Detection probe for engase acting on sugar chains on antibody

Abstract

The present invention addresses the problem of developing a highly sensitive, simple, and high-throughput system for detecting the activity of Endo-S, which is an ENGase specifically acting on sugar chains on an antibody. The present invention provides, for example: a sugar compound having a specific structure recognized as a substrate by Endo-S; a method for producing the sugar compound; a composition for detecting the activity of an endo-β-N-acetylglucosaminidase (ENGase) using the sugar compound; a method for measuring the activity of an ENGase using the sugar compound; a method for screening for an ENGase activity inhibitor using the sugar compound; and a method for screening for an ENGase using the sugar compound.

Description

Exploratory probe for ENGase acting on antibody sugar chains

[0001] The present invention relates to a sugar compound having a specific structure that is recognized as a substrate by Endo-S and a method for producing the same, a composition for detecting endo-β-N-acetylglucosaminidase (ENGase) activity using the sugar compound, a method for measuring ENGase activity using the sugar compound, a method for screening for ENGase activity inhibitors using the sugar compound, and a method for screening ENGase using the sugar compound.

[0002] Because glycans on antibodies affect the efficacy of antibody drugs, identification of glycan structures is required. Endo-β-N-acetylglucosaminidase (ENGase) is a glycosidase that acts on asparagine-linked glycans on proteins and cleaves glycans from glycoproteins. This activity has led to its use in analyzing the glycan structures of proteinaceous biopharmaceuticals. However, the only ENGase that can act on antibodies to cleave glycans is Endo-S, an ENGase derived from Streptococcus pyogenes. While Endo-S specifically acts on glycans on antibodies, its activity cannot be detected using fluorescently labeled glycopeptides, which have previously been used to detect ENGase activity. Therefore, a method currently used is to use the antibody itself as a substrate and confirm the change in molecular weight due to glycan cleavage using SDS-PAGE. Furthermore, because the sugar chain structures on antibodies are heterogeneous, when using antibodies from different production lots as substrates, differences in substrate recognition due to differences in sugar chain structure must be taken into consideration, leaving ambiguity in the evaluation of enzyme activity.

[0003] Furthermore, ENGase that acts on antibodies is limited to Endo-S, and there is a need to obtain ENGase with higher activity than Endo-S. However, as mentioned above, the only method available for detecting enzyme activity is a cumbersome method using SDS-PAGE, which has low throughput, and therefore research into discovering new ENGases has not progressed.

[0004] The present inventors have previously developed an ENGase detection molecular probe (FIG. 3, Patent Documents 1 and 2) that glows upon cleavage and operates on the principle of eliminating FRET quenching, as a substrate for a highly sensitive, simple, and high-throughput method for detecting ENGase activity. However, the activity of Endo-S could not be detected with this substrate developed by the present inventors, and the development of a highly sensitive, simple, and high-throughput method for detecting the activity of ENGase that acts on antibodies remained an issue.

[0005] Patent No. 6798085 Patent No. 7198482

[0006] In view of the above circumstances, an object of the present invention is to develop a highly sensitive, simple, and high-throughput activity detection system for Endo-S, an ENGase that specifically acts on sugar chains on antibodies.

[0007] The present inventors have conducted extensive research to solve the above-mentioned problems. They have found that Endo-S has high substrate specificity, and that the fluorescent substituents hinder the recognition of Endo-S as a substrate in previously developed probes that operate on the principle of eliminating FRET quenching (they glow when cleaved). Therefore, they have carefully examined the position at which the fluorescent substituents should be introduced on the sugar chain, and have developed a molecular probe that can easily detect the activity of Endo-S. Based on these findings, the present invention has been completed. Specifically, the gist of the present invention relates to the following:

[0008] [1] A sugar compound represented by the following formula (I):

[0009]

[0010] (In formula (I), Z １ and Z ２ one of which is a group represented by -R"-ZF having a fluorescent group ZF where fluorescence resonance energy transfer (FRET) occurs, and the other is a group represented by -R"-ZQ having a quenching group ZQ corresponding to the fluorescent group, １ are each independently either a group having a fluorescent group or a group having a quenching group, or —OR, and two Z １at least one of the groups is a group having a fluorescent group or a group having a quenching group, each R independently represents a hydrogen atom, a protecting group for a hydroxyl group, or a glycosyl residue, each R' independently represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group, each R'' independently represents a single bond, a secondary or tertiary amino group (-NR'-), an amide group (-NR'CO-), an oxy group (-O-), a carbonyl group (-CO-), an oxycarbonyl group (-OCO-), or a divalent hydrocarbon group having 1 to 6 carbon atoms which may have a substituent and which may contain at least one group selected from the group consisting of a secondary or tertiary amino group (-NR'-), an oxy group (-O-), and a carbonyl group (-CO-), and R''' represents a hydrogen atom or an optionally protected fucose residue. １ is a group having a fluorescent group or —OR, and Z ２ is a group having a quenching group. [3] The sugar compound, wherein the combination of the fluorescent group and the quenching group is any one of the following (i) to (iv): (i) A combination of a fluorescent group represented by the following formula (d-1) and a quenching group represented by the following formula (a-1):

[0011]

[0012] (In formula (d-1), R' represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group.) (ii) A combination of a fluorescent group represented by the following formula (d-2) and a quenching group represented by the following formula (a-1):

[0013]

[0014] (In formula (d-2), R represents a hydrogen atom, a protecting group for a hydroxyl group, or a glycosyl residue.) (iii) A combination of a fluorescent group represented by the following formula (d-3) and a quenching group represented by the following formula (a-1):

[0015]

[0016] (In formula (d-3), R represents a hydrogen atom, a protecting group for a hydroxyl group, or a glycosyl residue, and R′ represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group.) (iv) A combination of a fluorescent group represented by the following formula (d-4) and a quenching group represented by the following formula (a-2):

[0017]

[0018] (In formulas (d-4) and (a-2), R' each independently represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group.) [4] A composition for detecting endo-β-N-acetylglucosaminidase (ENGase) activity, comprising the sugar compound. [5] The composition, which is for detecting endo-β-N-acetylglucosaminidase (ENGase) S activity. [6] A method for producing a sugar compound, comprising a reaction step of producing a compound represented by the following formula (I) from a compound represented by the following formula (II).

[0019]

[0020] (In formula (I), Z １ and Z ２ one of which is a group represented by -R"-ZF having a fluorescent group ZF where fluorescence resonance energy transfer (FRET) occurs, and the other is a group represented by -R"-ZQ having a quenching group ZQ corresponding to the fluorescent group, １ are each independently either a group having a fluorescent group or a group having a quenching group, or —OR, and two Z １at least one of the groups is a group having a fluorescent group or a group having a quenching group, each R independently represents a hydrogen atom, a protecting group for a hydroxyl group, or a glycosyl residue, each R' independently represents a hydrogen atom, a hydrocarbon group of 1 to 6 carbon atoms, or a protecting group for an amino group, each R'' independently represents a single bond, a secondary or tertiary amino group (-NR'-), an amido group (-NR'CO-), an oxy group (-O-), a carbonyl group (-CO-), an oxycarbonyl group (-OCO-), or a divalent hydrocarbon group of 1 to 6 carbon atoms which may have a substituent and which may contain at least one group selected from the group consisting of a secondary or tertiary amino group (-NR'-), an oxy group (-O-), and a carbonyl group (-CO-), and R''' represents a hydrogen atom or an optionally protected fucose residue. [7] A method for producing a sugar compound, comprising a sugar transfer reaction step of reacting a compound represented by the following formula (VI) with a compound represented by the following formula (V) to produce a compound represented by the following formula (IV):

[0021]

[0022] [8] A method for measuring endo-β-N-acetylglucosaminidase (ENGase) activity, comprising: an activity confirmation step of contacting the sugar compound with endo-β-N-acetylglucosaminidase (ENGase) to confirm the decomposition activity of the sugar compound. [9] A method for screening for an endo-β-N-acetylglucosaminidase (ENGase) activity inhibitor, comprising: a contacting step of contacting a test compound with endo-β-N-acetylglucosaminidase (ENGase), and an activity confirmation step of contacting the sugar compound with the endo-β-N-acetylglucosaminidase (ENGase) that has been contacted with the test compound to confirm the decomposition activity of the sugar compound.

[10] A method for screening for endo-β-N-acetylglucosaminidase (ENGase), comprising: a contacting step of contacting a test compound with a candidate endo-β-N-acetylglucosaminidase (ENGase); and an activity confirmation step of contacting the sugar compound with the candidate endo-β-N-acetylglucosaminidase (ENGase) that has been contacted with the test compound, and confirming the decomposition activity of the sugar compound.

[0023] According to the present invention, a highly sensitive, simple and high-throughput system for detecting the activity of Endo-S is provided.

[0024] Fig. 1 shows the results (HPLC chromatogram) of the hydrolysis reaction of novel probe 1 of the present invention by Endo-S, which was carried out in Example 2. Fig. 2 shows the results of tracking the hydrolysis reaction of novel probe 1 of the present invention by Endo-S, which was carried out in Example 2, using a microplate reader. Fig. 3 shows an existing molecular probe for detecting ENGase activity. Fig. 4 shows the results of tracking the hydrolysis reaction of novel probes 1c and 1d of the present invention by Endo-S, which was carried out in Example 3, using a microplate reader.

[0025] The present invention will be described below.

[0026] In explaining the details of the present invention, specific examples will be given, but the present invention is not limited to the following content and can be modified as appropriate without departing from the spirit of the present invention. Furthermore, although the present specification describes a sugar compound with a heptasaccharide structure as an example, this is merely one example and the present invention may also be a sugar compound with an octasaccharide structure.

[0027] <Sugar Compound> A sugar compound according to one embodiment of the present invention (hereinafter sometimes abbreviated as "sugar compound of the present invention") is a compound represented by the following formula (I).

[0028]

[0029] (In formula (I), Z １ and Z ２ represents a group represented by -R"-ZF, one of which has a fluorescent group ZF where fluorescence resonance energy transfer (FRET) occurs, and the other a group represented by -R"-ZQ, which has a quenching group ZQ corresponding to the fluorescent group; Z １ are each independently either a group having a fluorescent group or a group having a quenching group, or —OR; １at least one of the groups is a group having a fluorescent group or a group having a quenching group, each R independently represents a hydrogen atom, a protecting group for a hydroxyl group, or a glycosyl residue, each R' independently represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group, each R'' independently represents a single bond, a secondary or tertiary amino group (-NR'-), an amido group (-NR'CO-), an oxy group (-O-), a carbonyl group (-CO-), an oxycarbonyl group (-OCO-), or a divalent hydrocarbon group having 1 to 6 carbon atoms which may have a substituent and which may contain at least one group selected from the group consisting of a secondary or tertiary amino group (-NR'-), an oxy group (-O-), and a carbonyl group (-CO-), and R''' represents a hydrogen atom or an optionally protected fucose residue.

[0030] The present inventors synthesized a sugar compound of formula (I) and confirmed that this compound is recognized as a substrate by Endo-S and can actually be used as a FRET probe for detecting Endo-S activity. For example, in one example of a sugar compound in a reaction represented by the following formula, two Z １ and an N-methylanthranyl group as a fluorescent group at the position of Z in formula (I). ２ When the sugar chain is cleaved by Endo-S, the distance between the fluorescent group and the quencher group increases, causing a change in the intensity of the fluorescent emission of the fluorescent group, allowing Endo-S activity to be detected (Figure 1).

[0031] The terms "fluorescent group that generates fluorescence resonance energy transfer (FRET)" and "quenching group corresponding to the fluorescent group" mean any combination of a fluorescent group and a quenching group that generates fluorescence resonance energy transfer (FRET).

[0032] The sugar compound of the present invention is a compound represented by the above formula (I), but the specific type is not particularly limited as long as it corresponds to formula (I) and exhibits the effects of the present invention, and can be appropriately selected depending on the purpose of use, etc.

[0033] Each R in formula (I) independently represents a "hydrogen atom," a "hydroxyl-protecting group," or a "glycosyl residue." Examples of the hydroxyl-protecting group include ether-based protecting groups such as methyl, benzyl, p-methoxybenzyl, and tert-butyl; acyl-based protecting groups such as acetyl, pivaloyl, and benzoyl; and silyl ether-based protecting groups such as trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, triisopropylsilyl, and tert-butyldiphenylsilyl. Examples of the glycosyl residue include residues of glucose, galactose, fructose, and the like.

[0034] In formula (I), R' each independently represents a "hydrogen atom," a "hydrocarbon group having 1 to 6 carbon atoms," or a "protecting group for an amino group." The "hydrocarbon group" is not limited to a linear saturated hydrocarbon group, but refers to a group consisting of only carbon atoms and hydrogen atoms, which may have a carbon-carbon unsaturated bond, a branched structure, or a cyclic structure.

[0035] The hydrocarbon group includes a methyl group (-CH ３ , -Me), ethyl group (-C ２ H ５ , -Et), n-propyl group (- ｎ C ３ H ７ ,- ｎ Pr), i-propyl group (- ｉ C ３ H ７ ,- ｉ Pr), n-butyl group (- ｎ C ４ H ９ ,- ｎ Bu), t-butyl group (- ｔ C ４ H ９ ,- ｔ Bu), n-pentyl group (- ｎ C ５ H １１ ), n-hexyl group (- ｎ C ６ H １３ ,- ｎ Hexyl), cyclohexyl group (- ｃ C ６ H１１ , -Cy), phenyl group (-C ６ H ５ , -Ph) and the like.

[0036] Examples of the protecting group for an amino group include alkoxycarbonyl protecting groups such as t-butoxycarbonyl group (Boc), benzyloxycarbonyl group (Cbz), 9-fluorenylmethyloxycarbonyl group (Fmoc), 2,2,2-trichloroethoxycarbonyl group (Troc), and allyloxycarbonyl group (Alloc); acyl protecting groups such as acetyl group and trifluoroacetyl group (Tfa); and alkyl(aryl)sulfonyl groups such as p-toluenesulfonyl group (Ts) and 2-nitrobenzenesulfonyl group (Ns).

[0037] R″ in formula (I) each independently represents a “single bond”, a “secondary or tertiary amino group (—NR′—)”, an “amide group (—NR′CO—)”, an “oxy group (—O—)”, a “carbonyl group (—CO—)”, an “oxycarbonyl group (—OCO—)”, or a “divalent hydrocarbon group having 1 to 6 carbon atoms which may contain at least one group selected from the group consisting of a secondary or tertiary amino group (—NR′—), an oxy group (—O—), and a carbonyl group (—CO—) and which may have a substituent”, and the “single bond” refers to a group represented by the formula Z １ and Z ２ is directly bonded to the six-membered ring of the sugar. １ is a group represented by -R''-ZF, Z ２ The following description will be given of an example of a group represented by -R''-ZQ, but the present invention is not limited thereto.

[0038]

[0039] The terms "secondary or tertiary amino group (-NR'-)", "amide group (-NR'CO-)", "oxy group (-O-)", "carbonyl group (-CO-)", and "oxycarbonyl group (-OCO-)" refer to the Z group described below, such as the structure represented by the following formula: １ and Z ２ is attached to the six-membered ring of the sugar via these groups.

[0040]

[0041] The term "divalent hydrocarbon group" refers to a hydrocarbon group having two bonding positions, and is not limited to a linear saturated hydrocarbon group, and may have a carbon-carbon unsaturated bond, a branched structure, or a cyclic structure. Furthermore, the term "may contain at least one group selected from the group consisting of a secondary or tertiary amino group (-NR'-), an oxy group (-O-), and a carbonyl group (-CO-)" means that the hydrocarbon group may contain such a group inside and / or at the end of the carbon skeleton, as in the structure represented by the following formula. Furthermore, the term "may contain a substituent" means that the hydrocarbon group may have a substituent on the carbon skeleton, as in the structure represented by the following formula. The substituent may be any substituent as long as the reaction of the present invention proceeds, and examples thereof include a hydroxyl group, an amino group, and a carboxyl group. R" may also be a structure derived from an amino acid.

[0042]

[0043] R''' in formula (I) represents a "hydrogen atom" or an "optionally protected fucose residue", and examples of the protecting group for the fucose residue include the same as the above-mentioned protecting group for the hydroxyl group.

[0044] Z １ and Z ２ represents "a group represented by -R"-ZF, one of which has a fluorescent group ZF whereby fluorescence resonance energy transfer (FRET) occurs," and "a group represented by -R"-ZQ, the other of which has a quenching group ZQ corresponding to the fluorescent group," and two Z １ are each independently either a group having a fluorescent group or a group having a quenching group, or —OR, and two Z １ At least one of the groups is a group having a fluorescent group or a group having a quenching group. １ is a group having a fluorescent group or —OR, and Z ２ is a group having a quenching group corresponding to the fluorescent group, and particularly, １is a group having a fluorescent group, and Z ２ is a group having a quenching group corresponding to the fluorescent group. Examples of R'' in -R''-ZF and -R''-ZQ include those similar to those mentioned above.

[0045] Examples of combinations of fluorescent groups and quenching groups include structures described in "FRET SUBSTRATES" published by Bachem et al. and Angew. Chem. Int. Ed. 2006, 45, 4562-4588. Among these, the following combinations of fluorescent groups and quenching groups (i) to (iv) are preferred, with (i) being particularly preferred.

[0046] (i) A combination of a fluorescent group represented by the following formula (d-1) and a quenching group represented by the following formula (a-1):

[0047]

[0048] (In formula (d-1), R' represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group.)

[0049] (ii) A combination of a fluorescent group represented by the following formula (d-2) and a quenching group represented by the following formula (a-1):

[0050]

[0051] (In formula (d-2), R represents a hydrogen atom, a protecting group for a hydroxyl group, or a glycosyl residue.)

[0052] (iii) A combination of a fluorescent group represented by the following formula (d-3) and a quenching group represented by the following formula (a-1):

[0053]

[0054] (In formula (d-3), R represents a hydrogen atom, a protecting group for a hydroxyl group, or a glycosyl residue, and R′ represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group.)

[0055] (iv) A combination of a fluorescent group represented by the following formula (d-4) and a quenching group represented by the following formula (a-2):

[0056]

[0057] (In formulas (d-4) and (a-2), R' each independently represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group.) Examples of R and R' in formulas (d-1) to (d-4) and formulas (a-1) and (a-2) include the same as those described above.

[0058] The sugar compounds of the present invention particularly include those represented by the following formula:

[0059]

[0060] The method for producing the sugar compound of the present invention is not particularly limited, and the compound can be produced by combining known organic synthesis reactions, chemoenzymatic methods, etc., with reference to the methods described in the Examples below.

[0061] <Composition for detecting endo-β-N-acetylglucosaminidase (ENGase) activity> As described above, ENGase has the specificity to selectively cleave a specific position in the heptasaccharide or octasaccharide structure of formula (I). When the sugar chain is cleaved by ENGase, a change in the intensity of fluorescence emission occurs, and ENGase activity can be detected (Figure 1). The use of the sugar compound of the present invention is not particularly limited, but an example is its use in detecting ENGase activity. Note that a composition for detecting ENGase activity containing the sugar compound of the present invention represented by formula (I) (hereinafter, sometimes abbreviated as "the composition of the present invention") also constitutes one aspect of the present invention. Note that "detection of ENGase activity" may also be "measurement of ENGase activity."

[0062] The content of the sugar compound represented by formula (I) in the composition of the present invention is, in terms of the amount of substance, usually 500% or more, preferably 2000% or more, and usually 40000% or less, preferably 4000% or less, relative to the target ENGase.

[0063] Examples of ENGases that are the subject of the composition of the present invention include those described in Glycobiology, vol. 23, no. 6, pp. 736-744, 2013, and specific examples include Endo-S, Endo-M, Endo-A, Endo-D, Endo-CC, Streptococcus pyogenes ENGase, human ENGase, mouse ENGase, yeast ENGase, and basidiomycete ENGase, and particularly Endo-S derived from streptococci.

[0064] The composition of the present invention may further contain other components, such as a buffer, a surfactant, etc., as long as it contains the sugar compound of the present invention as a component.

[0065] <Method 1 for producing sugar compound> As described above, the method for producing the sugar compound of the present invention is not particularly limited, and the sugar compound may be produced by combining known organic synthesis reactions, chemoenzymatic methods, etc., but it is preferable to produce the sugar compound by a method including a reaction step of producing a compound represented by the following formula (I) from a compound represented by the following formula (II).

[0066] (In formula (I), Z １ and Z ２ one of which is a group represented by -R"-ZF having a fluorescent group ZF where fluorescence resonance energy transfer (FRET) occurs, and the other is a group represented by -R"-ZQ having a quenching group ZQ corresponding to the fluorescent group, １ are each independently either a group having a fluorescent group or a group having a quenching group, or —OR, and two Z １at least one of the groups is a group having a fluorescent group or a group having a quenching group, each R independently represents a hydrogen atom, a protecting group for a hydroxyl group, or a glycosyl residue, each R' independently represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group, each R'' independently represents a single bond, a secondary or tertiary amino group (-NR'-), an amido group (-NR'CO-), an oxy group (-O-), a carbonyl group (-CO-), an oxycarbonyl group (-OCO-), or a divalent hydrocarbon group having 1 to 6 carbon atoms which may have a substituent and which may contain at least one group selected from the group consisting of a secondary or tertiary amino group (-NR'-), an oxy group (-O-), and a carbonyl group (-CO-), and R''' represents a hydrogen atom or an optionally protected fucose residue.

[0067] The reaction step is carried out by reacting a compound represented by formula (II) with Z １ and Z ２ The reaction step is usually carried out in a solvent. Suitable solvents are those that form a homogeneous phase with the reaction raw materials, and include, but are not limited to, ether solvents such as tetrahydrofuran, dimethoxyethane, 1,4-dioxane, and cyclopentyl methyl ether; alcohol solvents such as methanol and tert-butanol; aromatic solvents such as toluene and xylene; and aprotic polar solvents such as dichloromethane, dimethyl sulfoxide, and dimethylformamide.

[0068] As for details of the reaction step, for example, assuming that the compound represented by formula (II) is the following compound 3 and the compound represented by formula (I) is the following compound 1, for example, compound 3 is dissolved in water, sodium bicarbonate is added, and then DNPF dissolved in 1,4-dioxane is added, and the mixture is stirred at room temperature, followed by washing, purification, etc. to obtain compound 2, compound 2 is dissolved in DMF, DIPEA is added, and then N-methylanthranil and HATU are added, and the mixture is stirred at room temperature, followed by washing, purification, etc. to obtain compound 1.

[0069]

[0070]

[0071] The method for preparing the compound represented by formula (II) used in the above reaction step is not particularly limited. For example, when compound 3 is assumed as the compound represented by formula (II), a method following the reaction pathway described in the Examples below can be mentioned.

[0072] <Method for producing sugar compounds 2> One aspect of the present invention is a method for producing sugar compounds, comprising a glycosylation reaction step of reacting a compound represented by the following formula (VI) with a compound represented by the following formula (V) to produce a compound represented by the following formula (IV). A preferred aspect of this production method is a method for producing sugar compounds, comprising the glycosylation reaction step of producing compound (I) from compound (II), as described above in <Method for producing sugar compounds 1>. In other words, the sugar compounds produced by this production method can also be considered to be intermediate products in the production of the sugar compounds of the present invention.

[0073]

[0074] The reaction step is usually carried out in a solvent. Suitable solvents are those that form a homogeneous phase with the reaction raw materials, and include, but are not limited to, ether solvents such as tetrahydrofuran, dimethoxyethane, 1,4-dioxane, and cyclopentyl methyl ether, alcohol solvents such as tert-butanol, aromatic solvents such as toluene and xylene, and aprotic polar solvents such as dichloromethane, dimethyl sulfoxide, and dimethylformamide.

[0075] As for details of the above reaction step, assuming that the following compound 6 is the compound represented by formula (VI), the following compound 5 is the compound represented by formula (V), and the following compound 4 is the compound represented by formula (IV), for example, compound 6 and compound 5 are dissolved in dichloromethane, and the solution is added to dry molecular sieves, iodosuccinimide, and silver trifluoromethanesulfonate using a canola, followed by stirring at room temperature, terminating the reaction with triethylamine, and then diluting, filtering, washing, purifying, and the like to obtain compound 4.

[0076]

[0077] The method for preparing the compound represented by formula (VI) used in the above reaction step is not particularly limited. For example, when compound 6 is assumed as the compound represented by formula (VI), a method following the reaction pathway described in the Examples below can be mentioned.

[0078] The method for preparing the compound represented by formula (V) used in the above reaction step is not particularly limited. For example, when compound 5 is assumed to be the compound represented by formula (V), a method following the reaction pathway described in the Examples below can be mentioned.

[0079] <Method for measuring endo-β-N-acetylglucosaminidase (ENGase) activity> One aspect of the present invention is a method for measuring endo-β-N-acetylglucosaminidase (ENGase) activity (hereinafter sometimes abbreviated as "method for measuring ENGase activity of the present invention"), which comprises an activity confirmation step of contacting an endo-β-N-acetylglucosaminidase (ENGase) with a sugar compound represented by formula (I) of the present invention and confirming the decomposition activity of the sugar compound. The method for measuring ENGase activity of the present invention can use the sugar compound of the present invention and the composition of the present invention described above.

[0080] Since the sugar compounds of the present invention allow for easy detection of ENGase activity, ENGase activity can be efficiently measured by contacting ENGase with the sugar compounds of the present invention and confirming the decomposition activity of the sugar compounds of the present invention.

[0081] The activity confirmation step is a step of contacting a sugar compound represented by formula (I) with ENGase to confirm the sugar compound decomposition activity. The mass of the sugar compound represented by formula (I) to be contacted is usually 0.5 ng or more, preferably 5 ng or more, and usually 500 μg or less, preferably 300 μg or less, per 1 ng of ENGase.

[0082] The method for confirming the decomposition activity of the sugar compound represented by formula (I) in the activity confirmation step is not particularly limited, but includes a method of observing a change in the intensity of fluorescence emission based on the fluorescent group of the sugar compound represented by formula (I), and a method of observing the ultraviolet (UV) absorption wavelength based on the quenching group of the sugar compound represented by formula (I).

[0083] <Method for screening endo-β-N-acetylglucosaminidase (ENGase) activity inhibitors> ENGase activity inhibitors are useful for completely inhibiting the remaining enzyme activity after ENGase cleaves the sugar chain on an antibody and the reaction has progressed, when performing glycomodeling of an antibody drug. Because the sugar compound of the present invention allows for easy detection of ENGase activity, ENGase activity inhibitors can be efficiently screened by contacting ENGase, which has been contacted with a test compound, with the sugar compound of the present invention and confirming the decomposition activity of the sugar compound of the present invention. In addition, a screening method for ENGase activity inhibitors (hereinafter sometimes abbreviated as "the screening method for ENGase activity inhibitors of the present invention") which includes a contacting step (hereinafter sometimes abbreviated as "contacting step") of contacting a test compound with ENGase, and an activity confirmation step (hereinafter sometimes abbreviated as "activity confirmation step") of contacting a sugar compound represented by formula (I) of the present invention with the ENGase that has been contacted with the test compound, and confirming the decomposition activity of the sugar compound is also one aspect of the present invention.

[0084] The contacting step is a step of contacting a test compound with ENGase. The mass of the test compound to be contacted is usually 7.5 μg or more, preferably 15 μg or more, and usually 150 μg or less, preferably 75 μg or less, per 1 ng of ENGase.

[0085] The activity confirmation step is a step of contacting a sugar compound represented by formula (I) with ENGase that has been contacted with a test compound, and confirming the sugar compound decomposition activity. Other aspects of the activity confirmation step in this embodiment are similar to those of the activity confirmation step in the above-mentioned <Method for measuring endo-β-N-acetylglucosaminidase (ENGase) activity>. However, a method for confirming the sugar compound decomposition activity represented by formula (I) in the activity confirmation step can be, for example, by comparing the sugar compound decomposition activity of ENGase not contacted with the test compound with the sugar compound decomposition activity of ENGase contacted with the test compound, and if the sugar compound decomposition activity of ENGase contacted with the test compound is inferior, the test compound can be determined to have an inhibitory effect on ENGase activity (to be an ENGase activity inhibitor).

[0086] <Method for screening endo-β-N-acetylglucosaminidase (ENGase)> One aspect of the present invention is a method for screening endo-β-N-acetylglucosaminidase (ENGase) (hereinafter sometimes abbreviated as "the method for screening ENGase of the present invention"), comprising a contacting step of contacting a test compound with a candidate endo-β-N-acetylglucosaminidase (ENGase), and an activity confirmation step of contacting a sugar compound represented by formula (I) of the present invention with the candidate endo-β-N-acetylglucosaminidase (ENGase) that has been contacted with the test compound, and confirming the decomposition activity of the sugar compound.

[0087] Because the sugar compound of the present invention allows for simple detection of ENGase activity, novel ENGases can be efficiently screened by contacting a candidate ENGase with the sugar compound of the present invention and confirming the decomposition activity of the sugar compound of the present invention. Furthermore, the contacting step and activity confirmation step in this embodiment are similar to the contacting step in the above-mentioned <Method for Measuring Endo-β-N-Acetylglucosaminidase (ENGase) Activity> and the activity confirmation step in the above-mentioned <Method for Screening for Endo-β-N-Acetylglucosaminidase (ENGase) Activity Inhibitor>. However, as a method for confirming the decomposition activity of the sugar compound represented by formula (I) in the activity confirmation step, for example, the decomposition activity of a candidate ENGase contacted with a test compound is measured, and if significant activity is observed compared to the control, it can be determined that the sugar compound is useful as an ENGase, a novel ENGase, or the like.

[0088] The present invention will be specifically described below with reference to examples, but these are merely examples of the present invention and the scope of the present invention is not limited to these examples.

[0089] (Method and equipment for measuring physical properties) Solvents and reagents used in the reaction were purchased from Kanto Chemical Co., Ltd., Tokyo Chemical Industry Co., Ltd., Wako Pure Chemical Industries, Ltd., and Sigma-Aldrich. Furthermore, mili-Q water was used as the solvent and developing solvent. Unless otherwise noted, the reaction was carried out under an argon atmosphere, and the progress of the reaction was monitored by thin-layer chromatography (TLC) using Merck silica gel 60 F. 254 A CE plate was used. Silica gel column chromatography was performed using KANTO CHEMICAL Silica gel 60 N (40-100 mesh or 100-210 mesh) or a YAMAZEN Universal column (Yamazen Corporation). Gel filtration chromatography was performed using a Japan Analytical Industry LaboACE (JAIGEL-HR Series). High-performance liquid chromatography (HPLC) was performed using a Shimadzu Nexera system fluorescence detector RF-20Axz (Shimadzu Corporation) analytical system and a UV detector UV / Vis-156 (Gilson) preparative system. A SpectraMax i3x microplate reader (Molecular Devices) was used. The molecular sieves used in the reaction were powdered Molecular Sieves 4A (Nacalai Tesque), activated by heating and drying overnight at 200°C under a vacuum pump. Celite (Cica-Reagent) Celite® 545 was used. Amberlyst was a catalytic ion exchange resin, Amberlyst (registered trademark), manufactured by ORGANO Inc. Endo-S, the enzyme used in the enzymatic reaction, was purchased from New England Biolabs.

[0090] 1 H-NMR spectra were measured using a JEOL-JNM-ECS 400 MHz model and a JEOL-JNM-ECS 600 MHz model nuclear magnetic resonance spectrometer (JEOL Ltd.). 13 ​C-NMR spectra were measured using a JEOL-JNM-ECA 600 MHz nuclear magnetic resonance spectrometer. Unless otherwise noted, the solvent used was tetramethylsilane as the internal standard for deuterated chloroform solutions, and the peak of deuterium oxide as the internal standard for deuterated water solutions. Mass spectrometry was performed in high-resolution mode using a matrix-assisted laser ionization time-of-flight mass spectrometer, MALDI-TOFMS AXIMA® Performance (Shimadzu Corporation). 2,5-dihydroxybenzoic acid (DHBA) was used as the matrix.

[0091] (Unit definition) 1 U: Defined as the amount of enzyme required to remove 95% or more of the sugars from 5 μg of IgG in 10 μl of total reaction solution at 37°C for 1 hour (New England Biolabs, Endo S (P0741) - New England Biolabs Japan (nebj.jp))

[0092] Example 1: Preparation of sugar compound represented by formula (I) (Synthesis of phenyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-D-galactopyranoside (12))

[0093]

[0094] Compound 11 (139 mg, 0.239 mmol) was dissolved in anhydrous dichloromethane (1 mL), followed by the addition of pyridine (55.5 μL, 0.688 mmol) and trifluoromethanesulfonic anhydride (56.4 μL, 0.344 mmol) and stirring at room temperature for 30 minutes. After confirming the completion of the reaction by TLC, the reaction solution was added to saturated aqueous sodium bicarbonate at 0°C and stirred. The organic layer was diluted with ethyl acetate and washed sequentially with saturated aqueous sodium bicarbonate, saturated brine, 1M hydrochloric acid, and saturated brine. After drying over anhydrous magnesium sulfate, the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 3 / 1, v / v). Compound 24 was dissolved in N,N-dimethylformamide (1 mL), followed by the addition of sodium nitrite (119 mg, 1.72 mmol) and stirring at room temperature overnight to give reaction intermediate 25. 1M hydrochloric acid (600 μL) was added to the reaction solution at 0°C, and the mixture was stirred at room temperature for 3 hours. The mixture was washed sequentially with 1M hydrochloric acid, saturated brine, saturated aqueous sodium bicarbonate, and saturated brine, and then dried over anhydrous magnesium sulfate. The solution was filtered, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (toluene / ethyl acetate = 7 / 1, v / v) to give compound 12 (98.5 mg, 0.169 mmol, 71%).

[0095] R f = 0.33 (toluene / EtOAc = 5 / 1), 1 H NMR (400 MHz, CDCl3) δ 7.39-6.92 (m, 20H), 5.51 (d, J = 10.5 Hz, 1H), 4.63-4.52 (m, 4H), 4.32-4.19 (m, 3H), 3.89-3.79 (m, 3H), 2.34 (s, 2H); MALDI-TOF MS m / z: [M+Na] + calcd for C 34 H 31 NO6S; 604.18; found 604.55.

[0096] (Synthesis of Phenyl 4-azide-3,6-di-O-benzyl-2,4-di-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside (13))

[0097]

[0098] Compound 12 (619 mg, 1.06 mmol) was dissolved in anhydrous dichloromethane (6 mL), and then pyridine (375 μL, 1.54 mmol) and trifluoromethanesulfonic anhydride (254 μL, 4.65 mmol) were added at 0 °C and stirred at room temperature for 30 minutes. After confirming the completion of the reaction by TLC, the reaction mixture was diluted with ethyl acetate and washed sequentially with 1 M hydrochloric acid, saturated brine, saturated aqueous sodium bicarbonate, and saturated brine, and then dried over anhydrous magnesium sulfate. The solvent was then removed under reduced pressure. The resulting residue was purified by silica gel column chromatography (toluene / ethyl acetate = 100 / 1, v / v). The resulting compound 26 was dissolved in N,N-dimethylformamide (6 mL), and then sodium azide (286 mg, 4.40 mmol) was added and stirred at 40 °C for 1 hour. After confirming the completion of the reaction by TLC, the reaction mixture was diluted with ethyl acetate and washed sequentially with water, saturated aqueous sodium bicarbonate, and saturated brine. The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate = 3 / 1, v / v) to give compound 13 (567 mg, 0.935 mmol, 88%).

[0099] R f = 0.67 (hexane / EtOAc = 3 / 1); 1 H NMR (600 MHz, CDCl3) δ 7.80-6.83 (m, 20H), 5.48-5.43 (m, 1H), 4.83-4.57 (dd, 2H), 4.68-4.56 (dd, 2H), 4.28-4.20 (m, 2H), 3.86-3.75 (m, 3H), 3.54 (dq, 1H); 13C NMR (151 MHz, CDCl3) δ 168.2, 167.2, 138.1, 137., 134.1, 134.0, 132.8, 131.9, 131.6, 131.5, 128.9, 128.5, 128.2, 128.1, MALDI-TOF MS m / z: [M+Na] + calcd for C 34 H 30 N4O5S; 629.18; found 629.71.

[0100] (Synthesis of Fluoride 4-azide-3,6-di-O-benzyl-2,4-di-deoxy-2-phthalimide-β-D-glucopyranoside (7))

[0101]

[0102] Compound 13 (74.4 mg, 0.123 mmol) was dissolved in anhydrous dichloromethane (3 mL) and transferred to N-bromosuccinimide (48 mg, 0.2698 mmol) azeotroped with toluene using a cannula. The mixture was placed in a -40 °C thermostatic bath, and N,N-diethylaminosulfur trifluoride (19.4 μL, 0.147 mmol) was added. The temperature was adjusted to -10 °C and the mixture was stirred for 2 h. After confirming the completion of the reaction by TLC, the reaction solution was diluted with ethyl acetate and added dropwise to a beaker containing aqueous sodium bicarbonate and ethyl acetate at 0 °C to quench the reaction. The mixture was washed sequentially with saturated aqueous sodium bicarbonate and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed under reduced pressure. Purification by silica gel column chromatography (hexane / ethyl acetate = 5 / 1, v / v) gave compound 7 (55.9 mg, 0.108 mmol, 88%).

[0103] R f = 0.57 (hexane / EtOAc = 2 / 1), 1H NMR (600 MHz, CDCl3) δ 7.75–6.84 (m, 15H), 5.86–5.72 (dq, 1H), 4.86–4.40 (q, 2H), 4.71–4.58 (q, 2H), 4.30–4.25 (m, 2H), 3.90–3.79 (m, 3H), 3.58(dt, 1H); 13 C NMR (151 MHz, CDCl3) δ 167.7, 137.7, 137.3, 134.1, 131.5, 128.5, 128.3, 128.1, 127.9; 77.3, 77.1, 76.9, 75.0, 74.2, 73.8, 68.4, 62.9, 55.7; MALDI-TOF MS m / z: [M+Na] + calcd for C 28 H 25 FN4O5; 539.17; found 539.98.

[0104] (Phenyl 4-azide-3,6-di-O-benzyl-2,4-di-deoxy-2-phthalimide-β-D-glucopyranosyl-(1-2)-4,6-O-benzylidene-3-naphthylmethyl-1-thio-α-D-mannopyranoside(14))

[0105]

[0106] Molecular sieves 4A (750 mg), hafnocenedichloride (73.7 mg, 0.194 mmol), and silver trifluoromethanesulfonate (100 mg, 0.388 mmol) were added to a two-bottle eggplant, followed by a solution of compound 7 (87.4 mg, 0.169 mmol) and compound 8 (63.3 mg, 0.147 mmol) in anhydrous dichloromethane (3 mL) at -40°C and stirring for 3 hours. After confirming the completion of the reaction by TLC, the reaction was quenched by the addition of triethylamine. The reaction mixture was diluted with ethyl acetate and filtered through silica gel and Celite. The filtrate was washed successively with 1 M hydrochloric acid, saturated brine, saturated aqueous sodium bicarbonate, and saturated brine. After drying over anhydrous magnesium sulfate, the solvent was removed under reduced pressure. The resulting residue was crudely purified by silica gel column chromatography, then purified using gel filtration chromatography (chloroform), and then purified by silica gel column chromatography (toluene / hexane / ethyl acetate=1 / 1 / 0.12, v / v / v) to obtain compound 14 (94.6 mg, 94.9 μmol, 65%).

[0107] R f = 0.51 (toluene / hexane / EtOAc = 1 / 1 / 0.3); 1 H NMR (600 MHz, CDCl3) δ 7.76-6.83 (m, 32H), 5.44 (s, 1H), 5.18 (d, 1H), 5.13 (d, 1H), 4.89-4.43 (q, 2H), 4.87-4.77 (q, 2H), 4.62-4.54 (q, 2H), 4.37-4.30 (m, 3H), 3.98-3.72 (m, 6H), 3.56-3.53 (m, 2H), 3.07 (t, 1H); 13C NMR (151 MHz, CDCl3) δ 138.0, 137.6, 137.5, 135.6, 133.9, 133.8, 133.3, 133.1, 130.4, 129.1, 129.0, 128.5, 128.3, 128.1, 128.0, 127.8, 127.7, 127.6, 127.3, 126.5, 126.2, 126.0, 125.9, 125.8, 123.1, 101.7, 96.5, 85.6, 78.3, 78.2, 77.3, 77.1, 76.9, 74.9, 74.7, 73.9, 73.8, 71.5, 69.7, 68.1, 65.0, 63.7, 55.8; MALDI-TOF MS m / z: [M+Na] + calcd for C 58 H 52 N4O 10 S; 1019.33; found 1020.14.

[0108] (Synthesis of Phenyl 4-azide-3,6-di-O-benzyl-2,4-di-deoxy-2-phthalimide-β-D-glucopyranosyl-(1-2)-4,6-O-benzylidene-1-thio-α-D-mannopyranoside (15))

[0109]

[0110] Compound 14 (227 mg, 0.228 mmol) was dissolved in dichloromethane (2.2 mL) and water (220 μL). 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (259 mg, 1.14 mmol) was added and the mixture was stirred vigorously at room temperature for 1.5 hours. The reaction mixture was diluted with chloroform and then added dropwise to a beaker containing saturated aqueous sodium bicarbonate and chloroform to quench the reaction. The reaction mixture was washed sequentially with saturated aqueous sodium bicarbonate and saturated brine. The resulting organic layer was dried over anhydrous magnesium sulfate, and the solvent was removed under reduced pressure. The resulting residue was purified by silica gel chromatography (toluene / ethyl acetate = 15 / 1, v / v) to give compound 15 (165 mg, 0.193 mmol, 84%).

[0111] Rf = 0.59 (toluene / EtOAc = 5 / 1); 1 1H NMR (600 MHz, CDCl3) δ 7.66 - 6.84 (m, 25H), 5.41 (s, 1H), 5.19 - 5.15 (m, 1H), 5.05 (d, 1H), 4.86 - 4.42 (q, 2H), 4.66 - 4.55 (q, 2H), 4.27 - 4.19 (m, 3H), 3.99 (td, 1H), 3.92 (dd, 1H), 3.86 - 3.80 (m, 1H), 3.78 - 3.74 (m, 2H), 3.69 - 3.62 (m, 2H), 3.50 (dt, 1H), 3.13 (t, 1H); 13 13C NMR (151 MHz, CDCl3) δ 137.7, 137.5, 137.2, 134.0, 133.6, 130.8, 129.2, 129.1, 128.6, 128.3, 128.3, 128.1, 127.9, 127.7, 127.5, 126.4, 123.3, 102.3, 97.2, 86.1, 80.6, 79.5, 78.1, 77.3, 77.1, 76.9, 74.9, 74.7, 73.7, 68.6, 68.1, 68.0, 64.7, 63.3, 55.8; MALDI - TOF MS m / z: [M+Na] + calcd for C 47 H 44 N4O 10 S; 879.27; found 879.83.

[0112] (Synthesis of Phenyl 4 - azide - 3,6 - di - O - benzyl - 2,4 - di - deoxy - 2 - phthalimide - β - D - glucopyranosyl-(1 - 2)-4,6 - O - benzylidene - 3 - O - pivaloyl - 1 - thio - α - D - mannopyranoside (5))

[0113]

[0114] Compound 15 (124 mg, 0.145 mmol) dissolved in dichloromethane (3 mL) was added with triethylamine (202 μL, 1.45 mmol), pivaloyl chloride (88 μL, 0.724 mmol), and 4-dimethylaminopyridine (88.5 mg, 0.724 mmol) and stirred at 50°C for 30 minutes. After confirming completion of the reaction by TLC, the reaction was quenched with methanol. The reaction solution was diluted with ethyl acetate and washed sequentially with 1 M hydrochloric acid, saturated brine, saturated aqueous sodium bicarbonate, and saturated brine. After drying over anhydrous magnesium sulfate, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate = 5 / 1, v / v) to give compound 5 (118 mg, 0.125 mmol, 86%).

[0115] R f = 0.70 (toluene / EtOAc = 7 / 1), 1 H NMR (600 MHz, CDCl3) δ 7.38-6.85 (m, 23H), 5.38 (s, 1H), 5.12 (d, 1H), 5.04 (d, 1H), 4.98 (dd, 1H), 4.85-4.43 (q, 2H), 4.59-4.48 (q, 2H), 4.39 (dd, 1H), 4.22 (m, 2H), 4.01 (td, 1H), 3.89-3.82 (m, 2H), 3.76-3.69 (m, 2H), 3.55 (q, 1H), 3.41 (dq, 1H), 2.97-2.94 (t, 1H), 1.18 (s, 9H); 13C NMR (151 MHz, CDCl3) δ 178.1, 137.8, 137.7, 137.2, 133.9, 133.8, 131.9, 131.7, 130.5, 129.1, 128.9, 128.5, 128.3, 128.1, 127.8, 127.7, 127.6, 127.3, 126.0, 123.1, 101.4, 95.7, 84.9, 77.9, 77.3, 77.1, 76.9, 75.9, 74.8, 74.3, 73.6, 69.2, 69.0, 68.0, 64.8, 63.4, 55.9, 38.9, 27.1; MALDI-TOF MS m / z: [M+Na] + calcd for C 52 H 52 N4O 11 S; 963.33; found 964.05.

[0116] 3-Azidopropyl [4-azide-3,6-di-O-benzyl-2,4-di-deoxy-2-phthalimide-β-D-glucopyranosyl-(1-2)-4,6-O-benzylidene-3-O-pivaloyl-α-D-mannopyranosyl-(1-3)]-[4-azide-3,6-di-O-benzyl-2,4-di-deoxy-2-phthalimide-β-D-glucopyranosyl-(1-2)-4,6-O-benzylidene-3-O-pivaloyl-α-D-mannopyranosyl-(1-6)]-β-D-galactopyranosyl-(1-4)-3,6-di-O-benzyl--2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1-4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside(4)

[0117]

[0118] Molecular sieves 4A (640 mg) and N-iodosuccinimide (27.4 mg, 0.112 mmol) were added to the eggplant, and compound 6 (45.0 mg, 37.3 μmol) and compound 5 (70.3 mg, 74.7 μmol) were dissolved in anhydrous dichloromethane (4 mL) and transferred to the eggplant using a cannula. The eggplant was placed in a -78°C thermostatic bath, and trifluoromethanesulfonic acid (3.3 μL, 0.388 mmol) was added. The mixture was stirred for 4 hours. After confirming the completion of the reaction by TLC, triethylamine was added to the reaction mixture to quench the reaction. The reaction mixture was diluted with ethyl acetate and filtered through Celite. The filtrate was washed sequentially with sodium thiosulfate solution, saturated brine, 1M hydrochloric acid, saturated brine, saturated sodium bicarbonate solution, and saturated brine. After drying over anhydrous magnesium sulfate, the solvent was removed under reduced pressure. The residue was purified using gel filtration chromatography (chloroform) and then by PTLC (toluene / ethyl acetate=3 / 1) to obtain compound 4 (12.5 mg, 4.36 μmol, 11%).

[0119] R f = 0.65 (toluene / EtOAc = 3 / 1); 1 H NMR (600 MHz, CDCl3) δ 7.86-6.72 (m, 83H), 5.40 (s, 1H), 5.37 (s, 1H), 5.24 (d, 1H), 5.12-5.08 (d, q, 2H), 4.99 (d, 1H), 4.95-4.90 (d, q, 2H), 4.86-4.82 (m, 3H), 4.71 (d, 1H), 4.61-4.40 (m, 13H), 4.38-4.06 (m, 15H), 3.92-3.65 (m, 16H), 3.54-3.25 (m, 12H), 3.15 (dd, 1H), 3.11-3.02 (m, 3H), 2.89 (dt, 2H), 1.66-1.52 (m, 2H), 1.16 (d, 20H); 13C NMR (151 MHz, CDCl3)δ 178.2, 178.1, 168.9, 168.3, 167.8, 138.6, 138.5, 138.4, 137.9, 137.8, 137.8, 137.7, 137.6, 137.4, 137.2, 134.2, 134.1, 134.0, 134.0, 133.9, 133.9, 133.9, 133.8, 133.7, 133.7, 131.8, 131.7, 131.7, 131.6, 131.6, 131.5, 131.5, 128.8, 128.7, 128.6, 128.4, 128.4, 128.3, 128.2, 128.1, 128.0, 128.0, 127.9, 127.8, 127.8, 127.7, 127.7, 127.6, 127.5, 127.4, 127.3, 127.1, 126.9, 126.0, 126.0, 123.6, 123.5, 123.4, 123.4, 123.2, 123.2, 123.0, 123.0, 102.7, 101.3, 98.2, 97.5, 97.5, 97.0, 97.0, 96.1, 82.3, 78.1, 78.0, 77.7, 77.5, 77.3, 77.1, 76.8, 76.6, 75.8, 75.6, 75.5, 74.9, 74.8, 74.7, 74.5, 74.4, 74.3, 74.1, 74.0, 73.9, 73.5, 73.5, 73.1, 73.0, 72.6, 71.8, 70.7, 69.1, 68.5, 68.3, 68.3, 68.1, 68.0, 68.0, 67.0, 65.8, 65.5, 64.4, 64.2, 63.6, 63.2, 63.2, 56.7, 55.9, 55.9, 55.7, 48.0, 38.9, 38.8, 28.8, 27.1; MALDI-TOF MS m / z: [M+Na] + calcd for C 157 H 159 N 13 O 40 ; 2889.07; found 2888.79.

[0120] Synthesis of (3-Azidopropyl [4-azide-3,6-di-O-benzyl-2,4-di-deoxy-2-phthalimide-β-D-glucopyranosyl-(1-2)-4,6-O-benzylidene-3-O-pivaloyl-α-D-mannopyranosyl-(1-3)]-[4-azide-3,6-di-O-benzyl-2,4-di-deoxy-2-phthalimide-β-D-glucopyranosyl-(1-2)-4,6-O-benzylidene-3-O-pivaloyl-α-D-mannopyranosyl-(1-6)]-β-D-galactopyranosyl-(1-4)-3,6-di-O-benzyl--2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1-4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside (4) (Synthesis Method 1))

[0121]

[0122] Molecular sieves 4A (700 mg), N-iodosuccinimide (31.6 mg, 0.140 mmol), and silver trifluoromethanesulfonate (26.3 mg, 0.102 mmol) were added to the eggplant. Compound 6 (50.8 mg, 42.3 μmol) and compound 5 (78.2 mg, 93.0 μmol) were dissolved in anhydrous dichloromethane (4 mL) and then transferred to the eggplant using a cannulator in a -20°C thermostatic bath. The eggplant was then placed in a 0°C thermostatic bath and stirred for 48 hours. After confirming the completion of the reaction by TLC, triethylamine was added to the reaction mixture to quench the reaction. The reaction mixture was diluted with ethyl acetate and filtered through silica gel and Celite. The filtrate was washed sequentially with sodium thiosulfate solution, saturated brine, 1M hydrochloric acid, saturated brine, saturated sodium bicarbonate solution, and saturated brine. After drying over anhydrous magnesium sulfate, the solvent was removed under reduced pressure. The residue was purified using gel filtration chromatography (chloroform) and then silica gel column chromatography (hexane / ethyl acetate=3 / 2, v / v) to give compound 4 (69.5 mg, 24.2 μmol, 57%).

[0123] R f = 0.65 (toluene / EtOAc = 3 / 1); 1 H NMR (600 MHz, CDCl3) δ 7.86-6.72 (m, 83H), 5.40 (s, 1H), 5.37 (s, 1H), 5.24 (d, 1H), 5.12-5.08 (d, q, 2H), 4.99 (d, 1H), 4.95-4.90 (d, q, 2H), 4.86-4.82 (m, 3H), 4.71 (d, 1H), 4.61-4.40 (m, 13H), 4.38-4.06 (m, 15H), 3.92-3.65 (m, 16H), 3.54-3.25 (m, 12H), 3.15 (dd, 1H), 3.11-3.02 (m, 3H), 2.89 (dt, 2H), 1.66-1.52 (m, 2H), 1.16 (d, 20H); 13C NMR (151 MHz, CDCl3)δ 178.2, 178.1, 168.9, 168.3, 167.8, 138.6, 138.5, 138.4, 137.9, 137.8, 137.8, 137.7, 137.6, 137.4, 137.2, 134.2, 134.1, 134.0, 134.0, 133.9, 133.9, 133.9, 133.8, 133.7, 133.7, 131.8, 131.7, 131.7, 131.6, 131.6, 131.5, 131.5, 128.8, 128.7, 128.6, 128.4, 128.4, 128.3, 128.2, 128.1, 128.0, 128.0, 127.9, 127.8, 127.8, 127.7, 127.7, 127.6, 127.5, 127.4, 127.3, 127.1, 126.9, 126.0, 126.0, 123.6, 123.5, 123.4, 123.4, 123.2, 123.2, 123.0, 123.0, 102.7, 101.3, 98.2, 97.5, 97.5, 97.0, 97.0, 96.1, 82.3, 78.1, 78.0, 77.7, 77.5, 77.3, 77.1, 76.8, 76.6, 75.8, 75.6, 75.5, 74.9, 74.8, 74.7, 74.5, 74.4, 74.3, 74.1, 74.0, 73.9, 73.5, 73.5, 73.1, 73.0, 72.6, 71.8, 70.7, 69.1, 68.5, 68.3, 68.3, 68.1, 68.0, 68.0, 67.0, 65.8, 65.5, 64.4, 64.2, 63.6, 63.2, 63.2, 56.7, 55.9, 55.9, 55.7, 48.0, 38.9, 38.8, 28.8, 27.1; MALDI-TOF MS m / z: [M+Na] + calcd for C 157 H 159 N 13 O 40 ; 2889.07; found 2888.79.

[0124] (3-Azidopropyl [4-azide-3,6-di-O-benzyl-2,4-di-deoxy-2-phthalimide-β-D-glucopyranosyl-(1-2)-4,6-O-benzylidene-3-O-pivaloyl -α-D-mannopyranosyl-(1-3)]-[4-azide-3,6-di-O-benzyl-2,4-di-deoxy-2-phthalimide-β-D-glucopyranosyl-(1-2)-4,6- O-benzylidene-3-O-pivaloyl-α-D-mannopyranosyl-(1-6)]-β-D-galactopyranosyl-(1-4)-3,6-di-O-benzyl--2-deoxy-2- Synthesis of phthalimido-β-D-glucopyranosyl-(1-4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside (4) (Synthesis method 2))

[0125]

[0126] Molecular sieves 4A (650 mg), N-iodosuccinimide (37.1 mg, 0.165 mmol), and silver trifluoromethanesulfonate (27.8 mg, 0.108 mmol) were added to a two-neck eggplant. Compound 6 (49.3 mg, 40.9 μmol) and compound 5 (80.8 mg, 85.9 μmol) were dissolved in anhydrous dichloromethane (5 mL) and then transferred to the eggplant using a cannulator in a thermostatic bath at -20°C. The mixture was then stirred at 20°C for 48 hours. After confirming the completion of the reaction by TLC, triethylamine was added to the reaction mixture to quench the reaction. The reaction mixture was diluted with ethyl acetate and filtered through silica gel and Celite. The filtrate was washed sequentially with sodium thiosulfate solution, saturated brine, 1M hydrochloric acid, saturated brine, saturated sodium bicarbonate solution, and saturated brine. After drying over anhydrous magnesium sulfate, the solvent was evaporated under reduced pressure. The residue was purified using gel filtration chromatography (chloroform) and then silica gel column chromatography (hexane / ethyl acetate=3 / 2, v / v) to give compound 4 (98.5 mg, 34.3 μmol, 84%).

[0127] R f = 0.65 (toluene / EtOAc = 3 / 1); 1 H NMR (600 MHz, CDCl3) δ 7.86-6.72 (m, 83H), 5.40 (s, 1H), 5.37 (s, 1H), 5.24 (d, 1H), 5.12-5.08 (d, q, 2H), 4.99 (d, 1H), 4.95-4.90 (d, q, 2H), 4.86-4.82 (m, 3H), 4.71 (d, 1H), 4.61-4.40 (m, 13H), 4.38-4.06 (m, 15H), 3.92-3.65 (m, 16H), 3.54-3.25 (m, 12H), 3.15 (dd, 1H), 3.11-3.02 (m, 3H), 2.89 (dt, 2H), 1.66-1.52 (m, 2H), 1.16 (d, 20H); 13C NMR (151 MHz, CDCl3)δ 178.2, 178.1, 168.9, 168.3, 167.8, 138.6, 138.5, 138.4, 137.9, 137.8, 137.8, 137.7, 137.6, 137.4, 137.2, 134.2, 134.1, 134.0, 134.0, 133.9, 133.9, 133.9, 133.8, 133.7, 133.7, 131.8, 131.7, 131.7, 131.6, 131.6, 131.5, 131.5, 128.8, 128.7, 128.6, 128.4, 128.4, 128.3, 128.2, 128.1, 128.0, 128.0, 127.9, 127.8, 127.8, 127.7, 127.7, 127.6, 127.5, 127.4, 127.3, 127.1, 126.9, 126.0, 126.0, 123.6, 123.5, 123.4, 123.4, 123.2, 123.2, 123.0, 123.0, 102.7, 101.3, 98.2, 97.5, 97.5, 97.0, 97.0, 96.1, 82.3, 78.1, 78.0, 77.7, 77.5, 77.3, 77.1, 76.8, 76.6, 75.8, 75.6, 75.5, 74.9, 74.8, 74.7, 74.5, 74.4, 74.3, 74.1, 74.0, 73.9, 73.5, 73.5, 73.1, 73.0, 72.6, 71.8, 70.7, 69.1, 68.5, 68.3, 68.3, 68.1, 68.0, 68.0, 67.0, 65.8, 65.5, 64.4, 64.2, 63.6, 63.2, 63.2, 56.7, 55.9, 55.9, 55.7, 48.0, 38.9, 38.8, 28.8, 27.1; MALDI-TOF MS m / z: [M+Na] + calcd for C 157 H 159 N 13 O 40 ; 2889.07; found 2888.79.

[0128] Synthesis of (3-Azidopropyl [4-azide-3,6-di-O-benzyl-2,4-di-deoxy-2-phthalimide-β-D-glucopyranosyl-(1-2)-4,6-O-benzylidene-3-O-pivaloyl-α-D-mannopyranosyl-(1-3)]-[4-azide-3,6-di-O-benzyl-2,4-di-deoxy-2-phthalimide-β-D-glucopyranosyl-(1-2)-4,6-O-benzylidene-3-O-pivaloyl-α-D-mannopyranosyl-(1-6)]-2,4-di-O-acethyl-β-D-mannopyranosyl-(1-4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1-4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside (3) (Synthesis Method 1))

[0129]

[0130] Compound 4 (99.9 mg, 34.8 μmol) was dissolved in anhydrous dichloromethane (2 mL), followed by the addition of pyridine (422 μL, 5.22 mmol) and trifluoromethanesulfonic anhydride (286 μL, 1.74 mmol) and stirring at room temperature for 20 hours. After confirming the completion of the reaction by TLC, the reaction mixture was diluted with dichloromethane, washed sequentially with saturated aqueous sodium bicarbonate and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed under reduced pressure. The mixture was purified by silica gel column chromatography (toluene / ethyl acetate = 5 / 1, v / v). The fraction containing compound 21 was removed under reduced pressure. The resulting residue was dissolved in anhydrous toluene (5.5 mL), added with tetrabutylammonium acetate (525 mg, 1.74 mmol), and reacted with ultrasound for 18 hours. After confirming the completion of the reaction by TLC, the reaction mixture was diluted with ethyl acetate, washed sequentially with saturated aqueous sodium bicarbonate and saturated brine, dried over anhydrous magnesium sulfate, and the resulting organic layer was removed under reduced pressure. Purification was carried out by preparative HPLC (hexane / ethyl acetate, ethyl acetate=38% to 50%, v / v) to obtain compound 3 (30 mg, 10.2 μmol, 29%).

[0131] R f = 0.44 (toluene / EtOAc = 4 / 1); 1 H NMR (600 MHz, CDCl3) δ 7.87-6.71 (m, 87H), 5.31 (d, 2H), 5.20 (d, 1H), 5.12 (d, 1H), 5.00-4.81 (m, 10H), 4.70-4.33 (m, 17H), 4.25-4.03 (m, 13H), 3.97 (q, 1H), 3.89-3.82 (m, 2H), 3.74-3.59 (m, 12H), 3.50-3.26 (m, 11H), 3.17-3.13 (m, 2H), 3.10-2.98 (m, 3H), 2.78-2.69 (t, 2H), 2.15 (s, 3H), 2.10 (s, 3H), 1.65-1.52 (m, 2H), 1.14 (d, 20H); 13C NMR (151 MHz, CDCl3) δ177.8, 177.5, 170.4, 169.9, 168.6, 168.5, 168.3, 167.9, 167.7, 138.7, 138.5, 138.3, 137.9, 137.8, 137.6, 137.5, 137.4, 137.4, 134.1, 134.0, 133.9, 133.9, 133.7, 131.8, 131.7, 131.6, 131.5, 128.8, 128.7, 128.5, 128.4, 128.3, 128.3, 128.2, 128.1, 128.1, 127.9, 127.8, 127.8, 127.7, 127.6, 127.4, 127.0, 126.2, 126.0, 123.6, 123.4, 123.3, 123.3, 123.0, 101.6, 101.3, 98.4, 98.2, 98.0, 97.1, 95.9, 95.6, 78.2, 78.0, 77.9, 77.3, 77.1, 76.9, 76.7, 76.6, 75.8, 75.6, 75.5, 74.9, 74.8, 74.6, 74.5, 74.4, 74.4, 74.1, 73.6, 73.5, 73.4, 73.2, 72.9, 72.7, 71.6, 71.0, 70.0, 69.8, 69.1, 69.0, 68.5, 68.3, 68.2, 67.9, 67.3, 65.9, 64.4, 63.5, 63.4, 63.4, 56.6, 55.9, 55.7, 48.1, 38.9, 38.8, 29.8, 28.9, 27.3, 27.2, 27.2, 21.2, 21.0; MALDI-TOF MS m / z: [M+Na] + calcd for C 161 H 163 N 13 O 42 ; 2973.09; found 2974.60.

[0132] Synthesis of (3-Azidopropyl [4-azide-3,6-di-O-benzyl-2,4-di-deoxy-2-phthalimide-β-D-glucopyranosyl-(1-2)-4,6-O-benzylidene-3-O-pivaloyl-α-D-mannopyranosyl-(1-3)]-[4-azide-3,6-di-O-benzyl-2,4-di-deoxy-2-phthalimide-β-D-glucopyranosyl-(1-2)-4,6-O-benzylidene-3-O-pivaloyl-α-D-mannopyranosyl-(1-6)]-2,4-di-O-acethyl-β-D-mannopyranosyl-(1-4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1-4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside (3) (Synthesis Method 2))

[0133]

[0134] Compound 4 (84.1 mg, 29.3 μmol) was dissolved in anhydrous dichloromethane (1.8 mL), followed by the addition of pyridine (355 μL, 4.40 mmol) and trifluoromethanesulfonic anhydride (240 μL, 1.47 mmol) and stirring at room temperature for 20 hours. After confirming the completion of the reaction by TLC, the reaction mixture was diluted with dichloromethane, washed sequentially with saturated aqueous sodium bicarbonate and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed under reduced pressure. The product was purified by silica gel column chromatography (toluene / ethyl acetate = 5 / 1, v / v). The fraction containing compound 21 was removed under reduced pressure. The resulting residue was dissolved in anhydrous toluene (5 mL), followed by the addition of cesium acetate (281 mg, 1.47 mmol) and 18-crown-6-ether (388 mg, 1.47 mmol), and the mixture was sonicated for 18 hours. After confirming the completion of the reaction by TLC, the reaction mixture was diluted with ethyl acetate, washed with saturated aqueous sodium bicarbonate and saturated brine, dried over anhydrous magnesium sulfate, and the organic layer was evaporated under reduced pressure. The residue was purified by preparative HPLC (hexane / ethyl acetate, ethyl acetate = 38% to 50%, v / v) to give compound 3 (23.9 mg, 8.10 μmol, 28%).

[0135] R f = 0.44 (toluene / EtOAc = 4 / 1); 1 H NMR (600 MHz, CDCl3) δ 7.87-6.71 (m, 87H), 5.31 (d, 2H), 5.20 (d, 1H), 5.12 (d, 1H), 5.00-4.81 (m, 10H), 4.70-4.33 (m, 17H), 4.25-4.03 (m, 13H), 3.97 (q, 1H), 3.89-3.82 (m, 2H), 3.74-3.59 (m, 12H), 3.50-3.26 (m, 11H), 3.17-3.13 (m, 2H), 3.10-2.98 (m, 3H), 2.78-2.69 (t, 2H), 2.15 (s, 3H), 2.10 (s, 3H), 1.65-1.52 (m, 2H), 1.14 (d, 20H); 13C NMR (151 MHz, CDCl3) δ177.8, 177.5, 170.4, 169.9, 168.6, 168.5, 168.3, 167.9, 167.7, 138.7, 138.5, 138.3, 137.9, 137.8, 137.6, 137.5, 137.4, 137.4, 134.1, 134.0, 133.9, 133.9, 133.7, 131.8, 131.7, 131.6, 131.5, 128.8, 128.7, 128.5, 128.4, 128.3, 128.3, 128.2, 128.1, 128.1, 127.9, 127.8, 127.8, 127.7, 127.6, 127.4, 127.0, 126.2, 126.0, 123.6, 123.4, 123.3, 123.3, 123.0, 101.6, 101.3, 98.4, 98.2, 98.0, 97.1, 95.9, 95.6, 78.2, 78.0, 77.9, 77.3, 77.1, 76.9, 76.7, 76.6, 75.8, 75.6, 75.5, 74.9, 74.8, 74.6, 74.5, 74.4, 74.4, 74.1, 73.6, 73.5, 73.4, 73.2, 72.9, 72.7, 71.6, 71.0, 70.0, 69.8, 69.1, 69.0, 68.5, 68.3, 68.2, 67.9, 67.3, 65.9, 64.4, 63.5, 63.4, 63.4, 56.6, 55.9, 55.7, 48.1, 38.9, 38.8, 29.8, 28.9, 27.3, 27.2, 27.2, 21.2, 21.0; MALDI-TOF MS m / z: [M+Na] + calcd for C 161 H 163 N 13 O 42 ; 2973.09; found 2974.60.

[0136] (Aminopropyl [2-acetoamido-4-azide-3,6-di-O-benzyl-2,4-di-deoxy-β-D-glucopyranosyl-(1-2)-α-D-mannopyr anosyl-(1-3)]-[2-acetoamido-4-azide-3,6-di-O-benzyl-2,4-di-deoxy-β-D-glucopyranosyl-(1-2) -α-D-mannopyranosyl(1-6)]-β-D-mannopyranosyl-(1-4)-2-acetoamido-3,6-di-O-benzyl-2-deoxy- Synthesis of β-D-glucopyranosyl-(1-4)-2-acetoamido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranoside (22))

[0137]

[0138] Compound 3 (24.6 mg, 8.33 μmol) was added with n-butanol (1 mL) and ethylenediamine (200 μL) and stirred under an argon atmosphere at 90°C for two nights. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in pyridine (1 mL). Acetic anhydride (200 μL) was added in an ice bath, and the mixture was stirred overnight at 40°C under an argon atmosphere. 200 μL of methanol was added to terminate the reaction, and the reaction mixture was concentrated under reduced pressure. The residue was diluted with chloroform, and the organic layer was washed sequentially with 1 M hydrochloric acid, saturated brine, saturated aqueous sodium bicarbonate, and saturated brine. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The residue was dissolved in acetic acid (1 mL), and methanol (200 μL) and water (200 μL) were added. The mixture was stirred overnight at 80°C. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in tetrahydrofuran (1 mL) and methanol (1 mL). 1M sodium methoxide methanol solution (100 μL) was added in an ice bath. The reaction mixture was allowed to warm to room temperature and stirred overnight at 40°C under an argon atmosphere. The reaction mixture was neutralized with Amberlyst and evaporated under reduced pressure. The residue was purified using reverse-phase column chromatography (water / methanol = 3 / 2) to give compound 22 (13.2 mg, 6.08 μmol, 73%).

[0139] 1 1H NMR (600 MHz, CD3OD) δ 7.35 - 7.11 (m, 60H), 5.04 (s, 1H), 4.98 (d, 1H), 4.96 (d, 1H), 4.79 - 4.35 (m, 27H), 4.08 (d, 1H), 4.05 - 4.05 (dd, 1H), 3.99 - 3.32 (m, 54H), 3.24 (dq, 1H), 3.13 - 3.11 (m, 2H), 1.94 - 1.85 (m, 8H), 1.90 (s, 3H), 1.89 (s, 3H), 1.82 (d, 7H), 1.75 (m, 1H); MALDI-TOF MS m / z: [M+Na] + calcd for C 109 H 135 N 13 O 34 ; 2192.91; found 2193.60.

[0140] (Synthesis of (Aminopropyl [2 - acetoamido - 4 - amino - 2,4 - di - deoxy - β - D - glucopyranosyl-(1 - 2)-α - D - mannopyranosyl-(1 - 3)]-[2 - acetoamido - 4 - amino - 2,4 - di - deoxy - β - D - glucopyranosyl-(1 - 2)-α - D - mannopyranosyl(1 - 6)]-β - D - mannopyranosyl-(1 - 4)-2 - acetoamido - 2 - deoxy - β - D - glucopyranosyl-(1 - 4)-2 - acetoamido - 2 - deoxy - β - D - glucopyranoside (23))

[0141]

[0142] Compound 22 (11.6 mg, 5.34 μmol) was dissolved in tert-butanol (2 mL) and water (1 mL) was added. The reaction vessel was purged with argon gas, palladium hydroxide (12 mg) was added, and the atmosphere was purged with argon gas again. The reaction system was purged with hydrogen gas, filled with hydrogen, and then stirred at 40°C for two nights. The palladium hydroxide was removed by filtration through Celite, and the solution was concentrated under reduced pressure. The residue was purified by reverse-phase column chromatography (100% water) and lyophilized to give compound 23 (5.18 mg, 3.77 μmol, 71%).

[0143] 1 H NMR (600 MHz, D2O) δ 4.97 (d, 1H), 4.78 (s, 1H), 4.63 (s, 1H), 4.51 (q, 2H), 4.46 (d, 1H), 4.36-4.34 (m, 1H), 4.11 (d, 1H), 4.05 (t, 1H), 3.97 (m, 1H), 3.89-3.31 (m, 47H), 3.18-3.15 (t, 2H), 2.93 (t, 2H), 1.96-1.87 (m, 15H), 1.80 (t, 2H), 1.20 (d, 1H); MALDI-TOF MS m / z: [M+Na] + calcd for C 53 H 93 N7O 34 ; 1394.57; found 1394.46.

[0144] (3-N-2,4-di-nitrophenyl-3-aminopropyl [2-acetoamido-4-amino-2,4-di-deoxy-β-D-glucopyranosyl-(1-2)-α-D-mannopyranosyl-(1-3)]-[2-acetoamido-4-amino-2,4-di-deoxy-β-D-glucopyranosyl-(1-2 )-α-D-mannopyranosyl(1-6)]-β-D-mannopyranosyl-(1-4)-2-acetoamido-2-deoxy-β-D-glucopyranosyl-(1-4)-2-acetoamido-2-deoxy-β-D-glucopyranoside (2))

[0145]

[0146] Compound 3 (2.0 mg, 1.46 μmol) was dissolved in water (40 μL) and sodium bicarbonate (1.22 mg, 14.6 μmol) in water (20 μL) was added. 1,4-Dioxane (32 μL) was added, followed by 2,4-dinitrofluorobenzene (0.326 mg, 1.75 μmol) in 1,4-dioxane (8 μL). The mixture was stirred at room temperature for 2 hours. Sodium bicarbonate (1.22 mg, 14.6 μmol) in water (20 μL) was added, followed by 2,4-dinitrofluorobenzene (0.326 mg, 1.75 μmol) in 1,4-dioxane (8 μL). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with water (4 mL) and extracted three times with diethyl ether. After freeze-drying, the product was purified by reverse-phase column chromatography (water / acetonitrile=75 / 25) to obtain Compound 2 (1.43 mg, 0.930 μmol, 64%).

[0147] 11H NMR (600 MHz, D2O) δ 9.00 (d, 1H), 8.17 (dd, 1H), 6.98 (d, 1H), 4.97 (s, 1H), 4.77 (s, 1H), 4.63 (s, 1H), 4.46 (d, 2H), 4.41 (d, 1H), 4.38 (d, 1H), 4.11 (s, 1H), 4.05 (m, 1H), 3.96 (m, 1H), 3.90 (m, 1H), 3.83 - 3.30 (m, 45H), 1.94 - 1.91 (m, 7H), 1.87 (t, 3H), 1.78 (s, 3H); MALDI-TOF MS m / z: [M+Na] + calcd for C 59 H 95 N9O 38 ; 1560.57; found 1561.61.

[0148] (Synthesis of (3-N-2,4-di-nitrophenyl-3-aminopropyl [2-acetoamido-2,4-di-deoxy-4-N-methylanthranyl-β-D-glucopyranosyl-(1-2)-α-D-mannopyranosyl-(1-3)]-[2-acetoamido-2,4-di-deoxy-4-N-methylanthranyl-β-D-glucopyranosyl-(1-2)-α-D-mannopyranosyl-(1-6)]-β-D-mannopyranosyl-(1-4)-2-acetoamido-2-deoxy-β-D-glucopyranosyl-(1-4)-2-acetoamido-2-deoxy-β-D-glucopyranoside (1))

[0149]

[0150] Compound 2 (0.34 mg, 0.221 μmol) was dissolved in N,N-dimethylformamide (12 μL), and diisopropylethylamine (0.256 μL, 1.47 μmol) in N,N-dimethylformamide (10 μL) was added. In a separate vessel, diisopropylethylamine (0.513 μL, 2.95 μmol) in N,N-dimethylformamide (20 μL), N-methylanthranil (0.347 mg, 2.30 μmol) in N,N-dimethylformamide (4 μL), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (0.840 mg, 2.21 μmol) in N,N-dimethylformamide (4 μL) were added in that order. After confirming that the mixture turned yellow, the mixture was added to the vessel containing compound 2. The mixture was stirred at room temperature for 3 hours. The mixture was diluted with water (4 mL) and extracted three times with diethyl ether. After freeze-drying, the product was purified by preparative HPLC (water / methanol=55 / 45, v / v) to obtain Compound 1 (0.0359 mg, 0.0199 μmol, 9%).

[0151] MALDI-TOF MS m / z: [M+Na] + calcd for C 75 H 108 N 11 O 40 ; 1826.67; found 1826.83.

[0152] Example 2: Enzyme experiment using Endo-S (activity measurement using novel molecular probe 1 of the present invention) (Method 1: detection of hydrolysis reaction activity using HPLC) 10 μL (100 U) of Endo-S (New England Biolabs) was added to a solution containing 10 μL of probe solution (100 μM), 25 μL of water, and 5 μL of sodium phosphate buffer (500 mM, pH 7.5), and the resulting reaction mixture (50 μL in total) was incubated at 37° C. for 2 hours. The reaction was stopped by dispensing 5 μL of the reaction solution at 0, 7.5, 15, 30, 60, and 120 minutes, and adding 15 μL of acetonitrile to the mixture. The reaction mixture was diluted with 20 μL of water and analyzed by HPLC (TOSOH ODS-80Ts (5 μL, 4.6 x 750 mm)), water / acetonitrile = 97 / 3 to 55 / 45, 0.1% TFA solution, flow rate 1 mL / min, 15 min, fluorescence detection ex) 340 nm, em) 440 nm, UV 360 nm. The results are shown in Figure 1. With the passage of reaction time, the peak of novel probe 1 decreased, and peaks of the hydrolysis products, hexasaccharide derivative 2 and monosaccharide derivative 3, appeared. These results demonstrate that novel probe 1 of the present invention serves as a substrate for Endo-S and enables detection of enzyme activity.

[0153] (Method 2: Detection of Hydrolysis Reaction Activity Using a Microplate Reader) 10 μL of Endo-S (New England Biolabs) (100 U, 50 U, 25 U, 0 U) was added to a solution containing 10 μL of probe solution (25 μM), 25 μL of water, and 5 μL of sodium phosphate buffer (500 mM, pH 7.5), and the resulting reaction mixture (50 μL in total) was incubated at 37°C for 2 hours. The reaction was monitored at an excitation wavelength of 340 nm and a monitoring wavelength of 440 nm. The results are shown in Figure 2. Novel molecular probe 1 increased its fluorescence intensity over time in an enzyme concentration-dependent manner. Therefore, it was demonstrated that the use of novel molecular probe 1 of the present invention enables real-time, highly sensitive detection of Endo-S activity.

[0154] Example 3: Enzyme Experiment 2 Using Endo-S (Activity Measurement Using Novel Molecular Probes 1c and 1d of the Present Invention) (Method: Detection of Hydrolysis Activity Using a Microplate Reader) 50 U, 25 U, 10 U, or 5 U of Endo-S was added to a solution (total volume: 50 μL) containing probe 1c, water, 5 mM calcium chloride, and sodium acetate buffer (50 mM, pH 5.5), and the reaction mixture was incubated at 37°C for 2 hours. The reaction was monitored at an excitation wavelength of 340 nm and a monitoring wavelength of 440 nm. A solution containing probe 1d was similarly prepared and tested. The results are shown in Figure 4. As a result, the novel sugar compound / probes 1c and 1d of the present invention, like probe 1, showed an enzyme concentration-dependent increase in fluorescence intensity over time, demonstrating that Endo-S activity could be detected in real time with high sensitivity without SDS-PAGE.

[0155] Previously, Endo-S activity detection was performed using an antibody as a substrate and SDS-PAGE analysis, which was cumbersome. The novel probe of the present invention allows activity to be easily detected using a microplate reader at the manufacturing site of antibody pharmaceuticals. Furthermore, the use of a microplate reader allows simultaneous and easy detection of multiple types of samples.

[0156] Furthermore, while previous antibody-based activity detection methods have made rigorous evaluation of enzyme activity difficult due to variations in the sugar chain structures attached to antibodies, the chemical synthesis of the probe of the present invention provides a substrate with a uniform sugar chain structure. Therefore, because enzyme activity can be rigorously evaluated, it could become a standard for enzyme activity detection in antibody drug production processes. Furthermore, the novel probe 1 developed in this invention enables high-throughput enzyme activity detection, facilitating research into the discovery of new ENGases that can replace Endo-S. If a new Endo-S-like ENGase that acts on antibodies is discovered, it is expected to lead to industrial applications such as drug discovery research.

Claims

1. A sugar compound represented by the following formula (I): (In formula (I), Z １ and Z ２ one of which is a group represented by -R"-ZF having a fluorescent group ZF where fluorescence resonance energy transfer (FRET) occurs, and the other is a group represented by -R"-ZQ having a quenching group ZQ corresponding to the fluorescent group, １ are each independently either a group having a fluorescent group or a group having a quenching group, or —OR, and two Z １ at least one of the groups is a group having a fluorescent group or a group having a quenching group, each R independently represents a hydrogen atom, a protecting group for a hydroxyl group, or a glycosyl residue, each R' independently represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group, each R'' independently represents a single bond, a secondary or tertiary amino group (-NR'-), an amido group (-NR'CO-), an oxy group (-O-), a carbonyl group (-CO-), an oxycarbonyl group (-OCO-), or a divalent hydrocarbon group having 1 to 6 carbon atoms which may have a substituent and which may contain at least one group selected from the group consisting of a secondary or tertiary amino group (-NR'-), an oxy group (-O-), and a carbonyl group (-CO-), and R''' represents a hydrogen atom or an optionally protected fucose residue.

2. The above Z １ is a group having a fluorescent group or —OR, and Z ２ The sugar compound according to claim 1 , wherein is a group having a quenching group.

3. The sugar compound according to claim 1, wherein the combination of the fluorescent group and the quenching group is any one of the following (i) to (iv): (i) a combination of a fluorescent group represented by the following formula (d-1) and a quenching group represented by the following formula (a-1): (In formula (d-1), R' represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group.) (ii) A combination of a fluorescent group represented by the following formula (d-2) and a quenching group represented by the following formula (a-1): (In formula (d-2), R represents a hydrogen atom, a protecting group for a hydroxyl group, or a glycosyl residue.) (iii) A combination of a fluorescent group represented by the following formula (d-3) and a quenching group represented by the following formula (a-1): (In formula (d-3), R represents a hydrogen atom, a protecting group for a hydroxyl group, or a glycosyl residue, and R′ represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group.) (iv) A combination of a fluorescent group represented by the following formula (d-4) and a quenching group represented by the following formula (a-2): (In formulas (d-4) and (a-2), each R' independently represents a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms, or a protecting group for an amino group.) 4. A composition for detecting endo-β-N-acetylglucosaminidase (ENGase) activity, comprising the sugar compound according to any one of claims 1 to 3.

5. The composition according to claim 4, which is used to detect endo-β-N-acetylglucosaminidase (ENGase) S activity.

6. A method for producing a sugar compound, comprising a reaction step of producing a compound represented by the following formula (I) from a compound represented by the following formula (II): (In formula (I), Z １ and Z ２ one of which is a group represented by -R"-ZF having a fluorescent group ZF where fluorescence resonance energy transfer (FRET) occurs, and the other is a group represented by -R"-ZQ having a quenching group ZQ corresponding to the fluorescent group, １ are each independently either a group having a fluorescent group or a group having a quenching group, or —OR, and at least one of the two Z1's is a group having a fluorescent group or a group having a quenching group; R's each independently represent a hydrogen atom, a protecting group for a hydroxyl group, or a glycosyl residue; R's each independently represent a hydrogen atom, a hydrocarbon group of 1 to 6 carbon atoms, or a protecting group for an amino group; R''s each independently represent a single bond, a secondary or tertiary amino group (—NR'—), an amido group (—NR'CO—), an oxy group (—O—), a carbonyl group (—CO—), an oxycarbonyl group (—OCO—); or a divalent hydrocarbon group of 1 to 6 carbon atoms which may have a substituent and which may contain at least one group selected from the group consisting of a secondary or tertiary amino group (—NR'—), an oxy group (—O—), and a carbonyl group (—CO—); and R''' represents a hydrogen atom or an optionally protected fucose residue.

7. A method for producing a sugar compound, comprising a sugar transfer reaction step of reacting a compound represented by the following formula (VI) with a compound represented by the following formula (V) to produce a compound represented by the following formula (IV).

8. A method for measuring endo-β-N-acetylglucosaminidase (ENGase) activity, comprising an activity confirmation step of contacting the sugar compound according to any one of claims 1 to 3 with endo-β-N-acetylglucosaminidase (ENGase) and confirming the activity of decomposing said sugar compound.

9. A method for screening for inhibitors of endo-β-N-acetylglucosaminidase (ENGase), comprising: a contacting step of contacting a test compound with endo-β-N-acetylglucosaminidase (ENGase); and an activity confirmation step of contacting the endo-β-N-acetylglucosaminidase (ENGase) that has been contacted with the test compound with a sugar compound according to any one of claims 1 to 3, and confirming the decomposition activity of the sugar compound.

10. A method for screening for endo-β-N-acetylglucosaminidase (ENGase), comprising: a contacting step of contacting a test compound with a candidate for endo-β-N-acetylglucosaminidase (ENGase); and an activity confirmation step of contacting the candidate for endo-β-N-acetylglucosaminidase (ENGase) that has been contacted with the test compound with a sugar compound according to any one of claims 1 to 3, and confirming the decomposition activity of the sugar compound.

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