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WO2024174680A1 - Building blocks for difficult peptide synthesis and method of making thereof - Google Patents

Building blocks for difficult peptide synthesis and method of making thereof Download PDF

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Publication number
WO2024174680A1
WO2024174680A1 PCT/CN2023/137784 CN2023137784W WO2024174680A1 WO 2024174680 A1 WO2024174680 A1 WO 2024174680A1 CN 2023137784 W CN2023137784 W CN 2023137784W WO 2024174680 A1 WO2024174680 A1 WO 2024174680A1
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unsubstituted
group
formula
substituted
hydrogen
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PCT/CN2023/137784
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French (fr)
Inventor
Xuechen Li
Hongxiang Wu
Zhenquan SUN
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Versitech Limited
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Publication of WO2024174680A1 publication Critical patent/WO2024174680A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
    • C07K1/065—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups for hydroxy functions, not being part of carboxy functions
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
    • C07K1/067—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups for sulfur-containing functions
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06052—Val-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
    • C07K5/06069—Ser-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06104—Dipeptides with the first amino acid being acidic

Definitions

  • This invention is generally in the field of protein/peptide synthesis.
  • SPPS solid phase peptide synthesis
  • NBDs N, O/S-benzylidene acetal dipeptides
  • the compounds can have the structure of Formula I:
  • R 1 can be a protected or unprotected side chain of an amino acid
  • R 2 can be an amine protecting group
  • R 3 -R 6 can be independently hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF 3 ) , -NO 2 , unsubstituted C 1 -C 6 alkyl, unsubsituted C 1 -C 6 alkenyl, unsubstituted aryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol;
  • R 7 and R 8 can be indepedently hydrogen, methyl, or other protected/unprotected side chains of amino acids
  • R 9 can be hydrogen or a functional group suitable for protecting and/or
  • the protection group can be a substituted or unsubstitited alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a carbonyl (e.g., carboxylic acid or carboxylic ester) , an amide, an amino, a substitute or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a substituted or unsubstituted heterocyclic ring.
  • the compounds can have the structure of Formula I’:
  • R 1 -R 9 and X can be as defined above, and R 10 can be hydrogen or a functional group suitable for hindering the reactivity of phenyl group, such as a C 1 -C 4 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) .
  • a C 1 -C 4 acyl group e.g., formyl, acetyl, propionyl
  • a carbonyl group e.g., alloc
  • carbamate group e.g., tert-butyl methyl (2- (methylamino) ethyl) carbamate
  • a L-or D-Amino acid with suitable protection on amine and optionally its sidechain functional groups can be firstly coupled with salicylaldehyde or a mono-/multiply- substituted form thereof. Then, the obtained salicylaldehyde ester reacts with serine/threonine/cysteine/penicillamine (Ser/Thr/Cys/Pen) or its ester in a suitable solvent. The obtained ester may be further derivatized to relevant acids with or without phenol protections.
  • the NBDs can be obtained in high yield (at least 40%) .
  • FIG. 1 shows a comparison of the synthesis of PD-L1 (121-123) using the disclosed method and conventional SPPS.
  • FIG. 2 shows a comparison of the synthesis of IL-2 (125-133) using the disclosed method and conventional SPPS.
  • FIG. 3 shows a comparison of the synthesis of Amylin using the disclosed method and conventional SPPS.
  • FIG. 4 shows a comparison of the synthesis of Rantes using the disclosed method and conventional SPPS.
  • NBD N, O/S-benzylidene acetal dipeptide
  • alkyl refers to a branched or straight-chain monovalent saturated aliphatic hydrocarbon radical of one to eight carbon atoms. This term is further exemplified by radicals such as methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl, n-pentyl, isopentyl, t-pentyl, n-hexyl, etc.
  • alkenyl refers to a branched or straight-chain hydrocarbon group of from two to eight carbon atoms and structural formula containing at least one carbon-carbon double bond.
  • alkynyl refers to a branched or straight-chain hydrocarbon group of from two to eight carbon atoms and structural formula containing at least one carbon-carbon triple bond.
  • aryl refers to C 5 -C 20 -membered aromatic or fused aromatic ring systems. Examples of aromatic groups are benzene, naphthalene, anthracene, phenanthrene, chrysene, pyrene, corannulene, coronene, etc.
  • cyclic group refers to a saturated or unsaturated monocyclic ring or a polycyclic ring (such as those formed from single or fused ring systems) , such as a cycloalkyl, a cycloalkenyl, or a cycloalkynyl, which have from three to ten carbon atoms, as geometric constraints permit.
  • heteroalkyl refers to straight or branched chain carbon-containing alkyl radicals containing at least one heteroatom on the carbon backbone. Suitable heteroatoms include, but are not limited to, O, N, Si, P and S, wherein the nitrogen, phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized.
  • heteroaryl refers to C 5 -C 20 -membered aromatic or fused aromatic ring systems, in which one or more carbon atoms on one or more aromatic ring structures have been substituted with a heteroatom. Suitable heteroatoms include, but are not limited to, oxygen, sulfur, and nitrogen. Examples of heteroaryl groups pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
  • heteroaryl rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H, 6H-1, 5, 2-dithiazinyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, is
  • heterocyclic group refers to a cyclic group as defined above where at least one of the carbon atoms of the ring is substituted with a heteroatom, such as, nitrogen, oxygen, sulfur, or phosphorus.
  • aralkyl refers to an aryl group or a heteroaryl group having an alkyl, alkynyl, or alkenyl group as defined above attached to the aromatic group, such as an aryl, a heteroaryl, a polyaryl, or a polyheteroaryl.
  • An example of an aralkyl group is a benzyl group.
  • alkoxyl refers to compounds represented by the formula -OR v , wherein R v includes, but is not limited to, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a heterocyclic, a cycloalkenyl, an aryl, a heteroaryl, an aralkyl, a heteroalkyl, etc.
  • exemplary alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like.
  • a “lower alkoxyl” group is an alkoxyl group containing from one to six carbon atoms.
  • An “ether” is two functional groups covalently linked by an oxygen as defined below.
  • the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O-alkenyl, -O-alkynyl, -O-aralkyl, -O-aryl, -O-heteroaryl, -O-cyclic, -O-heterocyclic, etc.
  • amino as used herein includes the group
  • 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, a substituted or unsubstituted polyaryl, a substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclyl, wherein independently of E, R x , R xi , and R xii each independently represent a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted carbonyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted al
  • R represents a hydroxyl group, a substituted or unsubstituted carbonyl group, a substituted or unsubstituted aryl, a substituted or unsubstituted cycloalkyl
  • quaternary amino also includes the groups where the nitrogen, R x , R xi , and R xii with the N + to which they are attached complete a heterocyclyl or heteroaryl having from 3 to 14 atoms in the ring structure. It is understood by those of ordinary skill in the art, that the E groups listed above are divalent (e.g., methylene, ethane-1, 2-diyl, ethene-1, 2-diyl, 1, 4-phenylene, cyclohexane-1, 2-diyl) .
  • amide or “amido” are used interchangeably, refer to both “unsubstituted amido” and “substituted amido” and are represented by the general formula:
  • E is absent, or E is a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted aralkyl, a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a substituted or unsubstituted polyaryl, a substituted or unsubstituted polyheteroaryl, or a substituted or unsubstituted heterocyclyl, wherein independently of E, R and R’ each independently represent a hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted carbonyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubsti
  • R” represents a hydroxyl group, a substituted or unsubstituted carbon
  • E when E is oxygen, a carbamate is formed. It is understood by those of ordinary skill in the art, that the E groups listed above are divalent (e.g., methylene, ethane-1, 2-diyl, ethene-1, 2-diyl, 1, 4-phenylene, cyclohexane-1, 2-diyl) .
  • Carbonyl, ” as used herein, is art-recognized and includes such moieties as can be represented by the general formula:
  • X is a bond, or represents an oxygen or a sulfur
  • R represents a hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted carbonyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted aralkyl (e.g.
  • E is absent, or E” is a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted aralkyl, a substituted or unsubsti
  • R represents a hydroxyl group, a substituted or unsubstituted aryl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted cycloalkenyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted arylalkyl) , a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted polyaryl, a substituted or unsubstituted polyheteroaryl, a substituted or unsubstituted heterocyclyl, a hydroxyl, an alkoxy, a phosphonium, a phosphanyl, an amido, an amino, or - (CH 2 ) m -R”; R” represents a hydroxyl group, a substituted or unsubstituted aryl, a substituted
  • substituents can be any substituents described above, e.g., halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl) , silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate) , alkoxyl, phosphonium, phosphanyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g., halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl) , silyl, ether,
  • E groups listed above are divalent (e.g., methylene, ethane-1, 2-diyl, ethene-1, 2-diyl, 1, 4-phenylene, cyclohexane-1, 2-diyl) .
  • X oxygen and R is defined as above, the moiety is also referred to as a carboxyl group.
  • R is hydrogen
  • the formula represents a “carboxylic acid. ”
  • X is oxygen and R’ is hydrogen
  • the formula represents a “formate. ”
  • X is oxygen and R or R’ is not hydrogen, the formula represents an "ester.
  • thiol are used interchangeably and are represented by -SR, where R can be a hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted aralkyl (e.g. a substituted or unsubstituted alkylaryl, a substituted or unsubstituted arylalkyl, etc.
  • R can be a hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted heterocyclyl, a substituted or un
  • a substituted or unsubstituted polyaryl a substituted or unsubstituted polyheteroaryl, a substituted or unsubstituted carbonyl, a phosphonium, a phosphanyl, an amido, an amino, an alkoxy, an oxo, a phosphonyl, a sulfinyl, or a silyl, described above.
  • substituents can be any substituents described above, e.g., halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl) , silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate) , alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g., halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl) , silyl, ether, ester, thiocarbonyl (such as
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, preferably 1-14 carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats.
  • substituents include a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted heterocyclic, a substituted or unsubstituted phenyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted polyaryl, a substituted or unsubstituted polyheteroaryl, a substituted or unsubstituted aralkyl, a halogen, a hydroxyl, an alkoxy, a phenoxy, an aroxy, a silyl, a thiol, an alkylthio, a substituted alkylthio, a phenylthio, an arylthio, a cyano, an isocyano, a nitro,
  • Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. It is understood that “substitution” or “substituted” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • compositions include a building block structure containing N,O/S-benzylidene acetal dipeptide (NBD) .
  • NBDs also referred to herein as “compounds”
  • compounds are simple and effective building blocks for syntheses of difficult peptides/proteins. Without being bound to any theories, it is believed the NBDs disclosed herein can act as a conformation-twisted dipeptide building block, which forms a pseudoproline turn to break down the interaction between peptides, and thereby can disrupt aggregation of difficult peptides/proteins on the resin (typically due to hydrophobic interaction of amino-acid side chains and hydrogen boding of amide bond) during SPPS process.
  • the disclosed compounds can have the structures of Formula I:
  • R 1 can be a protected or unprotected side chain of an amino acid
  • R 2 can be an amino protecting group
  • R 3 -R 6 can be independently hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF 3 ) , -NO 2 , unsubstituted C 1 -C 6 alkyl, unsubsituted C 1 -C 6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol;
  • R 7 and R 8 can be indepdently hydrogen, methyl, or other protected/unprotected side chains of amino acids
  • R 9 can be hydrogen or
  • the protection group can be a substituted or unsubstitited alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a carbonyl (e.g., carboxylic acid or carboxylic ester) , an amide, an amino, a substitute or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a heterocyclic ring.
  • R 1 is a protected side chain of an amino acid and the protection group can be an unsubstitited alkyl (e.g., unsubstitued C 1 -C 6 or C 1 -C 4 alkyl) , an unsubstituted alkenyl (e.g., unsubstitued C 1 -C 6 or C 1 -C 4 alkenyl) , an unsubstituted alkynyl (e.g., unsubstitued C 1 -C 6 or C 1 -C 4 alkynyl) , a carbonyl (e.g., carboxylic acid or carboxylic ester) , an amide, an amino, an unsubstituted aryl, an unsubstituted heteroaryl, or an unsubstituted heterocyclic ring.
  • an unsubstitited alkyl e.g., unsubstitued C 1 -C 6 or C 1 -C 4 al
  • the in the formulae described herein indicates a carbon stereocenter (to which the wave bond attaches) that can be either S-or R-configuration.
  • the acetyl carbon attaching to -C (O) OR 9 can be either S-or R-configuration.
  • R 1 can be a side chain of any suitable amino acid, natural or synthetic, and the amin acid can be either in its L-or D-configuration, or in various mixtures of their isomers.
  • R 1 may be a protected or unprotected side chain of any suitable alpha-amino acid, natural or synthetic.
  • R 1 is a side chain of an amino acid that carries a hydroxy group
  • the hydroxy group is optionally protected by a suitable hydroxy protecting group known in the art.
  • the amino group is optionally protected by a suitable amino protecting group known in the art.
  • the protection group can be t Bu, Boc, Thz, Acm, Trt, Cbz, Alloc, azido, Bn, Bz, Ac, or Pbf, or a combination thereof.
  • R 1 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 one of these amino acids can be either in its L-or D-configuration.
  • the protection group when R 1 is a protected side chain, can be t Bu, Boc, Thz, Acm, Trt, Cbz, Alloc, azido, Bn, Bz, Ac, or Pbf, or a combination thereof.
  • R 2 can be any substituents conventionally used to hinder the reactivity of the amino group.
  • suitable amino protecting groups are described in Green T., “Protective Groups in Organic Synthesis” , Chapter 7, John Wiley and Sons, Inc., 1991, 309-385.
  • R 2 can be Fmoc, Cbz, Moz, Boc, Troc, Teoc, Alloc, or Voc.
  • R 3 -R 6 can be independently hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF 3 ) , -NO 2 , unsubstituted alkyl (such as unsubstituted C 1 -C 8 alkyl) , unsubsituted alkenyl (unsubstituted C 1 -C 8 alkenyl) , unsubstituted alkynyl (such as unsubstituted C 1 -C 8 alkynyl) , unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol.
  • unsubstituted alkyl such as unsubstituted C 1 -C 8 alky
  • R 3 -R 6 can be independently hydrogen, halogen, -CN, -CF 3 , -NO 2 , unsubstituted C 1 -C 6 or C 1 -C 4 alkyl, unsubstituted C 1 -C 6 or C 1 -C 4 alkenyl, unsubstituted C 1 -C 6 or C 1 -C 4 alkynyl, alkoxyl, and unsubstituted aryl.
  • at least one of R 3 -R 6 is/are not hydrogen.
  • one of R 3 -R 6 , two of R 3 -R 6 , three of R 3 -R 6 , or all of R 3 -R 6 is/are not hydrogen.
  • R 7 and R 8 can be indepedently hydrogen or methyl. In some forms, R 7 and R 8 can be indepdently protected/unprotected side chains of amino acids.
  • R 9 can be hydrogen, benzyl, allyl, or unsubstituted C 1 -C 4 alkyl. In some forms, R 9 can be hydrogen. In some forms, R 9 can be any substituents conventionally used to protect the carboxylic acid group. 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 can also be any substituents conventionally used to activate the carboxylic acid group. For example, suitable amino protecting groups are described in Albericio F. “Peptide Coupling Reagents, More than a Letter Soup” , Chem. Rev. 2011, 111, 6557-6602.
  • R 9 can be benzyl, allyl, or unsubstituted C 1 -C 4 alkyl.
  • R 9 can be an activating group, such as l-hydroxy-7-azabenzotriazole (HOAt) , 1-hydroxybenzotriazole (HOBt) , ethyl 2-cyano-2- (hydroxyimino) acetate (Oxyma) , N-hydroxysuccinimide (NHS) , pentafluorophenol (Pfp) , etc.
  • HOAt l-hydroxy-7-azabenzotriazole
  • HBt 1-hydroxybenzotriazole
  • Oxyma ethyl 2-cyano-2- (hydroxyimino) acetate
  • NHS N-hydroxysuccinimide
  • Pfp pentafluorophenol
  • an activated ester group such as l-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.
  • the disclosed compounds can have the structures of Formula I':
  • R 1 -R 9 and X can be in any forms as defined above, and R 10 can be hydrogen or a functional group suitable for hindering the reactivity of phenyl group, such as a C 1 -C 4 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) .
  • a C 1 -C 4 acyl group e.g., formyl, acetyl, propionyl
  • a carbonyl group e.g., alloc
  • carbamate group e.g., tert-butyl methyl (2- (methylamino) ethyl) carbamate
  • R 10 can be hydrogen. In some forms, R 10 can be any substituents 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 can be a C 1 -C 4 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) .
  • acyl group e.g., formyl, acetyl, propionyl
  • carbonyl group e.g., alloc
  • carbamate group e.g., tert-butyl methyl (2- (methylamino) e
  • R 9 can be hydrogen and R 10 can be hydrogen.
  • R 9 can be benzyl, allyl, unsubstituted C 1 -C 4 alkyl, or an activating group, such as l-hydroxy-7-azabenzotriazole (HOAt) , 1-hydroxybenzotriazole (HOBt) , ethyl 2-cyano-2- (hydroxyimino) acetate (Oxyma) , N-hydroxysuccinimide (NHS) , pentafluorophenol (Pfp) , etc. and R 10 can be hydrogen.
  • HOAt l-hydroxy-7-azabenzotriazole
  • HBt 1-hydroxybenzotriazole
  • Oxyma ethyl 2-cyano-2- (hydroxyimino) acetate
  • NHS N-hydroxysuccinimide
  • Pfp pentafluorophenol
  • R 9 can be hydrogen and R 10 can be a C 1 -C 4 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) .
  • R 10 can be a C 1 -C 4 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) .
  • R 9 can be benzyl, allyl, unsubstituted C 1 -C 4 alkyl, or an activating group, such as l-hydroxy-7-azabenzotriazole (HOAt) , 1-hydroxybenzotriazole (HOBt) , ethyl 2-cyano-2- (hydroxyimino) acetate (Oxyma) , N-hydroxysuccinimide (NHS) , pentafluorophenol (Pfp) , etc.
  • an activating group such as l-hydroxy-7-azabenzotriazole (HOAt) , 1-hydroxybenzotriazole (HOBt) , ethyl 2-cyano-2- (hydroxyimino) acetate (Oxyma) , N-hydroxysuccinimide (NHS) , pentafluorophenol (Pfp) , etc.
  • R 10 can be a C 1 -C 4 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) .
  • acyl group e.g., formyl, acetyl, propionyl
  • carbonyl group e.g., alloc
  • carbamate group e.g., tert-butyl methyl (2- (methylamino) ethyl) carbamate
  • NBDs are shown below, where ⁇ represents indicates that attachment point, and R 3 -R 10 can be in any forms as defined above.
  • L-Thr-L-Thr NBD L-Asp-L-Ser NBD
  • L-Glu-L-Cys NBD L-Val-L-Pen NBD
  • L-Val-L-Cys NBD L-Val-L-Cys NBD
  • the disclosed NBDs are structurally different from of pseudo-prolines disclosed in et al., Pseudo-Prolines as a Solubilizing, Structure-Disrupting Protection Technique in Peptide Synthesis (J. Am. Chem. Soc. 1996, 118, 39, 9218-9227) .
  • the phenyl group of the disclosed NBDs provides more substitution sites for further functionalization, where in pseudo-proline, the symmetric dimethyl group was adopted without any other site for modification.
  • NBDs can cover broader range of peptide sequence and they can be easily prepared from mg- scale to sub-kg scale, whose estimated cost is only around 1 ⁇ 10%price of commercial competitors.
  • a L-or D-Amino acid with suitable protection on amine and optionally its sidechain functional groups can be firstly coupled with salicylaldehyde or a mono-/multiply-substituted form thereof. Then, the obtained salicylaldehyde ester reacts with serine/threonine/cysteine/penicillamine (Ser/Thr/Cys/Pen) or its ester in a suitable solvent. The obtained ester may be further derivatized to relevant acids with or without phenol protections.
  • the NBDs can be obtained in high yield (at least 40%) .
  • 76 exemplary NBDs (structures shown in the section above) were produced in moderate to excellent yield (i.e., 40%to 95%) .
  • Variants of those 76 NBDs that include D-amino acid version, different protecting groups on amine or side chain, other carboxylic derivatives and different substitutions of aryl could be also synthesized by following the developed synthetic strategy.
  • the disclosed method includes: (a) reacting a protected amino acid of Formula II:
  • R 1 can be a protected or unprotected side chain of an amino acid; and (ii) R 2 can be an amino protecting group,
  • R 3 -R 6 can be independently hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF 3 ) , -NO 2 , unsubstituted alkyl (such as unsubstituted C 1 -C 8 alkyl) , unsubsituted alkenyl (unsubstituted C 1 -C 8 alkenyl) , unsubstituted alkynyl (such as unsubstituted C 1 -C 8 alkynyl) , unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol,
  • R 7 and R 8 can be indepdently hydrogen, methyl, or other protected/unprotected side chains of amino acids;
  • R 9 can be hydrogen, benzyl, allyl, unsubstituted C 1 -C 4 alkyl, or a suitable activating group, such as N-hydroxysuccinimide (NHS) ;
  • X can be O or S,
  • R 1 -R 9 and X are as defined above for the reactants.
  • Activating groups suitable for R 9 of Formula V and Formula I are typically less active than HOAt and Oxyma, such as NHS, to reduce/prevent side reactions in step (b) .
  • the method disclosed herein may further include step (c) : deprotecting a protected carboxyl group of the NBDs; or step (d) acylating or activating a free acid group or a free phenol group of the NBDs; or a combination thereof.
  • a deprotected NBD with free acid form is obtained, for example, a deprotected NBD of Formula VI:
  • R 1 -R 8 , R 10 , and X can be in any forms as defined above.
  • R 9 and R 10 are hydrogen.
  • R 9 of the NBDs is hydrogen, such that this free carboxylic acid group can be activated in step (d) .
  • R 10 of the NBDs is hydrogen, such that this free hydroxyl group can be acylated in step (d) .
  • both R 9 and R 10 of the NBDs is hydrogen, such that the free carbocylic acid and/or the hydroxyl group can be acylated and/or activated, as needed, in step (d) .
  • an acylated NBD with or without a free carboxylic acid is obtained, for example, an acylated NBD of Formula I’:
  • R 1 -R 9 and X can be in any forms as defined above, and R 10 can be in any form as defined above expect for hydrogen.
  • the deprotection step (c) and acylation/activation step (d) can be performed in any order, and in any combination with steps (a) and (b) as described above.
  • the method includes steps (a) , (b) , and (c) , performed sequentially.
  • the method includes steps (a) , (b) , and (d) , performed sequentially.
  • the method includes steps (a) , (b) , (c) , and (d) , performed sequentially.
  • the method includes steps (a) , (b) , (d) , and (c) , performed sequentially.
  • the in the formulae described herein indicates a carbon stereocenter (to which the wave bond attaches) that can be either S-or R-configuration.
  • the acetyl carbon attaching to -C (O) OR 9 can be either S-or R-configuration.
  • the threonine, cysteine, or penicillamine, or an ester thereof used in step (b) can be either in its L-or D-configuration, as racemates, or in various mixtures of their isomers.
  • the protected amino acid used in step (a) can be either in its L-or D-configuration, as racemates, or in various mixtures of their isomers.
  • a protected amino acid of Formula II is reacted with a subsituted or unsubstituted salicylaldehyde of Formula III to obtain an intermediate comprising a salicylaldehyde ester of Formula IV:
  • R 1 -R 6 can be in any forms as defined above.
  • the protection group can be a substituted or unsubstitited alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a carbonyl (e.g., carboxylic acid or carboxylic ester) , an amide, an amino, a substitute or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a heterocyclic ring.
  • R 1 is a protected side chain of an amino acid and the protection group can be an unsubstitited alkyl, an unsubstituted alkenyl, an unsubstituted alkynyl, a carbonyl (e.g., carboxylic acid or carboxylic ester) , an amide, an amino, an unsubstituted aryl, an unsubstituted heteroaryl, or an unsubstituted heterocyclic ring.
  • the protection group can be an unsubstitited alkyl, an unsubstituted alkenyl, an unsubstituted alkynyl, a carbonyl (e.g., carboxylic acid or carboxylic ester) , an amide, an amino, an unsubstituted aryl, an unsubstituted heteroaryl, or an unsubstituted heterocyclic ring.
  • the protected amino acid of Formula II used in step (a) can be any natural amino acids or synthetic amino acids, which can be either in their L-or D-configuration.
  • 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 its L-or D-configuration.
  • the side chain of the amino acid used in step (a) may be protected using a protecting group known in the art.
  • Suitable protecting groups for the side chain include, but are not limited to, t Bu, Boc, Thz, Acm, Trt, Cbz, Alloc, Azido, Bn, Bz, Ac, and Pbf.
  • the terminal amino group of the amino acid used in step (a) are protected (i.e., 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 disclosed methods.
  • R 2 of the protected amino acids used in step (a) can be Fmoc, Cbz, Moz, Boc, Troc, Teoc, Alloc, or Voc.
  • the salicylaldehyde of Formula III that reacts with the protected amino acids of Formula II can be an unsubstituted salicylaldehyde or a mono-or poly-substituted salicylaldehyde.
  • the salicylaldehyde reacting with the protected amino acid of Formula II is substituted with one or more substituents (i.e., at least one of R 3 -R 6 , optionally two or more of R 3 -R 6 , is/are not hydrogen) .
  • one of R 3 -R 6 , two of R 3 -R 6 , three of R 3 -R 6 , or all of R 3 -R 6 , of the salicylaldehyde of Formula III used in step (a) is/are not hydrogen.
  • Suitable substituents of the substituted salicylaldehyde of Formula III are hydroxyl, halogen, -CN, haloalkyl (such as -CF 3 ) , -NO 2 , unsubstituted C 1 -C 6 alkyl, unsubsituted C 1 -C 6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, and thiol.
  • the substituents are independently halogen, -CN, -CF 3 , -NO 2 , unsubstituted C 1 -C 6 or C 1 -C 4 alkyl, unsubstituted C 1 -C 6 or C 1 -C 4 alkenyl, unsubstitued C 1 -C 6 or C 1 -C 4 alkynyl, alkoxyl, and unsubstituted aryl.
  • the reaction between the protected amino acid of Formula II and the subsituted or unsubstituted salicylaldehyde of Formula III is performed in a suitable organic solvent, and optionally in the presence of a condensation reagent and/or a base.
  • suitable organic solvents for performing the reaction in step (a) include, but are not limited to, DMF, DMSO, DMAc, NMP.
  • the organic solvent for performing the reaction in step (a) is DMF.
  • the condensation agent used in the reaction in step (a) is HATU.
  • Suitable base for performing the reaction in step (a) include, but are not limited to, DIPEA and Triethylamine.
  • the base used in the reaction in step (a) is DIPEA.
  • the reaction between the protected amino acid of Formula II and the subsituted or unsubstituted salicylaldehyde of Formula III can be performed in an suitable organic solvent, such as any one of thoes 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 time period ranging from 30 mins to 5 hours, preferably from 1 hour to 3 hours, such as about 1 hour or about 1.5 hour.
  • an organic solvent such as any one of thoes described above
  • a condensation reagent and/or a base at room temperature (i.e., 20 °C to 25 °C, at 1 atm) for a time period ranging from 30 mins to 5 hours, preferably from 1 hour to 3 hours, such as about 1 hour or about 1.5 hour.
  • the method disclosed herein further includes a step of purifying the intermediate containing the salicylaldehyde ester of Formula IV after step (a) and prior to step (b) .
  • the purification step can be performed using any suitable techniques known in the art, such as using extraction, washing, drying, column chromatography (such as by using a silica gel column) , or filtration, or a combination thereof.
  • the intermdeiate containing the salicylaldehyde ester of Formula IV is purified using the following techniques: extraction with an organic solvent that is different from the organic solvent used for performing the reaction, washing with an acid for one or more times, drying with a drying agent (such as sodium sulfate) and/or evaporation, and then separation using silica gel column.
  • a drying agent such as sodium sulfate
  • the salicylaldehyde ester of Formula IV obtained in step (a) has a yield of at least 40%, at least 50%, or in a range from about 50%to about 95%.
  • the yield of salicylaldehyde ester of Formula IV can be calculated using the formula: (The mole of the salicylaldehyde ester of Formula IV) / (The mole of the protected amino acid of Formula II) *100%. The yield may be determined either before or after purification of the intermediate.
  • the salicylaldehyde ester of Formula IV is reacted with serine, threonine, cysteine, or penicillamine, or an ester thereof of Formula V to obtain the compounds of Formula I:
  • R 1 -R 9 and X can be in any forms as defined above.
  • R 9 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 C 1 -C 4 alkyl.
  • the reaction between the salicylaldehyde ester of Formula IV and the serine, threonine, cysteine, or penicillamine, or ester thereof of Formula V is performed in a suitable solvent.
  • the solvent may be an organic solvent or water or a combination thereof, and typically contains a buffer at an amount ranging from 10 vol%to 20 vol%, such as 10 vol%to 20 vol%pyridine/HOAc (1: 1, molar ratio) .
  • Suitable solvents for preforming the reaction in step (b) include, but are not limited to, dichloromethane, trifluoroacetic acid, ethyl acetate, toluene, tetrahydrofuran, 1, 4-dixoane, acetonitrile, acetone, hexane, and water, and a combination thereof.
  • buffers suitable for use in the solvent for performing the reaction in 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-colidine-acetic acid buffer, and DMSO-picoline-acetic acid buffer.
  • a preferred buffer suitable for use is pyridine-acetic acid buffer, where the molar ratio of pyridine to acetic acid ranges from 1: 9 to 9: 1, such as 1: 1.
  • the reaction between the salicylaldehyde ester of Formula IV and the serine, threonine, cysteine, or penicillamine, or ester thereof of Formula V can be performed in an suitable solvent, such as any one of thoes described above, at room temperature for a time period ranging from 1 hour to 5 hours, preferably from 2 hours to 3 hours, such as about 2 hours or about 3 hours.
  • the method disclosed herein further includes a step of purifying the product containing the compound of Formula I after step (b) .
  • the purification step can be performed using any suitable techniques known in the art, such as any of those described above in step (a) .
  • the product containing the compounds of Formula I is purified using the following techniques: extraction with a suitable solvent and separation using column chromatography or filtration, washing with cod water and cold organic solvent (such as cold ethanol and/or cold ether) for one or more times, and separation by silica gel column.
  • the compound of Formula I obtained in step (b) has a yield of at least 40%or in a range from about 40%to about 95%.
  • the yield of compond of Formula I can be calculated using the formula: (The mole of the compound of Formula I) / (The mole of the salicylaldehyde ester Formula IV) *100%. The yield may be determined either before or after purification of the product.
  • the disclosed method further includes a step (c) : deprotecting a protected carboxyl group of the NBDs formed in step (b) or (d) as described herein.
  • the disclosed method includes steps (a) , (b) , and (c) performed sequentilly.
  • the disclosed method includes steps (a) , (b) , (c) , and (d) , performed sequentially.
  • the disclosed method includes steps (a) , (b) , (d) , and (c) , performed sequentially.
  • R 9 of the NBDs is typically not hydrogen, in particular when X of the formulae described herein is O.
  • the protected carboxyl group of the NBDs can be deprotected using reagents and reactions known in the art, for example, by using reagents such as Pd (PPh 3 ) 4 and/or PhSiH 3 for deallylation; H 2 and/or Pd/C for hydrogenation of Bn protection; LiOH for sponsification of alkyl protection, etc.
  • reagents such as Pd (PPh 3 ) 4 and/or PhSiH 3 for deallylation; H 2 and/or Pd/C for hydrogenation of Bn protection; LiOH for sponsification of alkyl protection, etc.
  • R 1 -R 8 , R 10 , and X can be in any forms as defined above.
  • the method disclosed herein further includes a step of purifying the product containing the compound of Formula VI after step (c) .
  • the purification step can be performed using any suitable techniques known in the art, such as any of those described above in step (a) or (b) .
  • the product containing the compounds of Formula VI is purified using the following techniques: extraction with a suitable solvent and separation using column chromatography or filtration, washing with cod water and cold organic solvent (such as cold ethanol and/or cold ether) for one or more times, and separation by silica gel column.
  • the compound of Formula VI obtained in step (c) has a yield of at least 40%or in a range from about 40%to about 95%.
  • the yield of compond of Formula VI can be calculated using the formula: (The mole of the compound of Formula VI) / (The mole of the compound of Formula I’) *100%. The yield may be determined either before or after purification of the product.
  • the disclosed method further includes a step (d) : acylating or activating a free acid or a free phenol group of the NBDs formed in step (b) or (c) .
  • a step (d) acylating or activating a free acid or a free phenol group of the NBDs formed in step (b) or (c) .
  • at least one of R 9 and R 10 of the reagent, such as an NBD of Formula I or VI is hydrogen.
  • R 9 of the NBDs is hydrogen, either in the NBD product of step (b) or in the deprotected NBD formed in the deprotection step (c) , such that this free carboxylic acid group can be activated in step (d) .
  • R 10 of the NBDs is hydrogen in the NBD product of step (b) and/or the deprotected NBD formed in step (c) , such that this free hydroxyl group can be acylated in step (d) .
  • both R 9 and R 10 of the NBDs is hydrogen, either in the NBD product of step (b) or in the deprotected NBD formed in the deprotection step (c) , such that the free carbocylic acid and/or the hydroxyl group can be acylated and/or activated, as needed, in step (d) .
  • both R 9 and R 10 of the NBDs is hydrogen, either in the NBD product of step (b) or in the deprotected NBD formed in the deprotection step (c) , such that the free carbocylic acid is activated, and/or the hydroxyl group is acylated, in step (d) .
  • an acylated NBD with or without a free carboxylic acid (e.g., -COOR 9 is carboxyl or ester) can be obtained, for example, an acylated NBD of Formula I’:
  • R 1 -R 9 and X can be in any forms as defined above, and R 10 can be in any form as defined above expect for hydrogen.
  • a free acid of the NBDs can be acylated and/or activated using reagents and reactions known in the art.
  • a free acid of the NBDs (-COOH and/or -OH) can be acylated using reagents such as acetyl anhydride, carbonate, or carbamate to obtain an acyl-capped NBD of Formula I’, where R 9 and/or R 10 , such as R 10 , is an acyl group.
  • a free acid of the NBDs e.g., -COOH
  • the method disclosed herein further includes a step of purifying the product containing acylated and/or activated NBDs after step (d) .
  • the purification step can be performed using any suitable techniques known in the art, such as any of those described above in step (a) , (b) or (c) .
  • the product containing the acylated and/or activated NBDs is purified using the following techniques: extraction with a suitable solvent and separation using column chromatography or filtration, washing with cod water and cold organic solvent (such as cold ethanol and/or cold ether) for one or more times, and separation by silica gel column.
  • the acylated and/or activated NBDs obtained in step (d) has a yield of at least 40%or in a range from about 40%to about 95%.
  • the yield of acylated and/or activated NBDs can be calculated using the formula: (The mole of the acylated and/or activated NBDs) / (The mole of the compound of Formula VI) *100%. The yield may be determined either before or after purification of the product.
  • R 1 -R 10 and X can be in any forms as defined above.
  • NBD of Formula I’ having a desired structure can be obtained following steps (a) and (b) ; following steps (a) , (b) , and step (c) ; following steps (a) , (b) , and (d) ; or following steps (a) , (b) , (c) , and (d) .
  • the peptide/protein synthesis methods disclosed herein are based at least on the application of NBD as a pre-formed dipeptide building block for synthesizing difficult peptides/proteins.
  • Difficult sequence and “difficult peptide/protein” are used interchangeably herein.
  • the concept of “difficult sequence” was introduced in the 80’s and was given distinction by Kent and co-workers for peptides that form strong inter-or intra molecular, non-covalent interactions which form insoluble peptide aggregates.
  • “Difficult sequences” are peptide sequences that contain high number of amino acids possessing hydrophobic side chains, so-called ⁇ -branched amino acids, including leucine, valine, phenylalanine or isoleucine. Additionally, glycine is known to induce ⁇ -sheet packing in combination with afore mentioned amino acids.
  • NBD-SPPS NBD-SPPS
  • difficult sequences include membrane proteins and their functional parts, for example, amylin, BM2 proton channel, influenza A, Copper storage protein 1 CSP-1, Interferon-induced transmembrane protein 3 (IFITM3) , NS4A, cofactor protein of serine protease from Hepatitis C virus; PD-L1 (programmed death-ligand 1) , IL-2 (INTERLEUCKIN -2) , Vasoactive intestinal peptide, Erythropoietin, Liraglutide, etc.
  • membrane proteins and their functional parts for example, amylin, BM2 proton channel, influenza A, Copper storage protein 1 CSP-1, Interferon-induced transmembrane protein 3 (IFITM3) , NS4A, cofactor protein of serine protease from Hepatitis C virus; PD-L1 (programmed death-ligand 1) , IL-2 (INTERLEUCKIN -2) , Vasoactive intestinal peptide, Erythropoi
  • the disclosed NBDs are typically used in SPPS methods known in the art.
  • Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is a preferred method for the chemical synthesis of the peptides.
  • Techniques for solid phase synthesis are known to those of skill 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.
  • Solid Phase Peptide Synthesis 2 nd ed. Pierce Chem. Co., Rockford, 111.
  • Such methods include bench scale solid phase synthesis and automated peptide synthesis in any one of the many commercially available peptide synthesizers. Solid phase synthesis is commonly used, and various commercial synthesizers are available, such as automated synthesizers by Applied Biosystems Inc., Foster City, CA; Beckman; MultiSyntech, Bochum, Germany etc.
  • Functional groups for conjugating the peptide to small molecules, label moieties, peptides, or proteins may be introduced into the molecule during chemical synthesis.
  • small molecules and label moieties/reporter units may be attached during the synthetic process. Introduction of the functional groups and conjugation to other molecules minimally affects the structure and function of the NBDs.
  • US 2008/0004451 describes the synthesis of dipeptides building blocks with acetonide protecting group to form a pseudoproline ring.
  • the disclosed methods utilizes the ligation intermediate of Serine/Threonine Ligation (STL) or Cysteine/Penicillamine Ligation (CPL) as the proline mimic i.e., NBDs, which allows the scale-up production of peptides in a very safe manner (NBDs are safe to handle) and it is environmentally friendly, because NBDs synthesis is a mild endothermic process and does not require heating or reflux conditions.
  • the disclosed NBDs are difficult to obtain by the methods disclosed in US 2008/0004451.
  • the disclosed methods have advantages over the methods disclosed in Sohma, et al., Novel and efficient synthesis of difficult sequence-containing peptides through O-N intramolecular acyl migration reaction of O-acyl isopeptides, Chem. Commun (Camb) , 1: 124-5 (2004) ( (DOI: 10.1039/B312129A) , which discloses isoacyl dipeptides (which are chemically distinct from the disclosed NBDs) . Further, the synthesis of isopeptide may have a risk of epimerization since the esterification required highly active condensation reagents.
  • the disclosed methods proceed via NBD; the ligation between two amino acid fragment is chemoselective and stereoselective, without the risk of epimerization.
  • the disclosed NBDs are much more stable under basic condition, which is important in Fmoc-SPPS for peptide synthesis. This advantage makes the disclosed NBDs much more suitable for long peptide or protein chemical synthesis.
  • R 1 is a protected or unprotected side chain of an amino acid
  • R 2 is an amine protecting group
  • R 3 -R 6 are independently hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF 3 ) , -NO 2 , unsubstituted C 1 -C 6 alkyl, unsubsituted C 1 -C 6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol;
  • R 7 and R 8 are indepdently hydrogen, methyl, or other protected/unprotected side chains of amino acids
  • R 9 is hydrogen or a functional group suitable for protecting and/or activating a carboxylic acid group
  • R 10 is hydrogen or a functional group suitable for hindering the reactivity of phenyl group, such as a C 1 -C 4 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
  • 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.
  • R 1 is a protected side chain
  • the protection group is selected from the group consisting of a substituted or unsubstitited alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a carbonyl (e.g., carboxylic acid or carboxylic ester) , an amide, an amino, a substitute or unsubstituted aryl, a substituted or unsubstituted heteroaryl, and a heterocyclic ring, optionally wherein the protection group is t Bu, Boc, Thz, Acm, Trt, Cbz, Alloc, Azido, Bn, Bz, Ac, and Pbf, or a combination thereof.
  • R 3 -R 6 are independently hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF 3 ) , -NO 2 , unsubstituted alkyl (such as unsubstituted C 1 -C 8 alkyl) , unsubsituted alkenyl (unsubstituted C 1 -C 8 alkenyl) , unsubstituted alkynyl (such as unsubstituted C 1 -C 8 alkynyl) , unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol, optionally wherein R 3 -R 6 are independently hydrogen, hydroxyl, halogen, -CN, hal
  • R 9 is hydrogen, benzyl, allyl, unsubstituted C 1 -C 4 alkyl; or is an activated ester group, such as l-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.
  • HOAt l-hydroxy-7-azabenzotriazole
  • HBt 1- hydroxybenzotriazole
  • NHS N-hydroxysuccinimide
  • Pfp pentafluorophenol
  • R 10 is hydrogen, a C 1 -C 4 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) .
  • a C 1 -C 4 acyl group e.g., formyl, acetyl, propionyl
  • a carbonyl group e.g., alloc
  • a carbamate group e.g., tert-butyl methyl (2- (methylamino) ethyl) carbamate
  • R 1 is a protected or unprotected side chain of an amino acid
  • R 2 is an amine protecting group
  • R 3 -R 6 are independently hydrogen, hdyroxyl, halogen, -CN, haloalkyl (such as -CF 3 ) , -NO 2 , unsubstituted C 1 -C 6 alkyl, unsubsituted C 1 -C 6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol;
  • R 7 and R 8 are indepdently hydrogen, methyl, or other protected/unprotected side chains of amino acids
  • R 9 is hydrogen, benzyl, allyl, unsubstituted C 1 -C 4 alkyl, or a functional group suitable for protecting and/or activating a carboxylic acid group;
  • R 1 and R 2 are as defined above for Formula I,
  • R 3 -R 6 are as defined above for Formula I,
  • 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.
  • R 1 is a protected side chain
  • the protection group is selected from the group consisting of a substituted or unsubstitited alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a carbonyl (e.g., carboxylic acid or carboxylic ester) , an amide, an amino, a substitute or unsubstituted aryl, a substituted or unsubstituted heteroaryl, and a heterocyclic ring, optionally wherein the protection group is t Bu, Boc, Thz, Acm, Trt, Cbz, Alloc, Azido, Bn, Bz, Ac, and Pbf, or a combination thereof.
  • R 2 is Fmoc, Cbz, Moz, Boc, Troc, Teoc, Alloc, or Voc.
  • R 3 -R 6 are independently hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF 3 ) , -NO 2 , unsubstituted alkyl (such as unsubstituted C 1 -C 8 alkyl) , unsubsituted alkenyl (unsubstituted C 1 -C 8 alkenyl) , unsubstituted alkynyl (such as unsubstituted C 1 -C 8 alkynyl) , unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol, optionally wherein R 3 -R 6 are independently hydrogen, hydroxyl, halogen, -CN, hal
  • step (a) is performed in the presence of a condensation agent, optionally wherein the condensation agent is HATU, PyBOP, HBTU, HCTU, COMU, TSTU, PyClock, PyOxim, EDCI, or DCC, or a combination thereof.
  • a condensation agent optionally wherein the condensation agent is HATU, PyBOP, HBTU, HCTU, COMU, TSTU, PyClock, PyOxim, EDCI, or DCC, or a combination thereof.
  • step (a) is performed in the presence of a base, optionally wherein the base is DIPEA or Triethylamine, or a combination thereof.
  • step (a) is performed in an organic solvent, optionally wherein the organic solvent is DMF, DMSO, DMAc, or NMP, or a combination thereof.
  • step (a) is performed at room temperature for a time period ranging from 30 mins to 5 hours, preferably from 1 hour to 3 hours.
  • step (a) has a yield of at least 40%, at least 50%, or in a range from about 50%to about 95%.
  • step (b) is performed 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.
  • the solvent is dichloromethane, trifluoroacetic acid, tetrahydrofuran, toluene, ethyl acetate, 1, 4-dioxane, acetonitrile, acetone, or water, or a combination thereof.
  • step (b) is performed at room temperature for a time period ranging from 1 hour to 5 hours, preferably from 2 hours to 3 hours.
  • step (b) The method of any one of paragraphs 9-22, wherein the compound of Formula I obtained in step (b) has a yield of at least 40%, such as in a range from about 40%to about 95%.
  • R 1 is a protected or unprotected side chain of an amino acid
  • R 2 is an amine protecting group
  • R 3 -R 6 are independently hydrogen, hdyroxyl, halogen, -CN, haloalkyl (such as -CF 3 ) , -NO 2 , unsubstituted C 1 -C 6 alkyl, unsubsituted C 1 -C 6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol;
  • R 7 and R 8 are indepdently hydrogen, methyl, or other protected/unprotected side chains of amino acids
  • R 10 is hydrogen or a functional group suitable for hindering the reactivity of phenyl group, such as a C 1 -C 4 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
  • a functional group suitable for hindering the reactivity of phenyl group such as a C 1 -C 4 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
  • X is O or S.
  • R 1 is a protected or unprotected side chain of an amino acid
  • R 2 is an amine protecting group
  • R 3 -R 6 are independently hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF 3 ) , -NO 2 , unsubstituted C 1 -C 6 alkyl, unsubsituted C 1 -C 6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol;
  • R 7 and R 8 are indepdently hydrogen, methyl, or other protected/unprotected side chains of amino acids
  • R 9 is hydrogen or a functional group suitable for protecting and/or activating a carboxylic acid group
  • R 10 is a functional group suitable for hindering the reactivity of phenyl group, such as a C 1 -C 4 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
  • step (c) is performed immediately following step (b) or immediately following 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 wherin R 9 of Formula I or Formula I’ is not hydrogen.
  • step (d) is performed immediately following step (b) or immediately following step (c)
  • the free-acid containing NBD is the NBD of Formula I formed in step (b) or the NBD of Formula VI formed in step (c) .
  • Step b
  • Step a & Step b
  • 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 under 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.
  • the first example synthesized Ac-capped L-Thr-L-Thr NBD the structure of which is shown below.
  • Step b
  • the Fmoc-Val-SAL ester was dissolved into 250 mL dichloromethane, and 10 mL pyridine/HOAc buffer containing 10.0 mmol L-H-Thr-Oall ester was added. The reaction mixture was stirred at room temperature for 2 h and extracted similarly as mentioned above. Later the crude product was purified by column chromatography, affording the unprotected NBD as pale solid in 91%yield.
  • NBD acid was then dissolved into 250 mL dichloromethane, and Ac 2 O (2.0 eq. ) and DIPEA (4.0 eq. ) were added.
  • the reaction mixture was stirred at room temperature for 3 h and extracted similarly as mentioned above. Then the crude product was purified by column chromatography, affording the desired Ac-capped NBD acid as pale solid in 90%yield.
  • the first example synthesized HOAt-activated L-Thr-L-Thr NBD the structure of which is shown below.
  • Step b
  • the Fmoc-Val-SAL ester was dissolved into 250 mL dichloromethane, and 10 mL pyridine/HOAc buffer containing 10.0 mmol L-H-Thr-OAll ester was added. The reaction mixture was stirred at room temperature for 2 h and extracted similarly as mentioned above. Later the crude product was purified by column chromatography, affording the unprotected NBD as pale solid in 91%yield.
  • NBD acid was then dissolved into 250 mL dichloromethane, and HATU (1.0 eq. ) and DIPEA (1.0 eq. ) were added.
  • the reaction mixture was stirred at room temperature for 0.5 h and extracted similarly as mentioned above. Then the crude product was purified by column chromatography, affording the desired HOAt-activated NBD acid as pale solid in 78%yield.
  • Step a & Step b
  • 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 under 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.
  • the NBD is already in acid form so that the step c could be skipped.
  • the unprotected NBD was dissolved into 250 mL dichloromethane, and tert-butyl methyl (2- (methylamino) ethyl) carbamate (2.0 eq. ) activated by 1, 1'-carbonyldiimidazole (2.0 eq. ) and DIPEA (4.0 eq. ) were added.
  • the reaction mixture was stirred at room temperature for 1 h and extracted similarly as mentioned above. Then the crude product was purified by column chromatography, affording the desired carbamate-capped NBD as pale solid in 82%yield.
  • the first case was the synthesis of PD-L1 (121-132) , a C-terminus of PD-L1 that was highly prone to form ⁇ -sheet structure.
  • the amino acids were installed by standard coupling procedure by using HATU as coupling reagent, sequentially, the resin was subjected to washing steps. Then the resin was treated with 20%piperidine/DMF to remove the Fmoc protecting group and allowed for the next coupling cycle.
  • Ile-Thr NBD was installed by a one-pot activation and coupling protocol. As shown in the Figure 1, the installation of Ile-Thr NBD improved the quality of final products significantly. Furthermore, without dipeptides, serious amino acid deletion was observed, indicating the powerfulness of dipeptide in solving difficult SPPS issue.
  • RANTES a natural HIV-suppressive factor containing 68 amino acids
  • the amino acids were installed by standard coupling procedure by using HATU as coupling reagent, sequentially, the resin was subjected to washing steps. Then the resin was treated with 20%piperidine/DMF to remove the Fmoc protecting group and enabled the next coupling cycle.
  • three corresponding NBDs were installed by a one-pot activation and coupling protocol. As shown in Figure 4, RANTES was successfully constructed when four NBDs were introduced and up to 21%isolated yield could be achieved after HPLC purification.

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Abstract

Building block structures containing N, O/S-benzylidene acetal dipeptide (NBD) are described. The unique core structure of NBD can effectively convert those difficult peptide/proteins sequence into the easy ones, allowing smooth synthesis of those challenging targets. Further, the production of NBDs is highly robust, and it can be scale up facilely, which can facilitate the research and development of tailor-made peptides/proteins in the future.

Description

BUILDING BLOCKS FOR DIFFICULT PEPTIDE SYNTHESIS AND METHOD OF MAKING THEREOF
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of and priority to U.S. Provisional Application No. 63/486,748 filed February 24, 2023, the entire content of which is incorporated herein by reference for all purpose in its entirety.
FIELD OF THE INVENTION
This invention is generally in the field of protein/peptide synthesis.
BACKGROUND OF THE INVENTION
Chemical protein synthesis plays an important role in the study of fundamental biochemistry and development of macromolecule-based therapy. It allows precise modifications at the atomic level, such as mirror image proteins. The advent of solid phase peptide synthesis (SPPS) has allowed peptides or small proteins to be synthesized chemically, allowing site-specific installation of chemical probes or post-translational modification of peptides to realize their biofunctions. Following SPPS, a variety of coupling reagents, orthogonal protecting groups and Fmoc-based SPPS have been well-developed to ensure peptide synthesis as a convenient and commonly used technique. SPPS remains the gold-standard method, and it 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 due to their aggregation tendency during the peptide synthesis, which classifies them as difficult peptides. The current strategies to solve this problem are less cost-effective and have limited application in industrial production. To overcome the problem of difficult peptide synthesis, three methods have been developed to inhibit hydrogen-bonded interchain association, including pseudoproline dipeptides, HMB (2-hydroxy-4-methoxybenzyl) , and isoacyl dipeptides and as the backbone-protecting group, 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) ) , respectively. These compounds can disrupt peptide aggregation during SPPS, but are not widely used in industries, as the synthesis of these building blocks are not very cost-effective.
There is a need for new and improved methods for synthesizing challenging peptides/proteins at low cost for fundamental chemical biology studies and therapeutic research & development.
SUMMARY OF THE INVENTION
Building block structures containing N, O/S-benzylidene acetal dipeptides (NBDs) are described herein. The unique core structure of NBD (also referred to herein as “compound” ) can effectively convert those difficult peptide/proteins sequences into the easier ones, allowing smooth synthesis of those challenging targets. Further, the production of NBDs is highly robust, and it can be scale up facilely, which can facilitate research and development of tailor-made peptides/proteins.
In some forms, the compounds can have the structure of Formula I: 
wherien: (i) R1 can be a protected or unprotected side chain of an amino acid; (ii) R2 can be an amine protecting group; (iii) R3-R6 can be independently hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF3) , -NO2, unsubstituted C1-C6 alkyl, unsubsituted C1-C6 alkenyl, unsubstituted aryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol;  (iv) R7 and R8 can be indepedently hydrogen, methyl, or other protected/unprotected side chains of amino acids; (v) R9 can 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, e.g., l-hydroxy-7-azabenzotriazole (HOAt) , 1-hydroxybenzotriazole (HOBt) , ethyl 2-cyano-2- (hydroxyimino) acetate (Oxyma) , N-hydroxysuccinimide (NHS) , pentafluorophenol (Pfp) , etc.; and (vi) X can be O or S.
When R1 is a protected side chain of an amino acid, the protection group can be a substituted or unsubstitited alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a carbonyl (e.g., carboxylic acid or carboxylic ester) , an amide, an amino, a substitute or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a substituted or unsubstituted heterocyclic ring.
In some forms, the compounds can have the structure of Formula I’:
wherein: R1-R9 and X can be as defined above, and R10 can be hydrogen or a functional group suitable for hindering the reactivity of 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) .
Methods of producing the compounds disclosed herein are described. Generally, the method includes two steps of reactions. Briefly, a L-or D-Amino acid with suitable protection on amine and optionally its sidechain functional groups, can be firstly coupled with salicylaldehyde or a mono-/multiply- substituted form thereof. Then, the obtained salicylaldehyde ester reacts with serine/threonine/cysteine/penicillamine (Ser/Thr/Cys/Pen) or its ester in a suitable solvent. The obtained ester may be further derivatized to relevant acids with or without phenol protections. Using the methods disclosed herein, the NBDs can be obtained in high yield (at least 40%) .
Methods of using the NBDs for peptide/protein synthesis as a pre-formed dipeptide building block for synthesizing difficult peptides/proteins are also disclosed.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a comparison of the synthesis of PD-L1 (121-123) using the disclosed method and conventional SPPS.
FIG. 2 shows a comparison of the synthesis of IL-2 (125-133) using the disclosed method and conventional SPPS.
FIG. 3 shows a comparison of the synthesis of Amylin using the disclosed method and conventional SPPS.
FIG. 4 shows a comparison of the synthesis of Rantes using the disclosed method and conventional SPPS.
DETAILED DESCRIPTION OF THE INVENTION
Difficult peptides/proteins are normally prone to aggregate on the resin through hydrophobic interaction of amino-acid side chains and hydrogen bonding of amide bond, which can cause incomplete coupling or deprotection during the SPPS process. Without being bound to any theories, it is believed that the disclosed N, O/S-benzylidene acetal dipeptide (NBD) can act as a conformation-twisted dipeptide building block, which forms a pseudoproline turn to disrupt the interaction between peptides. Therefore, the aggregation can be successfully prevented, and targeted sequence can be synthesized smoothly.
I. DEFINTIONS
The term “alkyl” refers to a branched or straight-chain monovalent saturated aliphatic hydrocarbon radical of one to eight carbon atoms. This term  is further exemplified by radicals such as methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl, n-pentyl, isopentyl, t-pentyl, n-hexyl, etc.
The term “alkenyl” refers to a branched or straight-chain hydrocarbon group of from two to eight carbon atoms and structural formula containing at least one carbon-carbon double bond.
The term “alkynyl” refers to a branched or straight-chain hydrocarbon group of from two to eight carbon atoms and structural formula containing at least one carbon-carbon triple bond.
The term “aryl” refers to C5-C20-membered aromatic or fused aromatic ring systems. Examples of aromatic groups are benzene, naphthalene, anthracene, phenanthrene, chrysene, pyrene, corannulene, coronene, etc.
The term “cyclic group” refers to a saturated or unsaturated monocyclic ring or a polycyclic ring (such as those formed from single or fused ring systems) , such as a cycloalkyl, a cycloalkenyl, or a cycloalkynyl, which have from three to ten carbon atoms, as geometric constraints permit.
The term “heteroalkyl” refers to straight or branched chain carbon-containing alkyl radicals containing at least one heteroatom on the carbon backbone. Suitable heteroatoms include, but are not limited to, O, N, Si, P and S, wherein the nitrogen, phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized.
The term “heteroaryl” refers to C5-C20-membered aromatic or fused aromatic ring systems, in which one or more carbon atoms on one or more aromatic ring structures have been substituted with a heteroatom. Suitable heteroatoms include, but are not limited to, oxygen, sulfur, and nitrogen. Examples of heteroaryl groups pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Examples of heteroaryl rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,  benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, 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, phenoxathinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 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 the rings can be substituted as defined below for “substituted heteroaryl. ” 
The term “heterocyclic group” refers to a cyclic group as defined above where at least one of the carbon atoms of the ring is substituted with a heteroatom, such as, nitrogen, oxygen, sulfur, or phosphorus.
The term “aralkyl” refers to an aryl group or a heteroaryl group having an alkyl, alkynyl, or alkenyl group as defined above attached to the aromatic group, such as an aryl, a heteroaryl, a polyaryl, or a polyheteroaryl. An example of an aralkyl group is a benzyl group.
The terms “alkoxyl” refers to compounds represented by the formula -ORv, wherein Rv includes, but is not limited to, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a heterocyclic, a cycloalkenyl, an aryl, a heteroaryl, an aralkyl, a heteroalkyl, etc. Exemplary alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. A “lower alkoxyl” group is an alkoxyl group  containing from one to six carbon atoms. An “ether” is two functional groups covalently linked by an oxygen as defined below. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O-alkenyl, -O-alkynyl, -O-aralkyl, -O-aryl, -O-heteroaryl, -O-cyclic, -O-heterocyclic, etc.
The term "amino" as used herein includes the group
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, a substituted or unsubstituted polyaryl, a substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclyl, wherein independently of E, Rx, Rxi, and Rxii each independently represent a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted carbonyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted aralkyl (e.g. a substituted or unsubstituted alkylaryl, a substituted or unsubstituted arylalkyl) , a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted polyaryl, a substituted or unsubstituted polyheteroaryl, a substituted or unsubstituted heterocyclyl, a hydroxyl, an alkoxy, a phosphonium, a phosphanyl, a phosphonyl, a sulfinyl, a silyl, a thiol, an amido, an amino, or - (CH2) m-R”’; R”’ represents a hydroxyl group, a substituted or unsubstituted carbonyl group, a substituted or unsubstituted aryl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted cycloalkenyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted aryl, a  substituted or unsubstituted heteroaryl, a substituted or unsubstituted polyaryl, a substituted or unsubstituted polyheteroaryl, an alkoxy, a phosphonium, a phosphanyl, an amido, or an amino; and m is zero or an integer ranging from 1 to 8. The term “quaternary amino” also includes the groups where the nitrogen, Rx, Rxi, and Rxii with the N+ to which they are attached complete a heterocyclyl or heteroaryl having from 3 to 14 atoms in the ring structure. It is understood by those of ordinary skill in the art, that the E groups listed above are divalent (e.g., methylene, ethane-1, 2-diyl, ethene-1, 2-diyl, 1, 4-phenylene, cyclohexane-1, 2-diyl) .
The terms “amide” or “amido” are used interchangeably, refer to both “unsubstituted amido” and “substituted amido” and are represented by the general formula:
wherein, E is absent, or E is a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted aralkyl, a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a substituted or unsubstituted polyaryl, a substituted or unsubstituted polyheteroaryl, or a substituted or unsubstituted heterocyclyl, wherein independently of E, R and R’ each independently represent a hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted carbonyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted aralkyl (e.g. a substituted or unsubstituted alkylaryl, a substituted or unsubstituted arylalkyl) , a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted polyaryl, a substituted or unsubstituted polyheteroaryl, a substituted or unsubstituted heterocyclyl, a hydroxyl, an alkoxy, a phosphonium, a phosphanyl, a  phosphonyl, a sulfinyl, a silyl, a thiol, an amido, an amino, or - (CH2) m-R”’, or R and R’ taken together with the N atom to which they are attached complete a heterocycle having from 3 to 14 atoms in the ring structure; R”’ represents a hydroxyl group, a substituted or unsubstituted carbonyl group, a substituted or unsubstituted aryl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted cycloalkenyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted polyaryl, a substituted or unsubstituted polyheteroaryl, an alkoxy, a phosphonium, a phosphanyl, an amido, or an 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 of ordinary skill in the art, that the E groups listed above are divalent (e.g., methylene, ethane-1, 2-diyl, ethene-1, 2-diyl, 1, 4-phenylene, cyclohexane-1, 2-diyl) .
“Carbonyl, ” as used herein, is art-recognized and includes such moieties as can be represented by the general formula:
wherein X is a bond, or represents an oxygen or a sulfur, and R represents a hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted carbonyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted aralkyl (e.g. a substituted or unsubstituted alkylaryl, a substituted or unsubstituted arylalkyl) , a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted polyaryl, a substituted or unsubstituted polyheteroaryl, a substituted or unsubstituted heterocyclyl, a hydroxyl, an alkoxy, a phosphonium, a phosphanyl, an amido, an amino, or - (CH2) m-R” , or a pharmaceutical acceptable salt; E” is absent, or E” is a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted aralkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a  substituted or unsubstituted polyaryl, a substituted or unsubstituted polyheteroaryl, a substituted or unsubstituted heterocyclyl; R’ represents a hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted aralkyl (e.g. a substituted or unsubstituted alkylaryl, a substituted or unsubstituted arylalkyl) , a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted polyaryl, a substituted or unsubstituted polyheteroaryl, a substituted or unsubstituted heterocyclyl, a hydroxyl, an alkoxy, a phosphonium, a phosphanyl, an amido, an amino, or - (CH2) m-R”; R” represents a hydroxyl group, a substituted or unsubstituted aryl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted cycloalkenyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted polyaryl, a substituted or unsubstituted polyheteroaryl, an alkoxy, a phosphonium, a phosphanyl, an amido, or an amino; and m is zero or an integer ranging from 1 to 8. Such substituents can be any substituents described above, e.g., halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl) , silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate) , alkoxyl, phosphonium, phosphanyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g. quarternized amino) , amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, and combinations thereof. It is understood by those of ordinary skill in the art, that the E” groups listed above are divalent (e.g., methylene, ethane-1, 2-diyl, ethene-1, 2-diyl, 1, 4-phenylene, cyclohexane-1, 2-diyl) . Where X is oxygen and R is defined as above, the moiety is also referred to as a carboxyl group. When X is oxygen and R is hydrogen, the formula represents a “carboxylic acid. ” Where X is oxygen and R’ is hydrogen,  the formula represents a “formate. ” Where X is oxygen and R or R’ is not hydrogen, the formula represents an "ester. ” In general, where the oxygen atom of the above formula is replaced by a sulfur atom, the formula represents a “thiocarbonyl” group. Where X is sulfur and R or R’ is not hydrogen, the formula represents a “thioester. ” Where X is sulfur and R is hydrogen, the formula represents a “thiocarboxylic acid. ” Where X is sulfur and R’ is hydrogen, the formula represents a “thioformate. ” Where X is a bond and R is not hydrogen, the above formula represents a “ketone. ” Where X is a bond and R is hydrogen, the above formula represents an “aldehyde. ”
The terms “thiol” are used interchangeably and are represented by -SR, where R can be a hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted aralkyl (e.g. a substituted or unsubstituted alkylaryl, a substituted or unsubstituted arylalkyl, etc. ) , a substituted or unsubstituted polyaryl, a substituted or unsubstituted polyheteroaryl, a substituted or unsubstituted carbonyl, a phosphonium, a phosphanyl, an amido, an amino, an alkoxy, an oxo, a phosphonyl, a sulfinyl, or a silyl, described above. Such substituents can be any substituents described above, e.g., halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl) , silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate) , alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g. quarternized 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.
Use of the term "about" is intended to describe values either above or below the stated value, which the term “about” modifies, to be within a range of approximately +/-10%. When the term "about" is used before a range of  numbers (i.e., about 1-5) or before a series of numbers (i.e., about 1, 2, 3, 4, etc. ) it is intended to modify both ends of the range of numbers and/or each of the numbers recited in the entire series, unless specified otherwise.
“Substituted, ” as used herein, refers to all permissible substituents of the compounds or functional groups described herein. In the broadest sense, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, preferably 1-14 carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats. Representative substituents include a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted heterocyclic, a substituted or unsubstituted phenyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted polyaryl, a substituted or unsubstituted polyheteroaryl, a substituted or unsubstituted aralkyl, a halogen, a hydroxyl, an alkoxy, a phenoxy, an aroxy, a silyl, a thiol, an alkylthio, a substituted alkylthio, a phenylthio, an arylthio, a cyano, an isocyano, a nitro, a substituted or unsubstituted carbonyl, a carboxyl, an amino, an amido, an oxo, a sulfinyl, a sulfonyl, a sulfonic acid, a phosphonium, a phosphanyl, a phosphoryl, a phosphonyl, an amino acid. Such a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted phenyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted polyaryl, a substituted or unsubstituted polyheteroaryl, a substituted or unsubstituted aralkyl, a halogen, a hydroxyl, an alkoxy, a phenoxy, an aroxy, a silyl, a thiol, an alkylthio, a substituted alkylthio, a phenylthio, an arylthio, a cyano, an isocyano, a nitro, a substituted or  unsubstituted carbonyl, a carboxyl, an amino, an amido, an oxo, a sulfinyl, a sulfonyl, a sulfonic acid, a phosphonium, a phosphanyl, a phosphoryl, a phosphonyl, and an amino acid can be further substituted.
Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. It is understood that “substitution” or “substituted” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
The full names of certain abbreviations as used herein is provided in the following table:

II. COMPOSITIONS
The disclosed compositions include a building block structure containing N,O/S-benzylidene acetal dipeptide (NBD) . The disclosed NBDs (also referred to herein as “compounds” ) are simple and effective building blocks for syntheses of difficult peptides/proteins. Without being bound to any theories, it is believed the NBDs disclosed herein can act as a conformation-twisted dipeptide building block, which forms a pseudoproline turn to break down the interaction between peptides, and thereby can disrupt aggregation of difficult peptides/proteins on the resin (typically due to hydrophobic interaction of amino-acid side chains and hydrogen boding of amide bond) during SPPS process.
In some forms, the disclosed compounds can have the structures of Formula I:
wherein: (i) R1 can be a protected or unprotected side chain of an amino acid; (ii) R2 can be an amino protecting group; (iii) R3-R6 can be independently hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF3) , -NO2, unsubstituted C1-C6 alkyl, unsubsituted C1-C6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol; (iv) R7 and R8 can be indepdently hydrogen, methyl,  or other protected/unprotected side chains of amino acids; (v) R9 can 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, e.g., 1-hydroxy-7-azabenzotriazole (HOAt) , 1-hydroxybenzotriazole (HOBt) , ethyl 2-cyano-2- (hydroxyimino) acetate (Oxyma) ; N-hydroxysuccinimide (NHS) , pentafluorophenol (Pfp) , etc.; and (vi) X can be O or S.
When R1 is a protected side chain of an amino acid, the protection group can be a substituted or unsubstitited alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a carbonyl (e.g., carboxylic acid or carboxylic ester) , an amide, an amino, a substitute or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a heterocyclic ring. In some forms, R1 is a protected side chain of an amino acid and the protection group can be an unsubstitited alkyl (e.g., unsubstitued C1-C6 or C1-C4 alkyl) , an unsubstituted alkenyl (e.g., unsubstitued C1-C6 or C1-C4 alkenyl) , an unsubstituted alkynyl (e.g., unsubstitued C1-C6 or C1-C4 alkynyl) , a carbonyl (e.g., carboxylic acid or carboxylic ester) , an amide, an amino, an unsubstituted aryl, an unsubstituted heteroaryl, or an unsubstituted heterocyclic ring.
Thein the formulae described herein indicates a carbon stereocenter (to which the wave bond attaches) that can be either S-or R-configuration. For example, the acetyl carbon attaching to -C (O) OR9 can be either S-or R-configuration. It is also understood that R1 can be a side chain of any suitable amino acid, natural or synthetic, and the amin acid can be either in its L-or D-configuration, or in various mixtures of their isomers.
R1 may be a protected or unprotected side chain of any suitable alpha-amino acid, natural or synthetic. When R1 is a side chain of an amino acid that carries a hydroxy group, the hydroxy group is optionally protected by a suitable hydroxy protecting group known in the art. For side chains that carry additional amino groups, the amino group is optionally protected by a suitable amino  protecting group known in the art. In some forms, when R1 is a protected side chain of an amino acid, the protection group can be tBu, Boc, Thz, Acm, Trt, Cbz, Alloc, azido, Bn, Bz, Ac, or Pbf, or a combination thereof.
In some forms, R1 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 one of these amino acids can be either in its L-or D-configuration. In these forms, when R1 is a protected side chain, the protection group can be tBu, Boc, Thz, Acm, Trt, Cbz, Alloc, azido, Bn, Bz, Ac, or Pbf, or a combination thereof.
R2 can be any substituents conventionally used to hinder the reactivity of the amino group. For example, suitable amino protecting groups are described in Green T., “Protective Groups in Organic Synthesis” , Chapter 7, John Wiley and Sons, Inc., 1991, 309-385. In some forms, R2 can be Fmoc, Cbz, Moz, Boc, Troc, Teoc, Alloc, or Voc.
R3-R6 can be independently hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF3) , -NO2, unsubstituted alkyl (such as unsubstituted C1-C8 alkyl) , unsubsituted alkenyl (unsubstituted C1-C8 alkenyl) , unsubstituted alkynyl (such as unsubstituted C1-C8 alkynyl) , unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol. In some forms, R3-R6 can be independently 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, alkoxyl, and unsubstituted aryl. In some forms, at least one of R3-R6, optionally two or more of R3-R6, is/are not hydrogen. For example, one of R3-R6, two of R3-R6, three of R3-R6, or all of R3-R6, is/are not hydrogen.
In some forms, R7 and R8 can be indepedently hydrogen or methyl. In some forms, R7 and R8 can be indepdently protected/unprotected side chains of amino acids.
In some forms, R9 can be hydrogen, benzyl, allyl, or unsubstituted C1-C4 alkyl. In some forms, R9 can be hydrogen. In some forms, R9 can be any substituents conventionally used to protect the carboxylic acid group. 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, R9 can also be any substituents conventionally used to activate the carboxylic acid group. For example, suitable amino protecting groups are described in Albericio F. “Peptide Coupling Reagents, More than a Letter Soup” , Chem. Rev. 2011, 111, 6557-6602. In some forms, R9 can be benzyl, allyl, or unsubstituted C1-C4 alkyl. In some forms, R9 can be an activating group, such as l-hydroxy-7-azabenzotriazole (HOAt) , 1-hydroxybenzotriazole (HOBt) , ethyl 2-cyano-2- (hydroxyimino) acetate (Oxyma) , N-hydroxysuccinimide (NHS) , pentafluorophenol (Pfp) , etc. In these forms, is an activated ester group, such as l-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.
In some forms, the disclosed compounds can have the structures of Formula I':

wherein R1-R9 and X can be in any forms as defined above, and R10 can be hydrogen or a functional group suitable for hindering the reactivity of 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) .
In some forms, R10 can be hydrogen. In some forms, R10 can be any substituents 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, R10 can be 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) .
In some forms, R9 can be hydrogen and R10 can be hydrogen. In some forms, R9 can be benzyl, allyl, unsubstituted C1-C4 alkyl, or an activating group, such as l-hydroxy-7-azabenzotriazole (HOAt) , 1-hydroxybenzotriazole (HOBt) , ethyl 2-cyano-2- (hydroxyimino) acetate (Oxyma) , N-hydroxysuccinimide (NHS) , pentafluorophenol (Pfp) , etc. and R10 can be hydrogen. In some forms, R9 can be hydrogen and R10 can be 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) . In some forms, R9 can be benzyl, allyl, unsubstituted C1-C4 alkyl, or an activating group, such as l-hydroxy-7-azabenzotriazole (HOAt) , 1-hydroxybenzotriazole (HOBt) , ethyl 2-cyano-2- (hydroxyimino) acetate (Oxyma) , N-hydroxysuccinimide (NHS) , pentafluorophenol (Pfp) , etc. and R10 can be 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) .
Representative examples of NBDs are shown below, where ● representsindicates that attachment point, and R3-R10 can be in any forms as defined above.
Additional 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 shown below:

The disclosed NBDs are structurally different from of pseudo-prolines disclosed inet al., Pseudo-Prolines as a Solubilizing, Structure-Disrupting Protection Technique in Peptide Synthesis (J. Am. Chem. Soc. 1996, 118, 39, 9218-9227) . The disclosed NBDs are also structurally different from pseudo-prolines supplied by CHEM IMPEX (Woodale IL, for example, catalog Number 46591 (www. chemimpex. com/product/productinfo/fmoc-arg-pbf-ser-psi-me-me-pro-oh/36788? cid=860) . For example, compared to the oxazolidine in pseudo-proline dipeptides, the phenyl group of the disclosed NBDs provides more substitution sites for further functionalization, where in pseudo-proline, the symmetric dimethyl group was adopted without any other site for modification.
III. METHODS
Compared to exiting protein/peptide synthesis methods, NBDs can cover broader range of peptide sequence and they can be easily prepared from mg- scale to sub-kg scale, whose estimated cost is only around 1～10%price of commercial competitors.
A. Production of NBD
Methods of producing the compounds disclosed herein are described. Generally, the method includes two steps of reactions. Briefly, a L-or D-Amino acid with suitable protection on amine and optionally its sidechain functional groups, can be firstly coupled with salicylaldehyde or a mono-/multiply-substituted form thereof. Then, the obtained salicylaldehyde ester reacts with serine/threonine/cysteine/penicillamine (Ser/Thr/Cys/Pen) or its ester in a suitable solvent. The obtained ester may be further derivatized to relevant acids with or without phenol protections. Using the methods disclosed herein, the NBDs can be obtained in high yield (at least 40%) .
Using the methods disclosed herein, 76 exemplary NBDs (structures shown in the section above) were produced in moderate to excellent yield (i.e., 40%to 95%) . Variants of those 76 NBDs that include D-amino acid version, different protecting groups on amine or side chain, other carboxylic derivatives and different substitutions of aryl could be also synthesized by following the developed synthetic strategy.
The disclosed method includes: (a) reacting a protected amino acid of Formula II:
wherein R1 can be a protected or unprotected side chain of an amino acid; and (ii) R2 can be an amino protecting group,
with a substituted or unsubstituted salicylaldehyde of Formula III:
wherein R3-R6 can be independently hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF3) , -NO2, unsubstituted alkyl (such as unsubstituted C1-C8 alkyl) , unsubsituted alkenyl (unsubstituted C1-C8 alkenyl) , unsubstituted alkynyl (such as unsubstituted C1-C8 alkynyl) , unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol,
to obtain an intermediate comprising a salicylaldehyde ester of Formula IV:
(b) reacting the salicylaldehyde ester of Formula IV with serine, threonine, cysteine, or penicillamine, or an ester thereof of Formula V:
wherein R7 and R8 can be indepdently hydrogen, methyl, or other protected/unprotected side chains of amino acids; (v) R9 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,
to obtain a product comprising the compound of Formula I:
wherein R1-R9 and X are as defined above for the reactants.
Activating groups suitable for R9 of Formula V and Formula I are typically less active than HOAt and Oxyma, such as NHS, to reduce/prevent side reactions in step (b) .
The method disclosed herein may further include step (c) : deprotecting a protected carboxyl group of the NBDs; or step (d) acylating or activating a free acid group or a free phenol group of the NBDs; or a combination thereof.
In some forms, in the deprotection step (c) , a deprotected NBD with free acid form is obtained, for example, a deprotected NBD of Formula VI:
wherein R1-R8, R10, and X can be in any forms as defined above.
In some forms, in the acylation/activation step (d) , at least one of R9 and R10 is hydrogen. For example, R9 of the NBDs is hydrogen, such that this free  carboxylic acid group can be activated in step (d) . For example, R10 of the NBDs is hydrogen, such that this free hydroxyl group can be acylated in step (d) . In some forms, both R9 and R10 of the NBDs is hydrogen, such that the free carbocylic acid and/or the hydroxyl group can be acylated and/or activated, as needed, in step (d) . In some forms, in step (d) , an acylated NBD with or without a free carboxylic acid is obtained, for example, an acylated NBD of Formula I’:
wherein R1-R9 and X can be in any forms as defined above, and R10 can be in any form as defined above expect for hydrogen.
The deprotection step (c) and 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 includes steps (a) , (b) , and (c) , performed sequentially. For example, the method includes steps (a) , (b) , and (d) , performed sequentially. For example, the method includes steps (a) , (b) , (c) , and (d) , performed sequentially. For example, the method includes steps (a) , (b) , (d) , and (c) , performed sequentially.
As noted above, thein the formulae described herein indicates a carbon stereocenter (to which the wave bond attaches) that can be either S-or R-configuration. For example, the acetyl carbon attaching to -C (O) OR9 can be either S-or R-configuration. It is thus understood that the threonine, cysteine, or penicillamine, or an ester thereof used in step (b) can be either in its L-or D-configuration, as racemates, or in various mixtures of their isomers. It is also  understood that the protected amino acid used in step (a) can be either in its L-or D-configuration, as racemates, or in various mixtures of their isomers.
1. Step (a) Producing Salicylaldehyde Ester
In the first step (a) of the disclosed methods, a protected amino acid of Formula II is reacted with a subsituted or unsubstituted salicylaldehyde of Formula III to obtain an intermediate comprising a salicylaldehyde ester of Formula IV:
wherein R1-R6 can be in any forms as defined above. When R1 is a protected side chain of an amino acid, the protection group can be a substituted or unsubstitited alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a carbonyl (e.g., carboxylic acid or carboxylic ester) , an amide, an amino, a substitute or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a heterocyclic ring. In some forms, R1 is a protected side chain of an amino acid and the protection group can be an unsubstitited alkyl, an unsubstituted alkenyl, an unsubstituted alkynyl, a carbonyl (e.g., carboxylic acid or carboxylic ester) , an amide, an amino, an unsubstituted aryl, an unsubstituted heteroaryl, or an unsubstituted heterocyclic ring.
The protected amino acid of Formula II used in step (a) can be any natural amino acids or synthetic amino acids, which can be either in their L-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 its L-or D-configuration.
The side chain of the amino acid used in step (a) may be protected using a protecting group known in the art. Suitable protecting groups for the side chain include, but are not limited to, tBu, Boc, Thz, Acm, Trt, Cbz, Alloc, Azido, Bn, Bz, Ac, and Pbf.
The terminal amino group of the amino acid used in step (a) are protected (i.e., R2) . Any amino protecting group known in the art can be used to protect the amino group of the amino acid used in the disclosed methods. For example, R2 of the protected amino acids used in step (a) can be Fmoc, Cbz, Moz, Boc, Troc, Teoc, Alloc, or Voc.
The salicylaldehyde of Formula III that reacts with the protected amino acids of Formula II can be an unsubstituted salicylaldehyde or a mono-or poly-substituted salicylaldehyde. In some forms of the methods, the salicylaldehyde reacting with the protected amino acid of Formula II is substituted with one or more substituents (i.e., at least one of R3-R6, optionally two or more of R3-R6, is/are not hydrogen) . For example, one of R3-R6, two of R3-R6, three of R3-R6, or all of R3-R6, of the salicylaldehyde of Formula III used in step (a) is/are not hydrogen. Examples of suitable substituents of the substituted salicylaldehyde of Formula III are hydroxyl, halogen, -CN, haloalkyl (such as -CF3) , -NO2, unsubstituted C1-C6 alkyl, unsubsituted C1-C6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, and thiol. Preferably, when the salicylaldehyde reacting with the protected amino acid of Formula II is substituted with one or more substituents, the substituents are independently halogen, -CN, -CF3, -NO2, unsubstituted C1-C6 or C1-C4 alkyl, unsubstituted C1-C6 or C1-C4 alkenyl, unsubstitued C1-C6 or C1-C4 alkynyl, alkoxyl, and unsubstituted aryl.
Typically, the reaction between the protected amino acid of Formula II and the subsituted or unsubstituted salicylaldehyde of Formula III is performed  in a suitable organic solvent, and optionally in the presence of a condensation reagent and/or a base.
Examples of suitable organic solvents for performing the reaction in step (a) include, but are not limited to, DMF, DMSO, DMAc, NMP. Preferably, the organic solvent for performing the reaction in step (a) is DMF.
Examples of suitable condensation agent for performing the reaction in step (a) include, but are not limited to, HATU, PyBOP, HBTU, HCTU, COMU, TSTU, PyClock, PyOxim, EDCI, and DCC. Preferably, the condensation agent used in the reaction in step (a) is HATU.
Examples of suitable base for performing 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.
The reaction between the protected amino acid of Formula II and the subsituted or unsubstituted salicylaldehyde of Formula III can be performed in an suitable organic solvent, such as any one of thoes described above, and optionally in the presence of a condensation reagent and/or a base, at room temperature (i.e., 20 ℃ to 25 ℃, at 1 atm) for a time period ranging from 30 mins to 5 hours, preferably from 1 hour to 3 hours, such as about 1 hour or about 1.5 hour.
Optionally, the method disclosed herein further includes a step of purifying the intermediate containing the salicylaldehyde ester of Formula IV after step (a) and prior to step (b) . The purification step can be performed using any suitable techniques known in the art, such as using extraction, washing, drying, column chromatography (such as by using a silica gel column) , or filtration, or a combination thereof. For example, the intermdeiate containing the salicylaldehyde ester of Formula IV is purified using the following techniques: extraction with an organic solvent that is different from the organic solvent used for performing the reaction, washing with an acid for one or more times, drying  with a drying agent (such as sodium sulfate) and/or evaporation, and then separation using silica gel column.
Generally, the salicylaldehyde ester of Formula IV obtained in step (a) has a yield of at least 40%, at least 50%, or in a range from about 50%to about 95%. The yield of salicylaldehyde ester of Formula IV can be calculated using the formula: (The mole of the salicylaldehyde ester of Formula IV) / (The mole of the protected amino acid of Formula II) *100%. The yield may be determined either before or after purification of the intermediate.
2. Step (b) Producing NBD
In the second step of the disclosed methods, the salicylaldehyde ester of Formula IV is reacted with serine, threonine, cysteine, or penicillamine, or an ester thereof of Formula V to obtain the compounds of Formula I:
wherein R1-R9 and X can be in any forms as defined above.
The serine, threonine, cysteine, or penicillamine, or an ester of Formula V can be either in their L-or D-configuration. Accordingly, R9 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.
Typically, the reaction between the salicylaldehyde ester of Formula IV and the serine, threonine, cysteine, or penicillamine, or ester thereof of Formula V is performed in a suitable solvent. The solvent may be an organic solvent or water or a combination thereof, and typically contains a buffer at an amount  ranging from 10 vol%to 20 vol%, such as 10 vol%to 20 vol%pyridine/HOAc (1: 1, molar ratio) .
Examples of suitable solvents for preforming the reaction in step (b) include, but are not limited to, dichloromethane, trifluoroacetic acid, ethyl acetate, toluene, tetrahydrofuran, 1, 4-dixoane, acetonitrile, acetone, hexane, and water, and a combination thereof.
Examples of buffers suitable for use in the solvent for performing the reaction in 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-colidine-acetic acid buffer, and DMSO-picoline-acetic acid buffer. A preferred buffer suitable for use is pyridine-acetic acid buffer, where the molar ratio of pyridine to acetic acid ranges from 1: 9 to 9: 1, such as 1: 1.
The reaction between the salicylaldehyde ester of Formula IV and the serine, threonine, cysteine, or penicillamine, or ester thereof of Formula V can be performed in an suitable solvent, such as any one of thoes described above, at room temperature for a time period ranging from 1 hour to 5 hours, preferably from 2 hours to 3 hours, such as about 2 hours or about 3 hours.
Optionally, the method disclosed herein further includes a step of purifying the product containing the compound of Formula I after step (b) . The purification step can be performed using any suitable techniques known in the art, such as any of those described above in step (a) . For example, the product containing the compounds of Formula I is purified using the following techniques: extraction with a suitable solvent and separation using column chromatography or filtration, washing with cod water and cold organic solvent (such as cold ethanol and/or cold ether) for one or more times, and separation by silica gel column.
Generally, the compound of Formula I obtained in step (b) has a yield of at least 40%or in a range from about 40%to about 95%. The yield of compond of Formula I can be calculated using the formula: (The mole of the compound of  Formula I) / (The mole of the salicylaldehyde ester Formula IV) *100%. The yield may be determined either before or after purification of the product.
3. Step (c) Deprotection of Protected Carboxyl Group
Optionally, the disclosed method further includes a step (c) : deprotecting a protected carboxyl group of the NBDs formed in step (b) or (d) as described herein. For example, the disclosed method includes steps (a) , (b) , and (c) performed sequentilly. For example, the disclosed method includes steps (a) , (b) , (c) , and (d) , performed sequentially. For example, the disclosed method includes steps (a) , (b) , (d) , and (c) , performed sequentially. When a deprotection step is included in the disclosed method, R9 of the NBDs is typically not hydrogen, in particular when X of the formulae described herein is O. The protected carboxyl group of the NBDs can be deprotected using reagents and reactions known in the art, for example, by using reagents such as Pd (PPh3) 4 and/or PhSiH3 for deallylation; H2 and/or Pd/C for hydrogenation of Bn protection; LiOH for sponsification of alkyl protection, etc. In step (c) , a deprotected NBD with free acid form is obtained, for example, a deprotected NBD of Formula VI:
wherein R1-R8, R10, and X can be in any forms as defined above.
Optionally, the method disclosed herein further includes a step of purifying the product containing the compound of Formula VI after step (c) . The purification step can be performed using any suitable techniques known in the art, such as any of those described above in step (a) or (b) . For example, the product containing the compounds of Formula VI is purified using the following  techniques: extraction with a suitable solvent and separation using column chromatography or filtration, washing with cod water and cold organic solvent (such as cold ethanol and/or cold ether) for one or more times, and separation by silica gel column.
Generally, the compound of Formula VI obtained in step (c) has a yield of at least 40%or in a range from about 40%to about 95%. The yield of compond of Formula VI can be calculated using the formula: (The mole of the compound of Formula VI) / (The mole of the compound of Formula I’) *100%. The yield may be determined either before or after purification of the product.
4. Step (d) Acylation or Activation of Acid Groups
Optionally, the disclosed method further includes a step (d) : acylating or activating a free acid or a free phenol group of the NBDs formed in step (b) or (c) . When an acylation/activation step is included in the disclosed method, at least one of R9 and R10 of the reagent, such as an NBD of Formula I or VI, is hydrogen. In some forms, R9 of the NBDs is hydrogen, either in the NBD product of step (b) or in the deprotected NBD formed in the deprotection step (c) , such that this free carboxylic acid group can be activated in step (d) . In the forms where the free carboxylic acid group is activated, exmples of the formed activated group include, but are not limited to NHS, HOAt, or Oxyma. In some forms, R10 of the NBDs is hydrogen in the NBD product of step (b) and/or the deprotected NBD formed in step (c) , such that this free hydroxyl group can be acylated in step (d) . In some forms, both R9 and R10 of the NBDs is hydrogen, either in the NBD product of step (b) or in the deprotected NBD formed in the deprotection step (c) , such that the free carbocylic acid and/or the hydroxyl group can be acylated and/or activated, as needed, in step (d) . For example, both R9 and R10 of the NBDs is hydrogen, either in the NBD product of step (b) or in the deprotected NBD formed in the deprotection step (c) , such that the free carbocylic acid is activated, and/or the hydroxyl group is acylated, in step (d) .
In some forms, in step (d) , an acylated NBD, with or without a free carboxylic acid (e.g., -COOR9 is carboxyl or ester) can be obtained, for example, an acylated NBD of Formula I’:
wherein R1-R9 and X can be in any forms as defined above, and R10 can be in any form as defined above expect for hydrogen.
A free acid of the NBDs can be acylated and/or activated using reagents and reactions known in the art. For example, a free acid of the NBDs (-COOH and/or -OH) can be acylated using reagents such as acetyl anhydride, carbonate, or carbamate to obtain an acyl-capped NBD of Formula I’, where R9 and/or R10, such as R10, is an acyl group. For example, a free acid of the NBDs (e.g., -COOH) can be activated by using reagents such as HATU, HBTU, Oxyma Pure to obtain an activated NBD of Formula I’, where R9 is an activating group.
Optionally, the method disclosed herein further includes a step of purifying the product containing acylated and/or activated NBDs after step (d) . The purification step can be performed using any suitable techniques known in the art, such as any of those described above in step (a) , (b) or (c) . For example, the product containing the acylated and/or activated NBDs is purified using the following techniques: extraction with a suitable solvent and separation using column chromatography or filtration, washing with cod water and cold organic solvent (such as cold ethanol and/or cold ether) for one or more times, and separation by silica gel column.
Generally, the acylated and/or activated NBDs obtained in step (d) has a yield of at least 40%or in a range from about 40%to about 95%. The yield of acylated and/or activated NBDs can be calculated using the formula: (The mole of the acylated and/or activated NBDs) / (The mole of the compound of Formula VI) *100%. The yield may be determined either before or after purification of the product.
An exemplary synthesis scheme of NBDs showing specific reaction conditions is shown below.
wherein R1-R10 and X can be in any forms as defined above.
Although not illustrated in the exemplary synthesis scheme, it is understood that obtaining the NBDs of Formula I’ above does not require all of the steps shown in this exemplary reaction scheme. An NBD of Formula I’ having a desired structure can be obtained following steps (a) and (b) ; following steps (a) , (b) , and step (c) ; following steps (a) , (b) , and (d) ; or following steps (a) , (b) , (c) , and (d) .
B. Peptide synthesis
The peptide/protein synthesis methods disclosed herein are based at least on the application of NBD as a pre-formed dipeptide building block for synthesizing difficult peptides/proteins.
The terms “difficult sequence” and “difficult peptide/protein” are used interchangeably herein. The concept of “difficult sequence” was introduced in  the 80’s and was given distinction by Kent and co-workers for peptides that form strong inter-or intra molecular, non-covalent interactions which form insoluble peptide aggregates. “Difficult sequences” are peptide sequences that contain high number of amino acids possessing hydrophobic side chains, so-called β-branched amino acids, including leucine, valine, phenylalanine or isoleucine. Additionally, glycine is known to induce β-sheet packing in combination with afore mentioned amino acids. These sequences tend to form β-sheet or α-helical structures within the molecule and therefore they have high aggregation potential and low solubility in aqueous or organic solvents. This results in a generally difficult handling, synthesis and purification. Peptide chains of “difficult sequences” exhibiting over 50-60 amino acids remain a challenge even when applying automated peptide synthesis protocols. With respect to SPPS “difficult sequences” are defined as peptides that are poorly solvated while attached to the solid support thus preventing complete deprotection and coupling steps. (Mueller, et al., Front Bioeng Biotechnol. 2020; 8: 162) Using the disclosed NBDs in SPPS (NBD-SPPS) , the peptide length (～50 AAs) in standard SPPS can be further extended to near 100 AAs, which was previously unobtainable in the field of peptide/protein synthesis.
Examples of difficult sequences include membrane proteins and their functional parts, for example, amylin, BM2 proton channel, influenza A, Copper storage protein 1 CSP-1, Interferon-induced transmembrane protein 3 (IFITM3) , NS4A, cofactor protein of serine protease from Hepatitis C virus; PD-L1 (programmed death-ligand 1) , IL-2 (INTERLEUCKIN -2) , Vasoactive intestinal peptide, Erythropoietin, Liraglutide, etc.
The disclosed NBDs are typically used in SPPS methods known in the art. Solid phase synthesis, in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is a preferred method for the chemical synthesis of the peptides. Techniques for solid phase synthesis are known to  those of skill 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 ed. Pierce Chem. Co., Rockford, 111. Such methods include bench scale solid phase synthesis and automated peptide synthesis in any one of the many commercially available peptide synthesizers. Solid phase synthesis is commonly used, and various commercial synthesizers are available, such as automated synthesizers by Applied Biosystems Inc., Foster City, CA; Beckman; MultiSyntech, Bochum, Germany etc. Functional groups for conjugating the peptide to small molecules, label moieties, peptides, or proteins may be introduced into the molecule during chemical synthesis. In addition, small molecules and label moieties/reporter units may be attached during the synthetic process. Introduction of the functional groups and conjugation to other molecules minimally affects the structure and function of the NBDs.
The disclosed methods are different and/or superior over previously described methods. For example, US 2008/0004451 describes the synthesis of dipeptides building blocks with acetonide protecting group to form a pseudoproline ring. By contrast, the disclosed methods utilizes the ligation intermediate of Serine/Threonine Ligation (STL) or Cysteine/Penicillamine Ligation (CPL) as the proline mimic i.e., NBDs, which allows the scale-up production of peptides in a very safe manner (NBDs are safe to handle) and it is environmentally friendly, because NBDs synthesis is a mild endothermic process and does not require heating or reflux conditions. Furthermore, the disclosed NBDs are difficult to obtain by the methods disclosed in US 2008/0004451. The much less reactive salicylaldehyde cannot fully react with the dipeptide, even in reflux temperature, resulting in low yield of the desired product. Also, the similar polarity of starting material and product made the  purification very challenging. Careful column chromatography and sophisticated chemists would be required, which dramatically increased the cost and the whole process was not effective enough. In contrast, using the methods disclosed herein, the NBDs could be easily prepared from milligram to kilogram scale without the need of heating or condensation apparatuses. The high reactivity of salicylaldehyde ester rendered complete conversion of starting materials and the purification process has also been simplified. Further, extra functional groups may be attached to the benzene ring of the NBDs to further facilitate the difficult peptide/protein synthesis.
The methods disclosed herein similar provide for simple and safer peptide synthesis than contemplated in previous disclosures, for example, 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 its dipeptides required harsh conditions such as anhydrous reflux with molecular sieves, which consumed more energy and had potential risk of explosion. By contrast, production of the disclosed NBD product do not need such complicated set up. NBDs produced as described herein j require mixing all ingredients together under room temperature, air atmosphere, moisture and high yield of product up to sub-kilogram can be obtained easily.
Similarly, the disclosed methods have advantages over the methods disclosed in Sohma, et al., Novel and efficient synthesis of difficult sequence-containing peptides through O-N intramolecular acyl migration reaction of O-acyl isopeptides, Chem. Commun (Camb) , 1: 124-5 (2004) ( (DOI: 10.1039/B312129A) , which discloses isoacyl dipeptides (which are chemically distinct from the disclosed NBDs) . Further, the synthesis of isopeptide may have a risk of epimerization since the esterification required highly active condensation reagents. By contrast, the disclosed methods proceed via NBD; the ligation between two amino acid fragment is chemoselective and stereoselective, without the risk of epimerization. In contrast, with acyl isopeptides, the  disclosed NBDs are much more stable under basic condition, which is important in Fmoc-SPPS for peptide synthesis. This advantage makes the disclosed NBDs much more suitable for long peptide or protein chemical synthesis.
The disclosed compounds and methods can be further understood through the following numbered paragraphs.
1. A compound having a structure of:
wherien:
(i) R1 is a protected or unprotected side chain of an amino acid;
(ii) R2 is an amine protecting group;
(iii) R3-R6 are independently hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF3) , -NO2, unsubstituted C1-C6 alkyl, unsubsituted C1-C6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol;
(iv) R7 and R8 are indepdently hydrogen, methyl, or other protected/unprotected side chains of amino acids;
(v) R9 is hydrogen or a functional group suitable for protecting and/or activating a carboxylic acid group;
(vi) R10 is hydrogen or a functional group suitable for hindering the reactivity of 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 paragraph 1, wherein R1 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 paragraph 1 or 2, wherein when R1 is a protected side chain, wherein the protection group is selected from the group consisting of a substituted or unsubstitited alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a carbonyl (e.g., carboxylic acid or carboxylic ester) , an amide, an amino, a substitute or unsubstituted aryl, a substituted or unsubstituted heteroaryl, and a heterocyclic ring, optionally wherein the protection group is tBu, Boc, Thz, Acm, Trt, Cbz, Alloc, Azido, Bn, Bz, Ac, and Pbf, or a combination thereof.
4. The compound of any one of paragraphs 1-3, wherein R2 is Fmoc, Cbz, Moz, Boc, Troc, Teoc, Alloc, or Voc.
5. The compound of any one of paragraphs 1-4, wherein R3-R6 are independently hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF3) , -NO2, unsubstituted alkyl (such as unsubstituted C1-C8 alkyl) , unsubsituted alkenyl (unsubstituted C1-C8 alkenyl) , unsubstituted alkynyl (such as unsubstituted C1-C8 alkynyl) , unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol, optionally wherein R3-R6 are independently hydrogen, hydroxyl, halogen, -CN, -CF3, -NO2, unsubstituted C1-C4 alkyl, alkoxyl, and unsubstituted aryl.
6. The compound of any one of paragraphs 1-5, wherein at least one of R3-R6, optionally two or more of R3-R6, is/are not hydrogen.
7. The compound of any one of paragraphs 1-6, wherein R9 is hydrogen, benzyl, allyl, unsubstituted C1-C4 alkyl; oris an activated ester group, such as l-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.
8. The comound of any one of paragraphs 1-7, wherein R10 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. A method of making the compound of Formula I:
wherien:
(i) R1 is a protected or unprotected side chain of an amino acid;
(ii) R2 is an amine protecting group;
(iii) R3-R6 are independently hydrogen, hdyroxyl, halogen, -CN, haloalkyl (such as -CF3) , -NO2, unsubstituted C1-C6 alkyl, unsubsituted C1-C6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol;
(iv) R7 and R8 are indepdently hydrogen, methyl, or other protected/unprotected side chains of amino acids;
(v) R9 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,
wherein the method comprises:
(a) reacting a protected amino acid of Formula II:
wherein R1 and R2 are as defined above for Formula I,
with a substituted or unsubstituted salicylaldehyde of Formula III:
wherein R3-R6 are as defined above for Formula I,
to obtain an intermediate comprising a salicylaldehyde ester of Formula IV:
(b) reacting the salicylaldehyde ester of Formula IV with serine, threonine, cysteine, or penicillamine, or an ester thereof to obtain a product comprising the compound of Formula I.
10. The method of paragraph 9, wherein R1 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 paragraph 9 or 10, wherein when R1 is a protected side chain, wherein the protection group is selected from the group consisting of a substituted or unsubstitited alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a carbonyl (e.g., carboxylic acid or  carboxylic ester) , an amide, an amino, a substitute or unsubstituted aryl, a substituted or unsubstituted heteroaryl, and a heterocyclic ring, optionally wherein the protection group is tBu, Boc, Thz, Acm, Trt, Cbz, Alloc, Azido, Bn, Bz, Ac, and Pbf, or a combination thereof..
12. The method of any one of paragraphs 9-11, wherein R2 is Fmoc, Cbz, Moz, Boc, Troc, Teoc, Alloc, or Voc.
13. The method of any one of paragraphs 9-12, wherein R3-R6 are independently hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF3) , -NO2, unsubstituted alkyl (such as unsubstituted C1-C8 alkyl) , unsubsituted alkenyl (unsubstituted C1-C8 alkenyl) , unsubstituted alkynyl (such as unsubstituted C1-C8 alkynyl) , unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol, optionally wherein R3-R6 are independently hydrogen, hydroxyl, halogen, -CN, -CF3, -NO2, unsubstituted C1-C4 alkyl, alkoxyl, and unsubstituted aryl.
14. The method of any one of paragraphs 9-13, wherein at least one of R3-R6, optionally two or more of R3-R6, is/are not hydrogen.
15. The method of any one of paragraphs 9-14, wherein step (a) is performed in the presence of a condensation agent, optionally wherein the condensation agent is HATU, PyBOP, HBTU, HCTU, COMU, TSTU, PyClock, PyOxim, EDCI, or DCC, or a combination thereof.
16. The method of any one of paragraphs 9-15, 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 method of any one of paragraphs 9-16, wherein step (a) is performed in an organic solvent, optionally wherein the organic solvent is DMF, DMSO, DMAc, or NMP, or a combination thereof.
18. The method of any one of paragraphs 9-17, wherein step (a) is performed at room temperature for a time period ranging from 30 mins to 5 hours, preferably from 1 hour to 3 hours.
19. The method of any one of paragraphs 9-18, wherein the salicylaldehyde ester of Formula IV obtained in step (a) has a yield of at least 40%, at least 50%, or in a range from about 50%to about 95%.
20. The method of any one of paragraphs 9-19, wherein step (b) is performed 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 paragraph 20, wherein the buffer is 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 method of any one of paragraphs 9-21, wherein step (b) is performed at room temperature for a time period ranging from 1 hour to 5 hours, preferably from 2 hours to 3 hours.
23. The method of any one of paragraphs 9-22, wherein the compound of Formula I obtained in step (b) has a yield of at least 40%, such as in a range from about 40%to about 95%.
24. The method of any one of paragraphs 9-23, further comprising purifying the intermediate to obtain the salicylaldehyde ester of Formula IV after step (a) and prior to step (b) .
25. The method of any one of paragraphs 9-24, further comprising purifying the product to obtain the compound of Formula I after step (b) .
26. The method of any one of paragraphs 9-25, further comprising:
(c) reacting a carboxyl protected NBD with a deprotection reagent to form a deprotected NBD of Formula VI:
(i) R1 is a protected or unprotected side chain of an amino acid;
(ii) R2 is an amine protecting group;
(iii) R3-R6 are independently hydrogen, hdyroxyl, halogen, -CN, haloalkyl (such as -CF3) , -NO2, unsubstituted C1-C6 alkyl, unsubsituted C1-C6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol;
(iv) R7 and R8 are indepdently hydrogen, methyl, or other protected/unprotected side chains of amino acids;
(v) R10 is hydrogen or a functional group suitable for hindering the reactivity of 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 paragraphs 9-26, further comprising:
(d) reacting a free-acid or free-phenol containing NBD with an acylation and/or activation reagent to form an acylated/activated NBD of Formula I’:
wherien:
(i) R1 is a protected or unprotected side chain of an amino acid;
(ii) R2 is an amine protecting group;
(iii) R3-R6 are independently hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF3) , -NO2, unsubstituted C1-C6 alkyl, unsubsituted C1-C6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol;
(iv) R7 and R8 are indepdently hydrogen, methyl, or other protected/unprotected side chains of amino acids;
(v) R9 is hydrogen or a functional group suitable for protecting and/or activating a carboxylic acid group;
(vi) R10 is a functional group suitable for hindering the reactivity of 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 paragraph 26 or 27, wherein step (c) is performed immediately following step (b) or immediately following 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 wherin R9 of Formula I or Formula I’ is not hydrogen.
29. The method of paragraph 27 or 28, wherein step (d) is performed immediately following step (b) or immediately following step (c) , and wherein the free-acid containing NBD is the NBD of Formula I formed in step (b) or the NBD of Formula VI formed in step (c) .
30. A method of using the compound of any one of paragraphs 1-8 for solid phase peptide synthesis.
31. The method of paragraph 30, comprising: mixing the compound with amino acids for forming the peptide.
The detailed synthetic procedures of exemplary NBDs are described below in the following non-limiting examples.
EXAMPLES
EXAMPLE 1. L-Thr-L-Thr NBD
The first example synthesized L-Thr-L-Thr NBD, the structure of which is shown below.
Step a:
Firstly, 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 DMF. Subsequently, DIPEA (2.58 g, 20.0 mmol) was added and the reaction mixture was stirred under 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 that, the mixture was extracted with 250 mL ethyl acetate and washed with 100 mL 1M HCl (aq) and 100 mL NaHCO3 (aq) for 3 times. The obtained organic phase was dried by sodium sulfate and evaporated under reduced pressure to give the crude product as a pink oil. After purification by silica gel column, Fmoc-Thr (tBu) -SAL ester could be obtained in 95%yield, 4.77 g.
Step b:
The Fmoc-Thr (tBu) -SAL ester was dissolved into 250 mL dichloromethane, and 10 mL pyridine/HOAc buffer containing 10.0 mmol L-H-Thr-OAll ester was added. The reaction mixture was stirred at room temperature for 2 h and extracted similarly as mentioned above. Later the crude product was purified by column chromatography, affording the desired NBD as pale solid in 93%yield.
Alternatively, the above procedure could be simplified in only one purification process:
After extraction and evaporation at step a., the crude L-Fmoc-Thr (tBu) -SAL ester was directly dissolved into 250 mL dichloromethane without any extra purification. Then, 10 mL pyridine/HOAc buffer containing 10.0 mmol L-H-Thr-OAll ester was added and the mixture reacted for 2 h to afford the desired L-Thr-L-Thr NBD in 85%yield over two steps. Furthermore, this simplified protocol has been robustly scale-up to sub-kg scale in the production of NBDs.
EXAMPLE 2. L-Val-L-Cys NBD
The second example synthesized L-Val-L-Cys NBD, the structure of which is shown below.
Step a & Step b:
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 under 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 that, the mixture was directly diluted by 100 mL 6M pyridine-acetic acid aqueous buffer, and 10 mL 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 precipitation was filtered by suction and washed by cold water, cold ethanol and cold ether. Later the product was further purified by column chromatography, affording the desired L-Val-L-Cys NBD as white solid in 83%yield. This protocol can also be scale-up to sub-kg scale in the production of Cys/Pen-based NBDs.
EXAMPLE 3. Ac-capped L-Val-L-Thr NBD
The first example synthesized Ac-capped L-Thr-L-Thr NBD, the structure of which is shown below.
Step a:
Firstly, 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 under 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 that, the mixture was extracted with 250 mL ethyl acetate and washed with 100 mL 1M HCl (aq) , 100 mL NaHCO3 (aq) for 3 times. The obtained organic phase was dried by sodium sulfate and evaporated under reduced pressure to give the crude product as a pink oil. After purification by silica gel column, Fmoc-Val-SAL ester could be obtained in 95%yield, 4.51 g.
Step b:
The Fmoc-Val-SAL ester was dissolved into 250 mL dichloromethane, and 10 mL pyridine/HOAc buffer containing 10.0 mmol L-H-Thr-Oall ester was  added. The reaction mixture was stirred at room temperature for 2 h and extracted similarly as mentioned above. Later the crude product was purified by column chromatography, affording the unprotected NBD as pale solid in 91%yield.
Step c:
The unprotected NBD was dissolved into 250 mL dichloromethane, and Pd (PPh3) 4 (5 mol%) , PhSiH3 (10 eq. ) were added. The reaction mixture was stirred at room temperature for 1 h and extracted similarly as mentioned above. Then the crude product was purified by column chromatography, affording the NBD acid form as pale solid in 95%yield.
Step d:
The NBD acid was then dissolved into 250 mL dichloromethane, and Ac2O (2.0 eq. ) and DIPEA (4.0 eq. ) were added. The reaction mixture was stirred at room temperature for 3 h and extracted similarly as mentioned above. Then the crude product was purified by column chromatography, affording the desired Ac-capped NBD acid as pale solid in 90%yield.
EXAMPLE 4. HOAt-Activated L-Thr-L-Thr NBD
The first example synthesized HOAt-activated L-Thr-L-Thr NBD, the structure of which is shown below.
Step a:
Firstly, 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 under 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 that, the mixture was extracted with 250 mL ethyl acetate and washed with 100 mL 1M HCl (aq) , 100 mL NaHCO3 (aq) for 3 times. The obtained organic phase was dried by sodium sulfate and evaporated under reduced pressure to give the crude product as a pink oil. After purification by silica gel column, Fmoc-Val-SAL ester could be obtained in 95%yield, 4.51 g.
Step b:
The Fmoc-Val-SAL ester was dissolved into 250 mL dichloromethane, and 10 mL pyridine/HOAc buffer containing 10.0 mmol L-H-Thr-OAll ester was added. The reaction mixture was stirred at room temperature for 2 h and extracted similarly as mentioned above. Later the crude product was purified by column chromatography, affording the unprotected NBD as pale solid in 91%yield.
Step c:
The unprotected NBD was dissolved into 250 mL dichloromethane, and Pd(PPh3) 4 (5 mol%) , PhSiH3 (10 eq. ) were added. The reaction mixture was stirred at room temperature for 1 h and extracted similarly as mentioned above. Then the crude product was purified by column chromatography, affording the NBD acid form as pale solid in 93%yield.
Step d:
The NBD acid was then dissolved into 250 mL dichloromethane, and HATU (1.0 eq. ) and DIPEA (1.0 eq. ) were added. The reaction mixture was stirred at room temperature for 0.5 h and extracted similarly as mentioned above. Then the crude product was purified by column chromatography, affording the desired HOAt-activated NBD acid as pale solid in 78%yield.
EXAMPLE 5. Carbamate-capped L-Val-L-Cys NBD
The second example synthesized carbamate-protected L-Val-L-Cys NBD, the structure of which is shown below.
Step a & Step b:
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 under 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 that, the mixture was directly diluted by 100 mL 6M pyridine-acetic acid aqueous buffer, and 10 mL 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 precipitation was filtered by suction and washed by cold water, cold ethanol and cold ether. Later the product was further purified by column chromatography, affording the desired L-Val-L-Cys NBD as white solid in 83%yield.
Step d:
In this case, the NBD is already in acid form so that the step c could be skipped. The unprotected NBD was dissolved into 250 mL dichloromethane, and tert-butyl methyl (2- (methylamino) ethyl) carbamate (2.0 eq. ) activated by 1, 1'-carbonyldiimidazole (2.0 eq. ) and DIPEA (4.0 eq. ) were added. The reaction mixture was stirred at room temperature for 1 h and extracted similarly as mentioned above. Then the crude product was purified by column chromatography, affording the desired carbamate-capped NBD as pale solid in 82%yield.
EXAMPLE 6. Peptide Synthesis
With these NBDs in hand, we further evaluated their effect in the construction of difficult peptides by SPPS. For this purpose, several difficult or even inaccessible peptide sequences reported from literature were selected.
The first case was the synthesis of PD-L1 (121-132) , a C-terminus of PD-L1 that was highly prone to form β-sheet structure. The amino acids were installed by standard coupling procedure by using HATU as coupling reagent, sequentially, the resin was subjected to washing steps. Then the resin was treated with 20%piperidine/DMF to remove the Fmoc protecting group and allowed for the next coupling cycle. During the synthesis, Ile-Thr NBD was installed by a one-pot activation and coupling protocol. As shown in the Figure 1, the installation of Ile-Thr NBD improved the quality of final products significantly. Furthermore, without dipeptides, serious amino acid deletion was observed, indicating the powerfulness of dipeptide in solving difficult SPPS issue. In the synthesis of IL-2 (125-133) , a Ser-Cys NBD was incorporated (Figure 2) . The amino acids were installed by standard coupling procedure by using HATU as coupling reagent, sequentially, the resin was subjected to washing steps. Then the resin was treated with 20%piperidine/DMF to remove the Fmoc protecting group and allowed for the next coupling cycle. During the synthesis, Ser-Cys NBD was installed by a one-pot activation and coupling protocol. Although SPPS with/without NBD both showed similar retention peak from the analytical HPLC data of the peptide, the one without using NBD showed messy mass spectrum. By contrast, with NBD insertion the mass quality of peptide was excellent. After that, synthesis of another difficult sequence, Amylin with 37 amino acids, was performed by introducing 3 NBDs during the Fmoc-SPPS (Figure 3) . The amino acids were installed by standard coupling procedure by using HATU as coupling reagent, sequentially, the resin was subjected to washing steps. Then the resin was treated with 20%piperidine/DMF to remove the Fmoc protecting group and enabled the next coupling cycle. During the synthesis, three corresponding NBDs were installed  by a one-pot activation and coupling protocol. With the installation of NBDs, the desired product could be achieved in high quality. However, no any desired product was observed in the synthesis without using NBDs. Finally, a natural HIV-suppressive factor containing 68 amino acids, RANTES which was also known as chemokine ligand 5, was chosen as an example of challenging targets with longer sequence. The amino acids were installed by standard coupling procedure by using HATU as coupling reagent, sequentially, the resin was subjected to washing steps. Then the resin was treated with 20%piperidine/DMF to remove the Fmoc protecting group and enabled the next coupling cycle. During the synthesis, three corresponding NBDs were installed by a one-pot activation and coupling protocol. As shown in Figure 4, RANTES was successfully constructed when four NBDs were introduced and up to 21%isolated yield could be achieved after HPLC purification. At the same time, the attempt to synthesized RANTES by conventional SPPS failed and only messy result was obtained, emphasizing the key role of our NBDs on preventing peptide aggregation during SPPS. Furthermore, only polystyrene-based resin was applied in all examples above, without the need to pursue modified and expensive polymer supports for difficult peptides/proteins.

Claims (31)

  1. A compound having a structure of:
    wherien:
    (i) R1 is a protected or unprotected side chain of an amino acid;
    (ii) R2 is an amine protecting group;
    (iii) R3-R6 are independently hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF3) , -NO2, unsubstituted C1-C6 alkyl, unsubsituted C1-C6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol;
    (iv) R7 and R8 are indepdently hydrogen, methyl, or other protected/unprotected side chains of amino acids;
    (v) R9 is hydrogen or a functional group suitable for protecting and/or activating a carboxylic acid group;
    (vi) R10 is hydrogen or a functional group suitable for hindering the reactivity of 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 R1 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 when R1 is a protected side chain, wherein the protection group is selected from the group consisting of a substituted or unsubstitited alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a carbonyl (e.g., carboxylic acid or carboxylic ester) , an amide, an amino, a substitute or unsubstituted aryl, a substituted or unsubstituted heteroaryl, and a heterocyclic ring, optionally wherein the protection group is tBu, Boc, Thz, Acm, Trt, Cbz, Alloc, Azido, Bn, Bz, Ac, and Pbf, or a combination thereof.
  4. The compound of any one of claims 1-3, wherein R2 is Fmoc, Cbz, Moz, Boc, Troc, Teoc, Alloc, or Voc.
  5. The compound of any one of claims 1-4, wherein R3-R6 are independently hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF3) , -NO2, unsubstituted alkyl (such as unsubstituted C1-C8 alkyl) , unsubsituted alkenyl (unsubstituted C1-C8 alkenyl) , unsubstituted alkynyl (such as unsubstituted C1-C8 alkynyl) , unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol, optionally wherein R3-R6 are independently hydrogen, hydroxyl, halogen, -CN, -CF3, -NO2, unsubstituted C1-C4 alkyl, alkoxyl, and unsubstituted aryl.
  6. The compound of any one of claims 1-5, wherein at least one of R3-R6, optionally two or more of R3-R6, is/are not hydrogen.
  7. The compound of any one of claims 1-6, wherein R9 is hydrogen, benzyl, allyl, unsubstituted C1-C4 alkyl; oris an activated ester group, such as l-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.
  8. The comound of any one of claims 1-7, wherein R10 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. A method of making the compound of Formula I:
    wherien:
    (i) R1 is a protected or unprotected side chain of an amino acid;
    (ii) R2 is an amine protecting group;
    (iii) R3-R6 are independently hydrogen, hdyroxyl, halogen, -CN, haloalkyl (such as -CF3) , -NO2, unsubstituted C1-C6 alkyl, unsubsituted C1-C6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol;
    (iv) R7 and R8 are indepdently hydrogen, methyl, or other protected/unprotected side chains of amino acids;
    (v) R9 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,
    wherein the method comprises:
    (a) reacting a protected amino acid of Formula II:

    wherein R1 and R2 are as defined above for Formula I,
    with a substituted or unsubstituted salicylaldehyde of Formula III: 
    wherein R3-R6 are as defined above for Formula I,
    to obtain an intermediate comprising a salicylaldehyde ester of Formula IV:
    (b) reacting the salicylaldehyde ester of Formula IV with serine, threonine, cysteine, or penicillamine, or an ester thereof to obtain a product comprising the compound of Formula I.
  10. The method of claim 9, wherein R1 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 R1 is a protected side chain, wherein the protection group is selected from the group consisting of a substituted or unsubstitited alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a carbonyl (e.g., carboxylic acid or carboxylic ester) , an amide, an amino, a substitute or unsubstituted aryl, a substituted or unsubstituted heteroaryl, and a heterocyclic ring, optionally  wherein the protection group is tBu, Boc, Thz, Acm, Trt, Cbz, Alloc, Azido, Bn, Bz, Ac, and Pbf, or a combination thereof.
  12. The method of any one of claims 9-11, wherein R2 is Fmoc, Cbz, Moz, Boc, Troc, Teoc, Alloc, or Voc.
  13. The method of any one of claims 9-12, wherein R3-R6 are independently hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF3) , -NO2, unsubstituted alkyl (such as unsubstituted C1-C8 alkyl) , unsubsituted alkenyl (unsubstituted C1-C8 alkenyl) , unsubstituted alkynyl (such as unsubstituted C1-C8 alkynyl) , unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol, optionally wherein R3-R6 are independently hydrogen, hydroxyl, halogen, -CN, -CF3, -NO2, unsubstituted C1-C4 alkyl, alkoxyl, and unsubstituted aryl.
  14. The method of any one of claims 9-13, wherein at least one of R3-R6, optionally two or more of R3-R6, is/are not hydrogen.
  15. The method of any one of claims 9-14, wherein step (a) is performed in the presence of a condensation agent, optionally wherein the condensation agent is HATU, PyBOP, HBTU, HCTU, COMU, TSTU, PyClock, PyOxim, EDCI, or DCC, or a combination thereof.
  16. The method of any one of claims 9-15, 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 method of any one of claims 9-16, wherein step (a) is performed in an organic solvent, optionally wherein the organic solvent is DMF, DMSO, DMAc, or NMP, or a combination thereof.
  18. The method of any one of claims 9-17, wherein step (a) is performed at room temperature for a time period ranging from 30 mins to 5 hours, preferably from 1 hour to 3 hours.
  19. The method of any one of claims 9-18, wherein the salicylaldehyde ester of Formula IV obtained in step (a) has a yield of at least 40%, at least 50%, or in a range from about 50%to about 95%.
  20. The method of any one of claims 9-19, wherein step (b) is performed 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 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 method of any one of claims 9-21, wherein step (b) is performed at room temperature for a time period ranging from 1 hour to 5 hours, preferably from 2 hours to 3 hours.
  23. The method of any one of claims 9-22, wherein the compound of Formula I obtained in step (b) has a yield of at least 40%, such as in a range from about 40%to about 95%.
  24. The method of any one of claims 9-23, further comprising purifying the intermediate to obtain the salicylaldehyde ester of Formula IV after step (a) and prior to step (b) .
  25. The method of any one of claims 9-24, further comprising purifying the product to obtain the compound of Formula I after step (b) .
  26. The method of any one of claims 9-25, further comprising:
    (c) reacting a carboxyl protected NBD with a deprotection reagent to form a deprotected NBD of Formula VI:
    (i) R1 is a protected or unprotected side chain of an amino acid;
    (ii) R2 is an amine protecting group;
    (iii) R3-R6 are independently hydrogen, hdyroxyl, halogen, -CN, haloalkyl (such as -CF3) , -NO2, unsubstituted C1-C6 alkyl, unsubsituted C1-C6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol;
    (iv) R7 and R8 are indepdently hydrogen, methyl, or other protected/unprotected side chains of amino acids;
    (v) R10 is hydrogen or a functional group suitable for hindering the reactivity of 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 9-26, further comprising:
    (d) reacting a free-acid or free phenol containing NBD with an acylation and/or activation reagent to form an acylated/activated NBD of Formula I’:
    wherien:
    (i) R1 is a protected or unprotected side chain of an amino acid;
    (ii) R2 is an amine protecting group;
    (iii) R3-R6 are independently hydrogen, hydroxyl, halogen, -CN, haloalkyl (such as -CF3) , -NO2, unsubstituted C1-C6 alkyl, unsubsituted C1-C6 alkenyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heteroalkyl, an unsubstituted cyclic group, an unsubstituted heterocyclic, an unsubstituted aralkyl, alkoxyl, amino, amido, carbonyl, or thiol;
    (iv) R7 and R8 are indepdently hydrogen, methyl, or other protected/unprotected side chains of amino acids;
    (v) R9 is hydrogen or a functional group suitable for protecting and/or activating a carboxylic acid group;
    (vi) R10 is a functional group suitable for hindering the reactivity of 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 following step (b) or immediately following 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 wherin R9 of Formula I or Formula I’ is not hydrogen.
  29. The method of claim 27 or 28, wherein step (d) is performed immediately following step (b) or immediately following step (c) , and wherein the free-acid containing NBD is the NBD of Formula I formed in step (b) or the NBD of Formula VI formed in step (c) .
  30. A method of using the compound of any one of claims 1-8 for solid phase peptide synthesis.
  31. The method of claim 30, comprising: mixing the compound with amino acids for forming the peptide.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102482320A (en) * 2009-08-12 2012-05-30 李学臣 Native chemical ligation at serine and threonine sites
CN112851761A (en) * 2019-11-12 2021-05-28 杭州多普源生物科技有限公司 Method for preparing plinacandin

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102482320A (en) * 2009-08-12 2012-05-30 李学臣 Native chemical ligation at serine and threonine sites
CN112851761A (en) * 2019-11-12 2021-05-28 杭州多普源生物科技有限公司 Method for preparing plinacandin

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ASADOLLAHI-BABOLI M., MANI-VARNOSFADERANI A.: "Molecular docking, molecular dynamics simulation, and QSAR model on potent thiazolidine-4-carboxylic acid inhibitors of influenza neuraminidase", MEDICINAL CHEMISTRY RESEARCH, BIRKHAEUSER, BOSTON., US, vol. 22, no. 4, 1 April 2013 (2013-04-01), US , pages 1700 - 1710, XP093203595, ISSN: 1054-2523, DOI: 10.1007/s00044-012-0175-y *
FREDERIC ZOLLER; ANNETTE MARKERT; PHILIPPE BARTHE; ULRIKE HEBLING; ANNETTE ALTMANN; THOMAS LINDNER; WALTER MIER; UWE HABERKORN: "A Disulfide‐Constrained Miniprotein with Striking Tumor‐Binding Specificity Developed by Ribosome Display", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, VERLAG CHEMIE, HOBOKEN, USA, vol. 52, no. 45, 20 September 2013 (2013-09-20), Hoboken, USA, pages 11760 - 11764, XP072077276, ISSN: 1433-7851, DOI: 10.1002/anie.201304603 *
FREDERIC ZOLLER; THIMON SCHWAEBEL; ANNETTE MARKERT; UWE HABERKORN; WALTER MIER: "Engineering and Functionalization of the Disulfide‐Constrained Miniprotein Min‐23 as a Scaffold for Diagnostic Application", CHEMMEDCHEM COMMUNICATIONS, WILEY-VCH, DE, vol. 7, no. 2, 23 December 2011 (2011-12-23), DE , pages 237 - 247, XP072423159, ISSN: 1860-7179, DOI: 10.1002/cmdc.201100497 *
XU YANYAN; QI CHENZE; WANG CHEN: "Mechanism, origin of diastereoselectivity and factors affecting reaction efficiency of serine/threonine ligation: A computational study", TETRAHEDRON, ELSEVIER SIENCE PUBLISHERS, AMSTERDAM, NL, vol. 76, no. 18, 17 March 2020 (2020-03-17), AMSTERDAM, NL , XP086132265, ISSN: 0040-4020, DOI: 10.1016/j.tet.2020.131143 *
XUECHEN LI, HIU YUNG LAM, YINFENG ZHANG, CHUN KEI CHAN: "Salicylaldehyde Ester-Induced Chemoselective Peptide Ligations: Enabling Generation of Natural Peptidic Linkages at the Serine/Threonine Sites", ORGANIC LETTERS, AMERICAN CHEMICAL SOCIETY, vol. 12, no. 8, 16 April 2010 (2010-04-16), pages 1724 - 1727, XP055076642, ISSN: 15237060, DOI: 10.1021/ol1003109 *

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