Compound for use as substrate for evaluating activity of γ-d-glutamyl-l-lysyl endopeptidase

Abstract

[Problem] To provide a novel compound for use as a substrate for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase and a method for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase using the compound. [Solution] The present invention relates to a compound represented by formula (1) or a stereoisomer thereof, which is useful as a substrate for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase. [Formula 1] (In formula (1), A represents formula (2) or an amino-protecting group, and Z represents a detection group which becomes detectable upon liberation.) [Formula 2] (In formula (2), B represents a hydrogen atom, an optionally substituted N-acetyl-muramoyl group, or an acyl- or amino-protecting group.)

Description

Compounds to be used as substrates for assessing the activity of gamma-D-glutamyl-L-lysyl endopeptidase

[0001] The present invention relates to a compound used as a substrate for assessing the activity of γ-D-glutamyl-L-lysyl endopeptidase, and a method for assessing the activity of γ-D-glutamyl-L-lysyl endopeptidase using the compound.

[0002] Lactic acid bacteria are known to have various functions, such as immune-stimulating and anti-inflammatory effects, and contribute to health, and cell wall polysaccharides are thought to play an important role in immune-stimulating effects.

[0003] Thus, structural analysis of cell wall polysaccharides has become important in elucidating the mechanism of action of the functionality of lactic acid bacteria. Conventionally, hot water extracts of cell walls have been used for structural analysis of cell wall polysaccharides. However, it was unclear whether hot water extracts maintained the original structure of cell wall polysaccharides.

[0004] Therefore, attention has been drawn to a method for analyzing the structure of cell wall polysaccharides using cell wall-lytic enzymes. Because enzyme treatment can be performed without high temperatures, it is expected that cell wall polysaccharides can be analyzed in their original structure.

[0005] International Publication No. 2007 / 045192 International Publication No. WO 2019 / 036433

[0006] Schleifer, KH, and Kandler, O. “Bacteriological Reviews.” American Society for Microbiology, 1972, pp. 407-477Regulski, K., et al., J Biol Chem, 2013, 288, pp. 20416-20426Munneke, 1972; S., et al., Carbohydrate Research, 2015, 414, p.1-7C. Gallo-Rodriguez et al., Carbohydrate Research, 1998, 305, p.163-170A. Babic, S. Pecar, Tetrahedron: Asymmetry. Babic, S. Pecar, Tetrahedron Letters, 2007, 48, p.4403-4405Imoto M., et al., Bull. Chem. Soc. Jpn., 1986, 59 p.3207-3212A. Nocentini et al., Bioorg. Med. Chem., 2015, June 23, p.6955-6966A. Srinivas, et al., Chem. Med. Chem., 2017, 12, p.1578-1584Wu, X., et al., Beilstein J. Org. Chem., 2011, 7, pp. 1030-1035Herve Aloysius and Longqin Hu, Chem Biol Drug Des, 2015, 86, pp. 837-843J. Tsuji, “Palladium Reagents and Catalysts,” Wiley, 2004, Chap. 4. pp. 431-5

[0007] The present inventors focused on γ-D-glutamyl-L-lysyl endopeptidase (Lc-Lys2), a cell wall-lytic enzyme of Lacticaceae Bacillus, for use in structural analysis of cell wall polysaccharides. To obtain active γ-D-glutamyl-L-lysyl endopeptidase, one possible method would be to clone γ-D-glutamyl-L-lysyl endopeptidase from Lacticaceae Bacillus, express it in Escherichia coli, and purify it. However, no method was known to accurately evaluate the activity of the produced γ-D-glutamyl-L-lysyl endopeptidase.

[0008] Therefore, an object of the present invention is to provide a novel compound that can be used as a substrate for assessing the activity of γ-D-glutamyl-L-lysyl endopeptidase, and a method for assessing the activity of γ-D-glutamyl-L-lysyl endopeptidase using the compound.

[0009] To solve the above problems, the present inventors searched for compounds that can be used to evaluate the activity of γ-D-glutamyl-L-lysyl endopeptidase (hereinafter also referred to as "Lc-Lys2"). A schematic diagram of the cell wall of the genus Lacticaceae is shown in Figure 1 (Non-Patent Documents 1 and 2). Lc-Lys2 was presumed to cleave the peptide portion of the cell wall between D-Glu or D-Gln and L-Lys.

[0010] To assess the activity of the Lc-Lys2 enzyme, we synthesized synthetic substrates containing the fluorescent substance 7-amino-4-methylcoumarin (AMC) or the chromogenic substance 4-nitroaniline (4NA) at the end of the Lc-Lys2 cleavage site, and conjugated them with MurNAc-L-Ala derivatives to enhance enzyme specificity. Specifically, we synthesized D-isoGln-MCA (MCA: 4-methylcoumarin-7-amide) and D-isoGln-pNA (pNA: p-nitroanilide) derivatives.

[0011] The specificity of these synthetic substrates for Lc-Lys2 was evaluated by measuring the AMC or 4NA released by reaction of these synthetic substrates with Lc-Lys2 using a fluorometer or UV-visible spectrophotometer. Based on these findings, the present inventors have discovered new compounds and methods that can be used to evaluate the activity of Lc-Lys2, and have completed the present invention.

[0012] The present invention provides a compound represented by the following formula (1) or a stereoisomer thereof:

[0013] ...(1) (In the above formula (1), A represents the following formula (2) or a protecting group for an amino group, and Z represents a detection group that becomes detectable when released.)

[0014] ...(2) (In the above formula (2), B represents a hydrogen atom, an N-acetyl-muramoyl group which may have a substituent, an acyl group, or an amino-protecting group.) The present invention also provides a compound or a stereoisomer thereof, wherein the amino-protecting group represented by B in the above formula (2) is an alkoxycarbonyl group, an alkenyloxycarbonyl group, or an aralkyloxycarbonyl group.

[0015] The present invention also provides a compound or a stereoisomer thereof, wherein the protecting group for the amino group represented by B in the above formula (2) is an Fmoc group (9-fluorenylmethyloxycarbonyl group), a Cbz group (benzyloxycarbonyl group), a Boc group (tert-butoxycarbonyl group), or an Alloc group (allyloxycarbonyl group).

[0016] The present invention also provides a compound or a stereoisomer thereof, wherein the optionally substituted N-acetyl-muramoyl group represented by B in the above formula (2) is an N-acetyl-1-benzylmuramoyl group or an N-acetyl-1-benzyl-4,6-benzylidenemuramoyl group.

[0017] The present invention also provides a compound or a stereoisomer thereof, wherein the acyl group represented by B in the above formula (2) is an acetyl group or a benzoyl group.

[0018] The present invention also provides a compound or a stereoisomer thereof, wherein the protecting group for the amino group represented by A in the above formula (1) is an alkoxycarbonyl group, an alkenyloxycarbonyl group or an aralkyloxycarbonyl group.

[0019] The present invention also provides a compound or a stereoisomer thereof, wherein the protecting group for the amino group represented by A in the above formula (1) is an Fmoc group (9-fluorenylmethyloxycarbonyl group), a Cbz group (benzyloxycarbonyl group), a Boc group (tert-butoxycarbonyl group), or an Alloc group (allyloxycarbonyl group).

[0020] The present invention also provides a compound or a stereoisomer thereof, wherein A in the above formula (1) is the following formula (3), (4), (5), (6), (7), (8) or (9):

[0021] ...(3) (In the above formula (3), Ac represents an acetyl group, and Bn represents a benzyl group.)

[0022] ...(4) (In the above formula (4), Ac represents an acetyl group, Bn represents a benzyl group, and Ph represents a phenyl group.)

[0023] ...(5)

[0024] ...(6)

[0025] ... (7)

[0026] ...(8)

[0027] ...(9)

[0028] The present invention also provides a compound or a stereoisomer thereof, wherein Z in the above formula (1) represents a chromophore that emits color upon liberation or a fluorescent group that emits fluorescence upon liberation.

[0029] The present invention also provides a compound or a stereoisomer thereof, wherein Z in the above formula (1) is the following formula (10), (11), (12), (13), (14) or (15):

[0030] ...(10)

[0031] ...(11)

[0032] ...(12)

[0033] ...(13)

[0034] ...(14)

[0035] ...(15)

[0036] The present invention also provides the above compound or a stereoisomer thereof, which is used as a substrate for assessing the activity of γ-D-glutamyl-L-lysyl endopeptidase.

[0037] The present invention also provides a method for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase, which comprises the steps of reacting γ-D-glutamyl-L-lysyl endopeptidase with a compound represented by the following formula (1) or a stereoisomer thereof, and detecting the liberated Z:

[0038] ...(1) (In the above formula (1), A represents the following formula (2) or a protecting group for an amino group, and Z represents a detection group that becomes detectable when released.)

[0039] ...(2) (In the above formula (2), B represents a hydrogen atom, an N-acetyl-muramoyl group which may have a substituent, an acyl group, or a protecting group for an amino group.)

[0040] The present invention can provide novel compounds that can be used as substrates for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase. The present invention also provides a method for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase using the compounds. Therefore, the present invention can be used to select γ-D-glutamyl-L-lysyl endopeptidases that have activity useful for structural analysis of cell wall polysaccharides of lactic acid bacteria.

[0041] A schematic diagram of the cell wall of Bacillus lactis is shown.

[0042] Terms used in this specification are defined below.

[0043] The term "alkyl group" refers to a straight-chain or branched-chain alkyl group having 1 to 20 carbon atoms. Examples of the alkyl group include straight-chain or branched-chain lower alkyl groups having 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, and 2-ethylbutyl, and straight-chain or branched-chain alkyl groups having 7 to 20 carbon atoms, such as heptyl, octyl, nonyl, and decyl. The alkyl group may partially form a ring.

[0044] The term "alkoxy group" refers to an alkoxy group having the above alkyl group. Examples of alkoxy groups include methyloxy (methoxy), ethyloxy (ethoxy), n-propyloxy (n-propoxy), isopropoxy, n-butyloxy (n-butoxy), isobutoxy, s-butoxy, tert-butoxy, and n-pentyloxy (n-pentoxy).

[0045] The term "acyl group" includes aliphatic acyl groups and aromatic acyl groups. Examples of the aliphatic acyl group include formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyloctanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, 1-methylpentadecanoyl, 14-methylpentadecanoyl, 13,13-dimethyltetradecanoyl, heptadecanoyl, 15-methylpentadecanoyl, 16-methylpentadecanoyl, 17-methylpentadecanoyl, 18-methylpentadecanoyl, 19-methylpentadecanoyl, 20-methylpentadecanoyl, 21-methylpentadecanoyl, 22-methylpentadecanoyl, 23-methylpentadecanoyl, 24-methylpentadecanoyl, 25-methylpentadecanoyl, 26-methylpentadecanoyl, 27-methylpentadecanoyl, 28-methylpentadecanoyl, 29-methylpentadecanoyl, 30-methylpentadecanoyl, 31-methylpentadecanoyl, 32-methylpentadecanoyl, 33-methylpentadecanoyl, 34-methylpentadecanoyl, 35-methylpentadecanoyl, 36-methylpentadecanoyl, 37-methylpentadecanoyl, 38-methylpentadecanoyl, 39-methylpentadecanoyl, 40-methylpentadecanoyl, 41-methylpentadecanoyl, 42-methylpentadecanoyl, 4 alkylcarbonyl groups such as -methylhexadecanoyl, octadecanoyl, 1-methylheptadecanoyl, nonadecanoyl, eicosanoyl, and henaicosanoyl; carboxylated alkylcarbonyl groups such as succinoyl, glutaroyl, and adipoyl; halogeno-lower alkylcarbonyl groups such as chloroacetyl, dichloroacetyl, trichloroacetyl, and trifluoroacetyl; lower alkoxy-lower alkylcarbonyl groups such as methoxyacetyl; and unsaturated alkylcarbonyl groups such as (E)-2-methyl-2-butenoyl. Examples of aromatic acyl groups include arylcarbonyl groups such as benzoyl, α-naphthoyl, and β-naphthoyl; halogenoarylcarbonyl groups such as 2-bromobenzoyl and 4-chlorobenzoyl; lower alkylated arylcarbonyl groups such as 2,4,6-trimethylbenzoyl and 4-toluoyl; lower alkoxylated arylcarbonyl groups such as 4-anisoyl; carboxylated arylcarbonyl groups such as 2-carboxybenzoyl, 3-carboxybenzoyl, and 4-carboxybenzoyl; nitrated arylcarbonyl groups such as 4-nitrobenzoyl and 2-nitrobenzoyl; lower alkoxycarbonylated arylcarbonyl groups such as 2-(methoxycarbonyl)benzoyl; and arylated arylcarbonyl groups such as 4-phenylbenzoyl.

[0046] The term "aralkyl group" refers to an alkyl group having 1 to 6 carbon atoms substituted with an aryl group. Examples of aralkyl groups include benzyl, α-naphthylmethyl, β-naphthylmethyl, indenylmethyl, phenanthrenylmethyl, anthracenylmethyl, diphenylmethyl, triphenylmethyl, α-naphthyldiphenylmethyl, 9-anthrylmethyl, 4-methylbenzyl, 2,4,6-trimethylbenzyl, 3,4,5-trimethylbenzyl, 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl, 4-cyanobenzyl, 1-phenethyl, 2-phenethyl, 1-naphthylethyl, 2-naphthylethyl, 1-phenylpropyl, 2-phenylpropyl, 3-phenylpropyl, 1-naphthylpropyl, 2-naphthylpropyl, and the like. Examples of alkyl groups include propyl, 3-naphthylpropyl, 1-phenylbutyl, 2-phenylbutyl, 3-phenylbutyl, 4-phenylbutyl, 1-naphthylbutyl, 2-naphthylbutyl, 3-naphthylbutyl, 4-naphthylbutyl, 1-phenylpentyl, 2-phenylpentyl, 3-phenylpentyl, 4-phenylpentyl, 5-phenylpentyl, 1-naphthylpentyl, 2-naphthylpentyl, 3-naphthylpentyl, 4-naphthylpentyl, 5-naphthylpentyl, 1-phenylhexyl, 2-phenylhexyl, 3-phenylhexyl, 4-phenylhexyl, 5-phenylhexyl, 6-phenylhexyl, 1-naphthylpentyl, 2-naphthylpentyl, 3-naphthylpentyl, 4-naphthylpentyl, 5-naphthylpentyl and 6-naphthylpentyl.

[0047] [Compound] The present invention provides a compound represented by the following formula (1) or a stereoisomer thereof:

[0048] ...(1)

[0049] In the above formula (1), A represents the following formula (2) or a protecting group for an amino group.

[0050] ...(2)

[0051] In the above formula (2), B represents a hydrogen atom, an N-acetyl-muramoyl group which may have a substituent, an acyl group, or a protecting group for an amino group.

[0052] When B in the above formula (2) represents an N-acetylmuramoyl group which may have a substituent, N-acetylmuramic acid (MurNAc) which may have a substituent is bound to B in formula (2) via an amide bond. The N-acetylmuramoyl group represented by B may have one or more substituents, or may have no substituent. The substituent may be, for example, an alkyl group having 1 to 6 carbon atoms, an aralkyl group having 7 to 10 carbon atoms, or a benzylidene group. The substituent may also be an alcohol-protecting group, such as a benzyl group (Bn), a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, a p-methoxybenzyl group, or a benzylidene group. The N-acetylmuramoyl group represented by B may have, for example, the 4-OH and 6-OH groups protected by the benzylidene carbon. B can preferably be an N-acetyl-1-benzylmuramoyl group or an N-acetyl-1-benzyl-4,6-benzylidenemuramoyl group.

[0053] The acyl group represented by B in the above formula (2) can be, for example, an aliphatic acyl group or an aromatic acyl group having 1 to 10 carbon atoms. The acyl group represented by B is preferably an acetyl group or a benzoyl group.

[0054] The amino-protecting group represented by B in the above formula (2) is not particularly limited, and examples thereof include amide-type protecting groups, phthalimide-type protecting groups, carbamate-type protecting groups, and sulfonamide-type protecting groups. The amino-protecting group represented by B is preferably a carbamate-type protecting group, such as an alkoxycarbonyl group, an alkenyloxycarbonyl group, and an aralkyloxycarbonyl group. Examples of carbamate-type protecting groups include the Fmoc group (9-fluorenylmethyloxycarbonyl group), the Cbz group (benzyloxycarbonyl group), the Boc group (tert-butoxycarbonyl group), the Alloc group (allyloxycarbonyl group), the Aoc group (tert-amyloxycarbonyl group), and the Troc group (2,2,2-triethoxycarbonyl group). Examples of amide-type protecting groups include a formyl group, an acetyl group (Ac), and a trifluoroacetyl group (TFA). Examples of phthalimide-type protecting groups include a phthaloyl group (Phth). Examples of sulfonamide-type protecting groups include a 3-nitro-2-pyridinesulfenyl group (Npys), a 2-nitrobenzenesulfonyl group (Ns), and a (2-trimethylsilyl)-ethanesulfonyl group (SES). The amino-protecting group represented by B is preferably an alkoxycarbonyl group, an alkenyloxycarbonyl group, or an aralkyloxycarbonyl group, more preferably an Fmoc group, a Cbz group, a Boc group, or an Alloc group, and even more preferably an Fmoc group.

[0055] In the above formula (1), the protecting group for the amino group represented by A is not particularly limited, and examples thereof include amide-type protecting groups, phthalimide-type protecting groups, carbamate-type protecting groups, and sulfonamide-type protecting groups. The protecting group for the amino group represented by A is preferably a carbamate-type protecting group, such as an alkoxycarbonyl group, an alkenyloxycarbonyl group, and an aralkyloxycarbonyl group. Examples of carbamate-type protecting groups include the Fmoc group (9-fluorenylmethyloxycarbonyl group), the Cbz group (benzyloxycarbonyl group), the Boc group (tert-butoxycarbonyl group), the Alloc group (allyloxycarbonyl group), the Aoc group (tert-amyloxycarbonyl group), and the Troc group (2,2,2-triethoxycarbonyl group). Examples of amide-type protecting groups include a formyl group, an acetyl group (Ac), and a trifluoroacetyl group (TFA). Examples of phthalimide-type protecting groups include a phthaloyl group (Phth). Examples of sulfonamide-type protecting groups include a 3-nitro-2-pyridinesulfenyl group (Npys), a 2-nitrobenzenesulfonyl group (Ns), and a (2-trimethylsilyl)-ethanesulfonyl group (SES). The amino-protecting group represented by A is preferably an alkoxycarbonyl group, an alkenyloxycarbonyl group, or an aralkyloxycarbonyl group, more preferably an Fmoc group, a Cbz group, a Boc group, or an Alloc group, and even more preferably an Fmoc group.

[0056] A in the above formula (1) can be, for example, the following formula (3), (4), (5), (6), (7), (8) or (9).

[0057] ...(3) (In the above formula (3), Ac represents an acetyl group, and Bn represents a benzyl group.)

[0058] ...(4) (In the above formula (4), Ac represents an acetyl group, Bn represents a benzyl group, and Ph represents a phenyl group.)

[0059] ...(5)

[0060] ...(6)

[0061] ... (7)

[0062] ...(8)

[0063] ...(9)

[0064] In the above formula (1), Z represents a detection group that becomes detectable upon release. Z can be, for example, a chromophore that develops color upon release or a fluorescent group that emits fluorescence upon release. Z can be, for example, a detection group that is colorless or non-fluorescent before cleavage by the enzyme, but becomes colored or fluorescent upon release following cleavage by the enzyme.

[0065] In the present invention, the chromophore represented by Z is not particularly limited as long as it is a compound that emits color upon liberation, and can be, for example, a paranitroaniline derivative. Furthermore, in the present invention, the fluorescent group represented by Z is not particularly limited as long as it is a compound that emits fluorescence upon liberation, and can be, for example, a coumarin derivative or a fluorescein derivative. Z is preferably represented by the following formula (10), (11), (12), (13), (14), or (15), and particularly preferably represented by (10), (11), or (12).

[0066] ...(10)

[0067] ...(11)

[0068] ...(12)

[0069] ...(13)

[0070] ...(14)

[0071] ...(15)

[0072] Z can be, for example, methylcoumarinamide (MCA) represented by the above formula (10) or paranitroanilide (pNA) represented by the above formula (12).

[0073] When aminomethylcoumarin (AMC) is released from a substrate containing MCA by an enzymatic reaction, it emits strong fluorescence, and the enzymatic reaction can be detected by detecting this change in fluorescence using a fluorometer or the like.

[0074] When 4-nitroaniline is released from a substrate containing pNA by an enzymatic reaction, it turns yellow, and the enzymatic reaction can be detected by detecting this color change using a UV-visible spectrophotometer or the like.

[0075] As used herein, the term "stereoisomer" refers to compounds that have the same chemical structure but differ in the arrangement of atoms or groups in space. When multiple stereoisomers exist, the compound of the present invention may be a mixture of two or more stereoisomers.

[0076] The compound of the present invention can preferably be benzyl-α-MurNAc-L-Ala-D-isoGln-MCA (compound 63B) or benzyl-α-MurNAc-L-Ala-D-isoGln-pNA (compound 64B).

[0077] [Substrate for Evaluating γ-D-Glutamyl-L-lysyl Endopeptidase Activity] The compounds of the present invention can be used as substrates for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase. The γ-D-glutamyl-L-lysyl endopeptidase can be, but is not limited to, γ-D-glutamyl-L-lysyl endopeptidase (Lc-Lys2) from lactic acid bacteria, particularly the genus Lacticaseibacillus. The genus Lacticaseibacillus includes, for example, Lacticaseibacillus casei, Lacticaseibacillus paracasei, and Lacticaseibacillus rhamnosus.

[0078] Lc-Lys2 is a cell wall lytic enzyme of the genus Lacticaceae. Lc-Lys2 has the activity of cleaving the D-isoGln site of the peptide portion of the cell wall. The compounds of the present invention contain Z, which is released and detectable upon cleavage by Lc-Lys2. Therefore, the activity of Lc-Lys2 can be evaluated by detecting Z.

[0079] [Method for assessing the activity of γ-D-glutamyl-L-lysyl endopeptidase] The present invention also provides a method for assessing the activity of γ-D-glutamyl-L-lysyl endopeptidase.

[0080] In the present invention, the "activity" of γ-D-glutamyl-L-lysyl endopeptidase means the activity of cleaving the D-isoGln site in the peptide portion of the cell wall of Lacticaceobacterium sp.

[0081] In the present invention, "evaluating activity" includes not only evaluating the presence or absence of substrate-cleaving activity but also evaluating the strength of the substrate-cleaving activity. In the present invention, "activity" can be expressed, for example, by the rate of substrate cleavage, the amount and proportion of products (liberated substances) generated by substrate cleavage, the time required for the substrate to be cleaved to a predetermined extent, etc.

[0082] The method of the present invention comprises the steps of reacting γ-D-glutamyl-L-lysyl endopeptidase with the compound of the present invention or a stereoisomer thereof, and detecting the liberated Z. In the method of the present invention, the compound or a stereoisomer thereof is used as a substrate for γ-D-glutamyl-L-lysyl endopeptidase.

[0083] As used herein, the expression "reacts" between an enzyme and a substrate means that the enzyme catalyzes the conversion of the substrate into one or more additional products. In the present invention, the expression "reacts" between γ-D-glutamyl-L-lysyl endopeptidase and a compound of the present invention means that γ-D-glutamyl-L-lysyl endopeptidase liberates the detection group represented by Z in formula (1) above from the compound of the present invention. The reaction can be achieved by contacting the enzyme with the substrate.

[0084] In the present invention, "detecting" Z includes detecting the presence or absence of free Z, and detecting changes such as increases and decreases in the amount and / or proportion of free Z.

[0085] The γ-D-glutamyl-L-lysyl endopeptidase used in the method of the present invention may be an enzyme purified from Escherichia coli or lactic acid bacteria, or may be an extract from bacteria that may contain γ-D-glutamyl-L-lysyl endopeptidase. Furthermore, the γ-D-glutamyl-L-lysyl endopeptidase used in the method of the present invention does not necessarily have to be an active enzyme, and may be a candidate substance whose function as γ-D-glutamyl-L-lysyl endopeptidase is unknown.

[0086] In the step of reacting γ-D-glutamyl-L-lysyl endopeptidase with the above-mentioned compound or its stereoisomer, the reaction can be carried out, for example, by contacting the γ-D-glutamyl-L-lysyl endopeptidase with the above-mentioned compound in a reaction solution for a certain period of time. The reaction temperature is not particularly limited, but can be, for example, 20 to 50°C, preferably 30 to 40°C, and more preferably 37°C. The reaction time is not particularly limited, and can be set appropriately between 5 minutes and 24 hours.

[0087] As used herein, "contacting" an enzyme with a substrate means bringing the enzyme and substrate close enough to each other so that they can catalyze. "Contacting" an enzyme with a substrate includes, for example, mixing the enzyme and substrate and adding the enzyme to the substrate. For example, the enzyme and substrate can be contacted by adding them to a reaction solution and mixing them by stirring or the like.

[0088] The reaction solution is not particularly limited, but for example, phosphate buffer and acetate buffer can be used.

[0089] In the step of detecting released Z, the presence, amount, and / or proportion of Z can be measured using an instrument capable of detecting Z, depending on the type of Z. When Z is a chromophore that emits color upon release, the presence, amount, and / or proportion of released Z can be measured using an ultraviolet-visible spectrophotometer or the like. When Z is a fluorescent group that emits fluorescence upon release, the presence, amount, and / or proportion of released Z can be measured using a fluorometer or the like.

[0090] In the methods of the present invention, the γ-D-glutamyl-L-lysyl endopeptidase used may be evaluated as having activity if the presence of Z is detected in the step of detecting released Z. Alternatively, the theoretical amount of Z produced may be calculated in advance, and the γ-D-glutamyl-L-lysyl endopeptidase used may be evaluated as having activity if the ratio (%) of the detected amount of Z to this theoretical amount exceeds a predetermined threshold. The threshold is not particularly limited, and can be set to any value, such as 6%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In the methods of the present invention, the activity of the γ-D-glutamyl-L-lysyl endopeptidase used may also be evaluated based on the amount and / or ratio of released Z.

[0091] [Production of Compounds] The compounds of the present invention or their stereoisomers can be produced by the methods described below, the methods described in the Examples, and methods modified therefrom. The reaction conditions can be optimized as appropriate depending on the compounds and solvents used.

[0092] (Scheme 1: Synthesis of benzyl-4,6-O-benzylidene-α-MurNAc-L,D-Ala-OH (Compounds 7 and 8)) Benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-OH (Compound 7) and benzyl-4,6-O-benzylidene-α-MurNAc-D-Ala-OH (Compound 8) can be produced according to Scheme 1 below.

[0093]

[0094] Benzyl 2-acetamido-2-deoxy-α-D-glucopyranoside (Compound 2) can be obtained by boiling N-acetyl-D-glucosamine (Compound 1) with benzyl alcohol (BnOH) and p-toluenesulfonic acid monohydrate (p-TSA·HO) in toluene under reflux (see Non-Patent Document 3).

[0095] Next, compound 2 is heated with benzaldehyde dimethyl acetal and p-TSA·HO in N,N-dimethylformamide (DMF) and dehydrated, and the hydroxyl groups at the 4 and 6 positions are benzylidene-protected to give benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-α-D-glucopyranoside (compound 3) (see Non-Patent Documents 4-6).

[0096] Next, (S)-(-)-2-chloropropionic acid is introduced into the 3-hydroxyl group of compound 3 using NaH as a base in DMF solvent under an argon atmosphere to synthesize 2-(2-(R)-(2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionic acid (benzyl-4,6-O-benzylidene-α-MurNAc) (compound 4) (see Patent Document 1 and Non-Patent Documents 4 to 7).

[0097] Protected MurNAc (compound 4) was condensed with L-alanine methyl ester hydrochloride or D-alanine methyl ester hydrochloride using N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) in the presence of N-methylmorpholine in DMF to give (S)-methyl 2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionamido)propionate (benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-OMe) (Compound 5) and (R)-methyl 2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionamido)-propionate (benzyl-4,6-O-benzylidene-α-MurNAc-D-Ala-OMe) (Compound 6) can be synthesized (see Non-Patent Documents 4-6).

[0098] Compounds 5 and 6 were hydrolyzed with 0.5 mol / L KOH in MeOH solvent to give (S)-2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionamido)propionic acid (benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-OH) (Compound 7) and (R)-2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionamido)propionic acid (benzyl-4,6-O-benzylidene-α-MurNAc-D-Ala-OH) (Compound 8).

[0099] (Scheme 2: Synthesis of D-Glu-MCA, D-Gln-MCA, D-isoGln-MCA, and D-isoGln-pNA Derivatives) D-Glu-MCA, D-Gln-MCA, D-isoGln-MCA, and D-isoGln-pNA derivatives can be synthesized according to Scheme 2 below.

[0100]

[0101] Sodium hydroselenide (NaSeH) can be prepared fresh from selenium (powder; Se) and sodium borohydride (NaBH4) in isopropanol (IPA). 7-Azido-4-methylcoumarin (compound 10) (see Non-Patent Documents 8-9) and 1-azido-4-nitrobenzene (compound 22) can be prepared from AMC and 4-nitroaniline hydrochloride.

[0102] tert-Butoxycarbonyl (Boc)-D-Glu(OBn)-OH (Compound 9), 9-fluorenylmethoxycarbonyl (Fmoc)-D-Glu(OAll)-OH (Compound 12), Fmoc-D-Glu(OH)-OAll (Compound 14), Boc-D-Gln-OH (Compound 16), Fmoc-D-Gln-OH (Compound 18), and Fmoc-D-isoGln-OH (Compound 20) were reacted with isopropyl chloroformate (toluene solution) in THF in the presence of N-methylpiperidine to give mixed acid anhydrides, which were then reacted with freshly prepared sodium hydroselenide (NaSeH) to give the selenium compounds. The nido intermediate can be reacted with 7-azido-4-methylcoumarin (Compound 10) and 1-azido-4-nitrobenzene (Compound 22) to synthesize Boc-D-Glu(OBn)-MCA (Compound 11), Fmoc-D-Glu(OAll)-MCA (Compound 13), Fmoc-D-Glu(MCA)-OAll (Compound 15), Boc-D-Gln-MCA (Compound 17), Fmoc-D-Gln-MCA (Compound 19), Fmoc-D-isoGln-MCA (Compound 21), and Fmoc-D-isoGln-pNA (Compound 23) (see Non-Patent Documents 10 and 11).

[0103] The Boc-protected compound (compound 11) can be treated with trifluoroacetic acid (TFA) in CHCl to give the Boc-deprotected HD-Glu(OBn)-MCA·TFA (compound 24), which can then be treated with aqueous NaHCO to give the free base (compound 25).

[0104] The Boc-protected compound (compound 17) can be reacted in TFA / CH2Cl2 / H2O at room temperature and treated with Amberlyst® A-26 to give HD-Gln-MCA (compound 26) as a free base.

[0105] Alternatively, the Fmoc-protected derivatives (compounds 13, 15, 19, 21, and 23) can be treated with 20% piperidine / DMF to give the free bases HD-Glu(OAll)-MCA (compound 28) (All; allyl), HD-Glu(MCA)-OAll (compound 27), HD-Gln-MCA (compound 26), HD-isoGln-MCA (compound 30), and HD-isoGln-pNA (compound 31). Upon Fmoc removal from compound 13, hydrolysis of the allyl ester occurs, yielding HD-Glu(OH)-MCA (compound 29) along with the free base (compound 28).

[0106] (Scheme 3: Synthesis of HL-Ala-D-Glu-MCA, HL-Ala-D-Gln-MCA, HL-Ala-D-isoGln-MCA, and HL-Ala-D-isoGln-pNA) HL-Ala-D-Glu-MCA, HL-Ala-D-Gln-MCA, HL-Ala-D-isoGln-MCA, and HL-Ala-D-isoGln-pNA can be synthesized according to Scheme 3 below.

[0107]

[0108] Boc-L-Ala-OH (Compound 32) can be reacted with isopropyl chloroformate in the presence of N-methylpiperidine in THF to give the mixed acid anhydride, which can then be reacted with HD-Glu(OBn)-MCA (Compound 25) and HD-Gln-MCA (Compound 26) to give Boc-L-Ala-D-Glu(OBn)-MCA (Compound 35) and Boc-L-Ala-D-Gln-MCA (Compound 36).

[0109] In addition, Fmoc-L-Ala-OH hydrate (Compound 33) was condensed with HD-Glu(MCA)-OAll (Compound 27), HD-Gln-MCA (Compound 26), HD-isoGln-MCA (Compound 30), and HD-isoGln-pNA (Compound 31) in the presence of 1-hydroxybenzotriazole monohydrate (HOBt·HO) in DMF using N,N-diisopropylethylamine (DIEA) as a base and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) as a condensing agent to give Fmoc-L-Ala-D-Glu(MCA)-OAll (Compound 37), Fmoc-L-Ala-D-Gln-MCA (Compound 38), Fmoc-L-Ala-D- isoGln-MCA (compound 39) and Fmoc-L-Ala-D-isoGln-pNA (compound 40) can be obtained.

[0110] The Boc-protected product (compound 35) can be deprotected using TFA in CHCl to give HL-Ala-D-Glu(OBn)-MCA·TFA (compound 41). The Fmoc-protected products (compounds 37, 38, 39, and 40) can be treated with 20% piperidine / DMF to give HL-Ala-D-Glu(MCA)-OAll (compound 42), HL-Ala-D-Gln-MCA (compound 44), HL-Ala-D-isoGln-MCA (compound 45), and HL-Ala-D-isoGln-pNA (compound 46).

[0111] During Fmoc removal of compound 37, hydrolysis of the allyl ester occurs, yielding HL-Ala-D-Glu(MCA)-OH (compound 43) as the main product. Compound 45 can also be converted to its hydrochloride salt by treatment with 5-10% HCl / MeOH.

[0112] HL-Ala-D-isoGln-pNA (compound 46) can be treated with acetyl chloride and benzoyl chloride in the presence of triethylamine (TEA) in CHCl to give Ac-L-Ala-D-isoGln-pNA (compound 47) and Bz-L-Ala-D-isoGln-pNA (compound 48).

[0113] (Scheme 4: Synthesis of D-Glu(OH)-MCA Derivative (Debenzylation)) A D-Glu(OH)-MCA derivative can be synthesized by debenzylation according to the following Scheme 4.

[0114]

[0115] Boc-D-Glu(OBn)-MCA (Compound 11), Boc-L-Ala-D-Glu(OBn)-MCA (Compound 35), HL-Ala-D-Glu(OBn)-MCA·TFA (Compound 41), and HD-Glu(OBn)-MCA·TFA (Compound 24) were hydrogenolyzed in EtOH under a hydrogen atmosphere using 5% Pd / C as a catalyst to give debenzylated Boc-D-Glu(OH)-MCA (Compound 50), Boc-L-Ala-D-Glu(OH)-MCA (Compound 51), HL-Ala-D-Glu(OH)-MCA·TFA (Compound 52), and HD-Glu(OH)-MCA·TFA (Compound 53).

[0116] (Scheme 5: Synthesis of benzyl-4,6-O-benzylidene-α-MurNAc-L, D-Ala-D-Glu-MCA, -D-Gln-MCA, -D-isoGln-MCA, and -D-isoGln-pNA derivatives) Benzyl-4,6-O-benzylidene-α-MurNAc-L, D-Ala-D-Glu-MCA, -D-Gln-MCA, -D-isoGln-MCA, and -D-isoGln-pNA derivatives can be synthesized according to Scheme 5 below.

[0117]

[0118] The protected MurNAc-L-Ala-D-Glu-MCA, protected MurNAc-L-Ala-D-Gln-MCA, protected MurNAc-L-Ala-D-isoGln-MCA, and protected MurNAc-L-Ala-D-isoGln-pNA derivatives can be synthesized via two synthetic routes.

[0119] Benzyl-4,6-O-benzylidene-α-MurNAc (compound 4) was condensed with HL-Ala-D-Glu(MCA)-OAll (compound 42), HL-Ala-D-Gln-MCA (compound 44), and HL-Ala-D-isoGln-pNA (compound 46) in DMF using N-methylmorpholine as a base and HBTU as a condensing agent (Method a) to synthesize benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Glu(MCA)-OAll (compound 54), benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Gln-MCA (compound 55), and benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-isoGln-pNA (compound 56).

[0120] In addition, benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-OH (Compound 7) was condensed with HD-Glu(OAll)-MCA (Compound 28), HD-Glu(MCA)-OAll (Compound 27), HD-isoGln-MCA (Compound 30), and HD-isoGln-pNA (Compound 31) in DMF in the presence of HOBt·HO using DIEA as a base and EDCI as a condensing agent. By combining these compounds (Method b), benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Glu(OAll)-MCA (Compound 57), benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Glu(MCA)-OAll (Compound 54), and benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-isoGln-MCA (Compound 58) can be obtained.

[0121] Condensation of benzyl-4,6-O-benzylidene-α-MurNAc-D-Ala-OH (compound 8) with compound 31 by method b gives benzyl-4,6-O-benzylidene-α-MurNAc-D-Ala-D-isoGln-pNA (compound 59). Condensation of compound 7 with compound 31 by method a gives benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-isoGln-pNA (compound 56).

[0122] (Scheme 6: Synthesis of benzyl-α-MurNAc-Ala-D-Glu-MCA, -D-Gln-MCA, -D-isoGln-MCA, and -D-isoGln-pNA derivatives) Benzyl-α-MurNAc-Ala-D-Glu-MCA, -D-Gln-MCA, -D-isoGln-MCA, and -D-isoGln-pNA derivatives can be synthesized by removing the 4,6-benzylidene and allyl groups according to Scheme 6 below.

[0123]

[0124] Benzyl-4,6-O-benzylidene-α-MurNAc derivatives (compounds 55, 57, 54, 58, 56, and 59) can be debenzylideneated by heating in 75% AcOH to give benzyl-α-MurNAc-L-Ala-D-Gln-MCA (compound 60), benzyl-α-MurNAc-L-Ala-D-Glu(OAll)-MCA (compound 61), benzyl-α-MurNAc-L-Ala-D-Glu(MCA)-OAll (compound 62), benzyl-α-MurNAc-L-Ala-D-isoGln-MCA (compound 63), benzyl-α-MurNAc-L-Ala-D-isoGln-pNA (compound 64), and benzyl-α-MurNAc-D-Ala-D-isoGln-pNA (compound 65).

[0125] The allyl-protected derivatives (compounds 61 and 62) can be deallylated using Pd(OAc)2-PPh3 catalyst in the presence of AcOH and 4-methylmorpholine as a nucleophile in CH3CN-HO (see Patent Document 2 and Non-Patent Document 12) to give benzyl-α-MurNAc-L-Ala-D-Glu(OH)-MCA (compound 66) and benzyl-α-MurNAc-L-Ala-D-Glu(MCA)-OH (compound 67).

[0126] In the following examples, commercially available reagents were used in the reactions without further purification.

[0127] The following abbreviations are used in the present specification and examples: 4NA 4-nitroaniline AcOEt ethyl acetate AcOH acetic acid AMC 7-amino-4-methylcoumarin Boc tert-butoxycarbonyl DIEA N,N-diisopropylethylamine DMF N,N-dimethylformamide DMSO dimethyl sulfoxide EDCI 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide eq. molar equivalent EtOH ethanol Fmoc 9-fluorenylmethoxycarbonyl HBTU O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HOBt hydroxybenzotriazole IPA isopropanol IPE isopropyl ether KOH potassium hydroxide LCMS liquid chromatography mass spectrometry Ph phenyl pNA p-nitroanilide p-TSA·H2O p-toluenesulfonic acid monohydrate Mass mass MeOH methanol NaSeH sodium hydroselenide n-Hex. normal hexane NMR nuclear magnetic resonance Pd / C palladium on carbon quant. quantitative yield THF tetrahydrofuran TFA trifluoroacetic acid TLC thin layer chromatography

[0128] The progress of the synthesis reaction was monitored by thin layer chromatography (TLC) (Merck TLC Silica gel 60 F 254 The analysis was carried out on a glass plate. Anisaldehyde was used for color development. Silica gel column chromatography was carried out using a flash automated purification system (Biotage Isolera One). The columns were Biotage® SNAP Cartridge KP-Sil 10g and SNAP Ultra 10g (HP-Sphere). TH 25μm), SNAP Ultra 25g (HP-Sphere TH 25μm), SNAP Ultra 50g (HP-Sphere TH25 μm), Sfar Silica HC D 25 g (High Capacity Duo 20 μm), Sfar Silica HC D 50 g (High Capacity Duo 20 μm), Sfar Silica HC D 100 g (High Capacity Duo 20 μm), Sfar Silica D 25 g (Duo 60 μm), or Shoko Science Purif-Pack (registered trademark)-EX SI-25 μm, SIZE 200 was used as appropriate.

[0129] 1 H nuclear magnetic resonance spectra were measured using a JEOL JNM-ECX-400P (400 MHz). Chemical shifts were measured using tetramethylsilane (0.00 ppm) as the internal standard in deuterated chloroform and deuterated dimethyl sulfoxide. Liquid chromatography-mass spectrometry (LCMS) spectra were measured using a Waters Acqity UPLC H-Class system [Column: Acquity UPLC BEH C18 61.7 μm 2.1*50 mm, Mobile phase: 0.1% HCOH (A) / CHCN (B), Flow rate: 0.7 mL / min, Gradient: 0 min (A: 91%) - 3.2 min (A: 19%) - 3.7 min (A: 19 > B: 91) - 5 min (B: 91)].

[0130] (Production of γ-D-glutamyl-L-lysyl endopeptidase) The γ-D-glutamyl-L-lysyl endopeptidase used in this example was Lc-Lys2 from Lacticaseibacillus paracasei strain BL23.

[0131] ​The gene encoding the cell wall lytic enzyme Lc-Lys2 (γ-D-glutamyl-L-lysyl endopeptidase) derived from an endogenous phage was cloned from Lacticaseibacillus paracasei BL23 strain and ligated into an E. coli expression vector to express the enzyme in E. coli (Lc-Lys2). Lc-Lys2 was purified by dissolving inclusion bodies in the insoluble fraction with a denaturant, and active enzyme was produced by normalizing the protein folding using the refolding method.

[0132] Example 1: Synthesis of synthetic substrates Each compound was synthesized by the following method.

[0133] (Synthesis of benzyl 2-acetamido-2-deoxy-α-D-glucopyranoside (compound 2))

[0134]

[0135] N-acetyl-D-glucosamine (compound 1) (25.0 g, 113 mmol) and p-TSA·HO (1.9 g, 10 mmol) were suspended in toluene (300 mL) and benzyl alcohol (180 mL). The suspension was attached to a Dean-Stark apparatus and boiled under reflux for 3 hours. The reaction mixture was cooled to room temperature, and NaHCO3 (1.26 g, 15 mmol) dissolved in HO (15 mL) was added. The toluene was distilled off under reduced pressure. The residue (black liquid) was cooled to room temperature, and AcOEt (120 mL) and n-Hexane (620 mL) were added and stirred. The precipitated light brown solid was collected by filtration and suspended in a mixture of n-Hexane (240 mL) and AcOEt (120 mL). The mixture was then washed with stirring.

[0136] The suspension was filtered, washed with n-Hex, and recrystallized from IPA (150 mL). The precipitate was filtered, washed with cold IPA (45 mL) and isopropyl ether (IPE; 50 mL x 2), and dried (50 °C, reduced pressure) to give 15.17 g of crude compound as a light brown solid. The crude material was dissolved in 20% MeOH / CHCl3, silica gel (FL100D; 50 g) was added, and the mixture was concentrated to dryness under reduced pressure. The mixture was purified by silica gel column chromatography (5% to 20% MeOH / CHCl3) to give compound 2 (10.32 g, 29.3%) as a white to slightly brown solid. 1 H-NMR (400 MHz, DMSO-d6) δ ppm: 7.81 (d, 1H, J = 8.0 Hz, NH; D2O exchange), 7.42-7.24 (m, 5H, Ph-H), 5.01 (d, 1H, J = 5.7 Hz; D2O exchange), 4.72 (d, 1H, J =5.7 Hz; D2O exchange), 4.70 (d, 1H, J = 3.7 Hz, C1-βH), 4.67 (d, 1H, Jgem = 12.6 Hz, Ph-CH2a), 4.54 (t, 1H, J = 6.0 Hz, C6-OH; D2O exchange), 4.42 (d, 1H, Jgem = 12.6 Hz, LCMS m / z: 312.1 [M+H] + (Exact Mass: 311.14) Purity (LCMS): 100.00%

[0137] (Synthesis of benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-α-D-glucopyranoside (Compound 3))

[0138]

[0139] Benzyl 2-acetamido-2-deoxy-α-D-glucopyranoside (compound 2) (10.32 g, 33.1 mmol) was dissolved in anhydrous DMF (80 mL) and benzaldehyde dimethyl acetal (6.9 mL, 46.5 mmol, 1.4 eq.) and p-TSA·HO (63 mg, 0.3 mmol, 0.01 eq.) were added. The mixture was placed on a rotary evaporator, and the solvent was gently removed under reduced pressure every hour (approximately 15 min). The mixture was then heated at 60 °C for 5 h. The solvent was removed under reduced pressure, leaving a white solid residue. The residue was suspended in 2% aqueous NaHCO (50 mL), crushed, filtered, washed with water and IPE, and dried at 60 °C under reduced pressure to give compound 3 (13.07 g, 98.7%) as a white solid. 1 H-NMR (400 MHz, DMSO-d6) δppm: 8.00 (d, 1H, J = 8.2 Hz, NH; D2O exchange), 7.49-7.27 (m, 10H, Ph-H X 2), 5.62 (s, 1H, PhCH), 5.19 (d, 1H, J = 3.4 Hz, C3-OH; D2O exchange), 4.79 (d, 1H, J = 3.4 Hz, C1-βH), 4.70 (d, 1H, Jgem = 12.6 Hz, Ph-CH2a), 4.49 (d, 1H, Jgem = 12.6 Hz, Ph-CH2b), 4.18-4.11 (m, 1H), 3.90-3.81(m, 1H), 3.78-3.65 (m, 3H), 3.51 (t, 1H, J = 8.9 Hz), 1.85 (s, 3H, NHCOCH3). LCMS m / z: 400.1 [M+H] + (Exact Mass: 399.17) Purity (LCMS): 92.27%

[0140] (Synthesis of 2-(2-(R)-(2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionic acid (Compound 4))

[0141]

[0142] Benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-α-D-glucopyranoside (compound 3) (13.07 g, 32.72 mmol) was suspended in toluene (220 mL) and concentrated to dryness under reduced pressure (60 °C). The residue was dissolved in anhydrous DMF (220 mL). Under an argon gas stream, NaH (60% in oil) (8.18 g, net 4.91 g, 204.5 mmol, 6.25 eq.) was added with stirring at room temperature and stirred for 30 minutes. (S)-(-)-2-chloropropionic acid (10.00 g, 92.15 mmol, 2.8 eq.) was added dropwise over 25 minutes to the reaction mixture under an argon gas atmosphere at room temperature and stirred for 30 minutes. Further, under an argon atmosphere, NaH (60% in oil) (8.18 g) was added with stirring at room temperature and stirred for 19 hours. Deionized water (33 mL) was carefully added to the reaction mixture with stirring at room temperature, and the mixture was concentrated to dryness under reduced pressure. The residue was suspended in water (500 mL) and adjusted to pH 3-4 with 2 mol / L hydrochloric acid (83 mL) with stirring at room temperature. The suspension was collected by filtration, washed with water, and dried by vacuum. Afterwards, it was dissolved in CHCl3, dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure to give a pale yellow-brown solid residue. The residue was crystallized from n-Hex / CHCl3 and dried (50 °C, reduced pressure) to give compound 4 (10.11 g, 65.5%) as a white solid. 1 H-NMR (400 MHz, DMSO-d6) δ ppm: 8.11 (br, 1H, NH; D2O exchange), 7.47-7.26 (m, 10H, Ph-H X2), 5.70 (s, 1H, PhCH), 5.05 (d, 1H, J = 3.2 Hz, C1-βH), 4.70 (d, 1H, Jgem = 12.4 Hz, Ph-CH2a), 4.49 (d, 1H, Jgem = 12.4 Hz, Ph-CH2b), 4.27 (q, 1H, J = 6.9 Hz, CH-CH3), 4.18-4.11 (m, 1H), 3.83-3.66(m, 5H), 1.85(s, 3H, NHCOCH3), 1.27 (d, 3H, J = 6.9 Hz, CH3). LCMS m / z: 472.2 [M+H] +(Exact Mass: 471.19) Purity (LCMS): 97.25%

[0143] Synthesis of (S)-methyl 2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionamido)propionate (Compound 5)

[0144]

[0145] 2-(2-(R)-(2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionic acid (compound 4) (1.89 g, 4.01 mmol) was dissolved in anhydrous DMF (40 mL). N-methylmorpholine (1.32 mL, 12.03 mmol, 3.0 eq.) and HBTU (3.04 g, 8.02 mmol, 2.0 eq.) were added under an argon atmosphere and the mixture was stirred at room temperature for 10 minutes. L-alanine methyl ester hydrochloride (1.12 g, 8.02 mmol, 2.0 eq.) was then added and the mixture was stirred for 17 hours. Water (36 mL) was added to the reaction mixture, which was then diluted with AcOEt and separated. The AcOEt layer was separated, washed with 1 mol / L HCl, saturated aqueous NaHCO3, and saturated aqueous NaCl, dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure to give a residue as an agar. The residue was purified by silica gel column chromatography (2% to 5% MeOH / CHCl3:MeOH) and dried (60 °C, reduced pressure) to give compound 5 (2.05 g, 91.9%) as a white solid. 1H-NMR (400 MHz, DMSO-d6) δ ppm: 8.11 (d, 1H, J = 7.6 Hz, NHCO; D2O exchange), 7.81 (d, 1H, J = 7.1 Hz, CONH; D2O exchange), 7.45-7.27 (m, 10H, Ph-5H X 2), 5.71 (s, 1H, PhCH), 4.94 (d, 1H, J = 3.4 Hz, C1-βH), 4.71 (d, 1H, Jgem = 12.4 Hz, Ph-CH2a), 4.51 (d, 1H, Jgem = 12.4 Hz, Ph- CH2b), 4.33-4.12(m, 3H), 4.03-3.93(m, 1H), 3.85-3.64 (m, 4H), 3.58 (s, 3H, CO2 CH3), 1.82 (s, 3H, NHCO CH3), 1.29 (d, 3H, J = 7.1 Hz, CH3), 1.22 (d, 3H, J = 6.6 Hz, CH3). LCMS m / z: 557.7 [M+H] + (Exact Mass: 556.24) Purity (LCMS): 100.00%

[0146] Synthesis of (S)-2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionamido)propionic acid (Compound 7) (benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-OH)

[0147]

[0148] (S)-Methyl 2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionamido)propionate (compound 5) (1.90 g, 3.41 mmol) was suspended in MeOH (190 mL), and 0.5 mol / L KOH (32 mL) was added and stirred at room temperature for 5 hours. After confirming the disappearance of the starting materials by TLC (10% MeOH / CHCl3), the reaction mixture was concentrated to dryness under reduced pressure to give a white solid. The white solid was suspended in AcOEt (500 mL), and 1 mol / L HCl (100 mL) was added, followed by separation. The AcOEt layer was separated, washed with saturated aqueous NaCl, dried over Na2SO4, filtered, concentrated to dryness under reduced pressure, and then dried (60°C, reduced pressure) to give Compound 7 (1.85 g, quant.) as a white solid. 1 H-NMR (400 MHz, DMSO-d6) δppm: 13.01-12.30 (br, 1H, COOH; D2O exchange), 8.14 (d, 1H, J = 8.0 Hz, NHCO; D2O exchange), 7.64 (d, 1H, J = 7.6 Hz, CONH; D2O exchange), 7.48-7.25 (m, 10H, Ph-H 12.6 Hz, Ph-CH2b), 4.27-4.10 (m, 3H), 4.04-3.93 (m, 1H), 3.83-3.64 (m, 4H), 1.81 (s, 3H, NHCO CH3), 1.27 (d, 3H, J= 7.3 Hz, CH3), 1.81 (d, 3H, J= 6.6 Hz, CH3). LCMS m / z: 543.7 [M+H] + (Exact Mass: 542.23) Purity (LCMS): 87.46%

[0149] Synthesis of (R)-methyl 2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionamido)propionate (Compound 6)

[0150]

[0151] D-Alanine methyl ester hydrochloride was condensed using 2-(2-(R)-(2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionic acid (Compound 4) (945 mg, 2.01 mmol) in the same manner as for Compound 5 to give Compound 6 (778 mg, 69.7%) as a white solid. 1 H-NMR (400 MHz, DMSO-d) δ ppm: 8.20 (d, 1H, J = 7.3 Hz, NHCO; DO exchange), 7.91 (d, 1H, J = 6.9 Hz, CONH; DO exchange), 7.47-7.26 (m, 10H, benzyl-Ph, benzylidene-Ph), 5.70 (s, 1H, benzylidene-CH), 4.99 (d, 1H, J = 3.7 Hz, MurNAc C1-βH), 4.71 (d, 1H, J = 12.4 Hz, Ph- CH2a), 4.51 (d, 1H, J = 12.4 Hz, Ph- CH2b), 4.30-4.12 (m, 3H), 3.97-3.88 (m, 1H), 3.83-3.58 (m, 4H), 3.61 (s, 3H, CO2 CH3), 1.87 (s, 3H, NHCO CH3), 1.28 (d, 3H, J = 7.1 Hz, MurNAc- CH3 or D-Ala- CH3), 1.24 (d, 3H, J = 6.6 Hz, MurNAc- CH3 or D-Ala- CH3), LCMS m / z: 557.6 [M+H] + (Exact Mass: 556.24) Purity (LCMS): 94.89%

[0152] Synthesis of (R)-2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionamido)propionic acid (Compound 8) (benzyl-4,6-O-benzylidene-α-MurNAc-D-Ala-OH)

[0153]

[0154] (R)-Methyl 2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionamido)propionate (Compound 6) (750 mg, 1.35 mmol) was hydrolyzed with 0.5 mol / L KOH in MeOH solvent in the same manner as for Compound 7 to give Compound 8 (755 mg, quant.) as a white solid. 1 H-NMR (400 MHz, DMSO-d6) δ ppm: 12.70 (s, 1H, COOH; DO exchange), 8.23 ​​(d, 1H, J = 7.1 Hz, NHCO; DO exchange), 7.78 (d, 1H, J = 7.1 Hz, CONH; DO exchange), 7.46-7.27 (m, 10H, benzyl-Ph, benzylidene-Ph), 5.70 (s, 1H, benzylidene-CH), 4.97 (d, 1H, J = 3.7 Hz, MurNAc C1-βH), 4.70 (d, 1H, J = 12.4 Hz, Ph- CH2a), 4.51 (d, 1H, J = 12.4 Hz, Ph- CH2b), 4.31-4.11 (m, 3H), 3.96-3.87 (m, 1H), 3.83-3.61 (m, 4H), 1.86 (s, 3H, NHCO CH3), 1.28 (d, 3H, J = 7.3 Hz, MurNAc- CH3 or D-Ala- CH3), 1.24 (d, 3H, J = 6.9 Hz, MurNAc- CH3 or D-Ala- CH3). LCMS m / z: 543.6 [M+H] + (Exact Mass: 542.23) Purity (LCMS): 96.51%

[0155] (Synthesis of 7-azido-4-methylcoumarin (compound 10))

[0156]

[0157] 7-Amino-4-methylcoumarin (4.00 g, 22.8 mmol) was suspended in 2 mol / L aqueous HCl (40 mL). NaNO2 (1.89 g, 27.4 mmol, 1.2 eq.) was added with stirring under ice cooling, and the mixture was stirred for 50 min. NaN3 (2.23 g, 34.2 mmol, 1.5 eq.) was then added and the mixture was stirred for 1 h. The precipitated solid was collected by filtration, washed with water, and dried (50 °C, reduced pressure) to give compound 10 (4.54 g, 98.8%) as a pale ochre solid. 1 H-NMR (400 MHz, DMSO-d6) δppm: 7.79 (d, 1H, J = 8.5 Hz, C5-H), 7.17 (d, 1H, J = 2.3 Hz, C8-H), 7.15 (dd, 1H, J = 2.3, 8.5 Hz, C6-H), 6.35 (d, 1H, J = 1. 1 Hz, C3-H), 2.42 (d, 3H, J = 1.1 Hz, C4- CH3). LCMS m / z: 202.4 [M+H] + (Exact Mass: 201.05) Purity (LCMS): 99.09%

[0158] (Synthesis of 1-azido-4-nitrobenzene (compound 22))

[0159]

[0160] 4-Nitroaniline hydrochloride (3.49 g, 20.0 mmol) was suspended in 6 mol / L aqueous HCl (13 mL). An aqueous solution (7 mL) of NaNO (1.40 g, 20.3 mmol, 1.02 eq.) was added with stirring under ice cooling, and the mixture was stirred for 5 min. Next, an aqueous solution (7 mL) of CHCONa (1.12 g, 13.7 mmol, 0.69 eq.) and NaN (1.31 g, 20.1 mmol, 1.01 eq.) were added, and the mixture was stirred for 1 h. The precipitated solid was collected by filtration, washed with water, and dried (50 °C, reduced pressure) to give compound 22 (2.58 g, 79.2%) as a yellow solid. 1 H-NMR (400 MHz, DMSO-d6) δppm: 8.26 (d, 2H, J = 9.2 Hz), 7.36 (d, 2H, J = 9.2 Hz). Purity (LCMS): 98.81%

[0161] (Synthesis of 4-methylcoumarin-7-amide (MCA) and p-nitroanilide (pNA) derivatives; synthesis of D-Glu(OBn)-MCA, D-Glu(OAll)-MCA, D-Glu(MCA)-OAll, D-Gln-MCA, D-isoGln-MCA, and D-isoGln-pNA (general synthesis method))

[0162] (Preparation of sodium hydroselenide (NaHSe)) Degassed and dried IPA (130 mL / g Ce) was added to Se (1.2 eq.) and NaBH4 (1.44 eq.), and the mixture was stirred at room temperature under an argon gas atmosphere for 1 to 2 hours to prepare NaHSe (prepared just before use).

[0163] (Preparation of mixed acid anhydride) N-protected amino acid (1.2 eq.) was dissolved in dehydrated THF (130 mL / g), N-methylpiperidine (1.2 eq.) was added, and isopropyl chloroformate (ca. 2 mol / L in toluene) (1.4 eq.) was added with stirring at −15° C. under an argon gas atmosphere, and the mixture was stirred for 30 minutes to 1 hour.

[0164] (Synthesis of N-protected amino acid 4-methylcoumarin-7-amide (MCA form) and p-nitroanilide (pNA form)) The above mixed acid anhydride was slowly added to a freshly prepared solution of NaHSe in IPA at -15°C and stirred for 30 minutes to 1 hour under an argon atmosphere. Next, a solution of 7-azido-4-methyl-coumarin (1.0 eq.) in anhydrous THF (30 mL / g) or a solution of 1-azido-4-nitrobenzene (1.0 eq.) in anhydrous THF (18 mL / g) was added under an argon atmosphere with stirring at room temperature, and the mixture was stirred overnight at room temperature. The black suspension was filtered off through a Celite pad and washed with 20% MeOH / CHCl3. The filtrate and washings were combined and concentrated to dryness under reduced pressure. The residue was dissolved in 20% MeOH / CHCl3, silica gel (FL100 D) was added, and the mixture was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography.

[0165] (Synthesis of Fmoc-D-Glu(OAll)-MCA (Compound 13))

[0166]

[0167] Degassed and dried IPA (120 mL) was added to Se (942 mg, 11.93 mmol, 1.2 eq.) and NaBH (542 mg, 14.32 mmol, 1.44 eq.), and the mixture was stirred at room temperature under an argon gas atmosphere for 2.5 hours to prepare NaHSe (11.99 mmol, 1.4 eq.) (prepared immediately before use). Fmoc-D-Glu(OAll)-OH (compound 12) (4.88 g, 11.93 mmol, 1.2 eq.) was dissolved in anhydrous THF (120 mL), N-methylpiperidine (1.45 mL, 11.93 mmol, 1.2 eq.) was added, and isopropyl chloroformate (ca. 2 mol / L in toluene) (6.0 mL, 11.93 mmol, 1.4 eq.) was added with stirring at −15°C under an argon atmosphere, and the mixture was stirred for 1.5 hours. The above mixed acid anhydride was slowly added to a freshly prepared solution of NaHSe in IPA at −15°C, and the mixture was stirred for 25 minutes under an argon atmosphere. Next, a solution of 7-azido-4-methylcoumarin (compound 10) (1.73 g, 8.60 mmol) in anhydrous THF (50 mL) was added with stirring at room temperature under an argon atmosphere, and the mixture was stirred overnight at room temperature. The suspended solid was filtered off using a Celite pad and washed with 20% MeOH / CHCl3. The combined filtrate and washings were concentrated to dryness under reduced pressure. The residue was dissolved in 20% MeOH / CHCl3, silica gel (FL100D; 20 g) was added, and the mixture was concentrated to dryness under reduced pressure. The mixture was purified by silica gel column chromatography (CHCl3 to 15% AcOEt / CHCl3). The fractions with Rf = 0.39 (25% AcOEt / CHCl3) were combined and concentrated to dryness under reduced pressure. The residue was washed with n-Hex. / CHCl3 to give compound 13 (2.20 g, 45.1%) as a pale yellow solid. 1H-NMR (400 MHz, DMSO-d6) δppm: 10.53 (s, 1H, クマリンC7-NH; D2O exchange), 7.90 (d, 2H, J = 7.6 Hz, Fmoc-arom.), 7.84 (d, 1H, Glu-NH; D2O exchange), 7.80-7.70 (m, 4H, Fmoc-arom2H, クマリンC5-H & C8-H [7.78 (d, 1H, J = 2.1 Hz]), 7.51 (dd, 1H, J = 2.1, 8.7 Hz, クマリンC6-H), 7.46-7.29 (m, 2H, Fmoc-arom.), 6.28 (d, 1H, J = 1.1 Hz, クマリンC3-H), 5.96-5.83 (m, 1H, O- CH2-CH= CH2), 5.33-5.25 (m, 1H, O-CH2-CH= CH2a), 5.23-5.16 (m, 1H, O- CH2-CH= CH2b), 4.59-4.50 (m, 2H, O- CH2-CH= CH2), 4.36-4.14 (m, 4H, Fmoc-CH, CH2 & Glu-αH), 2.49-2.38 (m, 2H, Glu-βCH2), 2.41 (d, 3H, J = 1.1 Hz;クマリンC4- CH3), 2.12-1.87 (m, 2H, Glu-γCH2a,b). LCMS m / z: 567.5 [M+H] + .(Exact Mass: 566.21) Purity(LCMS): 99.58%

[0168] (Synthesis of Fmoc-D-Glu (MCA)-OAll (Compound 15))

[0169]

[0170] Fmoc-D-Glu (OH)-OAll (Compound 14) (4.72 g) was obtained by using the general synthesis method, reaction and post-treatment, and producing a yellow solid. Fmoc-D-Glu (MCA)-OAll (Compound 15) (2.47 g, 43.8%) was obtained. 1H-NMR (400 MHz, DMSO-d6) δppm: 10.38 (s, 1H, クマリンC7-NH; D2O exchange), 7.94-7.84 (m, 3H, Fmoc-arom2H, Glu-NH; D2O exchange), 7.77-7.67 (m, 4H, Fmoc-arom2H, クマリンC5-H & C8-H [7.75(d, 1H, J = 2.1 Hz; クマリンC8-H), 7.69(d, 1H, J = 8.7 Hz, クマリンC5-H)]), 7.47 (dd, 1H, J = 2.1, 8.7 Hz, クマリンC6-H), 7.45-7.39 (m, 2H, Fmoc-arom.), 7.37-7.30 (m, 2H, Fmoc-arom.), 6.24 (d, 1H, J = 1.1 Hz, クマリンC3-H), 5.97-5.86 (m, 1H, O- CH2-CH= CH2), 5.37-5.28 (m, 1H, O- CH2-CH= CH2a), 5.24-5.18 (m, 1H, O- CH2-CH= CH2b), 4.65-4.56 (m, 2H, O- CH2-CH= CH2), 4.38-4.11 (m, 4H, Fmoc-CH, CH2 & Glu-αCH), 2.56-2.47 (m, 2H, Glu-βCH2), 2.38 (d, 3H, J = 1.1 Hz, クマリンC4- CH3), 2.21-2.07 (m, 1H, Glu-γCH2a), 1.99-1.85 (m, 1H, Glu-γCH2b). LCMS m / z: 567.5 [M+H] + .(Exact Mass: 566.21) Purity(LCMS): 99.44%

[0171] (Synthesis of Fmoc-D-Gln-MCA (Compound 19))

[0172]

[0173] Fmoc-D-Gln-OH (Compound 18) (4.41 g) was obtained by using the general synthesis method, reaction and post-treatment, and a light yellow solid was obtained. Fmoc-D-Gln-MCA (Compound 19) (477 mg, 9.1%) was obtained. 1H-NMR (400 MHz, DMSO-d6) δppm: 10.53 (s, 1H, クマリンC7-NH; D2O exchange), 7.90 (d, 2H, J = 7.6 Hz, Fmoc-arom.), 7.82 (d, 1H, J = 7.6 Hz, OCONH; D2O exchange) exchange), 7.80 (d, 1H, J = 2.1 Hz, クマリンC8-H), 7.78-7.72 (m, 3H, Fmoc-arom2H, クマリンC5-H), 7.51 (dd, 1H, J = 2.1, 8.7 Hz,クマリンC6-H), 7.46-7.39 (m, 2H, Fmoc-arom.), 7.38-7.30 (m, 3H, Fmoc-arom.2H, CONH2a; D2O exchange), 6.84 (s, 1H, CONH2b; D2O exchange), 6.28 (d, 1H, J = 1.1 Hz, クマリンC3-H), 4.31-4.20 (m, 3H, Fmoc-CH, CH2), 4.19-4.11 (m, 1H, Gln-αH), 2.41 (d, 3H, J = 1.1 Hz, クマリンC4- CH3), 2.29-2.10 (m, 2H, Gln-βCH2), 2.03-1.92 (m, 1H, Gln-γCH2a), 1.91-1.80 (m, 1H, Gln-γCH2b). LCMS m / z: 526.6 [M+H] + .(Exact Mass: 525.19)Purity(LCMS): 97.60%

[0174] (Synthesis of Fmoc-D-isoGln-MCA (Compound 21))

[0175]

[0176] Fmoc-D-isoGln-OH (Compound 20) (3.67 g) was obtained by reaction and post-treatment using a general synthesis method, and a white to slightly yellow solid was obtained. Fmoc-D-isoGln-MCA (Compound 21) (1.24 g, 28.4%) was obtained. 1H-NMR (400 MHz, DMSO-d) δ ppm: 10.38 (s, 1H, coumarin C7-NH; DO exchange), 7.89 (d, 2H, J = 7.6 Hz, Fmoc-arom.), 7.76 (d, 1H, J = 1.8 Hz, coumarin C8-H), 7.73 (d, 2H, J = 7.3 Hz, Fmoc-arom.), 7.69 (d, 1H, J = 8.7 Hz, coumarin C5-H), 7.51-7.38 (m, 4H, CONHCH; DO exchange, Fmoc-arom. 2H, coumarin C6-H), 7.36 (s, 1H, CONH2a; DO exchange), 7.32 (t, 2H, J = 7.3 Hz, Fmoc-arom.), 7.09 (s, 1H, CONH2b; D2O exchange), 6.25 (d, 1H, J = 1.1 Hz, Coumarin C3-H), 4.30-4.16 (m, 3H), 4.04-3.95 (m, 1H), 2.44 (t, 2H, J = 7.8 Hz, isoGln-βCH2), 2.38 (d, 3H, J = 1.1 Hz, Coumarin C4- CH3), 2.11-1.97 (m, 1H, isoGln-γCH2a), 1.93-1.79 (m, 1H, isoGln-γCH2b). LCMS m / z: 526.6 [M+H] + (Exact Mass: 525.19) Purity (LCMS): 97.84%

[0177] (Synthesis of Fmoc-D-isoGln-pNA (Compound 23))

[0178]

[0179] Fmoc-D-isoGln-OH (compound 20) (4.40 g) was reacted and worked up according to the general synthetic method to give Fmoc-D-isoGln-pNA (compound 23) (2.98 g, 61.5%) as a pale yellow solid. 1H-NMR (400 MHz, DMSO-d6) δppm: 10.56 (s, 1H, pNA-NH; D2O exchange), 8.20 (d, 2H, J = 9.2 Hz, pNA C3,5-H), 7.89 (d, 2H, J = 7.6 Hz, Fmoc-arom.), 7.83 (d, 2H, J = 9.2 Hz, pNA C2,6-H), 7.73 (d, 2H, J = 7.6 Hz, Fmoc-arom.), 7.46 (d, 1H, J = 8.2 Hz, isoGln-NH; D2O exchange), 7.42 (t, 2H, J = 7.6 Hz, Fmoc-arom.), 7.36(s, 1H, CONH2a; D2O exchange), 7.33 (t, 2H, J = 7.6 Hz; Fmoc-arom.), 7.09 (s, 1H, CONH2b; D2O exchange), 4.33-4.16 (m, 3H, Fmoc-CH, CH2), 3.99 (dt, 1H, J = 5.0, 8.7 Hz, isoGln-αH), 2.47 (t, 2H, J = 7.8 Hz, isoGln-βCH2), 2.11-1.96 (m, 1H, isoGln-γCH2a), 1.93-1.79 (m, 1H, isoGln-γCH2b). LCMS m / z: 489.6 [M+H] + (Exact Mass: 488.17) Purity (LCMS): 98.67%

[0180] <Fmoc-L-Ala Conversion (General Method)> Fmoc-L-Ala-OH (hydrate) (compound 33) (1.1 eq.) was dissolved in anhydrous DMF (40 mL / g). HOBt.HO (1.3 eq.) and EDCI (1.3 eq.) were added with stirring at 0°C and the mixture was stirred for 30 minutes. DIEA (2.2 eq.) and HD-Glu(MCA)-OAll (compound 27, etc.) (1.0 eq.) were then added to the reaction mixture and stirred for 1 hour, followed by further stirring at room temperature for 3 hours. The reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified to obtain the target compound.

[0181] (Synthesis of Fmoc-L-Ala-D-Glu(MCA)-OAll (Compound 37))

[0182]

[0183] HD-Glu(MCA)-OAll (compound 27) (952 mg, 2.76 mmol) was used in the reaction according to the general method, and the reaction mixture was concentrated to dryness under reduced pressure. AcOEt (200 mL) was added to the residue (there was insoluble material in both layers), and the mixture was washed with water, saturated aqueous NaHCO3, and then saturated aqueous NaCl, and the AcOEt layer (containing suspended material) was separated. The mixture was dried over Na2SO4, filtered, and washed two or three times with 20% MeOH / CHCl3. The combined filtrate and washings were concentrated under reduced pressure. The precipitate was collected by filtration, washed with n-Hex. / AcOEt, and dried (50 °C, reduced pressure) to give compound 37 (1.79 g, quant.) as a white to pale yellow solid (Rf = 0.57, 0.64 [10% MeOH / CHCl3]). 1 The ratio of the major component was 0.64 (the ratio is unknown because there are no separated peaks in the H-NMR spectrum). 1H-NMR (400 MHz, DMSO-d6) δppm: 10.50 (s, 1H, クマリン-C7NH; D2O exchange), 8.41 (d,1H, J = 7.8 Hz, NH; D2O exchange), 7.91-7.84 (m, 2H, Fmoc-arom.), 7.78-7.66 (m, 4H, Fmoc-arom2H, クマリンC5-H & C8-H [7.76 (d, 1H, J = 1.8 Hz, クマリンC8-H), 7.69 (d, 1H, J = 8.7 Hz,クマリンC5-H)]), 7.53 (d, 1H, J = 8.0 Hz, NH; D2O exchange), 7.48 (dd, 1H, J = 1.8, 8.7 Hz, クマリンC6-H), 7.44-7.37 (m, 2H, Fmoc-arom.), 7.33-7.28 (m, 2H, Fmoc-arom.), 6.25 (d, 1H, J = 1.1Hz, クマリンC3-H), 5.97-5.84 (m, 1H, O- CH2-CH= CH2), 5.36-5.27 (m, 1H, O- CH2-CH= CH2a), 5.23-5.16 (m, 1H, O- CH2-CH= CH2b), 4.62-4.54 (m, 2H, O- CH2-CH= CH2), 4.38-4.29 (m, 1H), 4.28-4.09 (m, 4H), 2.52-2.45 (m, 2H, Glu-βCH2), 2.39 (d, 3H, J =1.1 Hz, クマリンC4- CH3), 2.18-2.06 (m, 1H, Glu-γCH2a), 2.01-1.88 (m, 1H, Gln-γCH2b), 1.26 (d, 3H, J = 7.1 Hz, Ala- CH3). LCMS m / z: 638.6 [M+H] + .(Exact Mass: 637.24) Purity(LCMS): 99.21%

[0184] (Synthesis of Fmoc-L-Ala-D-Gln-MCA (Compound 38))

[0185]

[0186] HD-Gln-MCA (compound 26) (220 mg, 0.73 mmol) was reacted according to the general method, and the reaction mixture was concentrated to dryness under reduced pressure. The residue was suspended in CHCl3 (10 mL), and n-Hex (10 mL) was added. The suspension was collected by filtration, washed with water, and dried (50 °C, reduced pressure) to give compound 38 (374 mg; Rf = 0.15, 0.25 [mixture of 10% MeOH / CHCl3]) as a white solid. 1 From the H-NMR spectrum, it was estimated to be a mixture of two isomers (abundance ratio: approximately 4:1). 1 H-NMR (400 MHz, DMSO-d) δ ppm: 10.29 (s, 0.2H, coumarin C7-NH; DO exchange), 10.23 (s, 0.8H, coumarin C7-NH; DO exchange), 8.42 (d, 1H, J = 8.0 Hz, NH; DO exchange), 7.86 (t, 2H, J = 6.9 Hz, Fmoc-arom.), 7.83 (d, 0.2H, J = 1.8 Hz, coumarin C8-H), 7.80 (d, 0.8H, J = 1.8 Hz, coumarin C8-H), 7.76-7.64 (m, 4H; 1H- DO exchange), 7.56 (dd, 0.2H, J = 2.1, 8.7 Hz, coumarin C6-H), 7.52 (dd, 0.8H, J = 2.1, 8.7 Hz, coumarin C6-H), 7.44-7.27 (m, 5H; 1H- DO exchange), 6.81 (br-s, 1H, CONH2b), 6.28 (s, 1H, coumarin C3-H), 4.45-4.35 (m, 1H), 4.33-4.05 (m, 4H), 2.39 (s, 3H, coumarin C4- CH3), 2.18-1.96 (m, 3H, Gln-βCH2, Gln-γCH2a), 1.90-1.77 (m, 1H, Gln-γCH2b), 1.26 (d, 2.4H, J = 7.1 Hz; Ala- CH3), 1.22 (d, 0.6H, J = 7.1 Hz; Ala- CH3). LCMS m / z: 597.6 [M+H] +(Exact Mass: 596.23) Purity (LCMS): 96.36%

[0187] (Synthesis of Fmoc-L-Ala-D-isoGln-pNA (Compound 40))

[0188]

[0189] HD-isoGln-pNA (compound 31) (500 mg, 1.88 mmol) was reacted according to the general method, and the reaction mixture was concentrated to dryness under reduced pressure. The residue was dissolved in 20% MeOH / CHCl3, silica gel (FL100D 20 g) was added, and the mixture was concentrated to dryness and purified by silica gel column chromatography (5% to 10%, MeOH / CHCl3). The target fractions were combined, concentrated to dryness, and dried (50 °C, reduced pressure) to give compound 40 (914 mg, 87.0%) as a white solid. 1 From the H-NMR spectrum, it was estimated to be a mixture of two isomers (abundance ratio: approximately 9:1). 1H-NMR(400 MHz, DMSO-d6)δppm: 10.53(s, 1H, pNA-NH; D2O exchange), 8.20(d, 0.2H, J = 9.2 Hz, pNA C3,5-H), 8.19(d, 1.8H, J = 9.2 Hz, pNA C3,5-H), 8.14(d, 0.1H, J = 7.3 Hz, NH; D2O exchange), 8.09(d, 0.9H, J = 8.2 Hz, NH; D2O exchange), 7.98(d, 0.1H, J = 8.0 Hz, NH; D2O exchange), 7.85(d, 2H, J =7.3 Hz, Fmoc-arom.), 7.82(d, 0.2H, J = 9.2Hz, pNA C2,6-H), 7.81(d, 1.8H, J = 9.2 Hz, pNA C2,6-H), 7.72(t, 2H, J = 6.9 Hz, Fmoc-arom.), 7.61(d, 1H, J = 7.1 Hz, NH; D2O exchange), 7.41(t, 2H, J =6.9 Hz, Fmoc-arom.), 7.36-7.27(m, 3H, Fmoc-arom. & isoGln CONH2a; D2O exchange), 7.16(s, 1H, isoGln CONH2b; D2O exchange), 4.32-4.16(m, 4H), 4.08(dq, 1H, J = 7.1, 7.1 Hz, Ala-αH), 2.41(t, 2H, J = 8.0 Hz, isoGln-βCH2), 2.16-2.00(m, 1H, isoGln-γCH2a), 1.91-1.75(m, 1H, isoGln-γCH2b), 1.24(d, 0.3H, J = 7.1 Hz, Ala- CH3), 1.23(d, 2.7H, J = 7.1 Hz, Ala- CH3). LCMS m / z: 560.6 [M+H] + .(Exact Mass: 559.21) Purity(LCMS): 100.00%(11.38%, 88.62%).

[0190] <Fmoc removal (general method)> The Fmoc compound (1.0 eq.) was dissolved in a 20% piperidine / DMF solution (22 mL / g) and stirred at room temperature for 20 minutes. After confirming the disappearance of the starting material by TLC (10% MeOH / CHCl3), the reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified to obtain the target compound.

[0191] Synthesis of HD-Glu(OAll)-MCA (Compound 28) and HD-Glu(OH)-MCA (Compound 29)

[0192]

[0193] Fmoc-D-Glu(OH)-MCA (compound 13) (1.80 g, 3.18 mmol) was reacted according to the general procedure, and the reaction mixture was concentrated to dryness under reduced pressure. The residue was reprecipitated with n-Hexane (30 mL) / CHCl3 (15 mL), stirred, filtered, and dried (60 °C, reduced pressure) to give HD-Glu(OH)-MCA (compound 29) (680 mg) as a pale yellow solid. The filtrate was concentrated to dryness under reduced pressure, dissolved in CHCl3, and purified by silica gel column chromatography (CHCl3 to 8% MeOH / CHCl3). The fraction with an Rf = 0.40 (10% MeOH / CHCl3) was concentrated to dryness under reduced pressure, and the residue was washed with n-Hexane / CHCl3 to give compound 28 (110 mg, 11.0%) as a pale yellow solid. The fraction with Rf = 0.28 (10% MeOH / CHCl3) was concentrated to dryness under reduced pressure, and the residue was washed with n-Hex. / CHCl3 to give compound 29 (132 mg; total yield: 812 mg, 74.2%) as a pale yellow to white solid. HD-Glu(OAll)-MCA: 1H-NMR(400 MHz, DMSO-d6)δppm: 7.83(d, 1H, J = 2.1 Hz, クマリンC8-H), 7.72(d, 1H, J = 8.7 Hz, クマリンC5-H), 7.57(dd, 1H, J = 2.1, 8.7 Hz, クマリンC6-H), 6.28(d, 1H, J = 1.1 Hz, クマリンC3-H), 5.98-5.84(m, 1H, O- CH2-CH= CH2), 5.34-5.26(m, 1H, O- CH2-CH= CH2a), 5.23-5.17(m, 1H, O- CH2-CH= CH2b), 4.55-4.52(m, 2H, O- CH2-CH= CH2), 3.36(dd, 1H, J = 5.3, 8.2 Hz, Glu-αH), 2.53-2.43(m, 2H, Glu-βCH2), 2.40(d, 3H, J = 1.1 Hz, クマリンC4- CH3), 2.02-1.90(m, 1H, Glu-γCH2a), 1.80-1.66(m, 1H, Glu-γCH2b). LCMS m / z: 345.5 [M+H] + .(Exact Mass: 344.14) Purity(LCMS): 98.961%(71.48% + 27.48% [M-OAll]) H-D-Glu(OH)-MCA: 1H-NMR (400 MHz, DMSO-d) δ ppm: 10.50 (s, 1H, coumarin C7-NH; DO exchange), 7.93 (s, 1H, COOH; DO exchange), 7.78 (d, 1H, J = 2.1 Hz, coumarin C8-H), 7.74 (d, 1H, J = 8.7 Hz, coumarin C5-H), 7.53 (dd, 1H, J = 2.1, 8.7 Hz, coumarin C6-H), 6.28 (d, 1H, J = 1.1 Hz, coumarin C3-H), 4.23 (dd, 1H, J = 4.4, 8.7 Hz, Glu-αH), 2.44-2.32 (m, 1H). 2.40 (d, 3H, J = 1.1 Hz, Coumarin C4- CH3), 2.31-2.11 (m, 2H), 2.08-1.97 (m, 1H). LCMS m / z: 287.5 [M-OH]+. (Exact Mass: 304.11) Purity (LCMS): 100.00%

[0194] (Synthesis of HD-Glu(MCA)-OAll (Compound 27))

[0195]

[0196] Fmoc-D-Glu(MCA)-OAll (compound 15) (1.80 g, 3.18 mmol) was reacted according to the general procedure, and the reaction mixture was concentrated to dryness under reduced pressure. The residue was reprecipitated with n-Hex (30 mL) / CHCl (30 mL), stirred, filtered, and dried (50 °C, reduced pressure) to give compound 27 (983 mg, 89.9%) as a pale yellow solid. 1H-NMR (400 MHz, DMSO-d6) δ ppm: 10.38 (s, 1H, coumarin C7-NH; DO exchange), 7.76 (d, 1H, J = 2.1 Hz, coumarin C8-H), 7.70 (d, 1H, J = 8.7 Hz, coumarin C5-H), 7.48 (dd, 1H, J = 2.1, 8.7 Hz, coumarin C6-H), 6.25 (d, 1H, J = 1.1 Hz, coumarin C3-H), 6.00-5.87 (m, 1H, O- CH2-CH= CH2), 5.37-5.29 (m, 1H, O- CH2-CH= CH2a), 5.25-5.19 (m, 1H, O- CH2-CH= CH2b), 4.61-4.56 (m, 2H, O- CH2-CH= CH2), 3.39 (dd, 1H, J = 5.3, 8.5 Hz, Glu-αH), 2.52-2.47 (m, 2H, Glu-βCH2), 2.39 (d, 3H, J = 1.1 Hz, Coumarin C4- CH3), 2.04-1.92 (m, 1H, Glu-γCH2a), 1.92-1.79 (br, 2H, NH2; D2O exchange), 1.79-1.67 (m, 1H, Glu-γCH2b). LCMS m / z: 345.5 [M+H] + (Exact Mass: 344.14) Purity (LCMS): 97.36%

[0197] (Synthesis of HD-Gln-MCA (Compound 26))

[0198]

[0199] Fmoc-D-Gln-MCA (compound 19) (450 mg, 0.86 mmol) was reacted according to the general method, and the reaction mixture was concentrated to dryness under reduced pressure. The residue was suspended in n-Hexane (10 mL) / CHCl3 (10 mL), stirred, and filtered. The filtered product was dried (50 °C, reduced pressure) to give compound 26 (249 mg, 95.8%) as a pale ochre solid. 1H-NMR (400 MHz, DMSO-d6) δ ppm: 7.85 (d, 1H, J = 2.1 Hz, coumarin C8-H), 7.73 (d, 1H, J = 8.7 Hz, coumarin C5-H), 7.57 (dd, 1H, J = 2.1, 8.7 Hz, coumarin C6-H), 7.30 (br-s, 1H, CONH2a; DO exchange), 6.76 (br-s, 1H, CONH2b; DO exchange), 6.27 (d, 1H, J = 1.1 Hz, coumarin C3-H), 3.41-3.29 (m, 1H, Gln-αH), 2.40 (d, 3H, J = 1.1 Hz, Coumarin C4- CH3), 2.25-2.09 (m, 2H, Gln-βCH2), 1.94-1.82 (m, 1H, Gln-γCH2a), 1.73-1.61 (m, 1H, Gln-γCH2b). LCMS m / z: 304.5 [M+H] + (Exact Mass: 303.12) Purity (LCMS): 74.88%

[0200] (Synthesis of HL-Ala-D-Gln-MCA (Compound 44))

[0201]

[0202] Fmoc-L-Ala-D-Gln-MCA (compound 38) (350 mg, 0.59 mmol) was reacted according to the general method, and the reaction mixture was concentrated to dryness under reduced pressure. The residue was suspended in n-hexane (20 mL) / CHCl3 (10 mL), stirred, and collected by filtration (precipitate; moisture absorption). The collected material was dissolved in 20% MeOH / CHCl3 and concentrated to dryness under reduced pressure. The residue was triturated with n-hexane and dried (50 °C, reduced pressure) to give compound 44 (213 mg, 96.8%) as a white solid. 1 The H-NMR spectrum indicated that it was a mixture of two isomers (abundance ratio approximately 5:1). 1H-NMR (400 MHz, DMSO-d6) δppm: 10.53 (s, 0.84H, クマリンC7-NH; D2O exchange), 10.31 (s, 0.16H, クマリンC7-NH; D2O exchange), 8.41-7.91 (br, 1H, NH; D2O exchange), 7.83 (d, 0.16H, J = 2.1 Hz, クマリンC8-H), 7.78 (d, 0.84H, J = 2.1 Hz, クマリンC8-H), 7.74 (d, 1H, J = 8.7 Hz, クマリンC5-H), 7.59 (dd, 0.16H, J = 2.1, 8.7 Hz, クマリンC6-H), 7.50 (dd, 0.84H, J = 2.1, 8.7 Hz, クマリンC6-H), 7.32 (br-s, 1H, CONH2a; D2O exchange), 6.79 (br-s, 1H, CONH2b; D2O exchange), 6.28 (d, 1H, J = 1.1 Hz, クマリンC3-H), 4.42 (br-dd, 1H, J = 5.5, 8.0 Hz, Gln-αH), 3.35 (q, 0.84H, J = 6.9 Hz, Ala-αCH), 3.30 (q, 0.16H, J = 6.9 Hz, Ala-αCH), 2.40 (d, 3H, J = 1.1 Hz, クマリンC4- CH3), 2.20-2.07 (m, 2H, Gln-βCH2), 2.03-1.81 (m, 2H, Gln-γCH2), 1.14 (d, 2.52H, J = 6.9 Hz, Ala- CH3), 1.13 (d, 0.48H, J = 6.9 Hz, Ala- CH3). LCMS m / z: 375.6 [M+H] + .(Exact Mass: 374.16) Purity(LCMS): 94.76%(83.03%, 11.73%).

[0203] (Synthesis of HD-isoGln-MCA (Compound 30))

[0204]

[0205] Fmoc-D-isoGln-MCA (compound 21) (1.14 g, 2.17 mmol) was reacted according to the general method, and the reaction mixture was concentrated to dryness under reduced pressure. The residue was suspended in n-Hex (25 mL) / CHCl3 (25 mL), stirred, and collected by filtration. The collected material was dried (60 °C, reduced pressure) to give compound 30 (602 mg, 91.5%) as a white solid. 1 H-NMR (400 MHz, DMSO-d) δ ppm: 10.40 (br-s, 1H, CONH; DO exchange), 7.77 (d, 1H, J = 1.8 Hz, coumarin C8-H), 7.70 (d, 1H, J = 8.7 Hz, coumarin C5-H), 7.48 (dd, 1H, J = 2.1, 8.7 Hz, coumarin C6-H), 7.33 (br-s, 1H, CONH2a; DO exchange), 6.97 (br-s, 1H, CONH2b; DO exchange), 6.25 (d, 1H, J = 1.1 Hz, coumarin C3-H), 3.17-3.11 (m, 1H, isoGln-αH), 2.44 (t, 2H, J = 7.8 Hz, isoGln-γCH2), 2.39 (d, 3H, J = 1.1 Hz, Coumarin C4- CH3), 1.94-1.83 (m, 1H, isoGln-βCH2a), 1.80 (br, 2H, isoGln-αNH2; D2O exchange), 1.73-1.65 (m, 1H, isoGln-βCH2b). LCMS m / z: 304.5 [M+H] + (Exact Mass: 303.12) Purity (LCMS): 97.81%

[0206] Synthesis of HL-Ala-D-isoGln-MCA.HCl (Compound 45)

[0207]

[0208] Fmoc-L-Ala-D-isoGln-MCA (compound 39) (80 mg, 0.13 mmol) was reacted according to the general method, and the reaction mixture was concentrated to dryness under reduced pressure. The residue was suspended in n-Hex (15 mL) / CHCl3 (7 mL), stirred, and collected by filtration. The collected material was dried (50 °C, reduced pressure) to give HL-Ala-D-isoGln-MCA (52 mg, quant.) as a pale ochre solid. LCMS m / z: 375.2 [M+H] + (Exact Mass: 374.16), Purity (LCMS): 95.65% HL-Ala-D-isoGln-MCA (50 mg, 0.13 mmol) was dissolved in a mixture of CHCl3 (10 mL) and MeOH (5 mL), and 5% HCl / MeOH (0.2 mL) was added and mixed. The mixture was concentrated to dryness under reduced pressure to give a pale ochre solid residue. The residue was washed with n-Hex (5 mL) / CHCl3 (5 mL) and dried (50 °C, reduced pressure) to give compound 45 (52 mg, 94.5%). 1 H-NMR (400 MHz, DMSO-d6) δppm: 10.52 (s, 1H, coumarin C7-NH; D2O exchange), 8.62 (d, 1H, J = 8.0 Hz, isoGln-NH; D2O exchange), 7.96-7.41 (br, 3H, Ala- NH3 +; DO exchange), 7.78 (d, 1H, J = 1.8 Hz, coumarin C8-H), 7.71 (d, 1H, J = 8.7 Hz, coumarin C5-H), 7.53 (s, 1H, CONH2a; DO exchange), 7.50 (dd, 1H, J = 1.8, 8.7 Hz, coumarin C6-H), 7.19 (s, 1H, CONH2b; DO exchange), 6.26 (d, 1H, J = 1.1 Hz, coumarin C3-H), 4.34-4.23 (m, 1H, Ala-αCH), 3.91-3.80 (m, 1H, isoGln-αH), 2.43 (t, 2H, J = 8.0 Hz, isoGln-βCH2), 2.40 (d, 3H, J = 1.1 Hz, Coumarin C4- CH3), 2.15-2.03 (m, 1H, isoGln-γCH2a), 1.94-1.80 (m, 1H, isoGln-γCH2b), 1.35 (d, 3H, J = 6.9 Hz, Ala- CH3). LCMS m / z: 375.3 [M+H] + (Exact Mass: 374.16) Purity (LCMS): 95.46%

[0209] (Synthesis of HD-isoGln-pNA (Compound 31))

[0210]

[0211] Fmoc-D-isoGln-pNA (compound 23) (2.96 g, 6.06 mmol) was reacted according to the general method, and the reaction mixture was concentrated to dryness under reduced pressure. The residue was crystallized from n-Hex (60 mL) / CHCl (30 mL), filtered, and dried (50 °C, reduced pressure) to give compound 31 (1.51 g, 93.5%) as a pale gray-white solid. 1H-NMR (400 MHz, DMSO-d6) δppm: 11.18-10.01 (br, 1H, pNA-NH; D2O exchange), 8.21 (d, 2H, J = 9.4 Hz, pNA C3,5-H), 7.84 (d, 2H, J = 9.4 Hz, pNA C2,6-H), 7.34 (s, 1H, CONH2a; D2O exchange), 6.98 (s, 1H, CONH2b; D2O exchange), 3.14 (dd, 1H, J = 5.5, 7.8 Hz, isoGln-αH), 2.47 (t, 2H, J = 7.8 Hz, isoGln-βCH2), 2.04-1.53(m, 4H [2.04-1.53 ​​(br, 2H, NH2; D2O exchange), 1.94-1.82 (m, 1H, isoGln-γCH2a), 1.76-1.63 (m, 1H, isoGln-γCH2b]). LCMS m / z: 267.5 [M+H] + (Exact Mass: 266.10) Purity (LCMS): 97.63%

[0212] (Synthesis of HL-Ala-D-isoGln-pNA (Compound 46))

[0213]

[0214] Fmoc-L-Ala-D-isoGln-pNA (compound 40) (885 mg, 1.58 mmol) was reacted according to the general method, and the reaction mixture was concentrated to dryness under reduced pressure. n-Hex (15 mL) / CHCl3 (15 mL) was added to the residue, and the mixture was stirred, filtered, and dried (50 °C, reduced pressure) to give compound 46 (474 ​​mg, 88.8%) as a white solid. 1 From the H-NMR spectrum, it was estimated to be a mixture of two isomers (abundance ratio: approximately 9:1). 1H-NMR(400 MHz, DMSO-d6)δppm: 10.56(s, 0.9H, pNA-NH; D2O exchange), 10.54(s, 0.1H, pNA-NH; D2O exchange), 8.21(d, 2H, J = 9.4 Hz, pNA C3,5-H), 8.11-7.91(br, 1H, D-isoGln-NH; D2O exchange), 7.83(d, 2H, J = 9.4 Hz, pNA C2,6-H), 7.47(s, 0.9H, isoGln- CONH2a; D2O exchange), 7.38(s, 0.1H, isoGln- CONH2a; D2O exchange), 7.15(s, 0.9H, isoGln- CONH2b; D2O exchange), 7.12(s, 0.1H, isoGln- CONH2b; D2O exchange), 4.26(br-dd, 1H, J = 5.7, 7.3 Hz, isoGln-αH), 3.29(q, 1H, J = 6.9 Hz, Ala-αH), 2.40(t, 2H, J = 8.0 Hz, isoGln-βCH2), 2.14-1.73(m, 4H, isoGln-γCH2a,b, Ala- NH2; D2O exchenge), 1.122(d, 2.7H, J = 6.9 Hz, Ala- CH3), 1.116(d, 0.3H, J = 6.9 Hz, Ala- CH3). LCMS m / z: 338.5 [M+H] + .(Exact Mass: 337.14) Purity(LCMS): 95.02%(84.76% + 10.26%)

[0215] Synthesis of benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Glu(OAll)-MCA and -D-Glu(MCA)-OAll (EDCI-HOBt method) (General Method) Benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-OH (compound 7) (1.1 eq.) was dissolved in anhydrous DMF (10 mL / g), HOBt·HO (1.3 eq.) and EDCI (1.3 eq.) were added, and the mixture was stirred on ice for 30 min. DIEA (74 mg, 0.57 mmol, 2.2 eq.) and HD-Glu(OAll)-MCA or HD-Glu(MCA)-OAll (1.0 eq.) were then added to the reaction mixture, followed by stirring for 1 h and then at room temperature for another 2 h. The reaction mixture was concentrated to dryness under reduced pressure.

[0216] Synthesis of Benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Glu(OAll)-MCA (Compound 57)

[0217]

[0218] HD-Glu(OAll)-MCA (compound 28) (90 mg, 0.26 mmol) was used in the reaction according to the general method, and the reaction mixture was concentrated to dryness under reduced pressure. The residue was dissolved in CHCl3 (60 mL) and washed with water, 1 mol / L HCl, saturated aqueous NaHCO3, and saturated aqueous NaCl. It was then dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure. The residue was suspended in CHCl3, and n-Hex was added with stirring. The mixture was stirred at room temperature. The suspension was filtered and dried (60 °C, reduced pressure) to give 209 mg of a white solid. The solid was then dissolved in 5% MeOH / CHCl3 and purified by silica gel column chromatography (CHCl3 to 10% MeOH / CHCl3). The mixed fraction with Rf = 0.66 and 0.62 (10% MeOH / CHCl3) was concentrated to dryness under reduced pressure, and the residue was washed with n-Hex. / CHCl3 and dried (60 °C, reduced pressure) to give compound 57 (144 mg, 63.4%) as a white solid.

[0219] 1 ​From the H-NMR spectrum, it was estimated to be a mixture of five isomers in the estimated ratio of 5:17:6:50:22. LCMS showed two retention times. 1H-NMR(400 MHz, DMSO-d6)δppm: 10.50, 10.46, 10.41, 10,36, 10,25(each s, 1H, クマリンC7-NH; D2O exchange), 8.65-8.10(m, 2H, NH; D2O exchange [8.61(d, J = 7.8 Hz), 8.50(d, J = 7.8 Hz), 8.43(d, J = 7.3 Hz), 8.35(d, J = 7.3 Hz), 8.30(d, J = 7.8 Hz), 8.28(d, J = 7.8 Hz), 8.15(d, J = 8.5 Hz)]), 7.84-7.47(m, 4H, クマリンC8-H [7.82(d, J = 2.1 Hz,), 7.79(d, J = 2.1 Hz), 7.77(d, J = 2.1 Hz), 7.75(d, J = 2.1 Hz)], クマリンC5-H [7.724(d, J = 8.7 Hz,), 7.718(d, J = 8.7 Hz), 7.69(d, J = 8.7 Hz)], 7.63(d, J = 6.9 Hz, NH; D2O exchange), クマリンC6-H [7.55(dd, J = 2.1, 8.7 Hz), 7.53(dd, J = 2.1, 8.7 Hz), 7.50(dd, J = 2.1, 8.7 Hz)]), 7.46-7.20(m, 10H, OCH2Ph, ベンジリデン-Ph), 6.28(d, 1H, J = 1,1 Hz, クマリンC3-H), 5.95-5.82(m, 1H, OCH2-CH= CH2), 5.71, 5.70, 5.69(each s, 1H, ベンジリデン-CH), 5.32-5.23(m, 1H, O CH2a -CH= CH2), 5.22-5.15(m, 1H, O CH2b -CH= CH2), 4.92-4.83(m, 1H, MurNAc C1-βH [4.89(d, J = 3.7 Hz), 4.87(d, J = 3.7 Hz), 4.86(d, J = 3.7 Hz)]), 4.74-4.62(m, 1H, Ph CH2a [4.71(d, Jgem = 12.6 Hz,), 4.69(d, Jgem = 12.6 Hz), 4.65 (d, Jgem = 12.6 Hz)]), 4.58-4.09 (m, 7H), 4.06-3.86 (m, 1H), 3.83-3.57 (m, 4H), 2.48-2.35 (m, 5H, Glu-βCH2, Coumarin C4- CH3 [2.40 (d, J = 1,1 Hz), 2.37 (d, J = 1,1 Hz)]), 2.19-1.75 (m, 5H, Glu-γCH2, NHCO CH3 [1.81(s), 1.80(s)]), 1.31-1.18(m, 6H, Ala- CH3 or MurNAc- CH3 [1.27(d, J = 7,1 Hz), 1.26(d, LCMS m / z: 869.6 [M+H]. + (Exact Mass: 868.35) Purity (LCMS; 326 nm): 91.67% (52.98%, 38.69%).

[0220] Synthesis of Benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Glu(MCA)-OAll (Compound 54)

[0221]

[0222] HD-Glu(MCA)-OAll (compound 27) (325 mg, 0.94 mmol) was used in the reaction according to the general method, and the reaction mixture was concentrated to dryness under reduced pressure. The residue was dissolved in CHCl3 (200 mL) and washed with water, 1 mol / L HCl, saturated aqueous NaHCO3, and saturated aqueous NaCl. It was then dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure. The residue was suspended in CHCl3 (20 mL), and n-Hexane (40 mL) was added with stirring and the mixture was stirred at room temperature. The suspension was collected by filtration, washed with n-Hexane (40 mL) / CHCl3 (10 mL), and dried (50 °C, reduced pressure) to give compound 54 (741 mg, 90.4%) as a pale yellow solid. 1 From the H-NMR spectrum, it was estimated to be a mixture of two isomers (abundance ratio: approximately 3:1). 1H-NMR (400 MHz, DMSO-d6) δppm: 10.37 (s, 0.25H, Coumarin C7-NH; D2O exchange), 10.35 (s, 0.75H, Coumarin C7-NH; D2O exchange), 8.63 (d, 0.25H, J = 7.6 Hz, NH; D2O exchange), 8.51 (d, 0.75H, J = 7.6 Hz, NH; D2O exchange), 8.40 (d, 0.25H, J = 8.0 Hz, NH; D2O exchange), 8.16 (d, 0.75H, J = 8.0 Hz, NH; D2O exchange), 7.75 (d, 0.25H, J = 2.1 Hz, Coumarin C8-H), 7.74 (d, 0.75H, J = 2.1 Hz, coumarin C8-H), 7.70 (d, 0.75H, J = 8.7 Hz, coumarin C5-H), 7.695 (d, 0.25H, J = 8.7 Hz, coumarin C5-H), 7.65 (d, 0.25H, J = 7.8 Hz, NH; DO exchange), 7.56 (d, 0.75H, J = 7.8 Hz, NH; DO exchange), 7.50-7.24 (m, 11H, OCH2Ph, benzylidene-Ph, coumarin C6-H), 6.26 (d, 1H, J = 1.1 Hz, coumarin C3-H), 5.95-5.82 (m, 1H, O CH2-CH= CH2), 5.701 (s, 0.25H, benzylidene-CH), 5.700 (s, 0.75H, benzylidene-CH), 5.31 (ddt, 0.25H, J = 1.6, 3.2, 17.2 Hz, O CH2a -CH= CH2), 5.29 (ddt, 0.75H, J = 1.6, 3.2, 17.2 Hz, O CH2a -CH= CH2), 5.23-5.17 (m, 1H), 4.89 (d, 0.25H, J = 3.7 Hz, MurNAc C1-βH), 4.87 (d, 0.75H, J = 3.7 Hz, MurNAc C1-βH), 4.70 (d, 0.75H, Jgem = 12.6 Hz, Ph- CH2a), 4.69 (d, 0.25H, Jgem = 12.6 Hz, Ph- CH2a), 4.63-4.53 (m, 2H), 4.51 (d, 0.75H, Jgem = 12.6 Hz, Ph- CH2b), 4.47 (d, 0.25 H, Jgem = 12.6 Hz, Ph- CH2b), 4.45-4.12 (m, 4H), 4.03-3.91 (m, 1H), 3.84-3.61 (m, 4H), 2.46 (t, 2H, J = 7.3Hz, Glu-βCH2), 2.394 (d, 0.75H, J = 1,1 Hz,クマリンC4- CH3), 2.391 (d, 2.25H, J = 1,1 Hz, クマリンC4- CH3), 2.21 (m, 1H, Glu-γCH2a), 2.02-1.84 (m, 1H, Glu-γCH2b), 1.81 (s, 0.75H, NHCO CH3), 1.79 (s, 2.25H, NHCO CH3), 1.27 (d, 0.75H, J = 7.1 Hz, Ala- CH3 or MurNAc- CH3), 1.25 (d, 2.25H, J = 7.1 Hz, Ala- CH3 or MurNAc- CH3), 1.24 (d, 0.75H, J = 6.6 Hz, Ala- CH3 or MurNAc- CH3), 1.21(d, 2.25H, J = 6.6 Hz, Ala- CH3 or MurNAc- CH3). LCMS m / z: 869.82 [M+H]. + .(Exact Mass: 868.35) Purity(LCMS): 89.94%(62.74% + 27.20%)

[0223] (Synthesis of ベンジル-4,6-O-ベンジリデン-α-MurNAc-L-Ala-D-isoGln-MCA (Compound 58))

[0224]

[0225] Benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-OH (compound 7) (750 mg, 1.38 mmol) was dissolved in a mixture of anhydrous CHCl (30 mL) and anhydrous DMF (2 mL). HOBt·HO (254 mg, 1.66 mmol, 1.2 eq.) and EDCI (318 mg, 1.66 mmol, 1.2 eq.) were added and stirred at room temperature for 30 min. DIEA (357 mg, 2.76 mmol, 2.0 eq.) and HD-isoGln-MCA (compound 30) (500 mg, 1.65 mmol, 1.2 eq.) were then added to the reaction mixture and stirred for 2 h. The reaction mixture was diluted with CHCl and washed with water, saturated aqueous NaHCO, and saturated aqueous NaCl. The mixture was then dried over NaSO, filtered, and concentrated to dryness under reduced pressure. The residue was dissolved in a 20% CHCl3 / MeOH mixture, silica gel (FL-100D, 20 g) was added, and the mixture was concentrated to dryness under reduced pressure. The mixture was purified by silica gel column chromatography (5% to 10% MeOH / CHCl3) and dried (50 °C, reduced pressure) to give compound 58 (910 mg, 79.8%) as a white solid. 1 From the H-NMR spectrum and LCMS spectrum, it was estimated to be a mixture of two isomers (abundance ratio: approximately 3:7). 1H-NMR(400 MHz, DMSO-d6)δppm: 10.37-10.32(s-like, 1H, クマリンC7-NH; D2O exchange), 8.39-8.11(m, 2H, NH; D2O exchange), 7.49(d, 1H, J = 2.1 Hz), 7.73-7.54(m, 2H; 1H- D2O exchange), 7.50-7.25(m, 12H; 1H- D2O exchange), 7.16-7.09(br-like, 1H, NH; D2O exchange), 6.26(d, 1H, J = 1.1 Hz), 5.71(s, 1H), 4.92(d, 0.7H, J = 3.7 Hz), 4.86(d, 0.3H, J = 3.7Hz), 4.71(d, 0.3H, Jgem = 12.6 Hz, Ph- CH2a), 4.70(d, 0.7H, Jgem = 12.6 Hz, Ph- CH2a), 4.52(d, 0.3H, Jgem = 12.6 Hz, Ph- CH2b), 4.50(d, 0.7H, Jgem = 12.6 Hz, Ph- CH2b), 4.42-4.12(m, 4H), 4.06-3.978(m, 0.3H), 3.975-3.89(m, 0.7H), 3.82-3.63(m, 4H), 2.46-2.35(m, 5H, CH2 & クマリンC4- CH3), 2.12-1.99(m, 1H), 1.95-1.81(m, 1H), 1.84(s, 0.7H, NHCO CH3), 1.81(s, 0.3H, NHCO CH3), 1.30-1.19(m, 6H, CH3 X 2). * 1.253(d, 2.1H, J = 6.6 Hz; CH3), 1.246(d, 2.1H, J = 6.6 Hz; CH3), 1.28-1.23(0.9H; CH3), 1.21(d, 0.9H, J = 6.6 Hz; CH3). LCMS m / z: 828.8 [M+H] + .(Exact Mass: 827.34) Purity(LCMS): 99.81%(29.36%, 70.45%).

[0226] (Synthesis of benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-isoGln-pNA (Compound 56))

[0227]

[0228] HD-isoGln-pNA (compound 31) (400 mg, 1.50 mmol) was reacted according to the general method, and the reaction mixture was concentrated to dryness under reduced pressure. The residue was dissolved in CHCl3 (180 mL), and water (80 mL) was added to separate the layers. The white solid precipitated in the aqueous layer was collected by filtration, dissolved in 20% MeOH / CHCl3, dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure to give a pale yellow, viscous oily residue. The residue was treated with n-Hex (25 mL) / CHCl3 (25 mL), and the resulting white gel was collected by filtration and dried (50 °C, reduced pressure) to give compound 56 (622 mg, 52.4%) as a pale yellow solid. 1 From the H-NMR spectrum and LCMS spectrum, it was estimated to be a mixture of two isomers (abundance ratio: approximately 5.7:1). 1H-NMR(400 MHz, DMSO-d6)δppm: 10.53(s, 1H, pNA-NH; D2O exchange), 8.36(d, 0.15H, J = 8.5 Hz, NH; D2O exchange), 8.24-8.17(m, 3H [8.21(d, 2H, J = 9.4 Hz, pNA C3,5-H), 8.20(d, 0.85H, J = 8.5 Hz, NH; D2O exchange), 0.15H, NH; D2O exchange]), 8.14(d, 0.85H, J = 8.5 Hz, NH; D2O exchange), 7.82(d, 2H, J = 9.4 Hz, pNA C2,6-H), 7.69(d, 0.15H, J = 7.1 Hz, NH; D2O exchange), 7.56(d, 0.85H, J = 7.1 Hz, NH; D2O exchange), 7.47-7.25(m, 11H, ベンジリデン-Ph, ベンジル-Ph, isoGln- NH2a; D2O exchange), 7.13(br-s, 0.15H, isoGln- NH2b; D2O exchange), 7.11(br-s, 0.85H, isoGln- NH2b; D2O exchange), 5.71(s, 1H, ベンジリデン-CH), 4.91(d, 0.15H, J = 3.7 Hz, MurNAc C1-βH), 4.86(d, 0.85H, J = 3.7 Hz, MurNAc C1-βH), 4.71(d, 0.85H, Jgem = 12.6 Hz, Ph- CH2a), 4.70(d, 0.15H, Jgem = 12.6 Hz, Ph- CH2a), 4.51(d, 0.85H, Jgem = 12.6 Hz, Ph- CH2b), 4.49(d, 0.15H, Jgem = 12.6 Hz, Ph- CH2b), 4.40-4.12(m, 4H), 4.06-3.89(m, 1H), 3.83-3.63(m, 4H), 2.44(t, 0.15H, J = 7.8 Hz, isoGln-βCH2), 2.41(t, 0.85H, J = 7.8 Hz, isoGln-βCH2), 2.12-2.01(m, 1H, isoGln-γCH2a), 1.86-1.74(m, 4H [1.86-1.74(m, 1H, isoGln-γCH2b), 1.84(s, 0.45H, NHCO CH3), 1.80(s, 2.55H, NHCO CH3]), 1.29-1.16 (m, 6H [1,244 (d, 0.45H, J = 6.9 Hz, Ala- CH3 or MurNAc- CH3), 1.237 (d, 2.55H, J = 6.9 Hz, Ala- CH3 or MurNAc- CH3), 1.20 (d, 2.55H, J = 6.6 Hz, Ala- CH3 or MurNAc- CH3]). LCMS m / z: 791.8 [M+H]. + (Exact Mass: 790.32) Purity (LCMS): 97.05% (79.14%, 17.91%).

[0229] (Synthesis of benzyl-4,6-O-benzylidene-α-MurNAc-D-Ala-D-isoGln-pNA (Compound 59))

[0230]

[0231] HD-isoGln-pNA (compound 31) (312 mg, 1.17 mmol) was reacted according to the general method, and the reaction mixture was concentrated to dryness under reduced pressure. A pale yellow, syrup-like residue was obtained. The residue was suspended in CHCl3 (10 mL), and n-Hex. (40 mL) was added and stirred. The suspension was collected by filtration, washed with water, and dried (50 °C, reduced pressure) to obtain compound 59 (973 mg, quant.) as a white solid. 1 From the H-NMR spectrum and LCMS spectrum, it was estimated to be a mixture of two isomers (abundance ratio: approximately 3:1). 1H-NMR(400 MHz, DMSO-d6)δppm: 10.53(s, 1H, pNA-NH; D2O exchange), 8.37(d, 0.75H, J = 7.6 Hz, CONH; D2O exchange), 8.24-8.11(m, 3.25H [8.20(d, 2H, J = 9.4 Hz, pNA C3,5-H), 8.15(d, 0.25H, J = 8.2 Hz, CONH; D2O exchange), 0.25H, CONH; D2O exchange]), 8.14(d, 0.75H, J = 8.2 Hz, CONH; D2O exchange), 7.82(d, 2H, J = 9.4 Hz, pNA C2,6-H), 7.70(d, 0.75H, J = 7.3 Hz, CONH; D2O exchange), 7.69(d, 0.25H, J = 7.4 Hz, CONH; D2O exchange), 7.47-7.25(m, 11H, ベンジリデン-Ph, ベンジル-Ph, isoGln- CONH2a; D2O exchange), 7.13(br-s, 0.75H, isoGln- CONH2b; D2O exchange), 7.11(br-s, 0.25H, isoGln- CONH2b; D2O exchange), 5.71(s, 1H, ベンジリデン-CH), 4.92(d, 0.75H, J = 3.7 Hz, MurNAc C1-βH), 4.86(d, 0.25H, J = 3.7 Hz, MurNAc C1-βH), 4.71(d, 0.25H, Jgem = 12.6 Hz, Ph- CH2a), 4.70(d, 0.75H, Jgem = 12.6 Hz, Ph- CH2a), 4.51(d, 0.25H, Jgem = 12.6 Hz, Ph- CH2b), 4.50(d, 0.75H, Jgem = 12.6 Hz, Ph- CH2b), 4.41-4.11(m, 4H), 4.05-3.86(m, 1H), 3.84-3.62(m, 4H), 2.44(t, 0.75H, J = 8.0 Hz, isoGln-βCH2), 2.41(t, 0.25H, J = 8.0 Hz, isoGln-βCH2), 2.12 - 1.96 (m, 1H, isoGln-γCH2a), 1.94 - 1.77 (m, 4H [1.94 - 1.77 (m, 1H, isoGln-γCH2b), 1.84 (s, 2.25H, NHCO CH3), 1.81 (s, 0.75H, NHCO CH3)]), 1.28 - 1.18 (m, 6H [1.25 (d, 2.25H, J = 7.1 Hz, Ala-CH3 or MurNAc-CH3), 1.24 (d, 2.25H, J = 6.6 Hz, Ala-CH3 or MurNAc-CH3), 1.20 (d, 0.75H, J = 6.6 Hz, Ala-CH3 or MurNAc-CH3)]). LCMS m / z: 791.8 [M+H]. + . (Exact Mass: 790.32) Purity (LCMS): 81.26% (20.68% + 60.58%)

[0232] <Synthesis of H-L-Ala-D-isoGln-MCA・HCl> (Synthesis of Fmoc-L-Ala-D-isoGln-MCA (Compound 39))

[0233]

[0234] Fmoc-L-Ala-OH (compound 33) (87 mg, 0.28 mmol) was dissolved in a mixture of anhydrous CHCl (6 mL) and anhydrous DMF (0.4 mL). HOBt·HO (51 mg, 0.33 mmol, 1.2 eq.) and EDCI (63 mg, 0.33 mmol, 1.2 eq.) were added and the mixture was stirred at room temperature for 30 min. DIEA (72 mg, 0.56 mmol, 2.0 eq.) and HD-isoGln-MCA (compound 30) (100 mg, 0.33 mmol, 1.2 eq.) were then added to the reaction mixture and the mixture was stirred for 3 h. The reaction mixture was concentrated to dryness under reduced pressure. The residue was dissolved in 20% CHCl3 / MeOH, silica gel (FL-100D, 4 g) was added, and the mixture was concentrated to dryness under reduced pressure. The mixture was purified by silica gel column chromatography (5% to 10% MeOH / CHCl3). The target fractions were collected and concentrated to dryness under reduced pressure. The residue was washed with n-Hex. / CHCl3 and dried (50 °C, reduced pressure) to give compound 39 (98 mg, 58.7%) as a white solid. 1 H-NMR (400 MHz, DMSO-d6) δppm: 10.36 (s, 1H, Coumarin C7-NH; D2O exchange), 8.09 (d, 1H, J = 8.2 Hz, NH; D2O exchange), 7.88 (d, 2H, J = 7.6 Hz), 7.78-7.65 (m, 4H), 7.61 (d, 1H, J = 7.1 Hz, NH; D2O exchang), 7.50-7.37 (m, 3H), 7.36-7.27 (m, 3H; 1H- D2O exchange), 7.15 (s, 1H, NH; D2O exchange), 6.25 (d, 1H, J = 1.1 Hz, Coumarin C3-H), 4.31-4.17 (m, 4H), 4.13-4.04 (m, 1H), 2.43-2.36 (m, 2H), 2.39 (d, 3H, J = 1.1 Hz, Coumarin C4- CH3), 2.15-1.99 (m, 1H), 1.93-1.75 (m, 1H), 1.23 (d, 3H, J = 7.1 Hz, Ala- CH3). LCMS m / z: 597.6 [M+H] +(Exact Mass: 596.23) Purity (LCMS): 98.28%

[0235] Benzyl-4,6-O-benzylidene-α-MurNAc (general method) Benzyl-4,6-O-benzylidene-α-MurNAc (compound 4) (1.5 eq.) was dissolved in anhydrous DMF (75 mL / g), N-methylmorpholine (4.5 eq.) and HBTU (3.0 eq.) were added under an argon atmosphere, and the mixture was stirred at room temperature for 15 minutes. Subsequently, HL-Ala-D-Glu, HL-Ala-D-Gln, or HL-Ala-D-isoGln derivative (1.0 eq.) was added and the mixture was stirred for 24 hours. The reaction mixture was concentrated to dryness under reduced pressure.

[0236] Synthesis of benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Gln-MCA (Compound 55)

[0237]

[0238] HL-Ala-D-Gln-MCA (compound 44) ​​(186 mg, 0.50 mmol) was reacted according to the general method, and the reaction mixture was concentrated to dryness under reduced pressure. The residue was suspended in AcOEt and washed with water (80 mL). The AcOEt layer (suspension) was separated and washed with 1 mol / L HCl (100 mL), saturated aqueous NaHCO3 (100 mL), and then saturated aqueous NaCl (120 mL). The AcOEt layer (suspension) was separated and concentrated to dryness under reduced pressure. The residue was washed with n-Hex (25 mL) / CHCl3 (20 mL), then with water, and dried (50 °C, reduced pressure) to give compound 55 (412 mg, quant.) as a pale yellow to white solid. 1 From the H-NMR spectrum, it was estimated to be a mixture of two isomers (abundance ratio: approximately 5:1). 1H-NMR (400 MHz, DMSO-d) δ ppm: 10.35 (s, 0.85H, coumarin C7-NH; DO exchange), 10.31 (s, 0.15H, coumarin C7-NH; DO exchange), 8.49 (d, 1H, J = 7.8 Hz, NH; DO exchange), 8.17 (d, 0.15H, J = 8.2 Hz, NH; DO exchange), 8.15 (d, 0.85H, J = 8.2 Hz, NH; DO exchange), 7.83 (d, 0.15H, J = 2.1 Hz, coumarin C8-H), 7.79 (d, 0.85H, J = 2.1 Hz, coumarin C8-H). 7.74 (d, 0.15H, J = 8.7 Hz; coumarin C5-H), 7.72 (d, 0.85H, J = 8.7 Hz; coumarin C5-H), 7.60 (d, 1H, J = 7.1 Hz, NH; DO exchange), 7.55 (dd, 1H, J = 2.1, 8.7 Hz, coumarin C6-H), 7.44-7.26 (m, 11H, Ph X 2, Gln-CONH2a; DO exchange), 6.78 (s, 1H, Gln-CONH2b; DO exchange), 6.28 (d, 1H, J = 1.1 Hz, coumarin C3-H), 5.69 (s, 0.85H, Benzylidene-CH), 5.67 (s, 0.15H, Benzylidene-CH), 4.87 (d, 0.15H, J = 3.7 Hz, MurNac C1-βH), 4.86 (d, 0.85H, J = 3.7 Hz, MurNAc C1-βH), 4.71 (d, 0.85H, Jgem = 12.6 Hz, Ph- CH2a), 4.69 (d, 0.15H, Jgem = 12.6 Hz, Ph- CH2a), 4.51 (d, 0.85H, Jgem = 12.6 Hz, Ph- CH2b), 4.48 (d, 0.15H, Jgem = 12.6 Hz, Ph- CH2b), 4.43-4.26 (m, 2H), 4.25-4.11 (m, 2H,), 4.06-3.96 (m, 1H), 3.82-3.64 (m, 4H), 2.40 (d, 3H, J = 1.1 Hz, Coumarin C4- CH3), 2.18-1.95 (m, 3H, Gln-βCH2, Gln-γCH2a), 1.87-1.76 (m, 1H, Gln-γCH2b), 1.84 (s, 0.45H, NHCO CH3), 1.80 (s, 2.55H, NHCO CH3), 1.27 (d, 2.55H, J = 6.9 Hz, CH3), 1.23 (d, 0.45H, J = 6.9 Hz, CH3), 1.21 (d, 2.55H, J = 6.9 Hz, CH3), 1.19 (d, 0.45H, J = 6.9Hz, CH3). LCMS m / z: 828.7 [M+H]. + (Exact Mass: 827.34) Purity (LCMS): 98.53% (86.66% + 11.87%)

[0239] (Synthesis of benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-isoGln-pNA (Compound 56))

[0240]

[0241] Benzyl-4,6-O-benzylidene-α-MurNAc (compound 4) (226 mg, 0.48 mmol) and HL-Ala-D-isoGln-pNA (compound 46) (210 mg, 0.62 mmol, 1.3 eq.) were reacted according to the general method. Water (5 mL) was added to the reaction mixture (suspension), and the mixture was separated by adding AcOEt. The AcOEt layer (suspension) was separated and washed with 1 mol / L HCl, saturated aqueous NaHCO3, and then water. The AcOEt layer (suspension) was separated and concentrated to dryness under reduced pressure. The residue was suspended in n-Hex (15 mL) / CHCl3 (15 mL), washed, and dried (60 °C, reduced pressure) to give compound 56 (399 mg, quant.) as a white solid. 1 From the 1 H-NMR spectrum and LCMS, it was estimated to be a mixture of two isomers (abundance ratio: approximately 5.7:1). 1H-NMR(400 MHz, DMSO-d6)δppm: 10.57(s, 1H, pNA-NH; D2O exchange), 8.30-8.18(br, 1H, NH; D2O exchange), 8.20(d, 2H, J = 9.4 Hz, pNA C3,5-H), 8.15(br-d, 1H, J = 8.2 Hz, NH; D2O exchange), 7.82(d, 2H, J = 9.4 Hz, pNA C2,6-H), 7.75(br-d, 1H, J = 6.2 Hz , NH; D2O exchange), 7.47-7.26(m, 11H), ベンジリデン-Ph, ベンジル-Ph, isoGln- NH2a; D2O exchange), 7.10(br-s, 1H, isoGln- NH2b; D2O exchange), 5.71(s, 0.85H, ベンジリデン-CH), 5.70(s, 0.15H, ベンジリデン-CH), 4.88(d, 0.15H, J = 3.7 Hz, MurNAc C1-βH), 4.86(d, 0.85H, J = 3.7 Hz, MurNAc C1-βH), 4.71(d, 1H, Jgem = 12.6 Hz, Ph- CH2a), 4.51(d, 1H, Jgem = 12.6 Hz, Ph- CH2b), 4.26-4.12(m, 4H), 4.06-3.95(m, 1H), 3.84-3.66(m, 4H), 2.41(t, 2H, J = 7.8 Hz, isoGln-βCH2), 2.13-2.00(m, 1H, isoGln-γCH2a), 1.87- 1.75(m, 4H, isoGln-γCH2b [1.81(s, 0.45H, NHCO CH3), 1.80(s, 2.55H, NHCO CH3)]), 1.24(d, 3H, J = 6.9 Hz, L-Ala- CH3 or MurNAc- CH3), 1.20(d, 3H, J = 6.6 Hz, L-Ala- CH3 or MurNAc- CH3). LCMS m / z: 791.8 [M+H] +(Exact Mass: 790.32) Purity (LCMS): 98.61% (80.66% + 17.95%)

[0242] <Debenzylidene conversion (general method)> The benzylidene derivative was suspended in 75% AcOH (75-170 mL / g) and stirred for 5-23 hours at 50-60°C. After confirming the disappearance of the raw materials by TLC (10% MeOH / CHCl3), the reaction mixture was concentrated to dryness under reduced pressure to obtain a colorless, starch-like residue.

[0243] Synthesis of Benzyl-α-MurNAc-L-Ala-D-Glu(OAll)-MCA (Compound 61)

[0244]

[0245] Benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Glu(OAll)-MCA (compound 57) (130 mg, 0.15 mmol) was reacted according to the general procedure and concentrated to dryness under reduced pressure. The residue was crystallized from n-Hex. / AcOEt (3:1) and dried (60 °C, reduced pressure) to give compound 61 (115 mg, 98.3%) as a white solid. 1 From H-NMR spectroscopy and LCMS, it was estimated to be a mixture of five isomers in the ratio of 2:17:7:50:24. 1H-NMR(400 MHz, DMSO-d6)δppm: 10.51, 10.46, 10.42, 10,35, 10,32(each s, 1H, クマリンC7-NH; D2O exchange), 8.59-8.06(m, 2H, NH; D2O exchange [8.55(d, J = 8.0 Hz), 8.50(d, J = 8.0 Hz), 8.83(d, J = 7.3 Hz), 8.31(d, J = 7.3 Hz), 8.30(d, J = 7.3 Hz), 8.28(d, J = 7.3 Hz), 8.10(d, J = 8.0 Hz)]), 7.82-7.47(m, 4H, クマリンC8-H [7.80(d, J = 2.1 Hz)], クマリンC5-H [7.73(d, J = 8.7 Hz)], 7.63(d, J = 6.9 Hz, NH; D2O exchange), クマリンC6-H [7.56(dd, J = 2.1, 8.7 Hz)]), 7.41-7.20(m, 5H, O CH2Ph), 6.30-6.27(m, 1H, クマリンC3-H), 5.95-5.83(m, 1H, O CH2-CH= CH2), 5.41-5.16(m, 3H, MurNAc C4-OH; D2O exchange [5.38(d, J = 6.9 Hz), 5.37(d, J = 6.9 Hz), 5.35(d, J = 6.9 Hz), 5.28(d, J = 6.9 Hz)], O CH2a -CH= CH2 [5.32-5.24(m, 1H)], O CH2b -CH= CH2 [5.22-5.16(m, 1H)]), 4.93-4.22(m, 9H, MurNAc C6-OH; D2O exchange, MurNAc C1-βH [4.82(d, J = 3.4 Hz), 4.79(d, J = 3.4 Hz), 4.74(d, J = 3.4 Hz)], Ph CH2a [4.67(d, Jgem = 12.6 Hz)]), 3.88-3.59(m, 2H), 3.57-3.40(m, 3H), 3.39-3.26(m, 1H), 2.48-2.38(m, 5H, Glu-βCH2, クマリンC4- CH3 [2.40 (d, J = 1,1 Hz)]), 2.19-2.02 (m, 1H, Glu-γCH2a), 1.99-1.75 (m, 1H, Glu-γCH2b), 1.80, 1.794, 1.786 (each s, 3H, NHCO CH3), 1.34-1.20 (m, 6H, Ala- CH3 or MurNAc- CH3 [1.27 (d, J = 7,1 Hz), 1.26 (d, J = 6.6 Hz)]). LCMS m / z: 781.6 [M+H]. + (Exact Mass: 780.32) Purity (LCMS; 326 nm): 89.08% (6.61%, 44.59%, 15.07%, 21.34%, 1.47%).

[0246] Synthesis of Benzyl-α-MurNAc-L-Ala-D-Glu(MCA)-OAll (Compound 62)

[0247]

[0248] Benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Glu(MCA)-OAll (compound 54) (500 mg, 0.58 mmol) was reacted according to the general procedure and concentrated to dryness under reduced pressure. The residue was crystallized from n-Hex. / AcOEt (1:1) and dried (50 °C, reduced pressure) to give compound 62 (440 mg, 98.0%) as a pale ochre solid. 1 From the H-NMR spectrum, it was estimated to be a mixture of two isomers (abundance ratio: approximately 3:1). 1H-NMR(400 MHz, DMSO-d6)δppm: 10.38(s, 0.25H, クマリンC7-NH; D2O exchange), 10.36(s, 0.75H, クマリンC7-NH; D2O exchange), 8.59(d, 0.25H, J = 7.6 Hz, NH; D2O exchange), 8.50(d, 0.75H, J = 7.6 Hz, NH; D2O exchange), 8.36(d, 0.25H, J = 8.0 Hz, NH; D2O exchange), 8.12(d, 0.75H, J = 8.0 Hz, NH; D2O exchange), 7.76(d, 0.25H, J = 2.1 Hz, クマリンC8-H), 7.74(d, 0.75H, J = 2.1 Hz, クマリンC8-H), 7.71(d, 0.75H, J = 8.7 Hz, クマリンC5-H), 7.70(d, 0.25H, J = 8.7 Hz, クマリンC5-H), 7.69(d, 0.25H, J = 7.8 Hz, NH; D2O exchange), 7.58(d, 0.75H, J = 7.8 Hz, NH; D2O exchange), 7.47(dd, 0.75H, J = 2.1, 8.7 Hz, クマリンC6-H), 7.45(dd, 0.25H, J = 2.1, 8.7 Hz, クマリンC6-H), 7.40-7.23(m, 5H, O CH2Ph), 6.26(d, 1H, J = 1.1 Hz, クマリンC3-H), 5.96-5.83(m, 1H, O CH2-CH= CH2), 5.37(d, 0.25H, J = 6.9Hz, MurNAc C4-OH; D2O exchange), 5.31(d, 0.75H, J = 6.9 Hz, MurNAc C4-OH; D2O exchange), 5.34-5.27(m, 1H, O CH2-CH= CH2a), 5.24-5.18(m, 1H, O CH2-CH= CH2b), 4.80(d, 0.25H, J = 3.4 Hz, MurNAc C1-βH), 4.75(d, 0.75H, J = 3.4 Hz, MurNAc C1-βH), 4.67(d, 0.75H, Jgem = 12.6 Hz, Ph- CH2a), 4.64 (d, 0.25H, Jgem = 12.6 Hz, Ph- CH2a), 4.63-4.55 (m, 3H, [1H-MurNAc C6-OH; D2O exchange]), 4.52-4.26 (m, 4H, [4.43(d, 0.75H, Jgem = 12.6 Hz, Ph- CH2b), 4.41(d, 0.25H, Jgem = 12.6 Hz, Ph- CH2b)]), 3.85-3.60(m, 2H), 3.57-3.42(m, 3H), 3.40-3.26(m, 1H), 2.47(t, 2H, J = 7.6 Hz, Glu-βCH2), 2.40 (d, 3H, J = 1.1 Hz, Coumarin C4- CH3), 2.21-2.06 (m, 1H, Gln-γCH2a), 2.00-1.83 (m, 1H, Gln-γCH2b), 1.80 (s, 0.75H, NHCO CH3), 1.73(s, 2.25H, NHCO CH3), 1.30-1.22(m, 6H [1.26(d, 2.25H, J = 6.6 Hz, Ala- CH3 or MurNAc- CH3), 1.25(d, 2.25H, J = 6.6 Hz, Ala- CH3 or MurNAc-CH3)).LCMS m / z: 781.8 [M+H]. + (Exact Mass: 780.32) Purity (LCMS): 84.68% (60.31% + 24.37%)

[0249] Synthesis of Benzyl-α-MurNAc-L-Ala-D-Gln-MCA (Compound 60)

[0250]

[0251] Benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Gln-MCA (compound 55) (384 mg, 0.46 mmol) was reacted according to the general procedure and concentrated to dryness under reduced pressure. The residue was crystallized from n-Hex. / CHCl3 (1:1) and dried (50 °C, reduced pressure) to give compound 60 (327 mg, 95.3%) as a white solid. 1From the H-NMR spectrum, it was estimated to be a mixture of two isomers (abundance ratio: approximately 4:1). 1H-NMR(400 MHz, DMSO-d6)δppm: 10.35(s, 0.8H, クマリンC7-NH; D2O exchange), 10.31(s, 0.2H, クマリンC7-NH; D2O exchange), 8.49(d, 1H, J = 8.0 Hz, NH; D2O exchange), 8.28(d, 0.2H, J = 8.2 Hz, NH; D2O exchange), 8.11(d, 0.8H, J = 8.2 Hz, NH; D2O exchange), 7.85(d, 0.2H, J = 1.8 Hz, クマリンC8-H), 7.81(d, 0.8H, J = 1.8 Hz, クマリンC8-H), 7.75(d, 0.2H, J = 8.7 Hz, クマリンC5-H), 7.73(d, 0.8H, J = 8.7 Hz, クマリンC5-H), 7.61(d, 1H, J = 7.1 Hz, NH; D2O exchange), 7.57(dd, 1H, J = 1.8, 8.7 Hz, クマリンC6-H), 7.40-7.24(m, 6H, Ph, CONH2a; D2O exchange), 6.79(s, 1H, CONH2b; D2O exchange), 6.28(d, 1H, J = 1.1 Hz; クマリンC3-H), 5.52(d, 0.2H, J = 7.1 Hz, MurNAc C4-OH; D2O exchange), 5.29(d, 0.8H, J = 7.1 Hz, MurNAc C4-OH; D2O exchange), 4.76(d, 0.2H, J = 3.4 Hz), 4.74(d, 0.8H, J = 3.4 Hz), 4.66(d, 1H, Jgem = 12.6 Hz, Ph- CH2a), 4.62(t, 0.2H, J = 6.0 Hz, MurNAc C6-OH; D2O exchange), 4.59(t, 0.8H, J = 6.0 Hz, MurNAc C6-OH; D2O exchange), 4.47-4.23(m, 3H), 4.442(d, 0.2H, Jgem = 12.6 Hz, Ph- CH2b), 4.436(d, 0.8H, Jgem = 12.6 Hz, Ph- CH2b), 3.86-3.78 (m, 1H), 3.69-3.60 (m, 1H), 3.86-3.44 (m, 2H), 3.35-3.26 (m, 1H), 2.40 (d, 3H, J = 1.1 Hz, Coumarin C4- CH3), 2.18-2.10 (m, 2H, Gln-βCH2), 2.07-1.97 (m, 1H, Gln-γCH2a), 1.88-1.78 (m, 1H, Gln-γCH2b), 1.82 (s, 0.6H, NHCO CH3), 1.79 (s, 2.4H, NHCO CH3), 1.27(d, 3H, J = 6.9 Hz, CH3), 1.26 (d, 3H, J = 6.6 Hz, CH3). LCMS m / z: 740.7 [M+H]. + (Exact Mass: 739.31) Purity (LCMS): 79.34%

[0252] (Synthesis of benzyl-α-MurNAc-L-Ala-D-isoGln-MCA (Compound 63))

[0253]

[0254] Benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-isoGln-MCA (Compound 58) (500 mg, 0.60 mmol) was reacted according to the general method, and the reaction mixture was concentrated to dryness under reduced pressure. The residue was crystallized from n-hexane (10 mL) / CHCl3 (10 mL), dissolved in 20% MeOH / CHCl3, added with silica gel (FL 100D, 10 g), concentrated to dryness under reduced pressure, and purified by silica gel column chromatography (5% to 12% MeOH / CHCl3). Drying (50 °C, reduced pressure) gave compound 63 (63A: Rf = 0.25 [10% MeOH / CHCl3] 198 mg, 63B: Rf = 0.19 [10% MeOH / CHCl3] 54 mg, 86 mg as a mixture) as a white solid (total yield 338 mg, 75.6%).

[0255] (Compound 63A) 1H-NMR(400 MHz, DMSO-d6)δppm: 10.35(s, 1H, クマリンC7-NH; D2O exchange), 8.33(d, 1H, J = 7.1 Hz, isoGln-NH; D2O exchange), 8.10(d, 1H, J = 8.0 Hz, NH-Ac; D2O exchange), 7.79(d, 1H, J = 7.1 Hz, Ala-NH; D2O exchange), 7.76(d, 1H, J = 2.1 Hz, クマリンC8-H), 7.69(d, 1H, J = 8.7 Hz, クマリンC5-H), 7.45(dd, 1H, J = 2.1, 8.7 Hz, クマリンC6-H), 7.38-7.33(m, 5H, Ph-4H, CONH2a; D2O exchange), 7.31-7.24(m, 1H, Ph-1H), 7.12(s, 1H, CONH2b; D2O exchange), 6.26(d, 1H, J = 1.1 Hz, クマリンC3-H), 5.38(d, 1H, J = 6.9 Hz, MurNAc C4-OH; D2O exchange), 4.83(d, 1H, J = 3.4 Hz), 4.66(d, 1H, Jgem = 12.6 Hz, Ph- CH2a), 4.60(t, 1H, J = 6.0 Hz, MurNAc C6-OH; D2O exchange), 4.46(q, 1H, J = 6.6 Hz, MurNAc-OCH CH3), 4.42(d, 1H, Jgem = 12.6 Hz, Ph- CH2b), 4.37(pent., 1H, J = 7.1Hz, Ala-αH), 4.29-4.20(m, 1H), 3.75-3.60(m, 2H), 3.57-3.42(m, 3H), 3.40-3.33(m, 1H), 2.46-2.37(m, 2H), 2.39(d, 3H, J = 1.1 Hz, クマリンC4- CH3), 2.12-1.98(m, 1H), 1.94-1.81(m, 1H), 1.82(s, 3H, NHCO CH3), 1.27(d, 3H, J = 6.6 Hz, MurNAc- CH3), 1.26(d, 3H, J = 7.1 Hz, Ala- CH3). LCMS m / z: 740.5 [M+H]. + .(Exact Mass: 739.31) Purity(LCMS): 99.09%

[0256] (Compound 63B) 1H-NMR(400 MHz, DMSO-d6)δppm: 10.35(s, 1H, クマリンC7-NH; D2O exchange), 8.19(d, 1H, J = 8.2 Hz, isoGln-NH or NH-Ac; D2O exchange), 8.10(d, 1H, J = 8.2 Hz, isoGln-NH or NH-Ac; D2O exchange), 7.75(d, 1H, J = 2.1 Hz, クマリンC8-H), 7.70(d, 1H, J = 8.7 Hz, クマリンC5-H), 7.57(d, 1H, J = 6.9 Hz, Ala-NH; D2O exchange), 7.47(dd, 1H, J = 2.1, 8.7 Hz, クマリンC6-H), 7.41-7.32(m, 5H, Ph-4H, CONH2a; D2O exchange), 7.31-7.25(m, 1H, Ph-H), 7.10(s, 1H, CONH2b; D2O exchange), 6.26(d, 1H, J = 1.1 Hz; クマリンC3-H), 5.30(d, 1H, J = 6.9 Hz, MurNAc C4-OH; D2O exchange), 4.74(d, 1H, J = 3.4 Hz), 4.67(d, 1H, Jgem = 12.6 Hz, Ph- CH2a), 4.60(t, 1H, J = 6.0 Hz, MurNAc C6-OH; D2O exchange), 4.44(d, 1H, Jgem = 12.6 Hz, Ph- CH2b), 4.35-4.17(m, 3H), 3.86-3.77(m, 1H), 3.70-3.61(m, 1H), 3.57-3.44(m, 3H), 3.36-3.27(m, 1H), 2.44-2.35(m, 2H), 2.39(d, 3H, J = 1.1 Hz, クマリンC4- CH3), 2.13-2.00(m, 1H), 1.88-1.74(m, 1H), 1.79(s, 3H, NHCO CH3), 1.25(d, 3H, J = 6.6 Hz, MurNAc- CH3), 1.24(d, 3H, J = 7.1 Hz, Ala- CH3). LCMS m / z: 740.5 [M+H]+ (Exact Mass: 739.31) Purity (LCMS): 99.62%

[0257] (Synthesis of benzyl-α-MurNAc-L-Ala-D-isoGln-pNA (Compound 64))

[0258]

[0259] Benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-isoGln-pNA (compound 56) (596 mg, 0.75 mmol) was reacted according to the general procedure and concentrated to dryness under reduced pressure. The residue was dissolved in 10% MeOH / CHCl3 and purified by silica gel column chromatography (5% to 14% MeOH / CHCl3). The combined fractions were concentrated to dryness and dried (50 °C, reduced pressure) to give compound 64 (64A: pale yellow solid, Rf = 0.12 [10% MeOH / CHCl3], 44 mg; 64B: white solid, Rf = 0.08 [10% MeOH / CHCl3], 166 mg; and a mixture of white solids, 222 mg) (total yield 432 mg, 81.5%).

[0260] (Compound 64A) 1H-NMR(400 MHz, DMSO-d6)δppm: 10.54(s, 1H, pNA-NH; D2O exchange), 8.34(d, 1H, J = 7.7 Hz, NH; D2O exchange), 8.20(d, 2H, J = 9.2 Hz, pNA C3,5-H), 8.11(d, 1H, J = 8.0 Hz, NH; D2O exchange), 7.82(d, 2H, J = 9.2 Hz, pNA C2,6-H), 7.79(d, 1H, J = 7.3 Hz, NH; D2O exchange), 7.38-7.24(m, 6H, ベンジル-Ph, CONH2a; D2O exchange), 7.13(s, 1H, CONH2b; D2O exchange), 5.39(d, 1H, J = 6.9 Hz, MurNAc C4-OH; D2O exchange), 4.83(d, 1H, J = 3.4 Hz, MurNAc C1-βH), 4.65(d, 1H, Jgem = 12.4 Hz, Ph- CH2a), 4.61(br-t, 1H, J = 5.3 Hz, MurNAc C6-OH; D2O exchange), 4.50-4.33(m, 3H [4.22(d, 1H, Jgme = 12.4 Hz, ベンジル- CH2b), 4.36(t, 1H, J = 7.1 Hz)]), 4.29-4.19(m, 1H), 3.75-3.60(m, 2H), 3.57-3.42(m, 3H), 3.40-3.30(m, 1H), 2.44(t, 2H, J = 8.0 Hz, isoGln-βCH2), 2.11-1.98(m, 1H, isoGln-γCH2a), 1.94-1.77(m, 1H, isoGln-γCH2b), 1.81(s, 3H, NHCO CH3), 1.26(d, 3H, J = 6.6 Hz, Ala- CH3 or MurNAc- CH3), 1.25(d, 3H, J = 6.9 Hz, Ala- CH3 or MurNAc- CH3). LCMS m / z: 703.7 [M+H] + .(Exact Mass: 702.29) Purity(LCMS): 97.63%

[0261] (Compound 64B) 1 H-NMR (400 MHz, DMSO-d6) δ ppm: 10.54 (s, 1H, pNA-NH; D2O exchange), 8.21 (d, 2H, J = 9.2 Hz, pNA C3,5-H), 8.20 (d, 1H, J = 8.0 Hz, NH; D2O exchange), 8.11 (d, 1H, J = 8.2 Hz, NH; D2O exchange), 7.83 (d, 2H, J = 9.2 Hz, pNA C2,6-H), 7.57 (d, 1H, J = 6.9 Hz, NH; D2O exchange), 7.42 - 7.25 (m, 6H, benzyl-Ph, CONH2a; D2O exchange), 7.11 (s, 1H, CONH2b; D2O exchange), 5.31 (d, 1H, J = 6.9 Hz, MurNAc C4-OH; D2O exchange), 4.74 (d, 1H, J = 3.4 Hz, MurNAc C1-βH), 4.67 (d, 1H, Jgem = 12.6 Hz, Ph-CH2a), 4.61 (t, 1H, J = 6.0 Hz, MurNAc C6-OH; D2O exchange), 4.44 (d, 1H, Jgme = 12.6 Hz, Ph-CH2b), 4.35 - 4.17 (m, 3H), 3.87 - 3.77 (m, 1H), 3.71 - 3.60 (m, 1H), 3.57 - 3.44 (m, 3H), 3.36 - 3.26 (m, 1H), 2.41 (t, 2H, J = 7.8 Hz, isoGln-βCH2), 2.13 - 2.00 (m, 1H, isoGln-γCH2a), 1.86 - 1.73 (m, 1H, isoGln-γCH2b), 1.79 (s, 3H, NHCO CH3), 1.25 (d, 3H, J = 7.1 Hz, Ala-CH3 or MurNAc-CH3), 1.23 (d, 3H, J = 7.6 Hz, Ala-CH3 or MurNAc-CH3). LCMS m / z: 703.7 [M+H] +(Exact Mass: 702.29) Purity (LCMS): 99.45%

[0262] (Synthesis of benzyl-α-MurNAc-D-Ala-D-isoGln-pNA (Compound 65))

[0263]

[0264] Benzyl-4,6-O-benzylidene-α-MurNAc-D-Ala-D-isoGln-pNA (compound 59) (900 mg, 1.14 mmol) was reacted according to the general procedure and concentrated to dryness under reduced pressure. The residue was dissolved in 10% MeOH / CHCl3 and purified by silica gel column chromatography (5% to 15% MeOH / CHCl3). The combined fractions were concentrated to dryness and dried (50 °C, reduced pressure) to give compound 65 (65A: Rf = 0.14 [10% MeOH / CHCl3] 440 mg, 65B: Rf = 0.09 [10% MeOH / CHCl3] 72 mg, 109 mg as a mixture) as a white solid (total yield 621 mg, 77.6%).

[0265] (Compound 65A) 1H-NMR(400 MHz, DMSO-d6)δppm: 10.54(s, 1H, pNA-NH; D2O exchange), 8.21(d, 2H, J = 9.2 Hz, pNA C3,5-H), 8.20(d, 1H, J = 8.2 Hz, NH; D2O exchange), 8.11(d, 1H, J = 8.2 Hz, NH; D2O exchange), 7.83(d, 2H, J = 9.2 Hz, pNA C2,6-H), 7.57(d, 1H, J = 6.9 Hz, NH; D2O exchange), 7.43-7.25(m, 6H, ベンジル-Ph, CONH2a; D2O exchange), 7.12(s, 1H, CONH2b; D2O exchange), 5.31(d, 1H, J = 6.9 Hz, MurNAc C4-OH; D2O exchange), 4.74(d, 1H, J = 3.4 Hz, MurNAc C1-βH), 4.67(d, 1H, Jgem = 12.6 Hz, Ph- CH2a), 4.61(t, 1H, J = 6.0 Hz, MurNAc C6-OH; D2O exchange), 4.44(d, 1H, Jgem = 12.6 Hz, Ph- CH2b), 4.35-4.17(m, 3H), 3.87-3.77(m, 1H), 3.74-3.60(m, 1H), 3.59-3.42(m, 3H), 3.37-3.25(m, 1H), 2.42(t, 2H, J = 7.8 Hz, isoGln-βCH2), 2.13-1.99(m, 1H, isoGln-γCH2a), 1.89-1.74(m, 1H, isoGln-γCH2b), 1.79(s, 3H, NHCO CH3), 1.26(d, 3H, J = 7.1 Hz, D-Ala- CH3 or MurNAc- CH3), 1.24(d, 3H, J = 7.6 Hz, Ala- CH3 or MurNAc- CH3). LCMS m / z: 703.6 [M+H] + .(Exact Mass: 702.29) Purity(LCMS): 100.00%

[0266] (Compound 65B) 1 H-NMR (400 MHz, DMSO-d6) δ ppm: 10.53 (s, 1H, pNA-NH; D2O exchange), 8.34 (d, 1H, J = 7.1 Hz, NH; D2O exchange), 8.20 (d, 2H, J = 9.2 Hz, pNA C3,5-H), 8.10 (d, 1H, J = 8.0 Hz, NH; D2O exchange), 7.82 (d, 2H, J = 9.2 Hz, pNA C2,6-H), 7.79 (d, 1H, J = 7.6 Hz, NH; D2O exchange), 7.41 - 7.22 (m, 6H, benzyl-Ph, CONH2a; D2O exchange), 7.13 (s, 1H, CONH2b; D2O exchange), 5.39 (d, 1H, J = 6.9 Hz, MurNAc C4-OH; D2O exchange), 4.83 (d, 1H, J = 3.4 Hz, MurNAc C1-βH), 4.66 (d, 1H, Jgem = 12.6 Hz, Ph-CH2a), 4.61 (t, 1H, J = 5.9 Hz, MurNAc C6-OH; D2O exchange), 4.51 - 4.31 (m, 3H), 4.29 - 4.19 (m, 1H), 3.76 - 3.59 (m, 2H), 3.58 - 3.42 (m, 3H), 3.40 - 3.28 (m, 1H), 2.44 (t, 2H, J = 8.0 Hz, isoGln-βCH2), 2.11 - 1.96 (m, 1H, isoGln-γCH2a), 1.94 - 1.76 (m, 1H, isoGln-γCH2b), 1.82 (s, 3H, NHCO CH3), 1.27 (d, 3H, J = 6.6 Hz, D-Ala-CH3 or MurNAc-CH3), 1.25 (d, 3H, J = 6.9 Hz, D-Ala-CH3 or MurNAc-CH3)). LCMS m / z: 703.6 [M+H] + . (Exact Mass: 702.29) Purity (LCMS): 92.54%

[0267] <Deallylation (General Method)> Allyl ester (1 mmol) was dissolved in CH3CN (75 mL) and water (3 mL), degassed under reduced pressure, and AcOH (5.0 eq.) and 4-methylmorpholine (4.0 eq.) were added. Next, Pd(OAc)2 (0.5 eq.) and PPh3 (1.0 eq.) were added, and the mixture was stirred at 50 °C for 6 hours. The disappearance of the raw materials was confirmed by TLC (10% MeOH / CHCl3), and the reaction solution was filtered (Millipore Milex-HV 0.45 μm) and concentrated to dryness under reduced pressure.

[0268] Synthesis of Benzyl-α-MurNAc-L-Ala-D-Glu(OH)-MCA (Compound 66)

[0269]

[0270] Benzyl-α-MurNAc-L-Ala-D-Glu(OAll)-MCA (compound 61) (100 mg, 0.13 mmol) was reacted according to the general procedure. The suspension was filtered off and concentrated to dryness under reduced pressure. The residue was crystallized from n-hexane (3 mL) / CHCl3 (3 mL). The supernatant was removed, and the residue was crystallized again from n-hexane (3 mL) / CHCl3 (3 mL). The solid was collected by filtration, washed with n-hexane / CHCl3 (1:1), and dried (60 °C, reduced pressure) to give compound 66 (84 mg, 88.4%) as a pale yellow solid. 1 From the H-NMR and LCMS spectra, it was estimated to be a mixture of three isomers (abundance ratio: approximately 3:1:1). 1H-NMR (400 MHz, DMSO-d6) δppm: 10.38, 10.36 (each s, 1H, クマリンC7-NH; D2O exchange), 8.35-8.18 (br-m, 1H, NH; D2O exchange), 7.87-7.67 (m, 3H, NH; D2O exchange, クマリンC8-H [7.80(d, J = 2.1 Hz), 7.78(d, J = 2.1 Hz)], クマリンC5-H [7.73(d, J = 8.7 Hz), 7.71(d, J = 8.7 Hz)]), 7.59-7.47 (m, 1H, クマリンC6-H [7.56 (dd, J = 2.1, 8.7 Hz]), 7.41-7.19 (m, 5H, O CH2Ph), 6.27 (d, 1H, J = 1.1 Hz, クマリンC3-H), 4.84-3.90 (m, 8H; 2H D2O exchange [4.11 (d, 0.35H, J = 7.3 Hz, MurNAc C4-OH; D2O exchange), 3.93 (br-t like, 0.65H, MurNAc C6-OH; D2O exchange)]), 3.82-3.17 (m, 6H), 2.40 (d, 3H, J = 1,1 Hz,クマリンC4- CH3), 2.32-2.20 (br-m, 2H, Glu-βCH2), 2.12-1.73 (m, 5H, Glu-γCH2, NHCO CH3 [1.81, 1.80, 1.79 (each s, NHCO CH3)]), 1.34-1.17 (m, 6H, Ala- CH3 and MurNAc- CH3). LCMS m / z: 741.5 [M+H] + .(Exact Mass: 740.29) Purity (LCMS; 326 nm): 91.63% (53.12%, 19.61%, 18.90%).

[0271] (Synthesis of ベンジル-α-MurNAc-L-Ala-D-Glu(MCA)-OH (Compound 67))

[0272]

[0273] Benzyl-α-MurNAc-L-Ala-D-Glu(MCA)-OAll (compound 62) (200 mg, 0.26 mmol) was reacted according to the general procedure. The suspension was filtered off and concentrated to dryness under reduced pressure. The residue was crystallized from n-hexane (4 mL) / CHCl3 (4 mL). The supernatant was removed, and the residue was crystallized again from n-hexane (4 mL) / CHCl3 (4 mL). The solid was collected by filtration, washed with n-hexane, and dried (50 °C, reduced pressure) to give compound 67 (189 mg, 89.5%) as a pale ochre solid. 1 H-NMR indicated that it was a mixture of two isomers (abundance ratio: approximately 3:1). 1H-NMR(400 MHz, DMSO-d6)δppm: 10.52(s, 0.75H, クマリンC7-NH; D2O exchange), 10.51(s, 0.25H, クマリンC7-NH; D2O exchange), 8.41(d, 0.25H, J = 7.6 Hz, NH; D2O exchange), 8.26(d, 0.25H, J = 7.6 Hz, NH; D2O exchange), 8.16(d, 0.75H, J = 7.6 Hz, NH; D2O exchange), 8.12(d, 0.75H, J = 8.0 Hz, NH; D2O exchange), 7.76(d, 0.25H, J = 2.1 Hz, クマリンC8-H), 7.75(d-, 0.75H, J = 2.1 Hz, クマリンC8-H), 7.72(d, 0.25H, J = 7.8 Hz, NH; D2O exchange), 7.692(d, 0.75H, J = 8.7 Hz, クマリンC5-H), 7.686(d, 0.25H, J = 8.7 Hz, クマリンC5-H), 7.65(d, 0.75H, J = 7.8 Hz, NH; D2O exchange), 7.46(dd, 0.75H, J = 2.1, 8.7 Hz, クマリンC6-H), 7.45(dd, 0.25H, J = 2.1, 8.7 Hz, クマリンC6-H), 7.40-7.23(m, 5H, O CH2Ph), 6.25(d, 1H, J = 1.1 Hz; クマリンC3-H), 4.81(d, 0.25H, J = 3.4 Hz, MurNAc C1-βH), 4.75(d, 0.75H, J = 3.4 Hz, MurNAc C1-βH), 4.66(d, 0.75H, Jgem = 12.6 Hz, Ph- CH2a), 4.65(d, 0.25H, Jgem = 12.6 Hz, Ph- CH2a), 4.52-4.26(m, 3H, [4.49(q, 0.25H, J = 6.6 Hz), 4.43(d, 0.75H, Jgem = 12.6 Hz, Ph- CH2b), 4.42(d, 0.25H, Jgem = 12.6 Hz, Ph- CH2b), 4.32(q, 0.75H, J = 6.6 Hz)]), 4.22-4.08(m, 1H), 3.99-3.90(br, 1H), 3.85-3.25(m, 8H), 2.48-2.35(m, 5H, Glu-βCH2, [2.39(d, 3H, J = 1.1 Hz, Coumarin C4- CH3]), 2.13-2.01 (m, 1H, Gln-γCH2a), 1.95-1.75 (m, 1H, Gln-γCH2b), 1.81 (s, 0.75H, NHCO CH3), 1.75 (s, 2.25H, NHCO CH3), 1.31-1.19 (m, 6H [1.26 (d, 2.25H, J = 6.6 Hz, Ala- CH3 or MurNAc- CH3), 1.24 (d, 2.25H, J = 6.9 Hz, Ala- CH3 or MurNAc- CH3]). LCMS m / z: 741.6 [M+H]. + (Exact Mass: 740.29) Purity (LCMS): 90.39%

[0274] Acylation (acetylation, benzylation) (general method) HL-Ala-D-isoGln-pNA (compound 46) (1 mmol) was suspended in CHCl (100 mL), and TEA (1.05 eq.) and acid chloride (1.05 eq.) were added under ice cooling. The mixture was stirred for 15 minutes and then at room temperature for 2.5 hours. MeOH (1 mL) was added, and the mixture was concentrated to dryness under reduced pressure.

[0275] (Synthesis of Ac-L-Ala-D-isoGln-pNA (Compound 47))

[0276]

[0277] H-Ala-D-isoGln-pNA (compound 46) (120 mg, 0.36 mmol) and acetyl chloride (29 mg, 0.37 mmol) were reacted according to the general method and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (5% to 10% MeOH-CHCl3). The collected fractions were combined, concentrated to dryness, and dried (50 °C, reduced pressure) to give 122 mg of a white solid. This was suspended and washed with CHCl3 and dried (60 °C, reduced pressure) to give compound 47 (69 mg, 51.1%). 1 H-NMR indicated that it was a mixture of two isomers (abundance ratio: approximately 13:1). 1 H-NMR (400 MHz, DMSO-d6) δppm: 10.56 (s, 1H, pNA-NH; D2O exchange), 8.40 (d, 0.07H, J = 6.4 Hz, NH; D2O exchange), 8.21 (d, 2H, J = 9.4 Hz, pNA C3,5-H), 8.164 (d, 1H, J = 8.2 Hz, NH; D2O exchange), 8.155 (d, 0.93H, J = 6.4 Hz, NH; D2O exchange), 7.83 (d, 2H, J = 9.4 Hz, pNA C2,6-H), 7.32 (s, 1H, isoGln- CONH2a; D2O exchange), 7.13(s, 1H, isoGln- CONH2b; D2O exchange), 4.31-4.13 (m, 2H, L-Ala-αH and isoGln-αH), 2.42 (t, 2H, J = 7.8 Hz, D-isoGln-βCH2), 2.16-2.04 (m, 1H, isoGln-γCH2a), 1.88-1.75 (m, 4H, CH3CO [1.84 (s, 3H) and isoGln-γCH2b), 1.23 (d, 0.21H, J = 7.1 Hz, Ala- CH3), 1.19 (d, 2.79H, J = 7.1 Hz, Ala- CH3). LCMS m / z: 380.5 [M+H] +(Exact Mass: 379.15) Purity (LCMS): 96.53% (88.33% + 8.20%)

[0278] (Synthesis of Benzoyl-L-Ala-D-isoGln-pNA (Compound 48))

[0279]

[0280] H-Ala-D-isoGln-pNA (compound 46) (120 mg, 0.36 mmol) and benzoyl chloride (52 mg, 0.37 mmol) were reacted according to the general method and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (5% to 10% MeOH-CHCl3). The collected fractions were combined, concentrated to dryness, and dried (50 °C, reduced pressure) to give compound 48 (146 mg, 99.3%) as a white solid. 1 H-NMR indicated that it was a mixture of two isomers (abundance ratio: approximately 13:1). 1H-NMR(400 MHz, DMSO-d6)δppm: 10.54(s, 0.93H, pNA-NH; D2O exchange), 10.53(s, 0.07H, pNA-NH; D2O exchange), 8.63(d, 0.93H, J = 6.6 Hz, NH; D2O exchange), 8.57(d, 0.07H, J = 6.6 Hz, NH; D2O exchange), 8.22(d, 0.93H, J = 8.2 Hz, NH; D2O exchange), 8.20(d, 2H, J = 9.2 Hz, pNA C3,5-H), 7.98(d, 0.07H, J = 8.2 Hz, NH; D2O exchange), 7.92-7.86(m, 2H, Bz C2,6-H), 7.82(d, 2H, J = 9.2 Hz, pNA C2,6-H), 7.58-7.51(m, 1H, Bz C4-H), 7.50-7.43(m, 2H, Bz C3,5-H), 7.56(s, 0.93H, isoGln- CONH2a; D2O exchange), 7.30(s, 0.07H, isoGln- CONH2a; D2O exchange), 7.18(s, 0.93H, isoGln- CONH2b; D2O exchange), 7.13(s, 0.07H, isoGln- CONH2b; D2O exchange), 4.51-4.40(m, 1H, Ala-αH), 4.30-4.16(m, 1H, isoGln-αH), 2.44(t, 2H, J = 7.8 Hz, isoGln-βCH2), 2.19-2.05(m, 1H, isoGln-γCH2a), 1.90-1.77(m, 1H, isoGln-γCH2b), 1.35(d, 2.79H, J = 7.1 Hz, Ala- CH3), 1.25(d, 0.21H, J = 7.1 Hz, Ala- CH3). LCMS m / z: 442.5 [M+H] + .(Exact Mass: 441.16) Purity(LCMS): 100.00%

[0281] Example 2: Evaluation of specificity of synthetic substrates The synthesized synthetic substrates were reacted with Lc-Lys2 to evaluate the specificity of the enzyme reaction.

[0282] 50 μL of 0.244 mmol / L synthetic substrate (DMF) was added to 145 μL of 20 mmol / L phosphate buffer (pH 6.0) and mixed thoroughly. 5 μL of enzyme was added and the mixture was incubated at 37°C for reaction. The specificity of the fluorescent substrate was measured using a multiplate reader (excitation 355 nm / emission 460 nm, measurement time 0.1 seconds). The specificity of the chromogenic substrate was measured using a spectrophotometer (OD405).

[0283] (MCA substrate) MCA substrates 63, 67, 60, and 66 were used to react at 37°C for 4 hours, and the fluorescence was measured to evaluate specificity. The results of measuring the specificity of each compound are shown in Table 1. The specificity of the synthetic substrates was expressed as the ratio of AMC produced in each reaction to the theoretical amount of AMC produced (hydrolysis rate %).

[0284]

[0285] Benzyl-α-MurNAc-L-Ala-D-isoGln-MCA (compound 63) (63A: Rf = 0.25, 63B: Rf = 0.19; 10% MeOH / CHCl3), benzyl-α-MurNAc-L-Ala-D-Glu(MCA)-OH (compound 67) (isomer ratio approximately 3:1), benzyl-α-MurNAc-L-Ala-D-Gln-MCA (compound 60) (isomer ratio approximately 4:1), and benzyl-α-MurNAc-L-Ala-D-Glu(OH)-MCA 66 (isomer ratio approximately 4:1). The specificities (ratio of approximately 3:1:1) were 63A (19.1%), 63B (81.5%), 67 (5.2%), 60 (2.8%) and 66 (3.8%), respectively.

[0286] The D-Glu and D-Gln forms (compounds 67, 60, and 66) did not release AMC, indicating that they are not substrates for Lc-Lys2. On the other hand, the D-isoGln form (compound 63) was shown to be a good substrate. Compound 63 exists as two stereoisomers, 63A and 63B, and the more polar 63B was shown to be a better substrate.

[0287] These results strongly suggested that the amino acid at the cleavage site of Lc-Lys2 was D-isoGln.

[0288] Tables 2 and 3 show the results of measuring specificity when the MCA substrates, Compounds 63, 45, 21, 30, 39 and 58, were reacted at 37° C. for 60 minutes.

[0289]

[0290]

[0291] The specificity of compound 63 was 63A (7.3%), 63B (33.9%), HL-Ala-D-isoGln-MCA·HCl (compound 45) (20.0%), Fmoc-D-isoGln-MCA (compound 21) (23.4%), HD-isoGln-MCA (compound 30) (1.3%), Fmoc-L-Ala-D-isoGln-MCA (compound 39) (7.6%), and benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-isoGln-MCA (compound 58) (14.0%).

[0292] Compound 30 showed no specificity and was not a substrate. On the other hand, compounds 63, 45, 21, 39, and 58 showed specificity and could be used as substrates. These results indicate that compounds with an acyl group (Ala) or an acyloxy group (Fmoc group) of appropriate size at the amino group moiety can be used as substrates.

[0293] (pNA substrate) Since it was suggested that the amino acid at the cleavage site of Lc-Lys2 is D-isoGln, benzyl-α-MurNAc-L-Ala-D-isoGln-pNA (compound 64), in which 4NA was introduced into D-isoGln, was synthesized and its specificity was evaluated. Using the pNA substrate, the reaction was carried out at 37°C for 20 or 180 minutes, and the specificity was evaluated by spectrophotometric measurement. The results of the specificity measurement for each compound are shown in Table 4. The specificity of the synthetic substrate was expressed as the ratio of 4NA produced in each reaction to the theoretical amount of 4NA (hydrolysis rate %).

[0294]

[0295] Compound 64 existed in two putative stereoisomers, 64A and 64B (64A: Rf = 0.12, 64B: Rf = 0.08; 10% MeOH / CHCl3). The specificity of these was 64A (43.0%, 97.0%) and 64B (97.4%, 97.3%). As with the MCA substrate, the highly polar 64B was shown to be a better substrate.

[0296] To investigate the effect of the Ala enantiomer, benzyl-α-MurNAc-D-Ala-D-isoGln-pNA (compound 65) was synthesized and its specificity was examined. Compound 65 also contained two putative stereoisomers, 65A and 65B (65A: Rf = 0.14, 65B: Rf = 0.09; 10% MeOH / CHCl3). The specificities of these were 65A (41.4%, 98.0%) and 65B (106.8%, 113.0%). This suggests that the configuration of Ala has little effect on specificity.

[0297] The specificity of the benzylidene-protected compound of compound 64, benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-isoGln-pNA (compound 56) (isomer ratio approximately 17:3), was also evaluated. The specificity was 88.3% (20 min) and 106.8% (180 min), showing the same specificity as the debenzylidene derivative (compound 64B).

[0298] Furthermore, the specificity of the synthetic intermediates Fmoc-D-isoGln-pNA (compound 23), HD-isoGln-pNA (compound 31), Fmoc-L-Ala-D-isoGln-pNA (compound 40) (isomer ratio approximately 9:1), HL-Ala-D-isoGln-pNA (compound 46) (isomer ratio approximately 9:1), and the acyl derivatives of compound 46, Ac-L-Ala-D-isoGln-pNA (compound 47) (isomer ratio approximately 13:1), and Bz-L-Ala-D-isoGln-pNA (compound 48) (isomer ratio approximately 13:1) were also investigated. The reaction temperature and time were 37°C and 30 minutes, respectively. The specificity measurement results are shown in Table 5.

[0299]

[0300] Compound 31 showed no specificity and was not a suitable substrate. Compounds 23 (69.8%), 40 (75.6%), 46 (67.0%), 47 (62.2%), and 48 (62.9%) showed moderate specificity and were usable as substrates. These results suggest that compounds with an acyl group or an appropriately sized acyloxy group (Fmoc group) introduced into the amino moiety of compound 31 are likely to be substrates for Lc-Lys2.

[0301] (LCMS Results of Compounds 63A and 63B) The reaction of benzyl-α-MurNAc-L-Ala-D-isoGln-MCA (Compound 63A) (Rf = 0.25) and Compound 63B (Rf = 0.19) with Lc-Lys2 was monitored by LCMS. The measurement results are shown in Table 6.

[0302] [Substrate] A: Benzyl-α-MurNAc-L-Ala-D-isoGln-MCA (Rf = 0.25) Compound 63A Exact Mass: 739.31 B: Benzyl-α-MurNAc-L-Ala-D-isoGln-MCA (Rf = 0.19) Compound 63B Exact Mass: 739.31

[0303] [Product] C: AMC Exact Mass: 175.06 D: Benzyl-α-MurNAc-L-Ala-D-isoGln-OH Exact Mass: 582.20

[0304] [Measurement samples] a: Compound 63A reaction solution (substrate concentration: 61 μmol / L) and stock solution (0.244 mmol / L) b: Compound 63B reaction solution (substrate concentration: 61 μmol / L) and stock solution (0.244 mmol / L) c: AMC (0.244 mol / L)

[0305] [Measurement conditions] Column: Acquity UPLC BEH C18, 1.7 μm, 2.1 x 50 mm. Mobile phase: 0.1% HCO2H (A) / CH3CN (B). Flow rate: 0.7 mL / min. Gradient: 0 min. (A: 91%) - 3.2 min. (A: 19%) - 3.7 min. (A: 19 > B: 91) - 5 min. (B: 91). Injection volume: 10 μL of reaction solution a and b, 0.5 μL of DMF solution of a (stock solution), b (stock solution), and AMC. Measurement wavelength: 326 nm (stock solutions of a and b were measured at 270 nm).

[0306]

[0307] In LCMS measurements, the [M+H] values ​​of AMC and benzyl-α-MurNAc-L-Ala-D-isoGln-OH released by hydrolysis with Lc-Lys2 from the reaction solutions of both compounds 63A and 63B were + was observed. At the same time, unreacted compounds 63A and 63B were also confirmed. The specificity of these compounds was 19.0% for compound 63A and 95.6% for compound 63B, which was similar to the results measured by fluorescence detection (Table 1).

[0308] These results indicated that benzyl-α-MurNAc-L-Ala-D-isoGln-MCA (63B) and benzyl-α-MurNAc-L-Ala-D-isoGln-pNA (64B) are particularly preferred substrates for use in this activity evaluation method.

[0309] INDUSTRIAL APPLICABILITY The present invention has industrial applicability in that it provides a method for evaluating the activity of an enzyme used in the structural analysis of cell wall polysaccharides of lactic acid bacteria.

Claims

1. A compound represented by the following formula (1) or a stereoisomer thereof: ...(1) (In the above formula (1), A represents the following formula (2) or a protecting group for an amino group, and Z represents a detection group that becomes detectable when released.) ...(2) (In the above formula (2), B represents a hydrogen atom, an N-acetyl-muramoyl group which may have a substituent, an acyl group, or a protecting group for an amino group.) 2. The compound or stereoisomer thereof according to claim 1, wherein the amino-protecting group represented by B in the above formula (2) is an alkoxycarbonyl group, an alkenyloxycarbonyl group or an aralkyloxycarbonyl group.

3. The compound or stereoisomer thereof according to claim 1, wherein the protecting group for the amino group represented by B in the above formula (2) is an Fmoc group (9-fluorenylmethyloxycarbonyl group), a Cbz group (benzyloxycarbonyl group), a Boc group (tert-butoxycarbonyl group) or an Alloc group (allyloxycarbonyl group).

4. The compound or stereoisomer thereof according to claim 1, wherein the N-acetyl-muramoyl group, which may have a substituent and is represented by B in the above formula (2), is an N-acetyl-1-benzylmuramoyl group or an N-acetyl-1-benzyl-4,6-benzylidenemuramoyl group.

5. The compound or stereoisomer thereof according to claim 1, wherein the acyl group represented by B in the above formula (2) is an acetyl group or a benzoyl group.

6. The compound or stereoisomer thereof according to claim 1 or 2, wherein the protecting group for the amino group represented by A in the above formula (1) is an alkoxycarbonyl group, an alkenyloxycarbonyl group or an aralkyloxycarbonyl group.

7. The compound or stereoisomer thereof according to claim 1 or 2, wherein the protecting group for the amino group represented by A in the above formula (1) is an Fmoc group (9-fluorenylmethyloxycarbonyl group), a Cbz group (benzyloxycarbonyl group), a Boc group (tert-butoxycarbonyl group) or an Alloc group (allyloxycarbonyl group).

8. The compound or stereoisomer thereof according to claim 1 or 2, wherein A in the above formula (1) is the following formula (3), (4), (5), (6), (7), (8) or (9). ...(3) (In the above formula (3), Ac represents an acetyl group, and Bn represents a benzyl group.) ...(4) (In the above formula (4), Ac represents an acetyl group, Bn represents a benzyl group, and Ph represents a phenyl group.) ...(5) ...(6) ...(7) ...(8) ... (9) 9. The compound or stereoisomer thereof according to claim 1 or 2, wherein Z in the above formula (1) represents a chromophore that emits color when liberated or a fluorescent group that emits fluorescence when liberated.

10. The compound or stereoisomer thereof according to claim 1 or 2, wherein Z in the above formula (1) is the following formula (10), (11), (12), (13), (14) or (15). ...(10) ...(11) ...(12) ...(13) ...(14) ...(15) 11. The compound or stereoisomer thereof according to claim 1 or 2, which is used as a substrate for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase.

12. A method for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase, comprising the steps of reacting γ-D-glutamyl-L-lysyl endopeptidase with a compound represented by the following formula (1) or a stereoisomer thereof, and detecting the released Z: ...(1) (In the above formula (1), A represents the following formula (2) or a protecting group for an amino group, and Z represents a detection group that becomes detectable when released.) ...(2) (In the above formula (2), B represents a hydrogen atom, an N-acetyl-muramoyl group which may have a substituent, an acyl group, or a protecting group for an amino group.)

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