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WO2025089217A1 - COMPOSÉ DESTINÉ À ÊTRE UTILISÉ EN TANT QUE SUBSTRAT POUR ÉVALUER L'ACTIVITÉ DE LA γ-D-GLUTAMYL-L-LYSYL ENDOPEPTIDASE - Google Patents

COMPOSÉ DESTINÉ À ÊTRE UTILISÉ EN TANT QUE SUBSTRAT POUR ÉVALUER L'ACTIVITÉ DE LA γ-D-GLUTAMYL-L-LYSYL ENDOPEPTIDASE Download PDF

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WO2025089217A1
WO2025089217A1 PCT/JP2024/037335 JP2024037335W WO2025089217A1 WO 2025089217 A1 WO2025089217 A1 WO 2025089217A1 JP 2024037335 W JP2024037335 W JP 2024037335W WO 2025089217 A1 WO2025089217 A1 WO 2025089217A1
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isogln
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隆 八重樫
幸生 白澤
雅弘 小野
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Yakult Honsha Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
    • C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
    • C07D311/18—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted otherwise than in position 3 or 7
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to a compound used as a substrate for evaluating the activity of ⁇ -D-glutamyl-L-lysyl endopeptidase, and a method for evaluating the activity of ⁇ -D-glutamyl-L-lysyl endopeptidase using the compound.
  • the present inventors focused on ⁇ -D-glutamyl-L-lysyl endopeptidase (Lc-Lys2), one of the cell wall lytic enzymes of Lacticaceobacillus, for use in structural analysis of cell wall polysaccharides.
  • a possible method for obtaining active ⁇ -D-glutamyl-L-lysyl endopeptidase would be to clone ⁇ -D-glutamyl-L-lysyl endopeptidase from Lacticaceobacillus, express it in Escherichia coli, and purify it.
  • a method for accurately evaluating the activity of the produced ⁇ -D-glutamyl-L-lysyl endopeptidase was unknown.
  • the present invention therefore aims to provide a novel compound that can be used as a substrate for evaluating the activity of ⁇ -D-glutamyl-L-lysyl endopeptidase, and a method for evaluating the activity of ⁇ -D-glutamyl-L-lysyl endopeptidase using the compound.
  • Lc-Lys2 ⁇ -D-glutamyl-L-lysyl endopeptidase
  • the present inventors synthesized synthetic substrates by introducing the fluorescent substance 7-amino-4-methylcoumarin (AMC) or the chromogenic substance 4-nitroaniline (4NA) to the terminus of the cleavage site by Lc-Lys2 as a substrate for evaluating the activity of the Lc-Lys2 enzyme, and binding a MurNAc-L-Ala derivative to enhance the specificity with the enzyme.
  • AMC fluorescent substance 7-amino-4-methylcoumarin
  • 4NA 4-nitroaniline
  • the specificity of the 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 ultraviolet-visible spectrophotometer. Based on these findings, the inventors have discovered new compounds and methods of evaluating the activity of Lc-Lys2 that can be used to evaluate the activity of Lc-Lys2, and have completed the present invention.
  • the present invention provides a compound or a stereoisomer thereof represented by the following formula (1):
  • A represents the following formula (2) or a protecting group for an amino group
  • Z represents a detection group that becomes detectable when released.
  • 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.
  • the present invention also provides a compound or a stereoisomer thereof, wherein the protecting group for an amino group represented by B in the above formula (2) is an alkoxycarbonyl group, an alkenyloxycarbonyl group or an aralkyloxycarbonyl group.
  • the present invention also provides a compound or a stereoisomer thereof, in which 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).
  • 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).
  • the present invention also provides a compound or a stereoisomer thereof, in which 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.
  • the present invention also provides a compound or a stereoisomer thereof, in which the acyl group represented by B in the above formula (2) is an acetyl group or a benzoyl group.
  • the present invention also provides a compound or a stereoisomer thereof, in which 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.
  • the present invention also provides a compound or a stereoisomer thereof, in which 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).
  • 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).
  • the present invention also provides a compound or a stereoisomer thereof, in which A in the above formula (1) is the following formula (3), (4), (5), (6), (7), (8) or (9).
  • the present invention also provides a compound or a stereoisomer thereof in which Z in the above formula (1) represents a chromophore that emits color when released or a fluorescent group that emits fluorescence when released.
  • the present invention also provides a compound or a stereoisomer thereof, in which Z in the above formula (1) is the following formula (10), (11), (12), (13), (14) or (15).
  • the present invention also provides the above compound or a stereoisomer thereof for use as a substrate for evaluating the activity of ⁇ -D-glutamyl-L-lysyl endopeptidase.
  • the present invention also provides a method for evaluating the activity of ⁇ -D-glutamyl-L-lysyl endopeptidase, comprising the steps of reacting ⁇ -D-glutamyl-L-lysyl endopeptidase with a compound represented by the following formula (1) or a stereoisomer thereof, and detecting the liberated Z.
  • A represents the following formula (2) or a protecting group for an amino group
  • Z represents a detection group that becomes detectable when released.
  • 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.
  • the present invention can provide a novel compound that can be used as a substrate for evaluating the activity of ⁇ -D-glutamyl-L-lysyl endopeptidase.
  • the present invention can also provide a method for evaluating the activity of ⁇ -D-glutamyl-L-lysyl endopeptidase using the compound. Therefore, the present invention can be used to select ⁇ -D-glutamyl-L-lysyl endopeptidase that has activity useful for structural analysis of cell wall polysaccharides of lactic acid bacteria.
  • 1 shows a schematic diagram of the cell wall of Lacticaceobacter sp.
  • Alkyl group refers to a linear or branched alkyl group having 1 to 20 carbon atoms.
  • alkyl groups include linear or branched 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
  • Alkoxy group refers to an alkoxy group having the above alkyl group.
  • 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).
  • acyl group includes aliphatic acyl groups and aromatic acyl groups.
  • aliphatic acyl groups 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, 20-
  • Aromatic acyl groups include, for example, arylcarbonyl groups such as benzoyl, ⁇ -naphthoyl, and ⁇ -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-phenylbenzo
  • Alkyl group refers to an alkyl group having 1 to 6 carbon atoms substituted with an aryl group.
  • aralkyl groups include benzyl, ⁇ -naphthylmethyl, ⁇ -naphthylmethyl, indenylmethyl, phenanthrenylmethyl, anthracenylmethyl, diphenylmethyl, triphenylmethyl, ⁇ -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-naphthyl,
  • A represents the following formula (2) or a protecting group for an amino group.
  • 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.
  • N-acetyl-muramoyl group which may have a substituent
  • N-acetylmuramic acid (MurNAc) which may have a substituent is bonded to B in formula (2) through an amide bond.
  • the N-acetyl-muramoyl group represented by B may have one or more substituents, or may not have a 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, for example, a benzyl group (Bn), a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, a p-methoxybenzyl group, or a benzylidene group.
  • the N-acetyl-muramoyl group represented by B may have, for example, the 4-OH group and the 6-OH group protected by the benzylidene carbon.
  • B is preferably an N-acetyl-1-benzylmuramoyl group or an N-acetyl-1-benzyl-4,6-benzylidenemuramoyl group.
  • the acyl group represented by B in the above formula (2) can be, for example, an aliphatic acyl group having 1 to 10 carbon atoms and an aromatic acyl group.
  • the acyl group represented by B is preferably an acetyl group or a benzoyl group.
  • the amino protecting group represented by B in the above formula (2) is not particularly limited, and may be an amide protecting group, a phthalimide protecting group, a carbamate protecting group, or a sulfonamide protecting group.
  • the amino protecting group represented by B is preferably a carbamate protecting group, such as an alkoxycarbonyl group, an alkenyloxycarbonyl group, or an aralkyloxycarbonyl group.
  • carbamate 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).
  • amide protecting groups include the formyl group, the acetyl group (Ac), and the trifluoroacetyl group (TFA).
  • Phthalimide-type protecting groups include, for example, a phthaloyl group (Phth) and the like.
  • Sulfonamide-type protecting groups include, for example, a 3-nitro-2-pyridinesulfenyl group (Npys), a 2-nitrobenzenesulfonyl group (Ns) and a (2-trimethylsilyl)-ethanesulfonyl group (SES) and the like.
  • the protecting group for the amino 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.
  • the protecting group of the amino group represented by A is not particularly limited, and an amide type protecting group, a phthalimide type protecting group, a carbamate type protecting group, a sulfonamide type protecting group, etc. can be used.
  • the protecting group of the amino group represented by A can be preferably a carbamate type protecting group, such as an alkoxycarbonyl group, an alkenyloxycarbonyl group, and an aralkyloxycarbonyl group.
  • Examples of the carbamate type protecting group include, for example, 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 the amide type protecting group include, for example, a formyl group, an acetyl group (Ac), and a trifluoroacetyl group (TFA).
  • Phthalimide-type protecting groups include, for example, a phthaloyl group (Phth) and the like.
  • Sulfonamide-type protecting groups include, for example, a 3-nitro-2-pyridinesulfenyl group (Npys), a 2-nitrobenzenesulfonyl group (Ns) and a (2-trimethylsilyl)-ethanesulfonyl group (SES) and the like.
  • the protecting group for the amino 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.
  • a in the above formula (1) can be, for example, the following formula (3), (4), (5), (6), (7), (8) or (9).
  • Z represents a detection group that becomes detectable when released.
  • Z can be, for example, a chromophore that develops color when released or a fluorescent group that emits fluorescence when released.
  • Z can be, for example, a detection group that is colorless or non-fluorescent before cleavage by the enzyme, but becomes colored or fluorescent when released following cleavage by the enzyme.
  • the chromophore represented by Z is not particularly limited as long as it is a compound that emits color when released, but can be, for example, a paranitroaniline derivative.
  • the fluorescent group represented by Z is not particularly limited as long as it is a compound that emits fluorescence when released, but can be, for example, a coumarin derivative and a fluorescein derivative.
  • Z is preferably the following formula (10), (11), (12), (13), (14) or (15), and particularly preferably (10), (11) or (12).
  • Z can be, for example, methylcoumarinamide (MCA) represented by the above formula (10) or paranitroanilide (pNA) represented by the above formula (12).
  • MCA methylcoumarinamide
  • pNA paranitroanilide
  • AMC aminomethylcoumarin
  • stereoisomers refer 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.
  • the compound of the present invention can preferably be benzyl- ⁇ -MurNAc-L-Ala-D-isoGln-MCA (compound 63B) or benzyl- ⁇ -MurNAc-L-Ala-D-isoGln-pNA (compound 64B).
  • the compound of the present invention can be used as a substrate for evaluating the activity of ⁇ -D-glutamyl-L-lysyl endopeptidase.
  • the ⁇ -D-glutamyl-L-lysyl endopeptidase can be, but is not limited to, ⁇ -D-glutamyl-L-lysyl endopeptidase (Lc-Lys2) of lactic acid bacteria, particularly of the genus Lacticaceibacillus.
  • the genus Lacticaceibacillus includes, for example, Lacticaceibacillus casei, Lacticaceibacillus paracasei, and Lacticaceibacillus rhamnosus.
  • Lc-Lys2 is one of the cell wall lytic enzymes of the Lacticaceobacterium genus.
  • Lc-Lys2 has the activity of cleaving the D-isoGln site of the peptide portion of the cell wall.
  • the compound of the present invention contains Z, which is released and detectable when cleaved by Lc-Lys2, so the activity of Lc-Lys2 can be evaluated by detecting this Z.
  • the present invention also provides a method for assessing the activity of ⁇ -D-glutamyl-L-lysyl endopeptidase.
  • the "activity" of ⁇ -D-glutamyl-L-lysyl endopeptidase means the activity of cleaving the D-isoGln site in the cell wall peptide portion of Lacticaceobacter sp.
  • assessing activity includes not only assessing the presence or absence of activity to cleave a substrate, but also assessing the strength of the activity to cleave a substrate.
  • activity can be expressed, for example, by the rate at which the substrate is cleaved, the amount and proportion of products (liberated substances) generated by cleavage of the substrate, the time it takes for the substrate to be cleaved to a specified proportion, etc.
  • the method of the present invention includes a step of reacting ⁇ -D-glutamyl-L-lysyl endopeptidase with the compound of the present invention or its stereoisomer, and a step of detecting the released Z.
  • the compound or its stereoisomer is used as a substrate for ⁇ -D-glutamyl-L-lysyl endopeptidase.
  • the expression “reacts” between an enzyme and a substrate means that the enzyme catalyzes the conversion of the substrate into one or more different products.
  • the expression “reacts” between ⁇ -D-glutamyl-L-lysyl endopeptidase and a compound of the present invention means that ⁇ -D-glutamyl-L-lysyl endopeptidase liberates the detection group represented by Z in the above formula (1) from the compound of the present invention.
  • the reaction can be caused by contacting the enzyme with the substrate.
  • 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.
  • the ⁇ -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 ⁇ -D-glutamyl-L-lysyl endopeptidase. Furthermore, the ⁇ -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 ⁇ -D-glutamyl-L-lysyl endopeptidase is unknown.
  • the reaction can be carried out, for example, by contacting ⁇ -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 appropriately set between 5 minutes and 24 hours, for example.
  • contacting means bringing the enzyme and substrate close enough to each other to perform catalytic reactions.
  • Contacting includes, for example, mixing the enzyme with the substrate and adding the enzyme to the substrate.
  • the enzyme and substrate can be brought into contact by adding them to a reaction solution and mixing them by stirring or the like.
  • the reaction solution is not particularly limited, but for example, phosphate buffer and acetate buffer can be used.
  • the presence, absence, amount and/or proportion of Z can be measured using an instrument capable of detecting Z. If Z is a chromophore that emits color upon release, the presence, absence, amount and/or proportion of released Z can be measured using an ultraviolet-visible spectrophotometer or the like. If Z is a fluorescent group that emits fluorescence upon release, the presence, absence, amount and/or proportion of released Z can be measured using a fluorometer or the like.
  • the ⁇ -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.
  • the theoretical amount of Z produced may be calculated in advance, and the ⁇ -D-glutamyl-L-lysyl endopeptidase used may be evaluated as having activity if the ratio (%) of the amount of Z detected to this theoretical amount of production exceeds a preset threshold.
  • the threshold is not particularly limited, but may be set to any value, such as 6%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%.
  • the strength of activity of the ⁇ -D-glutamyl-L-lysyl endopeptidase used may be evaluated based on the amount and/or ratio of released Z.
  • the compound of the present invention or its stereoisomer can be produced by the following methods, the methods described in the Examples, or methods modified therefrom.
  • the reaction conditions can be optimized as appropriate depending on the compound and solvent used.
  • Benzyl 2-acetamido-2-deoxy- ⁇ -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 ⁇ H 2 O) in toluene under reflux (see Non-Patent Document 3).
  • compound 2 is heated with benzaldehyde dimethyl acetal and p-TSA ⁇ H 2 O 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- ⁇ -D-glucopyranoside (compound 3) (see non-patent documents 4-6).
  • DMF N,N-dimethylformamide
  • Protected MurNAc (compound 4) was condensed with L-alanine methyl ester hydrochloride or D-alanine methyl ester hydrochloride in the presence of N-methylmorpholine in DMF using N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) to give (S)-methyl 2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3- ⁇ -D-glucopyranosyl)oxy)propionate.
  • HBTU N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate
  • Compound 5 and compound 6 can be hydrolyzed with 0.5 mol/L KOH in MeOH solvent to obtain (S)-2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3- ⁇ -D-glucopyranosyl)oxy)propionamido)propionic acid (benzyl-4,6-O-benzylidene- ⁇ -MurNAc-L-Ala-OH) (compound 7) and (R)-2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3- ⁇ -D-gluco-pyranosyl)oxy)propionamido)propionic acid (benzyl-4,6-O-benzylidene- ⁇ -MurNAc-D-Ala-OH) (compound 8).
  • 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.
  • Sodium hydroselenide (NaSeH) can be prepared from selenium (powder; Se) and sodium borohydride (NaBH 4 ) in isopropanol (IPA) immediately before use.
  • 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.
  • 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
  • Fmoc-D-isoGln-pNA compound 23
  • the Boc-protected form (compound 11) can be treated with trifluoroacetic acid (TFA) in CH2Cl2 to give the Boc-deprotected form HD-Glu(OBn)-MCA ⁇ TFA (compound 24), which can then be treated further with aqueous NaHCO3 to give the free base (compound 25).
  • TFA trifluoroacetic acid
  • Boc-protected form (compound 17) can be reacted in TFA/CH 2 Cl 2 /H 2 O at room temperature and treated with Amberlyst® A-26 to give HD-Gln-MCA (compound 26) as a free base.
  • Fmoc-protected forms (compounds 13, 15, 19, 21, 23) can also be treated with 20% piperidine/DMF to give the free bases H-D-Glu(OAll)-MCA (compound 28) (All; allyl), H-D-Glu(MCA)-OAll (compound 27), H-D-Gln-MCA (compound 26), H-D-isoGln-MCA (compound 30) and H-D-isoGln-pNA (compound 31).
  • H-D-Glu(OAll)-MCA compound 28
  • All; allyl H-D-Glu(MCA)-OAll
  • H-Gln-MCA compound 26
  • H-D-isoGln-MCA compound 30
  • H-D-isoGln-pNA compound 31
  • compound 29 When compound 13 is de-Fmoc-converted, hydrolysis of the allyl ester occurs together with the free base (compound 28) to give H-D-Glu(OH)
  • Boc-L-Ala-OH (compound 32) is reacted with isopropyl chloroformate in the presence of N-methylpiperidine in THF to give a mixed acid anhydride, which is then reacted with H-D-Glu(OBn)-MCA (compound 25) and H-D-Gln-MCA (compound 26) to give Boc-L-Ala-D-Glu(OBn)-MCA (compound 35) and Boc-L-Ala-D-Gln-MCA (compound 36).
  • 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 ⁇ H 2 O) in DMF using N,N-diisopropylethylamine (DIEA) as a base and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) as a condensation 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-
  • the Boc-protected form (compound 35) can be de-Bocated using TFA in CH2Cl2 to give HL-Ala-D-Glu(OBn)-MCA ⁇ TFA (compound 41).
  • the Fmoc -protected forms (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).
  • HL-Ala-D-isoGln-pNA (compound 46) can be treated with acetyl chloride and benzoyl chloride in the presence of triethylamine (TEA) in CH2Cl2 to give Ac-L-Ala-D-isoGln-pNA (compound 47) and Bz-L-Ala-D-isoGln-pNA (compound 48).
  • TAA triethylamine
  • Boc-D-Glu(OBn)-MCA (compound 11), Boc-L-Ala-D-Glu(OBn)-MCA (compound 35), H-L-Ala-D-Glu(OBn)-MCA ⁇ TFA (compound 41) and H-D-Glu(OBn)-MCA ⁇ TFA (compound 24) can be hydrogenolyzed in EtOH under a hydrogen atmosphere using 5% Pd/C as a catalyst to obtain the debenzylated forms Boc-D-Glu(OH)-MCA (compound 50), Boc-L-Ala-D-Glu(OH)-MCA (compound 51), H-L-Ala-D-Glu(OH)-MCA ⁇ TFA (compound 52) and H-D-Glu(OH)-MCA ⁇ TFA (compound 53).
  • Scheme 5 Synthesis of benzyl-4,6-O-benzylidene- ⁇ -MurNAc-L, D-Ala-D-Glu-MCA, -D-Gln-MCA, -D-isoGln-MCA and -D-isoGln-pNA derivatives.
  • Benzyl-4,6-O-benzylidene- ⁇ -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.
  • 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 by two synthetic routes.
  • Benzyl-4,6-O-benzylidene- ⁇ -MurNAc (compound 4) was condensed with H-L-Ala-D-Glu(MCA)-OAll (compound 42), H-L-Ala-D-Gln-MCA (compound 44) and H-L-Ala-D-isoGln-pNA (compound 46) in DMF solvent using N-methylmorpholine as a base and HBTU as a condensing agent (method a), to give the vector.
  • benzyl-4,6-O-benzylidene- ⁇ -MurNAc-L-Ala-OH was reacted 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 with HOBt ⁇ H 2 Condensation using DIEA as a base and EDCI as a condensing agent in the presence of O (Method b) can give benzyl-4,6-O-benzylidene- ⁇ -MurNAc-L-Ala-D-Glu(OAll)-MCA (Compound 57), benzyl-4,6-O-benzylidene- ⁇ -MurNAc-L-Ala-D-Glu(MCA)-OAll (Compound 54), and benzyl-4,6-O-benzylidene
  • Benzyl-4,6-O-benzylidene- ⁇ -MurNAc-D-Ala-OH (compound 8) can be condensed with compound 31 by method b to obtain benzyl-4,6-O-benzylidene- ⁇ -MurNAc-D-Ala-D-isoGln-pNA (compound 59).
  • benzyl-4,6-O-benzylidene- ⁇ -MurNAc-L-Ala-D-isoGln-pNA (compound 56) can be obtained by condensing compound 7 with compound 31 by method a.
  • Scheme 6 Synthesis of benzyl- ⁇ -MurNAc-Ala-D-Glu-MCA, -D-Gln-MCA, -D-isoGln-MCA and -D-isoGln-pNA derivatives.
  • the 4,6-benzylidene and allyl groups can be removed to synthesize benzyl- ⁇ -MurNAc-Ala-D-Glu-MCA, -D-Gln-MCA, -D-isoGln-MCA and -D-isoGln-pNA derivatives according to Scheme 6 below.
  • the benzyl-4,6-O-benzylidene- ⁇ -MurNAc derivatives can be debenzylidene-substituted by heating in 75% AcOH to give benzyl- ⁇ -MurNAc-L-Ala-D-Gln-MCA (compound 60), benzyl- ⁇ -MurNAc-L-Ala-D-Glu(OAll)-MCA (compound 61), benzyl- ⁇ -MurNAc-L-Ala-D-Glu(MCA)-OAll (compound 62), benzyl- ⁇ -MurNAc-L-Ala-D-isoGln-MCA (compound 63), benzyl- ⁇ -MurNAc-L-Ala-D-isoGln-pNA (compound 64) and benzyl- ⁇ -MurNAc-D-Ala-D-isoGln-p
  • the allyl-protected derivatives (compounds 61 and 62) can be de-allylated using Pd(OAc) 2 - PPh3 catalyst in the presence of AcOH and 4-methylmorpholine as a nucleophile in CH3CN - H2O (see Patent Document 2 and Non-Patent Document 12) to give benzyl- ⁇ -MurNAc-L-Ala-D-Glu(OH)-MCA (compound 66) and benzyl- ⁇ -MurNAc-L-Ala-D-Glu(MCA)-OH (compound 67).
  • the ⁇ -D-glutamyl-L-lysyl endopeptidase used in this example was Lc-Lys2 contained in Lacticaseibacillus paracasei strain BL23.
  • Lc-Lys2 ⁇ -D-glutamyl-L-lysyl endopeptidase derived from an endogenous phage was cloned from the Lacticaseibacillus paracasei BL23 strain, and the enzyme was expressed in E. coli by ligating it to an E. coli expression vector (Lc-Lys2).
  • Lc-Lys2 was purified by dissolving the inclusion bodies of the insoluble fraction with a denaturant, and active enzyme was produced by normalizing the protein folding using the unfolding method.
  • Example 1 Synthesis of synthetic substrates Each compound was synthesized by the method described below.
  • N-acetyl-D-glucosamine (compound 1) (25.0 g, 113 mmol), p-TSA ⁇ H 2 O (1.9 g, 10 mmol) were suspended in toluene (300 mL) and benzyl alcohol (180 mL), and the suspension was boiled and refluxed for 3 hours with a Dean-Stark apparatus attached. The reaction mixture was cooled to room temperature, and NaHCO 3 (1.26 g, 15 mmol) was dissolved in H 2 O (15 mL) and added, and the toluene was distilled off under reduced pressure. The residue (black liquid) was cooled to room temperature, and AcOEt (120 mL) and n-Hex.
  • 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 obtain 15.17 g of crude compound as a light brown solid.
  • the crude was dissolved in 20% MeOH/CHCl 3 , 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/CHCl 3 ) to obtain compound 2 (10.32 g, 29.3%) as a white to slightly brown solid.
  • Benzyl 2-acetamido-2-deoxy- ⁇ -D-glucopyranoside (compound 2) (10.32 g, 33.1 mmol) was dissolved in dehydrated DMF (80 mL), and benzaldehyde dimethyl acetal (6.9 mL, 46.5 mmol, 1.4 eq.) and p-TSA ⁇ H 2 O (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 (about 15 min), and heated at 60 °C for 5 h. The solvent was removed under reduced pressure to obtain a white solid residue.
  • Benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy- ⁇ -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), and the residue was dissolved in dehydrated DMF (220 mL). NaH (60% in oil) (8.18 g, net 4.91 g, 204.5 mmol, 6.25 eq.) was added with stirring at room temperature under argon gas flow and stirred for 30 minutes.
  • L-alanine methyl ester hydrochloride (1.12 g, 8.02 mmol, 2.0eq.) was added and 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 obtain a residue in the form of agar.
  • the white solid was suspended in AcOEt (500 mL), and 1 mol/L HCl (100 mL) was added to separate the mixture.
  • the AcOEt layer was separated, washed with saturated NaCl water, dried over Na 2 SO 4 , filtered, concentrated to dryness under reduced pressure, and then dried (60° C., reduced pressure) to obtain compound 7 (1.85 g, quant.) as a white solid.
  • 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.
  • the black suspension was removed by filtration using a Celite pad and washed with 20% MeOH/CHCl 3.
  • the filtrate and washings were combined and concentrated to dryness under reduced pressure, the residue was dissolved in 20% MeOH/CHCl 3 , silica gel (FL100 D) was added, and the mixture was concentrated to dryness under reduced pressure, and purified by silica gel column chromatography.
  • Fmoc-D-Glu(OAll)-OH (compound 12) (4.88 g, 11.93 mmol, 1.2 eq.) was dissolved in dehydrated 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 argon gas atmosphere, and the mixture was stirred for 1.5 hours.
  • Fmoc-D-Glu(OH)-OAll (compound 14) (4.72 g) was reacted and worked up according to the general synthetic method to obtain Fmoc-D-Glu(MCA)-OAll (compound 15) (2.47 g, 43.8%) as a yellow solid.
  • Fmoc-D-Gln-OH (compound 18) (4.41 g) was reacted and worked up according to the general synthetic method to obtain Fmoc-D-Gln-MCA (compound 19) (477 mg, 9.1%) as a pale yellow solid.
  • Fmoc-D-isoGln-OH (compound 20) (3.67 g) was reacted and worked up according to the general synthetic method to obtain Fmoc-D-isoGln-MCA (compound 21) (1.24 g, 28.4%) as a white to pale yellow solid.
  • Fmoc-D-isoGln-OH (compound 20) (4.40 g) was reacted and worked up according to the general synthetic method to obtain Fmoc-D-isoGln-pNA (compound 23) (2.98 g, 61.5%) as a pale yellow solid.
  • 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. The residue was added with AcOEt (200 mL) (both layers contained insoluble matter), washed with water, saturated NaHCO 3 water, and then saturated NaCl water, and the AcOEt layer (containing suspended matter) was separated. It was dried over Na 2 SO 4 , filtered off, washed 2 and 3 times with 20% MeOH/CHCl 3, and the filtrate and washings were combined and concentrated under reduced pressure.
  • HD-isoGln-pNA (compound 31) (500 mg, 1.88 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 20% MeOH/CHCl 3 , silica gel (FL100D 20 g) was added, concentrated to dryness, and purified by silica gel column chromatography (5% to 10%, MeOH/CHCl 3 ). The target fractions were combined, concentrated to dryness, and dried (50°C, reduced pressure) to obtain compound 40 (914 mg, 87.0%) as a white solid. 1H -NMR spectrum suggested that it was a mixture of two isomers (abundance ratio approximately 9:1).
  • Fmoc-D-Glu(OAll)-MCA (compound 13) (1.80 g, 3.18 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. The residue was reprecipitated with n-Hex. (30 mL)/CHCl 3 (15 mL), stirred, filtered, and dried (60°C, reduced pressure) to obtain HD-Glu(OH)-MCA (compound 29) (680 mg) as a pale yellow solid. The filtrate was concentrated to dryness under reduced pressure, dissolved in CHCl 3 , and purified by silica gel column chromatography (CHCl 3 to 8%MeOH/CHCl 3 ).
  • Fmoc-D-Glu(MCA)-OAll (compound 15) (1.80 g, 3.18 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. The residue was reprecipitated with n-Hex. (30 mL)/CHCl 3 (30 mL), stirred, filtered, and dried (50°C, reduced pressure) to obtain compound 27 (983 mg, 89.9%) as a pale yellow solid.
  • Fmoc-D-Gln-MCA (compound 19) (450 mg, 0.86 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. The residue was suspended in n-Hex. (10 mL)/CHCl 3 (10 mL), stirred, and filtered. The filtered product was dried (50°C, reduced pressure) to obtain compound 26 (249 mg, 95.8%) as a pale ochre solid.
  • Fmoc-L-Ala-D-Gln-MCA (compound 38) (350 mg, 0.59 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. The residue was suspended in n-Hex. (20 mL)/CHCl 3 (10 mL), stirred, and filtered (precipitate; moisture absorption). The filtered product was dissolved in 20% MeOH/CHCl 3 and concentrated to dryness under reduced pressure. The residue was powdered in n-Hex. and dried (50°C, reduced pressure) to obtain compound 44 (213 mg, 96.8%) as a white solid.
  • Fmoc-D-isoGln-MCA (compound 21) (1.14 g, 2.17 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. The residue was suspended in n-Hex. (25 mL)/CHCl 3 (25 mL), stirred, and filtered. The filtered product was dried (60°C, reduced pressure) to obtain compound 30 (602 mg, 91.5%) as a white solid.
  • Fmoc-L-Ala-D-isoGln-MCA (compound 39) (80 mg, 0.13 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. The residue was suspended in n-Hex. (15 mL)/CHCl 3 (7 mL), stirred, and filtered. The filtered product was dried (50°C, reduced pressure) to obtain HL-Ala-D-isoGln-MCA (52 mg, quant.) as a pale ochre solid. LCMS m/z: 375.2 [M+H] + .
  • Fmoc-D-isoGln-pNA (compound 23) (2.96 g, 6.06 mmol) was reacted according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. The residue was crystallized from n-Hex. (60 mL)/CHCl 3 (30 mL), filtered, and dried (50°C, reduced pressure) to obtain compound 31 (1.51 g, 93.5%) as a pale gray solid.
  • Fmoc-L-Ala-D-isoGln-pNA (compound 40) (885 mg, 1.58 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. n-Hex. (15 mL)/CHCl 3 (15 mL) was added to the residue, which was stirred, filtered, and dried (50°C, reduced pressure) to obtain compound 46 (474 mg, 88.8%) as a white solid. From the 1 H-NMR spectrum, it was estimated to be a mixture of two isomers (abundance ratio approximately 9:1).
  • HD-Glu(OAll)-MCA (compound 28) (90 mg, 0.26 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure.
  • the residue was dissolved in CHCl 3 (60 mL), washed with water, 1 mol/L HCl, saturated NaHCO 3 aqueous solution, and then saturated NaCl water, dried over Na 2 SO 4 , filtered, and concentrated to dryness under reduced pressure.
  • the residue was suspended in CHCl 3 , and n-Hex. was added under stirring and stirred at room temperature. The suspension was filtered and dried (60°C, reduced pressure) to obtain 209 mg of a white solid.
  • HD-Glu(MCA)-OAll (compound 27) (325 mg, 0.94 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure.
  • the residue was dissolved in CHCl 3 (200 mL), washed with water, 1 mol/L HCl, saturated NaHCO 3 aqueous solution, and then saturated NaCl water, dried over Na 2 SO 4 , filtered, and concentrated to dryness under reduced pressure.
  • the residue was suspended in CHCl 3 (20 mL), and n-Hex. (40 mL) was added under stirring and stirred at room temperature. The suspension was filtered, washed with n-Hex.
  • Benzyl-4,6-O-benzylidene- ⁇ -MurNAc-L-Ala-OH (compound 7) (750 mg, 1.38 mmol) was dissolved in a mixture of dehydrated CH2Cl2 (30 mL) and dehydrated DMF (2 mL), and HOBt.H2O (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.
  • HD-isoGln-pNA (compound 31) (400 mg, 1.50 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. The residue was dissolved in CHCl 3 (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/CHCl 3 , dried over Na 2 SO 4 , filtered, and concentrated to dryness under reduced pressure to obtain a pale yellow viscous oily residue. The residue was treated with n-Hex.
  • HD-isoGln-pNA (compound 31) (312 mg, 1.17 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. A pale yellow syrup-like residue was obtained. The residue was suspended in CHCl 3 (10 mL), and n-Hex. (40 mL) was added and stirred. The suspension was filtered, washed with water, and dried (50°C, reduced pressure) to obtain compound 59 (973 mg, quant.) as a white solid. From the 1 H-NMR spectrum and LCMS spectrum, it was estimated to be a mixture of two isomers (abundance ratio approximately 3:1).
  • Fmoc-L-Ala-OH (compound 33) (87 mg, 0.28 mmol) was dissolved in a mixture of dehydrated CH2Cl2 (6 mL) and dehydrated DMF (0.4 mL), and HOBt.H2O (51 mg, 0.33 mmol, 1.2 eq.) and EDCI (63 mg, 0.33 mmol, 1.2 eq.) were added and stirred at room temperature for 30 minutes. 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 stirred for 3 hours.
  • reaction mixture was concentrated to dryness under reduced pressure.
  • the residue was dissolved in 20% CHCl 3 /MeOH, silica gel (FL-100D, 4 g) was added, and the mixture was concentrated to dryness under reduced pressure and purified by silica gel column chromatography (5% to 10% MeOH/CHCl 3 ).
  • the target fraction was collected and concentrated to dryness under reduced pressure.
  • the residue was washed with n-Hex./CHCl 3 and dried (50°C, reduced pressure) to give compound 39 (98 mg, 58.7%) as a white solid.
  • HL-Ala-D-Glu, HL-Ala-D-Gln, or HL-Ala-D-isoGln derivatives (1.0 eq.) were added and stirred for 24 hours, and the reaction mixture was concentrated to dryness under reduced pressure.
  • HL-Ala-D-Gln-MCA (compound 44) (186 mg, 0.50 mmol) was used in the reaction according to the general method, and the reaction solution 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 NaHCO 3 aqueous solution (100 mL), and then saturated NaCl water (120 mL).
  • the AcOEt layer (suspension) was separated and concentrated to dryness under reduced pressure. The residue was washed with n-Hex.
  • Benzyl-4,6-O-benzylidene- ⁇ -MurNAc (compound 4) (226 mg, 0.48 mmol) and HL-Ala-D-isoGln-pNA (compound 46) (210 mg, 0.62 mmol, 1.3eq.) were used to react according to the general method.
  • Water (5 mL) was added to the reaction mixture (suspension), and AcOEt was added and the layers were separated.
  • the AcOEt layer (suspension) was separated and washed with 1 mol/L HCl, saturated NaHCO 3 aqueous solution, and then water.
  • the AcOEt layer (suspension) was separated and concentrated to dryness under reduced pressure. The residue was suspended in n-Hex.
  • Benzyl-4,6-O-benzylidene- ⁇ -MurNAc-L-Ala-D-isoGln-MCA (compound 58) (500 mg, 0.60 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 crystallized from n-Hex. (10 mL)/CHCl 3 (10 mL), dissolved in 20% MeOH/CHCl 3 , silica gel (FL 100D, 10 g) was added, and the mixture was concentrated to dryness under reduced pressure.
  • Benzyl-4,6-O-benzylidene- ⁇ -MurNAc-L-Ala-D-isoGln-pNA (compound 56) was used in the reaction according to the general method, and the mixture was concentrated to dryness under reduced pressure. The residue was dissolved in 10% MeOH/CHCl 3 and purified by silica gel column chromatography (5% to 14% MeOH/CHCl 3 ).
  • Benzyl-4,6-O-benzylidene- ⁇ -MurNAc-D-Ala-D-isoGln-pNA (compound 59) (900 mg, 1.14 mmol) was used in the reaction according to the general method, and the mixture was concentrated to dryness under reduced pressure. The residue was dissolved in 10% MeOH/CHCl 3 and purified by silica gel column chromatography (5% to 15% MeOH/CHCl 3 ).
  • Benzyl- ⁇ -MurNAc-L-Ala-D-Glu(OAll)-MCA (compound 61) (100 mg, 0.13 mmol) was used in the reaction according to the general method, the suspension was removed by filtration, and the mixture was concentrated to dryness under reduced pressure. The residue was crystallized from n-Hex. (3 mL)/CHCl 3 (3 mL), the supernatant was removed, and the residue was crystallized again from n-Hex. (3 mL)/CHCl 3 (3 mL).
  • Benzyl- ⁇ -MurNAc-L-Ala-D-Glu(MCA)-OAll (compound 62) (200 mg, 0.26 mmol) was used in the reaction according to the general method, the suspension was removed by filtration, and the mixture was concentrated to dryness under reduced pressure. The residue was crystallized from n-Hex. (4 mL)/CHCl 3 (4 mL), the supernatant was removed, and the residue was crystallized again from n-Hex. (4 mL)/CHCl 3 (4 mL). The solid was filtered, washed with n-Hex., and dried (50°C, reduced pressure) to obtain compound 67 (189 mg, 89.5%) as a pale ochre solid.
  • 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-CHCl 3 ). The 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 CHCl 3 and dried (60°C, reduced pressure) to give compound 47 (69 mg, 51.1%). 1H -NMR suggested that it was a mixture of two isomers (abundance ratio approximately 13:1).
  • Example 2 Evaluation of specificity of synthetic substrates The synthesized substrate was reacted with Lc-Lys2 to evaluate the specificity of the enzyme reaction.
  • MCA substrate The MCA substrates, compounds 63, 67, 60 and 66, were used to react at 37°C for 4 hours, and the fluorescence was measured to evaluate the specificity.
  • the measurement results of 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 production amount of AMC (hydrolysis rate %).
  • 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.
  • the D-Glu and D-Gln forms did not release AMC, indicating that they are not substrates for Lc-Lys2.
  • the D-isoGln form (compound 63) was shown to be usable as a good substrate.
  • Compound 63 exists as two presumed stereoisomers, 63A and 63B, and the highly polar 63B was shown to be a better substrate.
  • compound 63 The specificity of compound 63 was 63A (7.3%) and 63B (33.9%). Also, it was H-L-Ala-D-isoGln-MCA ⁇ HCl (compound 45) (20.0%), Fmoc-D-isoGln-MCA (compound 21) (23.4%), H-D-isoGln-MCA (compound 30) (1.3%), Fmoc-L-Ala-D-isoGln-MCA (compound 39) (7.6%), and benzyl-4,6-O-benzylidene- ⁇ -MurNAc-L-Ala-D-isoGln-MCA (compound 58) (14.0%).
  • Compound 30 showed no specificity and was not shown to be a substrate. On the other hand, compounds 63, 45, 21, 39 and 58 showed specificity and were shown to be usable as substrates. These results indicate that compounds with an acyl group (Ala) or an acyloxy group (Fmoc group) of appropriate size in the amino group moiety can be used as substrates.
  • pNA substrate Since it was suggested that the amino acid at the cleavage site of Lc-Lys2 is D-isoGln, benzyl- ⁇ -MurNAc-L-Ala-D-isoGln-pNA (compound 64) was synthesized in which 4NA was introduced into D-isoGln, and the specificity was also evaluated. The reaction was carried out using the pNA substrate at 37°C for 20 or 180 minutes, and the specificity was evaluated by measuring with a spectrophotometer. The measurement results of the specificity of 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 production amount of 4NA (hydrolysis rate %).
  • benzyl- ⁇ -MurNAc-D-Ala-D-isoGln-pNA (compound 65) was synthesized and its specificity was examined.
  • the specificities of these were 65A (41.4%, 98.0%) and 65B (106.8%, 113.0%). This suggested that the stereoconfiguration of Ala had little effect on the specificity.
  • benzyl- ⁇ -MurNAc-L-Ala-D-isoGln-MCA (63B) and benzyl- ⁇ -MurNAc-L-Ala-D-isoGln-pNA (64B) are particularly preferred substrates for use in this activity evaluation method.
  • the present invention has industrial applicability in that it provides a method for evaluating the activity of an enzyme for use in structural analysis of cell wall polysaccharides of lactic acid bacteria.

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Abstract

Le problème décrit par la présente invention est de fournir un nouveau composé destiné à être utilisé en tant que substrat pour évaluer l'activité de la γ-D-glutamyl-L-lysyl endopeptidase et un procédé d'évaluation de l'activité de la γ-D-glutamyl-L-lysyl endopeptidase à l'aide du composé. La solution selon la présente invention concerne un composé représenté par la formule (1) ou un stéréoisomère de celui-ci, qui est utile en tant que substrat pour évaluer l'activité de la γ-D-glutamyl-L-lysyl endopeptidase [Formule 1] (Dans la formule (1), A représente la formule (2) ou un groupe amino-protecteur, et Z représente un groupe de détection qui devient détectable lors de la libération.) [Formule 2] (Dans la formule (2), B représente un atome d'hydrogène, un groupe N-acétyle-muramoyle éventuellement substitué, ou un groupe acyle ou amino-protecteur.)
PCT/JP2024/037335 2023-10-23 2024-10-21 COMPOSÉ DESTINÉ À ÊTRE UTILISÉ EN TANT QUE SUBSTRAT POUR ÉVALUER L'ACTIVITÉ DE LA γ-D-GLUTAMYL-L-LYSYL ENDOPEPTIDASE Pending WO2025089217A1 (fr)

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JP2005529838A (ja) * 2001-04-18 2005-10-06 イーライ・リリー・アンド・カンパニー リピドｉｉの製造方法
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CN106589055A (zh) * 2016-11-03 2017-04-26 清华大学 取代的细胞酰二肽类化合物及其制备方法和用途
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JPH02250899A (ja) * 1989-03-22 1990-10-08 Dai Ichi Seiyaku Co Ltd ムラミルトリペプチド誘導体の製造法
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