group (e.g., formyl, acetyl, propionyl), a carbonyl group (e.g., alloc), or a carbamate group (e.g., methyl(2-(methylamino)ethyl)carbamate). 9 It can be benzyl, allyl, unsubstituted C1~C4 alkyl or activated group, such as 1-hydroxy-7-azabenzotriazole (HOAt), 1-hydroxybenzotriazole (HOBt), 2-cyano-2-(hydroxyimino)acetic acid ethyl ester (Oxyma), N-hydroxysuccinimide (NHS), pentafluorophenol (Pfp), etc., and R 10 It may be a C1-C4 acyl group (eg, formyl, acetyl, propionyl), a carbonyl group (eg, alloc), or a carbamate group (eg, tert-butyl methyl(2-(methylamino)ethyl)carbamate).

[0063] Representative examples of NBD are shown below, where represent , represents the connection point, and R 3 ~R 10 It can be in any of the forms defined above.

[0064]

[0065]

[0066]

[0067] Further examples of the compounds are L-Thr-L-Thr NBD, L-Asp-L-Ser NBD, L-Glu-L-Cys NBD, L-Val-L-Pen NBD and L-Val-L-Cys NBD as shown below:

[0068]

[0069] The disclosed NBD is structurally different from the pseudo-proline disclosed in Wöhr et al., Pseudo-Prolines as a Solubilizing, Structure-Disrupting Protection Technique in Peptide Synthesis (J. Am. Chem. Soc. 1996, 118, 39, 9218–9227). The disclosed NBD is also structurally different from the pseudo-proline provided in CHEM IMPEX (Woodale, IL, e.g., catalog number 46591 (www.chemimpex.com / product / productinfo / fmoc-arg-pbf-ser-psi-me-me-pro-oh / 36788?cid=860). For example, the phenyl group of the disclosed NBD provides more substitution sites for further functionalization compared to the oxazolidine in the pseudo-proline dipeptide, whereas the symmetrical dimethyl group employed in the pseudo-proline does not provide any additional sites for modification.

[0070] III. Methods

[0071] Compared with existing protein / peptide synthesis methods, NBDs can cover a wider range of peptide sequences, and they can be easily prepared from milligram to sub-kilogram scales at an estimated cost of only about 1-10% of the price of commercial competitors.

[0072] A. Production of NBD

[0073] The present invention describes a method for making a compound disclosed herein. Generally, the method comprises two reaction steps. In brief, first, an L- or D-amino acid with appropriate protection on an amine and optionally its side chain functional group is coupled with salicylaldehyde or its single / multiple substituted form. Then, the resulting salicylaldehyde ester is reacted with serine / threonine / cysteine / penicillamine (Ser / Thr / Cys / Pen) or its ester in a suitable solvent. The resulting ester can be further derivatized to a corresponding acid with or without phenol protection. Using the method disclosed herein, NBD can be obtained in high yield (at least 40%).

[0074] Using the methods disclosed herein, 76 exemplary NBDs (structures shown in the sections above) were produced in moderate to excellent yields (i.e., 40% to 95%). Variants of these 76 NBDs, including D-amino acid versions, different protecting groups on the amine or side chain, other carboxylic acid derivatives, and different substitutions of the aromatic groups, can also be synthesized by following the developed synthetic strategy.

[0075] The disclosed method comprises: (a) reacting a protected amino acid of formula II with a substituted or unsubstituted salicylaldehyde of formula III to obtain an intermediate comprising a salicylaldehyde ester of formula IV:

[0076] ,

[0077] Where: R 1 can be a protected or unprotected side chain of an amino acid; and (ii) R 2 It may be an amino protecting group,

[0078] ,

[0079] where R 3 ~R 6 It can independently be hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF3, etc.), -NO2, unsubstituted alkyl (such as unsubstituted C1~C8 alkyl, etc.), unsubstituted alkenyl (unsubstituted C1~C8 alkenyl), unsubstituted alkynyl (such as unsubstituted C1~C8 alkynyl, etc.), unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, unsubstituted cyclic group, unsubstituted heterocycle, unsubstituted aralkyl, alkoxy, amino, amide, carbonyl or sulfhydryl,

[0080] ,and

[0081] (b) reacting the salicylic aldehyde ester of formula IV with serine, threonine, cysteine ​​or penicillamine or its ester to obtain a product comprising a compound of formula I:

[0082] ,

[0083] where R 7 and R 8 can independently be hydrogen, methyl or other protected / unprotected side chains of amino acids; (v) R 9 can be hydrogen, benzyl, allyl, unsubstituted C1~C4 alkyl or a suitable activating group, such as N-hydroxysuccinimide (NHS); and (vi) X can be O or S,

[0084] ,

[0085] where R 1 ~R 9 and X are as defined above for the reactants.

[0086] Suitable for R in formula V and formula I 9 The activating group of is generally less active than HOAt and Oxyma (such as NHS, etc.) to reduce / prevent side reactions in step (b).

[0087] The methods disclosed herein may further comprise step (c): deprotecting the protected carboxyl group of the NBD; or step (d): acylation or activation of the free acid group or free phenol group of the NBD; or a combination thereof.

[0088] In some forms, in the deprotection step (c), the deprotected NBD is obtained in the free acid form, for example, the deprotected NBD of Formula VI:

[0089] ,

[0090] where R 1 ~R 8 、R 10 and X may be in any form as defined above.

[0091] In some forms, in the acylation / activation step (d), R 9 and R 10 At least one of them is hydrogen. For example, R 9 is hydrogen, so that the free carboxylic acid group can be activated in step (d). For example, R 10 is hydrogen, so that the free hydroxyl group can be acylated in step (d). In some forms, R 9 and R 10 are all hydrogen, so that the free carboxylic acid and / or hydroxyl groups can be acylated and / or activated in step (d) as needed. In some forms, in step (d), an acylated NBD with or without free carboxylic acid is obtained, for example, an acylated NBD of formula I':

[0092] ,

[0093] where R 1 ~R 9 and X may be in any form as defined above, and R 10 It may be in any form as defined above except hydrogen.

[0094] The deprotection step (c) and the acylation / activation step (d) can be performed in any order and in any combination with steps (a) and (b) as described above. For example, the method comprises steps (a), (b), and (c) performed sequentially. For example, the method comprises steps (a), (b), and (d) performed sequentially. For example, the method comprises steps (a), (b), (c), and (d) performed sequentially. For example, the method comprises steps (a), (b), (d), and (c) performed sequentially.

[0095] As mentioned above, the “ " represents a carbon stereogenic center (to which the wavy line is attached), which may be in S- or R-configuration. For example, attachment to -C(O)OR 9 The acetyl carbon of the threonine, cysteine ​​or penicillamine or its ester used in step (b) can be in its L- or D-configuration, as a racemate, or various mixtures of its isomers. It is understood that the protected amino acid used in step (a) can be in its L- or D-configuration, as a racemate, or various mixtures of its isomers.

[0096] 1. Step (a): Production of salicylaldehyde ester

[0097] In the first step (a) of the disclosed method, a protected amino acid of formula II is reacted with a substituted or unsubstituted salicylaldehyde of formula III to obtain an intermediate comprising a salicylaldehyde ester of formula IV:

[0098] ,

[0099] where R 1 ~R 6 Can be in any form as defined above. 1 When the protected side chain is an amino acid, the protecting group can be a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a carbonyl (e.g., a carboxylic acid or a carboxylic ester), an amide, an amino group, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a heterocycle. 1 is a protected side chain of an amino acid, and the protecting group may be an unsubstituted alkyl group, an unsubstituted alkenyl group, an unsubstituted alkynyl group, a carbonyl group (e.g., a carboxylic acid or a carboxylate), an amide, an amino group, an unsubstituted aryl group, an unsubstituted heteroaryl group, or an unsubstituted heterocycle.

[0100] The protected amino acid of formula II used in step (a) can be any natural amino acid or synthetic amino acid, and can be L-configuration or D-configuration. Preferably, the protected amino acid used in step (a) is glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, cysteine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, serine or threonine in L-configuration or D-configuration.

[0101] The amino acid side chains used in step (a) can be protected using protecting groups known in the art. Suitable side chain protecting groups include, but are not limited to: t Bu, Boc, Thz, Acm, Trt, Cbz, Alloc, Azide, Bn, Bz, Ac, and Pbf.

[0102] The terminal amino group of the amino acid used in step (a) is protected (ie, R 2 Any amino protecting group known in the art can be used to protect the amino group of the amino acid used in the method of the present disclosure. For example, the R 2 It can be Fmoc, Cbz, Moz, Boc, Troc, Teoc, Alloc or Voc.

[0103] The salicylaldehyde of formula III that is reacted with the protected amino acid of formula II can be an unsubstituted salicylaldehyde or a mono- or poly-substituted salicylaldehyde. In some forms of this method, the salicylaldehyde that is reacted with the protected amino acid of formula II is substituted with one or more substituents (i.e., R 3 ~R 6 At least one of, optionally R 3 ~R 6 For example, R of the salicylaldehyde of formula III used in step (a) is 3 ~R 6 One of the 3 ~R 6 Two of them, R 3 ~R 6 Three or R 3 ~R 6Wherein all are not hydrogen. Examples of suitable substituents for the substituted salicylaldehyde of formula III are hydroxyl, halogen, -CN, haloalkyl (such as -CF3, etc.), -NO2, unsubstituted C1~C6 alkyl, unsubstituted C1~C6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, unsubstituted cyclic group, unsubstituted heterocycle, unsubstituted aralkyl, alkoxy, amino, amide, carbonyl and sulfhydryl. Preferably, when the salicylaldehyde reacted with the protected amino acid of formula II is substituted with one or more substituents, these substituents are independently halogen, -CN, -CF3, -NO2, unsubstituted C1~C6 or C1~C4 alkyl, unsubstituted C1~C6 or C1~C4 alkenyl, unsubstituted C1~C6 or C1~C4 alkynyl, alkoxy and unsubstituted aryl.

[0104] Typically, the reaction between the protected amino acid of formula II and the substituted or unsubstituted salicylaldehyde of formula III is carried out in a suitable organic solvent, and optionally in the presence of a condensation agent and / or a base.

[0105] Examples of suitable organic solvents for carrying out the reaction in step (a) include, but are not limited to, DMF, DMSO, DMAc, NMP. Preferably, the organic solvent for carrying out the reaction in step (a) is DMF.

[0106] Examples of suitable condensing agents for carrying out the reaction in step (a) include, but are not limited to, HATU, PyBOP, HBTU, HCTU, COMU, TSTU, PyClock, PyOxim, EDCI and DCC. Preferably, the condensing agent used in the reaction in step (a) is HATU.

[0107] Examples of suitable bases for carrying out the reaction in step (a) include, but are not limited to, DIPEA and triethylamine. Preferably, the base used in the reaction in step (a) is DIPEA.

[0108] The reaction between the protected amino acid of formula II and the substituted or unsubstituted salicylaldehyde of formula III can be carried out in a suitable organic solvent (such as any of those described above), and optionally in the presence of a condensation reagent and / or a base, at room temperature (i.e., 20° C. to 25° C. at 1 atm), for a period of 30 minutes to 5 hours, preferably 1 hour to 3 hours, such as about 1 hour or about 1.5 hours, etc.

[0109] Optionally, method disclosed herein further includes the step of the intermediate of the salicylic aldehyde ester of formula IV that purifying is contained after step (a) and before step (b).Purification step can use any suitable technology known in the art to carry out, such as using extraction, washing, drying, column chromatography (such as by using silica gel column) or filtration, or its combination etc.For example, use the intermediate of the salicylic aldehyde ester of formula IV to purify with following technology: with the organic solvent extraction different from the organic solvent used for reacting, with acid wash one or many, with siccative (such as sodium sulfate etc.) drying and / or evaporation, then use silica gel column to separate.

[0110] Usually, the yield of the salicylic aldehyde ester of formula IV obtaining in step (a) is at least 40%, at least 50% or in the scope of approximately 50% to approximately 95%.The yield of the salicylic aldehyde ester of formula IV can be calculated using the following formula: (molar number of the salicylic aldehyde ester of formula IV) / (molar number of the protected amino acid of formula II)*100%.Yield can be determined before or after intermediate purification.

[0111] 2. Step (b): Production of NBD

[0112] In the second step of the disclosed method, a salicylaldehyde ester of formula IV is reacted with serine, threonine, cysteine ​​or penicillamine of formula V or an ester thereof to obtain a compound of formula I:

[0113] ,

[0114] where R 1 ~R 9 and X may be in any form as defined above.

[0115] The serine, threonine, cysteine ​​or penicillamine or ester of formula V may be in the L- or D-configuration. 9 It can be hydrogen or any suitable functional group that can form an ester with the carboxyl group of serine, threonine, cysteine ​​and penicillamine, such as benzyl, allyl and unsubstituted C1~C4 alkyl.

[0116] Usually, the reaction between the salicylaldehyde ester of formula IV and the serine, threonine, cysteine ​​or penicillamine or its ester of formula V is carried out in a suitable solvent. The solvent can be an organic solvent or water or its combination, and usually contains the buffer agent of the amount of 10 vol% to 20 vol%, such as the pyridine / HOAc (mol ratio 1:1) of 10 vol% to 20 vol%.

[0117] Examples of suitable solvents for carrying out the reaction in step (b) include, but are not limited to, dichloromethane, trifluoroacetic acid, ethyl acetate, toluene, tetrahydrofuran, 1,4-dioxane, acetonitrile, acetone, hexane, and water, and combinations thereof.

[0118] Examples of suitable buffers for the solvent used in the reaction of step (b) include, but are not limited to, pyridine-acetic acid buffer, colidine-acetic acid buffer, picoline-acetic acid buffer, DMSO-pyridine-acetic acid buffer, DMSO-collidine-acetic acid buffer, and DMSO-picoline-acetic acid buffer. A preferred buffer suitable for use is pyridine-acetic acid buffer, wherein the molar ratio of pyridine to acetic acid is in the range of 1:9 to 9:1, such as 1:1.

[0119] The reaction between the salicylaldehyde ester of formula IV and serine, threonine, cysteine ​​or penicillamine or its ester of formula V can be carried out in a suitable solvent (such as any of those described above) at room temperature over a period of 1 hour to 5 hours, preferably 2 hours to 3 hours, such as the period of approximately 2 hours or approximately 3 hours.

[0120] Optionally, the method disclosed herein further comprises a step of purifying the product containing the compound of formula I after step (b). The purification step can be performed using any suitable technique known in the art, such as any of those described in step (a) above. For example, the product containing the compound of formula I can be purified using the following techniques: extraction with a suitable solvent and separation using column chromatography or filtration, washing one or more times with cold water and a cold organic solvent (such as cold ethanol and / or cold ether), and then separating by silica gel column.

[0121] Usually, the yield of the compound of formula I obtaining in step (b) is at least 40%, or in the scope of approximately 40% to approximately 95%.The yield of compound of formula I can be calculated using the following formula: (moles of compound of formula I) / (moles of formula IV salicylic aldehyde ester) * 100%.Yield can be determined before or after product purification.

[0122] 3. Step (c): Deprotection of the protected carboxyl group

[0123] Optionally, the disclosed method further comprises step (c): deprotecting the protected carboxyl group of the NBD formed in step (b) or (d) as described herein. For example, the disclosed method comprises steps (a), (b), and (c) performed sequentially. For example, the disclosed method comprises steps (a), (b), (c), and (d) performed sequentially. For example, the disclosed method comprises steps (a), (b), (d), and (c) performed sequentially. When the disclosed method comprises a deprotection step, the R 9Typically, it is not hydrogen, particularly when X of the formula described herein is O. The protected carboxyl group of the NBD can be deprotected using reagents and reactions known in the art, for example, by deallylation using reagents such as Pd(PPh3)4 and / or PhSiH3, etc.; hydrogenation of Bn protection using H2 and / or Pd / C; sponsification of alkyl protection using LiOH, etc. In step (c), the deprotected NBD is obtained in the form of a free acid, for example, a deprotected NBD of formula VI:

[0124] ,

[0125] where R 1 ~R 8 、R 10 and X may be in any form as defined above.

[0126] Optionally, the method disclosed herein further comprises a step of purifying the product containing the compound of formula VI after step (c). The purification step can be performed using any suitable technique known in the art, such as any of those described in steps (a) or (b) above. For example, the product containing the compound of formula VI can be purified using the following techniques: extraction with a suitable solvent and separation using column chromatography or filtration, washing one or more times with cold water and a cold organic solvent (such as cold ethanol and / or cold ether, etc.), and separation by silica gel column.

[0127] Typically, the yield of the compound of formula VI obtained in step (c) is at least 40% or in the range of about 40% to about 95%. The yield of the compound of formula VI can be calculated using the following formula: (moles of compound of formula VI) / (moles of compound of formula I')*100%. The yield can be determined before or after product purification.

[0128] 4. Step (d): Acylation or activation of the acid group

[0129] Optionally, the disclosed method further comprises the step (d) of acylation or activation of the free acid or free phenolic group of the NBD formed in step (b) or (c). When the disclosed method comprises an acylation / activation step, the R 9 and R 10 In some forms, at least one of the NBDs in the NBD product of step (b) or in the deprotected NBD formed in the deprotection step (c) is hydrogen. 9is hydrogen, so that the free carboxylic acid group can be activated in step (d). In the form in which the free carboxylic acid group is activated, examples of the activated group formed include but are not limited to NHS, HOAt or Oxyma. In some forms, R of the NBD in the NBD product of step (b) and / or in the deprotected NBD formed in step (c) is 10 is hydrogen, so that the free hydroxyl group can be acylated in step (d). In some forms, in the NBD product of step (b) or in the deprotected NBD formed in the deprotection step (c), R 9 and R 10 are all hydrogen, so that in step (d) the free carboxylic acid and / or hydroxyl groups can be acylated and / or activated as needed. For example, in the NBD product of step (b) or in the deprotected NBD formed in the deprotection step (c), R 9 and R 10 Both are hydrogen, so that in step (d) the free carboxylic acid is activated and / or the hydroxyl group is acylated.

[0130] In some forms, in step (d), a mixture with or without a free carboxylic acid (e.g., -COOR 9 is a carboxyl group or an ester), for example, an acylated NBD of formula I':

[0131] ,

[0132] where R 1 ~R 9 and X can be any form as defined above, and R 10 It may be any of the forms defined above except hydrogen.

[0133] The free acid of NBD can be acylated and / or activated using reagents and reactions known in the art. For example, the free acid (-COOH and / or -OH) of NBD can be acylated using reagents such as acetic anhydride, carbonates, or carbamates to obtain an acyl-terminated NBD of Formula I', wherein R 9 and / or R 10 (such as R 10 For example, the free acid of NBD (e.g., -COOH) can be activated by using a reagent (such as HATU, HBTU, Oxyma Pure, etc.) to obtain an activated NBD of formula I', wherein R 9 is an activating group.

[0134] Optionally, the method disclosed herein further comprises a step of purifying the product containing the acylated and / or activated NBD after step (d). The purification step can be performed using any suitable technique known in the art, such as any of those described in steps (a), (b), or (c) above. For example, the product containing the acylated and / or activated NBD can be purified using the following techniques: extraction with a suitable solvent and separation using column chromatography or filtration, washing one or more times with cold water and a cold organic solvent (such as cold ethanol and / or cold ether), and separation by silica gel column.

[0135] Typically, the yield of the acylated and / or activated NBD obtained in step (d) is at least 40%, or in the range of about 40% to about 95%. The yield of the acylated and / or activated NBD can be calculated using the following formula: (moles of acylated and / or activated NBD) / (moles of compound of formula VI)*100%. The yield can be determined before or after product purification.

[0136] An exemplary synthetic scheme for NBD showing specific reaction conditions is shown below.

[0137] ,

[0138] where R 1 ~R 10 and X may be in any form as defined above.

[0139] Although not illustrated in the exemplary synthesis scheme, it should be understood that not all steps shown in the exemplary reaction scheme are required to obtain the NBD of Formula I' above. The NBD of Formula I' having the desired structure can be obtained after: after steps (a) and (b); after steps (a), (b) and step (c); after steps (a), (b) and (d); or after steps (a), (b), (c) and (d).

[0140] B. Peptide Synthesis

[0141] The peptide / protein synthesis methods disclosed herein are based at least on the use of NBD as a preformed dipeptide building block for the synthesis of difficult peptides / proteins.

[0142] The terms "difficult sequences" and "difficult peptides / proteins" are used interchangeably herein. The concept of "difficult sequences" was introduced in the 1980s by Kent and colleagues to distinguish peptides that form strong inter- or intramolecular non-covalent interactions, resulting in the formation of insoluble peptide aggregates. "Difficult sequences" are peptide sequences containing a large number of amino acids with hydrophobic side chains (so-called β-branched amino acids), including leucine, valine, phenylalanine, or isoleucine. In addition, glycine is known to induce β-sheet stacking in combination with the above amino acids. These sequences tend to form β-sheet or α-helical structures within the molecule, and therefore have a high aggregation potential and low solubility in aqueous or organic solvents. This often makes them difficult to handle, synthesize, and purify. Even when applying automated peptide synthesis protocols, peptide chains exhibiting "difficult sequences" of more than 50-60 amino acids remain a challenge. With respect to SPPs, "difficult sequences" are defined as peptides that are poorly solvated when attached to a solid support, thereby hindering complete deprotection and coupling steps. (Mueller et al., Front Bioeng Biotechnol. 2020; 8: 162.) Using the NBD disclosed in SPPS (NBD-SPPS), the peptide length in standard SPPS (~50 AA) can be further extended to nearly 100 AA, which was previously unattainable in the field of peptide / protein synthesis.

[0143] Examples of difficult sequences include membrane proteins and their functional parts, such as amylin, BM2 proton channel, influenza A, copper storage protein 1 CSP-1, interferon-induced transmembrane protein 3 (IFITM3), NS4A, cofactor protein of the serine protease from hepatitis C virus; PD-L1 (programmed death-ligand 1), IL-2 (interleukin-2), vasoactive intestinal peptide, erythropoietin, liraglutide, etc.

[0144] The disclosed NBD is generally used in SPPS methods known in the art. Solid phase synthesis is a preferred method for chemical synthesis of peptides, in which the C-terminal amino acid of the sequence is attached to an insoluble support, and the remaining amino acids in the sequence are then added in sequence. Solid phase synthesis techniques are known to those skilled in the art and are described, for example, by Barany and Merrifield (1963) Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A.; Merrifield et al. (1963) J. Am. Chem. Soc, 85: 2149-2156, and Stewart et al. (1984) Solid Phase Peptide Synthesis, 2nd Edition, Pierce Chem. Co., Rockford, 111. Such methods include laboratory-scale solid phase synthesis and automated peptide synthesis in any of many commercially available peptide synthesizers. Solid phase synthesis is commonly used, and various commercial synthesizers are available, such as Applied Biosystems Inc., Foster City, CA; Beckman; MultiSyntech, Bochum, Germany's automated synthesizers, etc. During chemical synthesis, functional groups for conjugating peptides to small molecules, labeling moieties, peptides, or proteins can be introduced into the molecule. In addition, small molecules and labeling moieties / reporter units can also be connected during the synthesis process. The introduction of functional groups and conjugation with other molecules minimizes the structure and function of the NBD.

[0145] The disclosed method is different from and / or superior to previously described methods. For example, US 2008 / 0004451 describes the synthesis of dipeptide building blocks with acetone protecting groups to form pseudoproline rings. In contrast, the disclosed method utilizes a serine / threonine linkage (STL) or a cysteine / penicillamine linkage (CPL) as a proline mimetic, i.e., NBD, which allows for scale-up of peptide production in a very safe manner (NBD is safe to handle) and is environmentally friendly because the synthesis of NBD is a mild endothermic process and does not require heating or reflux conditions. In addition, the disclosed NBD is difficult to obtain using the method disclosed in US 2008 / 0004451. Salicylaldehyde, which is much less reactive, cannot react completely with the dipeptide even at reflux temperature, resulting in a low yield of the desired product. In addition, the similar polarity of the starting material and the product makes purification very challenging. This requires sophisticated column chromatography and experienced chemists, which greatly increases costs and the overall process is not efficient. In contrast, using the method disclosed herein, NBD can be easily prepared on a milligram to kilogram scale without the need for heating or condensation equipment. The high reactivity of salicylaldehyde esters allows complete conversion of the starting material and simplifies the purification process. In addition, additional functional groups can be attached to the phenyl ring of NBD to further facilitate difficult peptide / protein synthesis.

[0146] The methods disclosed herein similarly provide simpler and safer peptide synthesis than those previously considered in the disclosure, e.g., Wöhr et al., Pseudo-Prolines as a Solubilizing, Structure-Disrupting Protection Technique in Peptide Synthesis (J. Am. Chem. Soc. 1996, 118, 39, 9218–9227); the production of their dipeptides requires harsh conditions (such as anhydrous reflux with molecular sieves), which consumes more energy and has the potential for explosion. In contrast, the production of the disclosed NBD products does not require such complex equipment. NBDs produced as described herein require mixing all ingredients together at room temperature, in an air atmosphere, and at humidity, and can be readily obtained in high yields up to sub-kilograms.

[0147] Similarly, the disclosed method has advantages over the method disclosed in Sohma et al., Novel and efficient synthesis of difficult sequence-containing peptides through O–N intramolecular acylmigration reaction of O-acyl isopeptides, Chem. Commun (Camb), 1:124-5 (2004) (DOI: 10.1039 / B312129A), which discloses isoacyl dipeptides (which are chemically different from the disclosed NBD). In addition, the synthesis of isopeptides may have the risk of epimerization because esterification requires a highly reactive condensation reagent. In contrast, the disclosed method proceeds through the NBD; the connection between the two amino acid fragments is chemoselective and stereoselective without the risk of epimerization. In contrast, for acyl isopeptides, the disclosed NBD is more stable under alkaline conditions, which is important in Fmoc-SPPS for peptide synthesis. This advantage makes the disclosed NBD more suitable for chemical synthesis of long peptides or proteins.

[0148] The disclosed compounds and methods can be further understood by reference to the following numbered paragraphs.

[0149] 1. A compound having the following structure:

[0150] ,

[0151] in:

[0152] (i) R 1 is the protected or unprotected side chain of an amino acid;

[0153] (ii) R 2 is an amine protecting group;

[0154] (iii) R 3 ~R 6 independently hydrogen, hydroxy, halogen, -CN, haloalkyl (such as -CF3), -NO2, unsubstituted C1~C6 alkyl, unsubstituted C1~C6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, unsubstituted cyclic group, unsubstituted heterocycle, unsubstituted aralkyl, alkoxy, amino, amide, carbonyl or thiol;

[0155] (iv) R 7 and R 8 independently hydrogen, methyl, or other protected / unprotected side chains of amino acids;

[0156] (v) R 9 is hydrogen or a functional group suitable for protecting and / or activating a carboxylic acid group;

[0157] (vi) R 10 is hydrogen or a functional group suitable for hindering the reactivity of the phenyl group, such as a C1-C4 acyl group (e.g., formyl, acetyl, propionyl), a carbonyl group (e.g., alloc), or a carbamate group (e.g., tert-butyl methyl(2-(methylamino)ethyl)carbamate); and

[0158] (vii) X is O or S.

[0159] 2. The compound of paragraph 1, wherein R 1 is a protected or unprotected side chain of glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, cysteine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, serine, or threonine.

[0160] 3. The compound of paragraph 1 or 2, wherein when R 1 When it is a protected side chain, wherein the protecting group is selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, carbonyl (e.g., carboxylic acid or carboxylic ester), amide, amino, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and heterocycle, optionally wherein the protecting group is t Bu, Boc, Thz, Acm, Trt, Cbz, Alloc, azido, Bn, Bz, Ac, and Pbf, or a combination thereof.

[0161] 4. The compound of any one of paragraphs 1 to 3, wherein R 2 is Fmoc, Cbz, Moz, Boc, Troc, Teoc, Alloc or Voc.

[0162] 5. The compound of any one of paragraphs 1 to 4, wherein R 3 ~R 6 are independently hydrogen, hydroxy, halogen, -CN, haloalkyl (such as -CF3), -NO2, unsubstituted alkyl (such as unsubstituted C1~C8 alkyl), unsubstituted alkenyl (unsubstituted C1~C8 alkenyl), unsubstituted alkynyl (such as unsubstituted C1~C8 alkynyl), unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, unsubstituted cyclic group, unsubstituted heterocycle, unsubstituted aralkyl, alkoxy, amino, amide, carbonyl or thiol, optionally wherein R 3 ~R 6independently hydrogen, hydroxy, halogen, -CN, -CF3, -NO2, unsubstituted C1~C4 alkyl, alkoxy and unsubstituted aryl.

[0163] 6. The compound of any one of paragraphs 1 to 5, wherein R 3 ~R 6 At least one of, optionally R 3 ~R 6 Two or more of are not hydrogen.

[0164] 7. The compound of any one of paragraphs 1 to 6, wherein R 9 is hydrogen, benzyl, allyl, or unsubstituted C1-C4 alkyl; or is an activated ester group, such as 1-hydroxy-7-azabenzotriazole (HOAt) ester, 1-hydroxybenzotriazole (HOBt) ester, ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) ester, N-hydroxysuccinimide (NHS) ester, pentafluorophenol (Pfp) ester, and the like.

[0165] 8. The compound of any one of paragraphs 1 to 7, wherein R 10 is hydrogen, a C1-C4 acyl group (e.g., formyl, acetyl, propionyl), a carbonyl group (e.g., alloc), or a carbamate group (e.g., tert-butyl methyl(2-(methylamino)ethyl)carbamate).

[0166] 9. Method for preparing the compound of formula I:

[0167] ,

[0168] in:

[0169] (i) R 1 is the protected or unprotected side chain of an amino acid;

[0170] (ii) R 2 is an amine protecting group;

[0171] (iii) R 3 ~R 6 independently hydrogen, hydroxy, halogen, -CN, haloalkyl (such as -CF3), -NO2, unsubstituted C1~C6 alkyl, unsubstituted C1~C6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, unsubstituted cyclic group, unsubstituted heterocycle, unsubstituted aralkyl, alkoxy, amino, amide, carbonyl or thiol;

[0172] (iv) R 7 and R 8 independently hydrogen, methyl, or other protected / unprotected side chains of amino acids;

[0173] (v) R 9 is hydrogen, benzyl, allyl, unsubstituted C1-C4 alkyl, or a functional group suitable for protecting and / or activating a carboxylic acid group; and

[0174] (vi) X is O or S,

[0175] The method includes:

[0176] (a) reacting the protected amino acid of formula II with the replacement or unsubstituted salicylaldehyde of formula III to obtain an intermediate comprising the salicylaldehyde ester of formula IV:

[0177] ,

[0178] where R 1 and R 2 is as defined above for formula I,

[0179] ,

[0180] where R 3 ~R 6 is as defined above for formula I,

[0181] ,

[0182] (b) the salicylic aldehyde ester of formula IV is reacted with serine, threonine, cysteine ​​or penicillamine or its ester to obtain the product comprising formula I compound.

[0183] 10. The method of paragraph 9, wherein R 1 is a protected or unprotected side chain of glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, cysteine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, serine, or threonine.

[0184] 11. The method of paragraph 9 or 10, wherein when R 1 When it is a protected side chain, wherein the protecting group is selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, carbonyl (e.g., carboxylic acid or carboxylic ester), amide, amino, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and heterocycle, optionally wherein the protecting group is t Bu, Boc, Thz, Acm, Trt, Cbz, Alloc, azido, Bn, Bz, Ac, and Pbf, or a combination thereof.

[0185] 12. The method of any one of paragraphs 9 to 11, wherein R 2is Fmoc, Cbz, Moz, Boc, Troc, Teoc, Alloc or Voc.

[0186] 13. The method of any one of paragraphs 9 to 12, wherein R 3 ~R 6 are independently hydrogen, hydroxy, halogen, -CN, haloalkyl (such as -CF3), -NO2, unsubstituted alkyl (such as unsubstituted C1~C8 alkyl), unsubstituted alkenyl (unsubstituted C1~C8 alkenyl), unsubstituted alkynyl (such as unsubstituted C1~C8 alkynyl), unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, unsubstituted cyclic group, unsubstituted heterocycle, unsubstituted aralkyl, alkoxy, amino, amide, carbonyl or thiol, optionally wherein R 3 ~R 6 independently hydrogen, hydroxy, halogen, -CN, -CF3, -NO2, unsubstituted C1~C4 alkyl, alkoxy and unsubstituted aryl.

[0187] 14. The method of any one of paragraphs 9 to 13, wherein R 3 ~R 6 At least one of, optionally R 3 ~R 6 Two or more of are not hydrogen.

[0188] 15. The process of any one of paragraphs 9 to 14, wherein step (a) is carried out in the presence of a condensing agent, optionally wherein the condensing agent is HATU, PyBOP, HBTU, HCTU, COMU, TSTU, PyClock, PyOxim, EDCI or DCC, or a combination thereof.

[0189] 16. The process of any one of paragraphs 9 to 15, wherein step (a) is performed in the presence of a base, optionally wherein the base is DIPEA or triethylamine, or a combination thereof.

[0190] 17. The process of any one of paragraphs 9 to 16, wherein step (a) is carried out in an organic solvent, optionally wherein the organic solvent is DMF, DMSO, DMAc or NMP, or a combination thereof.

[0191] 18. The process of any one of paragraphs 9 to 17, wherein step (a) is carried out at room temperature for 30 minutes to 5 hours, preferably 1 hour to 3 hours.

[0192] 19. method any one in paragraph 9 to 18, wherein the salicylic aldehyde ester of formula IV obtaining in step (a) has at least 40%, at least 50% or approximately 50% to approximately 95% yield.

[0193] 20. The method of any one of paragraphs 9 to 19, wherein step (b) is carried out in a solvent comprising a buffer, optionally wherein the solvent is dichloromethane, trifluoroacetic acid, tetrahydrofuran, toluene, ethyl acetate, 1,4-dioxane, acetonitrile, acetone or water, or a combination thereof.

[0194] 21. The method of paragraph 20, wherein the buffer is a pyridine-acetic acid buffer, optionally wherein the molar ratio of pyridine to acetic acid ranges from 1:9 to 9:1, such as 1:1.

[0195] 22. The process of any one of paragraphs 9 to 21, wherein step (b) is performed at room temperature for a period of 1 hour to 5 hours, preferably for a period of 2 hours to 3 hours.

[0196] 23. The process of any one of paragraphs 9 to 22, wherein the compound of formula I is obtained in step (b) in a yield ranging from at least 40%, such as from about 40% to about 95%.

[0197] 24. method any one in paragraph 9 to 23, it is further included in step (a) after and before step (b) purifying described intermediate to obtain the salicylic aldehyde ester of formula IV.

[0198] 25. The process of any one of paragraphs 9 to 24, further comprising purifying the product after step (b) to obtain a compound of formula I.

[0199] 26. The method of any one of paragraphs 9 to 25, further comprising:

[0200] (c) reacting the carboxyl-protected NBD with a deprotecting agent to form a deprotected NBD of Formula VI:

[0201] ,

[0202] (i) R 1 is the protected or unprotected side chain of an amino acid;

[0203] (ii) R 2 is an amine protecting group;

[0204] (iii) R 3 ~R 6 independently hydrogen, hydroxy, halogen, -CN, haloalkyl (such as -CF3), -NO2, unsubstituted C1~C6 alkyl, unsubstituted C1~C6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, unsubstituted cyclic group, unsubstituted heterocycle, unsubstituted aralkyl, alkoxy, amino, amide, carbonyl or thiol;

[0205] (iv) R 7and R 8 independently hydrogen, methyl, or other protected / unprotected side chains of amino acids;

[0206] (v) R 10 is hydrogen or a functional group suitable for hindering the reactivity of the phenyl group, such as a C1-C4 acyl group (e.g., formyl, acetyl, propionyl), a carbonyl group (e.g., alloc), or a carbamate group (e.g., tert-butyl methyl(2-(methylamino)ethyl)carbamate); and

[0207] (vi) X is O or S.

[0208] 27. The method of any one of paragraphs 9 to 26, further comprising:

[0209] (d) reacting the NBD containing the free acid or free phenol with an acylating and / or activating reagent to form an acylated / activated NBD of Formula I':

[0210] ,

[0211] in:

[0212] (i) R 1 is the protected or unprotected side chain of an amino acid;

[0213] (ii) R 2 is an amine protecting group;

[0214] (iii) R 3 ~R 6 independently hydrogen, hydroxy, halogen, -CN, haloalkyl (such as -CF3), -NO2, unsubstituted C1~C6 alkyl, unsubstituted C1~C6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, unsubstituted cyclic group, unsubstituted heterocycle, unsubstituted aralkyl, alkoxy, amino, amide, carbonyl or thiol;

[0215] (iv) R 7 and R 8 independently hydrogen, methyl, or other protected / unprotected side chains of amino acids;

[0216] (v) R 9 is hydrogen or a functional group suitable for protecting and / or activating a carboxylic acid group;

[0217] (vi) R 10 is a functional group suitable for hindering the reactivity of the phenyl group, such as a C1-C4 acyl group (e.g., formyl, acetyl, propionyl), a carbonyl group (e.g., alloc), or a carbamate group (e.g., tert-butyl methyl(2-(methylamino)ethyl)carbamate); and

[0218] (vii) X is O or S.

[0219] 28. The method of paragraph 26 or 27, wherein step (c) is performed immediately after step (b) or immediately after step (d), wherein the carboxyl-protected NBD is an NBD of formula I formed in step (b) or an NBD of formula I' formed in step (d), and wherein R of formula I or formula I' is 9 Not hydrogen.

[0220] 29. The process of paragraph 27 or 28, wherein step (d) is performed immediately after step (b) or immediately after step (c), and wherein the NBD containing the free acid is the NBD of formula I formed in step (b) or the NBD of formula VI formed in step (c).

[0221] 30. A method for solid phase peptide synthesis using the compound of any one of paragraphs 1 to 8.

[0222] 31. The method of paragraph 30, comprising: mixing the compound with an amino acid to form a peptide.

[0223] Detailed synthetic procedures for exemplary NBDs are described in the following non-limiting examples.

[0224] Example

[0225] Example 1. L-Thr-L-Thr NBD

[0226] The first example of the synthesis of L-Thr-L-Thr NBD, the structure of which is shown below.

[0227]

[0228] Step a:

[0229] First, L-Fmoc-Thr (tBu) -OH (3.97 g, 10.0 mmol) and HATU (3.80 g, 10.0 mmol) were dissolved in 50.0 mL of DMF. DIPEA (2.58 g, 20.0 mmol) was then added, and the reaction mixture was stirred at room temperature for 1 min. Salicylaldehyde (1.22 g, 10.0 mmol) was then added, and the reaction was stirred for 1.5 h. After this, the mixture was extracted with 250 mL of ethyl acetate and washed three times with 100 mL of 1M HCl (aq) and 100 mL of NaHCO 3 (aq). The resulting organic phase was dried over sodium sulfate and evaporated under reduced pressure to obtain the crude product as a pink oil. After purification on a silica gel column, Fmoc-Thr (tBu) -SAL ester, 4.77 g, was obtained in a 95% yield.

[0230] Step b:

[0231] Fmoc-Thr(tBu)-SAL ester was dissolved in 250 mL of dichloromethane and 10 mL of pyridine / HOAc buffer containing 10.0 mmol of LH-Thr-OAll ester was added. The reaction mixture was stirred at room temperature for 2 h and extracted in a manner similar to that described above. The crude product was purified by column chromatography to obtain the desired NBD as a pale solid in 93% yield.

[0232] Alternatively, the above procedure can be simplified to just one purification step:

[0233] After extraction and evaporation in step a., the crude L-Fmoc-Thr(tBu)-SAL ester was directly dissolved in 250 mL of dichloromethane without any additional purification. Then, 10 mL of pyridine / HOAc buffer containing 10.0 mmol of LH-Thr-OAll ester was added, and the mixture was reacted for 2 hours to provide the desired L-Thr-L-Thr NBD in a two-step yield of 85%. Furthermore, this simplified protocol has been robustly scaled up to subkilogram scale for NBD production.

[0234] Example 2. L-Val-L-Cys NBD

[0235] The second example of synthesizing L-Val-L-Cys NBD has a structure shown below.

[0236]

[0237] Step a and step b:

[0238] First, L-Fmoc-Val-OH (3.39 g, 10.0 mmol) and HATU (3.80 g, 10.0 mmol) were dissolved in 50.0 mL DMF. Then, DIPEA (2.58 g, 20.0 mmol) was added and the reaction mixture was stirred at room temperature for 1 min. Then, 4-methoxy salicylaldehyde (1.52 g, 10.0 mmol) was added and the reaction was stirred for 1.0 h. After this, the mixture was directly diluted with 100 mL 6M pyridine-acetic acid aqueous buffer and 10 mL of 10% TFA aqueous solution containing 12.0 mmol L-H-Cys-OH was added dropwise. After the reaction mixture was stirred at room temperature for 3 h, it was poured into 1-L ice-cold water. The white precipitate was filtered with suction and washed with cold water, cold ethanol and cold ether. The product was then further purified by column chromatography to afford the desired L-Val-L-Cys NBD as a white solid in 83% yield. This protocol can also be scaled up to sub-kilogram scale for Cys / Pen-based NBD production.

[0239] Example 3. Ac-terminated L-Val-L-Thr NBD

[0240] The first example of the synthesis of Ac-terminated L-Thr-L-Thr NBD is shown below.

[0241]

[0242] Step a:

[0243] First, L-Fmoc-Val-OH (3.39 g, 10.0 mmol) and HATU (3.80 g, 10.0 mmol) were dissolved in 50.0 mL DMF. Subsequently, DIPEA (2.58 g, 20.0 mmol) was added and the reaction mixture was stirred at room temperature for 1 min. Then, salicylaldehyde (1.22 g, 10.0 mmol) was added and the reaction was stirred for 1.5 hours. After this, the mixture was extracted with 250 mL ethyl acetate and washed 3 times with 100 mL 1M HCl (aq), 100 mL NaHCO (aq). The gained organic phase was dried over sodium sulfate and evaporated under reduced pressure to obtain a crude product as a pink oil. After purification on a silica gel column, Fmoc-Val-SAL ester, 4.51 g, was obtained in a 95% yield.

[0244] Step b:

[0245] Fmoc-Val-SAL ester was dissolved in 250 mL of dichloromethane, and 10 mL of pyridine / HOAc buffer containing 10.0 mmol of LH-Thr-Oal ester was added. The reaction mixture was stirred at room temperature for 2 hours and extracted in a manner similar to that described above. The crude product was then purified by column chromatography to obtain the unprotected NBD as an off-white solid in 91% yield.

[0246] Step c:

[0247] Unprotected NBD was dissolved in 250 mL of dichloromethane and Pd(PPh3)4 (5 mol%) and PhSiH3 (10 equivalents) were added. The reaction mixture was stirred at room temperature for 1 h and extracted in a manner similar to that described above. The crude product was then purified by column chromatography to obtain the NBD acid form as an off-white solid in 95% yield.

[0248] Step d:

[0249] Then NBD acid is dissolved in 250 mL dichloromethane, and Ac o (2.0 equivalents) and DIPEA (4.0 equivalents) are added. The reaction mixture is stirred at room temperature for 3 h, and is extracted in a manner similar to that mentioned above. The crude product is then purified by column chromatography to obtain the desired Ac-blocked NBD acid in 90% yield, which is a pale solid.

[0250] Example 4. HOAt-activated L-Thr-L-Thr NBD

[0251] The first example of the synthesis of HOAt-activated L-Thr-L-Thr NBD is shown below.

[0252]

[0253] Step a:

[0254] First, L-Fmoc-Val-OH (3.39 g, 10.0 mmol) and HATU (3.80 g, 10.0 mmol) were dissolved in 50.0 mL DMF. Subsequently, DIPEA (2.58 g, 20.0 mmol) was added, and the reaction mixture was stirred at room temperature for 1 min. Then, salicylaldehyde (1.22 g, 10.0 mmol) was added, and the reaction was stirred for 1.5 h. After this, the mixture was extracted with 250 mL ethyl acetate and washed 3 times with 100 mL 1M HCl (aq), 100 mL NaHCO (aq). The gained organic phase was dried over sodium sulfate and evaporated under reduced pressure to obtain a crude product, which was a pink oil. After purification on a silica gel column, Fmoc-Val-SAL ester, 4.51 g, was obtained in 95% yield.

[0255] Step b:

[0256] Fmoc-Val-SAL ester was dissolved in 250 mL of dichloromethane, and 10 mL of pyridine / HOAc buffer containing 10.0 mmol of LH-Thr-OAll ester was added. The reaction mixture was stirred at room temperature for 2 hours and extracted in a manner similar to that described above. The crude product was then purified by column chromatography to provide the unprotected NBD as an off-white solid in 91% yield.

[0257] Step c:

[0258] Unprotected NBD was dissolved in 250 mL of dichloromethane and Pd(PPh3)4 (5 mol%) and PhSiH3 (10 equivalents) were added. The reaction mixture was stirred at room temperature for 1 h and extracted in a manner similar to that described above. The crude product was then purified by column chromatography to obtain the NBD acid form as an off-white solid in 93% yield.

[0259] Step d:

[0260] Then NBD acid is dissolved in 250 mL dichloromethane, and HATU (1.0 equivalent) and DIPEA (1.0 equivalent) are added.Reaction mixture is stirred at room temperature for 0.5 h, and is extracted similarly to the above-mentioned mode.Then crude product, through column chromatography purification, obtains the NBD acid of desired HOAt activation in 78% yield, which is a pale solid.

[0261] Example 5. Carbamate-terminated L-Val-L-Cys NBD

[0262] The second example synthesized a carbamate-protected L-Val-L-Cys NBD, the structure of which is shown below.

[0263]

[0264] Step a and step b:

[0265] First, L-Fmoc-Val-OH (3.39 g, 10.0 mmol) and HATU (3.80 g, 10.0 mmol) were dissolved in 50.0 mL DMF. Then, DIPEA (2.58 g, 20.0 mmol) was added and the reaction mixture was stirred at room temperature for 1 min. Then, 4-methoxy salicylaldehyde (1.52 g, 10.0 mmol) was added and the reaction was stirred for 1.0 h. After this, the mixture was directly diluted with 100 mL 6M pyridine-acetic acid aqueous buffer and 10 mL of 10% TFA aqueous solution containing 12.0 mmol L-H-Cys-OH was added dropwise. After the reaction mixture was stirred at room temperature for 3 h, it was poured into 1-L ice-cold water. The white precipitate was filtered with suction and washed with cold water, cold ethanol and cold ether. Afterwards, the product was purified by column chromatography to obtain the desired L-Val-L-Cys NBD as a white solid in 83% yield.

[0266] Step d:

[0267] In this case, the NBD is already in the acid form, making it possible to skip step c. The unprotected NBD was dissolved in 250 mL of dichloromethane and tert-butyl methyl (2-(methylamino)ethyl)carbamate (2.0 equivalents) and DIPEA (4.0 equivalents) activated with 1,1'-carbonyldiimidazole (2.0 equivalents) were added. The reaction mixture was stirred at room temperature for 1 h and extracted in a manner similar to that described above. The crude product was then purified by column chromatography to give the desired carbamate-terminated NBD as an off-white solid in 82% yield.

[0268] Example 6. Peptide Synthesis

[0269] With these NBDs, we further evaluated their role in the construction of difficult peptides by SPPS. For this purpose, we selected several difficult or even inaccessible peptide sequences reported in the literature.

[0270] The first case is the synthesis of PD-L1 (121-132), which is the C-terminus of PD-L1 that is very prone to forming a β-sheet structure. The amino acid was installed by a standard coupling procedure using HATU as a coupling reagent. Continuing, the resin was subjected to a washing step. The resin was then treated with 20% piperidine / DMF to remove the Fmoc protecting group and allow the next coupling cycle to proceed. During the synthesis, the Ile-Thr NBD was installed by a one-pot activation and coupling scheme. Figure 1 As shown in , the installation of Ile-ThrNBD significantly improved the quality of the final product. In addition, in the absence of the dipeptide, severe amino acid deletions were observed, demonstrating the ability of dipeptides to solve difficult SPPS problems. In the synthesis of IL-2(125-133), the Ser-Cys NBD ( Figure 2 ). The amino acids were installed by a standard coupling procedure using HATU as a coupling reagent and, subsequently, the resin was subjected to a washing step. The resin was then treated with 20% piperidine / DMF to remove the Fmoc protecting group and allow the next coupling cycle to proceed. During the synthesis, the Ser-Cys NBD was installed by a one-pot activation and coupling protocol. Although SPPS with / without NBD showed similar retention peaks from the analytical HPLC data of the peptide, SPPS without NBD showed a chaotic mass spectrum. In contrast, the quality of the peptide was excellent in the case of NBD insertion. Following this, the synthesis of another difficult sequence (amylin with 37 amino acids) was performed by introducing 3 NBDs during Fmoc-SPPS ( Figure 3 ). Amino acids were installed by standard coupling procedures using HATU as a coupling reagent, followed by a washing step on the resin. The resin was then treated with 20% piperidine / DMF to remove the Fmoc protecting group, enabling the next coupling cycle. During the synthesis, three corresponding NBDs were installed by a one-pot activation and coupling protocol. In the case of installation of NBD, the desired product could be obtained with high quality. However, no desired product was observed in the synthesis without NBD. Finally, the natural HIV inhibitor RANTES (also known as chemokine ligand 5), which contains 68 amino acids, was selected as an example of a challenging target with a longer sequence. Amino acids were installed by standard coupling procedures using HATU as a coupling reagent, followed by a washing step on the resin. The resin was then treated with 20% piperidine / DMF to remove the Fmoc protecting group, enabling the next coupling cycle. During the synthesis, three corresponding NBDs were installed by a one-pot activation and coupling protocol. As Figure 4As shown in [ ] , RANTES was successfully constructed when four NBDs were introduced, and isolated yields as high as 21% could be achieved after HPLC purification. Meanwhile, attempts to synthesize RANTES via traditional SPPS failed, yielding only chaotic results, highlighting the critical role of our NBDs in preventing peptide aggregation during SPPS. Furthermore, in all of the above examples, only polystyrene-based resins were employed, eliminating the need to resort to modified and expensive polymeric supports for difficult peptides / proteins.

Claims

1. A compound having the following structure: ， in: (i) R 1 is the protected or unprotected side chain of an amino acid; (ii) R 2 is an amine protecting group; (iii) R 3 ~R 6 independently hydrogen, hydroxy, halogen, -CN, haloalkyl (such as -CF3), -NO2, unsubstituted C1~C6 alkyl, unsubstituted C1~C6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, unsubstituted cyclic group, unsubstituted heterocycle, unsubstituted aralkyl, alkoxy, amino, amide, carbonyl or thiol; (iv) R 7 and R 8 independently hydrogen, methyl, or other protected / unprotected side chains of amino acids; (v) R 9 is hydrogen or a functional group suitable for protecting and / or activating a carboxylic acid group; (vi) R 10 is hydrogen or a functional group suitable for hindering the reactivity of the phenyl group, such as a C1-C4 acyl group (e.g., formyl, acetyl, propionyl), a carbonyl group (e.g., alloc), or a carbamate group (e.g., tert-butyl methyl(2-(methylamino)ethyl)carbamate); and (vii) X is O or S.

2. The compound of claim 1, wherein R 1 is a protected or unprotected side chain of glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, cysteine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, serine, or threonine.

3. The compound of claim 1 or 2, wherein R 1 When it is a protected side chain, wherein the protecting group is selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, carbonyl (e.g., carboxylic acid or carboxylate), amide, amino, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and heterocycle, optionally wherein the protecting group is t Bu, Boc, Thz, Acm, Trt, Cbz, Alloc, azido, Bn, Bz, Ac, and Pbf, or a combination thereof.

4. The compound of any one of claims 1 to 3, wherein R 2 is Fmoc, Cbz, Moz, Boc, Troc, Teoc, Alloc or Voc.

5. The compound of any one of claims 1 to 4, wherein R 3 ~R 6 are independently hydrogen, hydroxy, halogen, -CN, haloalkyl (such as -CF3), -NO2, unsubstituted alkyl (such as unsubstituted C1~C8 alkyl), unsubstituted alkenyl (unsubstituted C1~C8 alkenyl), unsubstituted alkynyl (such as unsubstituted C1~C8 alkynyl), unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, unsubstituted cyclic group, unsubstituted heterocycle, unsubstituted aralkyl, alkoxy, amino, amide, carbonyl or thiol, optionally wherein R 3 ~R 6 independently hydrogen, hydroxy, halogen, -CN, -CF3, -NO2, unsubstituted C1~C4 alkyl, alkoxy and unsubstituted aryl.

6. The compound of any one of claims 1 to 5, wherein R 3 ~R 6 At least one of, optionally R 3 ~R 6 Two or more of are not hydrogen.

7. The compound according to any one of claims 1 to 6, wherein R 9 is hydrogen, benzyl, allyl, or unsubstituted C1-C4 alkyl; or is an activated ester group, such as 1-hydroxy-7-azabenzotriazole (HOAt) ester, 1-hydroxybenzotriazole (HOBt) ester, ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) ester, N-hydroxysuccinimide (NHS) ester, pentafluorophenol (Pfp) ester, and the like.

8. The compound of any one of claims 1 to 7, wherein R 10 is hydrogen, a C1-C4 acyl group (e.g., formyl, acetyl, propionyl), a carbonyl group (e.g., alloc), or a carbamate group (e.g., tert-butyl methyl(2-(methylamino)ethyl)carbamate).

9. Method for producing a compound of formula I: ， in: (i) R 1 is the protected or unprotected side chain of an amino acid; (ii) R 2 is an amine protecting group; (iii) R 3 ~R 6 independently hydrogen, hydroxy, halogen, -CN, haloalkyl (such as -CF3), -NO2, unsubstituted C1~C6 alkyl, unsubstituted C1~C6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, unsubstituted cyclic group, unsubstituted heterocycle, unsubstituted aralkyl, alkoxy, amino, amide, carbonyl or thiol; (iv) R 7 and R 8 independently hydrogen, methyl, or other protected / unprotected side chains of amino acids; (v) R 9 is hydrogen, benzyl, allyl, unsubstituted C1-C4 alkyl, or a functional group suitable for protecting and / or activating a carboxylic acid group; and (vi) X is O or S, The method comprises: (a) reacting the protected amino acid of formula II with the replacement or unsubstituted salicylaldehyde of formula III to obtain an intermediate comprising the salicylaldehyde ester of formula IV: ， where R 1 and R 2 is as defined above for formula I, ， where R 3 ~R 6 is as defined above for formula I, ， (b) the salicylic aldehyde ester of said formula IV is reacted with serine, threonine, cysteine ​​or penicillamine or its ester to obtain the product comprising said formula I compound.

10. The method of claim 9, wherein R 1 is a protected or unprotected side chain of glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, cysteine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, serine, or threonine.

11. The method of claim 9 or 10, wherein when R 1 When it is a protected side chain, wherein the protecting group is selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, carbonyl (e.g., carboxylic acid or carboxylate), amide, amino, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and heterocycle, optionally wherein the protecting group is t Bu, Boc, Thz, Acm, Trt, Cbz, Alloc, azido, Bn, Bz, Ac, and Pbf, or a combination thereof.

12. The method of any one of claims 9 to 11, wherein R 2 is Fmoc, Cbz, Moz, Boc, Troc, Teoc, Alloc or Voc.

13. The method of any one of claims 9 to 12, wherein R 3 ~R 6 are independently hydrogen, hydroxy, halogen, -CN, haloalkyl (such as -CF3), -NO2, unsubstituted alkyl (such as unsubstituted C1~C8 alkyl), unsubstituted alkenyl (unsubstituted C1~C8 alkenyl), unsubstituted alkynyl (such as unsubstituted C1~C8 alkynyl), unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, unsubstituted cyclic group, unsubstituted heterocycle, unsubstituted aralkyl, alkoxy, amino, amide, carbonyl or thiol, optionally wherein R 3 ~R 6 independently hydrogen, hydroxy, halogen, -CN, -CF3, -NO2, unsubstituted C1~C4 alkyl, alkoxy and unsubstituted aryl.

14. The method of any one of claims 9 to 13, wherein R 3 ~R 6 At least one of, optionally R 3 ~R 6 Two or more of are not hydrogen.

15. The process of any one of claims 9 to 14, wherein step (a) is carried out in the presence of a condensing agent, optionally wherein the condensing agent is HATU, PyBOP, HBTU, HCTU, COMU, TSTU, PyClock, PyOxim, EDCI or DCC, or a combination thereof.

16. The process of any one of claims 9 to 15, wherein step (a) is performed in the presence of a base, optionally wherein the base is DIPEA or triethylamine, or a combination thereof.

17. The process of any one of claims 9 to 16, wherein step (a) is carried out in an organic solvent, optionally wherein the organic solvent is DMF, DMSO, DMAc or NMP, or a combination thereof.

18. The process of any one of claims 9 to 17, wherein step (a) is performed at room temperature for a period of 30 minutes to 5 hours, preferably 1 hour to 3 hours.

19. method described in any one in claim 9 to 18, wherein the salicylic aldehyde ester of the described formula IV obtaining in step (a) has at least 40%, at least 50% or approximately 50% to the yield in approximately 95% scope.

20. The process of any one of claims 9 to 19, wherein step (b) is carried out in a solvent comprising a buffer, optionally wherein the solvent is dichloromethane, trifluoroacetic acid, tetrahydrofuran, toluene, ethyl acetate, 1,4-dioxane, acetonitrile, acetone or water, or a combination thereof.

21. The method of claim 20, wherein the buffer is a pyridine-acetic acid buffer, optionally wherein the molar ratio of pyridine to acetic acid ranges from 1:9 to 9:1, such as 1:

1.

22. The process of any one of claims 9 to 21, wherein step (b) is performed at room temperature for a period of 1 to 5 hours, preferably 2 to 3 hours.

23. The process of any one of claims 9 to 22, wherein the compound of formula I is obtained in step (b) in a yield ranging from at least 40%, such as from about 40% to about 95%.

24. the method described in any one in claim 9 to 23, it is further included in step (a) after and before step (b) the described intermediate of purification to obtain the salicylic aldehyde ester of described formula IV.

25. The process of any one of claims 9 to 24, further comprising purifying the product after step (b) to obtain the compound of formula I.

26. The method of any one of claims 9 to 25, further comprising: (c) reacting the carboxyl-protected NBD with a deprotecting agent to form a deprotected NBD of Formula VI: ， (i) R 1 is the protected or unprotected side chain of an amino acid; (ii) R 2 is an amine protecting group; (iii) R 3 ~R 6 independently hydrogen, hydroxy, halogen, -CN, haloalkyl (such as -CF3), -NO2, unsubstituted C1~C6 alkyl, unsubstituted C1~C6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, unsubstituted cyclic group, unsubstituted heterocycle, unsubstituted aralkyl, alkoxy, amino, amide, carbonyl or thiol; (iv) R 7 and R 8 independently hydrogen, methyl, or other protected / unprotected side chains of amino acids; (v) R 10 is hydrogen or a functional group suitable for hindering the reactivity of the phenyl group, such as a C1-C4 acyl group (e.g., formyl, acetyl, propionyl), a carbonyl group (e.g., alloc), or a carbamate group (e.g., tert-butyl methyl(2-(methylamino)ethyl)carbamate); and (vi) X is O or S.

27. The method of any one of claims 9 to 26, further comprising: (d) reacting the NBD containing the free acid or free phenol with an acylating and / or activating reagent to form an acylated / activated NBD of Formula I': ， in: (i) R 1 is the protected or unprotected side chain of an amino acid; (ii) R 2 is an amine protecting group; (iii) R 3 ~R 6 independently hydrogen, hydroxy, halogen, -CN, haloalkyl (such as -CF3), -NO2, unsubstituted C1~C6 alkyl, unsubstituted C1~C6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, unsubstituted cyclic group, unsubstituted heterocycle, unsubstituted aralkyl, alkoxy, amino, amide, carbonyl or thiol; (iv) R 7 and R 8 independently hydrogen, methyl, or other protected / unprotected side chains of amino acids; (v) R 9 is hydrogen or a functional group suitable for protecting and / or activating a carboxylic acid group; (vi) R 10 is a functional group suitable for hindering the reactivity of the phenyl group, such as a C1-C4 acyl group (e.g., formyl, acetyl, propionyl), a carbonyl group (e.g., alloc), or a carbamate group (e.g., tert-butyl methyl(2-(methylamino)ethyl)carbamate); and (vii) X is O or S.

28. The method of claim 26 or 27, wherein step (c) is performed immediately after step (b) or immediately after step (d), wherein the carboxyl-protected NBD is the NBD of formula I formed in step (b) or the NBD of formula I' formed in step (d), and wherein R of formula I or formula I' is 9 Not hydrogen.

29. The process of claim 27 or 28, wherein step (d) is performed immediately after step (b) or immediately after step (c), and wherein the NBD containing a free acid is the NBD of formula I formed in step (b) or the NBD of formula VI formed in step (c).

30. A method for solid phase peptide synthesis using the compound of any one of claims 1 to 8.

31. The method of claim 30, comprising: The compound is mixed with amino acids to form the peptide.

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