JPH0931095A - Production of complex carbohydrate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily produce a desired complex carbohydrate nonexistent in nature and useful for the production of physiologically active complex glycopeptide, etc., by transferring the sugar chain of a complex carbohydrate to a synthetic substrate having N-acetylglucosamine residue in the presence of an endoglycosidase. SOLUTION: The desired complex carbohydrate nonexistent in nature and usable for the easy preparation of a physiologically active complex glycopeptide taking advantage of the transfer reaction of carbohydrate is easily produced by transferring a sugar chain of a complex carbohydrate (sugar chain donor) to a synthetic substrate (sugar chain acceptor) having an N-acetylglucosamine (GlcNAc) residue and expressed by formula (R<1> is H, an amino-protecting group, an amino acid, a peptide or a peptide obtained by protecting the α-amino group of an N-terminal amino acid; R<2> is OH, a carboxyl-protecting group, an amino acid, a peptide or a peptide obtained by protecting the carboxyl group of a C-terminated amino acid; (n) is 1 or 2) in the presence of an endoglycosidase (e.g. endo-β-N-acetylglucosaminidase).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a physiologically active complex glycopeptide by combining a peptide synthesis reaction and an enzymatic sugar chain transfer reaction. The invention has application in the pharmaceutical field.

[0002]

2. Description of the Related Art Carbohydrates and complex carbohydrates are present in living cells, body fluids and the like, and are deeply involved in cell substrate recognition and cell-cell recognition. In addition, sugars are related to the rate of metabolism such as absorption and decomposition of substances in the body. Proteins having a sugar chain are known, and for example, there are erythropoietin and tissue plasminogen activator, and these glycoproteins genetically engineered using animal cells are used as medicines. Some peptide hormones having a sugar chain are also known, such as human chorionic gonadotropin (hCG). In these glycoproteins or glycopeptides, the sugar chain is bound to Asn of the peptide chain via GlcNAc as an N-linked sugar chain.

[0003] By attaching a sugar chain to a protein or a physiologically active peptide, or by replacing an existing sugar chain with another sugar chain, it is expected to be useful for enhancing physiological functions and modifying physiological activities. As a method for enzymatically modifying the sugar chain, 1) a method using transferase or exoglycosidase and 2) a method using endoglycosidase can be considered.

[0004] As the method 1), for example, D. H. DH Joziasse et al. [European Journal of Biochemistry (Eur. J. Bioche)
m.), 191, 75-83 (1990)], which is a sequential reaction from the non-reducing end of the sugar chain. Recently, M.S. M. Schuster et al. [Journal of American Chemical Society, Vol. 116, No. 1135
~ 1136 (1994)] report a solid phase synthesis method of sugar chains in which several glycosyltransferases are combined.

On the other hand, the glycosyltransfer reaction using endoglycosidase of 2) is described in B. Trim Bull (RB Tr
imble) et al. [Journal of Biological Chemistry (J. Biol. Chem.), Vol. 261, 1200]
0 to 12005 (1986)], Flavobacterium meningoseptic.
um) -derived endo-β-N-acetylglucosaminidase (endo-F); M. RM Bardales et al. [Journal of Biological Chemistry (J. Biol. Chem.), Vol.
198993-19897 (1989)], which relates to endo-α-N-acetylgalactosaminidase derived from Diprococcus pneumoniae. , Serine, threonine, etc. are reported to be receptors. Thereafter, Takekawa et al. [JP-A-5-64594 (1993)]
And K. K. Takegawa et al. [Journal
Of Biological Chemistry (J. Biol. Che
m.), vol. 270, pp. 3094-3099 (199
5)] is Arthrobact
er protophormiae) -derived endo-β-N-acetylglucosaminidase (endo-A) for the transglycosylation reaction to sugars. K. Yamamoto et al. [Biochem. Biophys. Res. Commun.], No. 2
03, pp. 244-252 (1994)] reported a transglycosylation reaction to a carbohydrate by endo-M derived from Mucor hiemalis.

[0006]

The complex carbohydrate is composed of a sugar chain portion and a protein, peptide or ceramide portion to which the sugar chain is added. A glycoconjugate with natural stability and biological activity in vivo due to so-called sugar chain modification (remodeling), in which a sugar chain is newly added to the sugar or replaced with another sugar chain It is useful when applied to pharmaceuticals if it has an enhanced biological function or is added to biological functions that are not found in nature. In addition, the physiological functions of sugar chains in glycoconjugates have not been fully elucidated because there has been no effective means of sugar chain modification, but they provide important means for analyzing their roles. .

As a method for newly adding or modifying a sugar chain to a sugar or a glycoconjugate, an enzymatic method of sequentially adding sugar residues one by one using exoglycosidase or glycosyltransferase can be considered. Also,
The method using endoglycosidase such as endo-A and endo-M provides a more efficient method of transferring a complex sugar chain as a block to a sugar or a complex sugar.

The sugar chains of natural glycoproteins or glycopeptides are usually Asn-X-Ser as N-linked sugar chains.
(Thr) [X represents an arbitrary amino acid, Ser (Thr) represents serine or threonine], and is bound via GlcNAc linked to the amide group of Asn of the peptide chain of the amino acid sequence. That is, Asn of this amino acid sequence,
A sugar chain having a GlcNAc residue is added to the end and further modified to biosynthesize an N-linked complex sugar chain. Therefore,
N-linked sugar chains other than the sugar chain linked to Asn of this amino acid sequence have not been found in nature.

To add or modify a sugar chain by the above-mentioned enzyme, G or G is added to a protein or peptide which is a sugar chain receptor.
It is necessary to have an lcNAc residue, and the addition or modification of a sugar chain is conventionally performed by cutting off the sugar chain of a natural glycoprotein or glycopeptide with an endoglycosidase or an exoglycosidase, leaving a GlcNAc residue, and then removing another sugar. It was limited to replacing sugar chains prepared from proteins.

Some proteins and peptides include Asn-X
Even if there is a -Ser (Thr) sequence, there are many that do not have a sugar chain, for example, calcitonin and the like.
In addition, even if Asn is present, without this sequence, sugar chains cannot be added at the biosynthesis stage.

If a glycopeptide having GlcNAc linked to an Asn residue (GlcNAc-Asn-peptide) can be chemically synthesized, G linked to Asn by the above-mentioned enzymatic method.
A new complex glycopeptide in which a sugar chain is added to the lcNAc residue can be synthesized. In this case, Asn-X- on the peptide side.
The sequence of Ser (Thr) is not always necessary and only Asn is required. Further, if a glycopeptide in which a GlcNAc residue is added to glutamine (Gln), which is an amino acid related to Asn, is synthesized, the same complex glycopeptide can be expected to be synthesized.

[0012] Peptide synthesis is industrially put to practical use by solid phase synthesis with up to several tens of residues.

The present invention chemically synthesizes a peptide containing at least one sugar-bonded amino acid, that is, a synthetic substrate,
It was developed based on the idea that a new complex glycopeptide can be synthesized by enzymatically transferring a sugar or sugar chain to the sugar residue of the foothold.

[0014]

Means for Solving the Problems The present invention can be summarized as follows: 1) synthesis of a synthetic substrate having a GlcNAc residue which serves as a sugar chain receptor; and 2) synthesis of a synthetic substrate having a GlcNAc residue (sugar chain). 2 of enzymatic transfer of sugar chain to receptor)
It consists of one configuration.

The synthetic substrate having a GlcNAc residue referred to in the present invention has the following formula (Formula 1).

[0016]

(Wherein R ¹ is H, an amino protecting group, an amino acid,
A peptide or a peptide in which the α-amino group of the N-terminal amino acid is protected is shown. R ² is OH, a carboxyl protecting group,
An amino acid, a peptide, or a peptide in which the carboxyl group of the C-terminal amino acid is protected is shown. n is 1 or 2. ).

The term "synthetic substrate" as used in the present invention refers to a substance that is artificially synthesized, and has a GlcNAc residue prepared by enzymatically decomposing naturally occurring glycoproteins or glycopeptides such as glycosidases and proteases. The peptide of (Chemical formula 1) is excluded from the substance of (Chemical formula 1).

The synthetic substrate having the GlcNAc residue shown in Chemical formula 1 may be synthesized by any method. T. Inazu et al. [Peptide Chemistry 1993], No. 101-
Page 104 (1994)].

For example, a partial peptide hCG (β12-16) [H-Ile-As of the β subunit of human chorionic gonadotropin (hCG) having a GlcNAc residue.
n-Ala-Thr-Leu-OH] (Ile is isoleucine, Ala is alanine, Thr is threonine, Le
u represents leucine. ) Is taken as an example, N
GlcN was added to the amide group of asparagine whose terminal was protected (9-fluorenylmethyloxycarbonyl, Fmoc).
When an Fmoc-Asn-GlcNAc derivative bound with Ac was synthesized and peptide synthesis was performed using Asn instead of Asn in the solid phase synthesis of the peptide, Glc was added to Asn.
HCG (β12-16) peptide [Fmoc-Ile-Asn (GlcNAc) -Ala bound to NAc residue]
-Thr-Leu-OH] is synthesized.

In the method of the present invention, Asn-X-Ser is used.
Even if there is no amino acid sequence of (Thr), if there is Asn in the amino acid sequence, peptide synthesis is performed using Asn (GlcNAc-Asn) in which GlcNAc is bonded to the amide group instead of Asn, thereby obtaining an arbitrary GlcNAc.
-Asn-peptide can be synthesized. By transferring a sugar chain to GlcNAc bound to Asn, it becomes possible to synthesize a non-natural complex glycopeptide.

A peptide in which GlcNAc is bound to Gln (GlcNAc-Gln-peptide) is synthesized by using glutamine in which GlcNAc is bound to an amide group (GlcNAc-Gln) in place of Gln.
This corresponds to the compound of Chemical formula 1 in which n is 2.

Examples of the protecting group for the α-amino group of the terminal amino acid at R ¹ in (Chemical formula 1) include 9-fluorenylmethyloxycarbonyl (Fmoc) group, tert-butyloxycarbonyl (Boc) group and 3 A -nitro-2-pyridinesulfenyl (Npys) group, a benzyloxycarbonyl (Z) group, a dansyl (DNS) group or the like is used.

The protecting group of the N-terminal amino acid may be removed by a conventional method after the transglycosylation reaction, or may be subjected to the transglycosylation reaction after removing the protecting group in advance.

Examples of the protective group for the carboxyl group represented by R ² in (Chemical Formula 1) include a tert-butyl (Bu ^t ) group and benzyl (B
zl) group or methyl (Me) group, but in many cases, the protective group is removed to increase solubility in water, and the free form is used.

The second constitution of the present invention is the addition of a sugar chain from a sugar chain donor to a synthetic substrate which is a sugar chain acceptor by a transfer reaction.

In the presence of endoglycosidase, the following formula (Formula 1): X-GlcNAc-Y + Z → X-G
lcNAc-Z + Y (Formula 1) wherein X is a complex sugar chain, Y is a carbohydrate or complex carbohydrate, and Z is a synthetic substrate represented by Chemical Formula 1. Features.

The endoglycosidase used in the present invention is endo-β-N-acetylglucosaminidase (EC 3.2.1.96), for example, endo-A.
And End-M are used. The enzyme is a chitobiose moiety of an asparagine (Asn) -linked sugar chain of the following formula (formula 2): R-GlcNAc-GlcNAc-Asn- (peptide or protein) (formula 2) (wherein R represents a complex sugar chain). The (GlcNAc-GlcNAc) is hydrolyzed. If an appropriate sugar chain receptor is present at this time, the sugar chain (R-GlcNAc) portion is transferred to the receptor. The reaction of (Equation 1) utilizes this.

It is usually Asn-X-Ser that serves as a sugar chain acceptor in the transglycosylation reaction by endoglycosidase.
Gl linked to Asn of peptide containing (Thr) sequence
cNAc residue.

Even if there is no Asn-X-Ser (Thr) sequence, a peptide having a GlcNAc residue bound to Asn (GlcNAc-Asn-peptide) is a sugar chain receptor for a transglycosylation reaction by endoglycosidase. It turns out that can be. Further, the reaction proceeded even if the N-terminal α-amino group and the C-terminal carboxyl group of the peptide were not in free form and either or both ends were protected.

Even more surprisingly, the Gl of a peptide containing a GlcNAc residue linked to a non-naturally occurring glutamine (Gln) (GlcNAc-Gln-peptide).
It was found that the sugar chain transfer reaction also occurs in the cNAc residue as in the case of Asn.

The synthetic substrate having the GlcNAc residue shown in (Chemical Formula 1) is prepared on the basis of such findings,
The present invention has been completed in which a sugar chain transfer reaction by an enzyme to this synthetic substrate is carried out.

Regarding the substrate specificity of the sugar chain donor of the enzyme, endo-A acts only on high-mannose type sugar chains, but endo-M is not only high-mannose type sugar chains but also complex type sugar chains and mixed type sugar chains. Also works.

The original function of these enzymes is hydrolysis, and instead of Z in (Formula 1), water enters and the hydrolysis reaction proceeds as a side reaction together with the transfer reaction. Also, (Equation 1)
The rearrangement reaction product (X-GlcNAc-Z) produced in the above step becomes a substrate for the hydrolysis reaction and undergoes re-decomposition.

In order to efficiently carry out the transglycosylation reaction, it is necessary to suppress the hydrolysis reaction and preferentially carry out the reaction of the formula (1). By decreasing the amount of the enzyme to be added and increasing the charged concentrations of the sugar chain donor (X-GlcNAc-Y) and the sugar chain acceptor (Z), which are the substrates, while keeping the molar ratio close to 1, the reaction The influence of water in the (active center of the enzyme) can be eliminated to allow the transfer reaction to proceed efficiently.

An important point in the reaction system is that the reaction is carried out under enzyme-controlled conditions. That is, the rate of the sugar chain transfer reaction from the sugar chain donor to the acceptor depends on the amount of the enzyme, and the amount of the enzyme added is limited so that the minimum amount of enzyme required to maximize the rate is obtained. react. For example, in the case of Endo-M, 500 units (U) / mole (donor) to the sugar chain donor
Below, an enzyme amount of about 80 to 400 U / mol (donor) is preferably added. Here, 1 unit (U) of the enzyme hydrolyzes the dansylated (DNS) derivative of the asialo complex type sugar chain derived from human transferrin to give 3
1 micromolar (μmol) N-acetylglucosaminyl-asparagine DNS derivative (G
It is the amount of enzyme required to generate lcNAc-Asn-DNS).

It is also important to significantly increase the concentration of the sugar chain donor and acceptor charged. By charging both substrates at a high concentration while limiting the amount of the enzyme, the sugar chain transfer reaction is promoted, side reactions are suppressed, and the reaction yield is dramatically improved. The concentration of the sugar chain donor is 10 mM or more, preferably 15 to 7
5 mM is good. Further, the sugar chain receptor is 2.5 mM or more, preferably about 7.5 to 35 mM. It is desirable that the concentration of the sugar chain receptor is high, but when the solubility of the synthetic substrate is low, it can be used even at a concentration of about 2.5 mM.

As the sugar chain donor used in the present invention, any of high mannose type, complex type and mixed type sugar chains can be used. The high-mannose type sugar chain is prepared from, for example, ovalbumin, and the complex type sugar chain containing sialic acid is prepared from, for example, human transferrin, bovine fetuin, etc., and asialo complex type sugar chain is prepared by removing sialic acid by sialidase treatment or the like. To be done. Enzymatically or chemically modified sugar chains or chemically synthesized sugar chains can also be used.

The reaction of the present invention is carried out by mixing the substrate sugar chain donor, the sugar chain acceptor and the enzyme endoglycosidase in a buffer solution. As described above, the concentration of the sugar chain donor is 10 mM or more, preferably 15 to 75 m.
M and sugar chain receptor are added so as to have a concentration of 2.5 mM or more, preferably 7.5 to 35 mM. Enzyme amount is 500
U / mol (donor) or less, preferably 80 to 400 U /
It is limited to about mol (donor), and for example, in the case of endo-M, it is used in an amount of about 2 to 10 mU / ml. As the buffer solution, an appropriate buffer solution having a pH of about 5 to 8 and a concentration of 25 to 200 mM, preferably 50 to 100 mM is used. In case of Endo-M, the pH is usually 5.5 to 6.5 and the concentration is 50 to
The reaction is performed in 100 mM acetic acid or phosphate buffer. An example of a basic reaction solution composition is a sugar chain donor 25 m
M, sugar chain receptor 10 mM, endo-M 4 mU / ml and 60 mM phosphate buffer (pH 6.25).

The reaction temperature is usually room temperature to about 50 ° C., preferably 30 to 40 ° C., and the reaction time is 1 to 24 hours. For example, in the case of the endo-M enzyme, usually 37 ° C
For about 3 to 18 hours.

The produced glycoconjugate can be easily separated and purified from the reaction-terminated liquid according to known means. For example, gel filtration column chromatography, ion exchange resin column chromatography, lectin column chromatography, high performance liquid chromatography (HPLC)
For example, the complex saccharide of the reaction product may be separated from the reaction-terminated liquid, and then concentrated, desalted, freeze-dried and the like.

[0041]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

[0042]

Example 1 GlcNAc-Asn-Fmoc of high-mannose type sugar chain
Transglycosylation reaction to: High-mannose type sugar chain (Man) _6- (GlcNA) obtained by treating ovalbumin as a sugar chain donor with pronase, Sephadex G-25 gel filtration and separating and purifying by Dowex 50 ion exchange chromatography.
c) _1- mol (final concentration 25 mM) of _2- Asn (molecular weight 1651), GlcNAc-Asn as a sugar chain receptor
-Fmoc (molecular weight 558) 400 nmol (same 10
mM) and Endo-M (160 μU (4 mU / ml)) were added and reacted in 40 μl of 60 mM phosphate buffer (pH 6.25) at 37 ° C. for 18 hours. After the reaction was stopped by heat treatment, the reaction solution was diluted to 1 ml with distilled water and the reaction product was diluted with H
Analyzed by PLC. A transfer reaction product to GlcNAc-Asn-Fmoc was obtained with a reaction yield of 4.0% (to sugar chain receptor, molar ratio). The reaction product was collected by HPLC, and as a result of mass spectrometry, a signal was observed at m / z [MH] 1732, and (Man) _6- (GlcNAc) ₂ -A.
Transfer reaction product from sn to GlcNAc-Asn-Fmoc, namely (Man) _6- (GlcNAc) ₂ -As
It was confirmed to be n-Fmoc (molecular weight 1734).

[0043]

Example 2 GlcNAc- of human transferrin-derived sialo-glycan
Transfer Reaction to Asn-Fmoc: As a sugar chain donor,
Sialoglycopeptides having only Asn residues (TF-SG) obtained by repeating human transferrin (Seikagaku Corporation) with pronase and Sephadex G-25 gel filtration.
P, molecular weight 2338) 1 μmol (final concentration 25 mM)
And GlcNAc-Asn-Fmoc 400 nmol
(The same 10 mM) as a 0.1 M phosphate buffer (pH 6.2)
5) Dissolved in 24 μl, added 16 μl of enzyme solution containing 160 μU of Endo-M, and reacted at 37 ° C. for 6 hours. After stopping the reaction, the reaction solution was diluted to 1 ml with distilled water, and the reaction product was analyzed by HPLC. The transfer reaction product was obtained in a yield of 4.5% in 6 hours of reaction. The reaction product was isolated by HPLC fractionation, and as a result of mass spectrometry, m / z [MH] 2
A signal was observed at 561 and it was confirmed that the compound was a compound (molecular weight 2560) in which a disialo double-chain complex type sugar chain was transferred to GlcNAc-Asn-Fmoc.

[0044]

Example 3 GlcNAc of human transferrin-derived asialo sugar chain
-Asn-Fmoc transfer reaction: As a sugar chain donor, a human transferrin-derived sialoglycopeptide (T
F-SGP) was treated with sialidase to remove sialic acid, and asialoglycopeptide (TF-ASGP, molecular weight 175)
6) 1 μmol (final concentration 25 mM) and GlcNAc-
400 nmol (10 mM) of Asn-Fmoc was dissolved in 24 μl of 0.1 M phosphate buffer (pH 6.25), 16 μl of enzyme solution containing 160 μU of endo-M was added, and the mixture was reacted at 37 ° C. for 6 hours. After stopping the reaction, the reaction solution was diluted to 1 ml with distilled water, and the reaction product was analyzed by HPLC. The transfer reaction product was obtained in a yield of 8.0% in 6 hours of the reaction. The reaction product was isolated by HPLC fractionation, and as a result of mass spectrometry, a signal was observed at m / z [MH] 1983, and the asialo double-chain complex type sugar chain was GlcNAc-.
Compound transferred to Asn-Fmoc (molecular weight 1978)
Was confirmed.

[0045]

[Example 4] GlcNAc of asialo sugar chain derived from human transferrin
-Glycosyl transfer reaction to Asn-Npys: 3-nitro-2-pyridinesulfenyl (Npys) derivative of GlcNAc-Asn as a sugar chain receptor (GlcNAc-As)
n-Npys) (molecular weight 489) was synthesized as a compound in which the α amino protecting group of Asn was changed to the Npys group. Asialoglycopeptide (TF-ASGP) prepared in the same manner as in Example 3 was used as the sugar chain donor. 1 μmol of asialoglycopeptide (TF-ASGP) (final concentration 25 mM) and 500 nmol of GlcNAc-Asn-Npys (12.5 mM) in 0.1M phosphate buffer (pH 6.2).
5) Dissolved in 24 μl, added 16 μl of enzyme solution containing 160 μU of Endo-M, and reacted at 37 ° C. for 3 hours.
After stopping the reaction, the reaction solution was diluted to 1 ml with distilled water, and the reaction product was analyzed by HPLC. As a result, a transfer reaction product was obtained in a yield of 7.0%. When the reaction product was collected by HPLC and subjected to mass spectrometry, an ion peak (m / z) corresponding to a molecular weight of 1910 was observed, and the transferrin-derived asialo double-chain complex sugar chain was GlcNAc-Asn-.
It was confirmed that the compound was a compound transferred to Npys.

[0046]

Example 5 Transfer Reaction of Human Transferrin-Derived Complex Type Sugar Chain to Synthetic Peptide: As a sugar chain receptor, a partial peptide hCG (β1) of β chain of human chorionic gonadotropin (hCG)
2-16) peptide [Fmoc-Ile-Asn (GlcNAc) -Al in which GlcNAc is attached to the Asn residue]
a-Thr-Leu-OH], [GlcNAc-hCG
(Β12-16) -Fmoc] (molecular weight 956) (Il
e is isoleucine, Ala is alanine, Thr is threonine, and Leu is leucine). It was synthesized according to the method of Inazu et al. And used. Sialoglycopeptide (TF-SGP), a sugar chain donor, was added at 1 μmol (final concentration 25 m
M), GlcNAc-hCG (β12-1 as a receptor
6) -Fmoc was added to 400 nmol (the same 10 mM) to 0.
Dissolve in 24 μl of 1M phosphate buffer (pH 6.25),
16 μl of an enzyme solution containing 160 μU of Endo-M was added and reacted at 37 ° C. for 3 hours. After the reaction was stopped, the reaction solution was diluted to 1 ml with distilled water, and the reaction product was analyzed by HPLC. As a result, the disialo sugar chain showed GlcNAc-hCG (β
12-16) -Fmoc transferred compound (molecular weight 29
59) was observed. When asialoglycopeptide (TF-ASGP) is used as a sugar chain donor, the asialoglycan is GlcNAc-hCG (β12-16) -Fmoc.
To give a compound (molecular weight 2376).

[0047]

Example 6 Transfer Reaction of High-Mannose-Type Sugar Chain to Synthetic Peptide: As a sugar-chain donor, a high-mannose-type sugar chain (Man) _6- (GlcNAc) ₂ -Asn (molecular weight 1651) derived from ovalbumin was used, Partial peptide hCG (β12-16) [GlcNAc-h of human chorionic gonadotropin (hCG) having a GlcNAc residue as a sugar chain receptor
CG (β12-16) -Fmoc] was reacted for 3 hours under the same reaction conditions as in Example 5, the high mannose type sugar chain was GlcNAc-hCG (β12-16)-.
Formation of a compound (molecular weight 2132) transferred to Fmoc was observed.

[0048]

Example 7 GlcNAc-Gln-Fmoc of high mannose type sugar chain
Glycotransfer Reaction to GlcNAc-Gln of Sugar Receptor
-Fmoc (molecular weight 572) is Gln instead of Asn
GlcNAc-A as a compound containing (glutamine)
It was synthesized according to the synthesis method of sn-Fmoc. The sodium salt was used for the enzymatic reaction. High-mannose type sugar chain (Man) _6- (GlcNAc) ₂ -Asn as a sugar chain donor
(Molecular weight 1651) 1 μmol (final concentration 25 mM),
200 nmol (5 mM) of GlcNAc-Gln-Fmoc (molecular weight 572) as an oligosaccharide receptor, endo-
160 μU of M (4 mU / ml) was added, and the mixture was reacted in 40 μl of 60 mM phosphate buffer (pH 6.25) at 37 ° C. for 3 hours. After stopping the reaction, the reaction solution was diluted to 1 ml with distilled water and the reaction product was analyzed by HPLC. The transfer reaction product was obtained in a yield of 7.5%. The transfer reaction product is HPL
As a result of C fractionation and mass spectrometry, an ion peak (m / z) corresponding to a molecular weight of 1748 was observed, and (Man) ₆
-(GlcNAc) ₂ -Asn to GlcNAc-Gl
Transfer reaction product to n-Fmoc, that is, (Man) ₆ −
It was confirmed to be (GlcNAc) ₂ -Gln-Fmoc.

[0049]

Example 8 GlcNAc- of human transferrin-derived complex sugar chain
Transfer Reaction to Gln-Fmoc: As a sugar chain donor,
Sialo and asialoglycopeptides (TF-SGP and TF-ASGP) prepared from human transferrin were added at 1 μmol (final concentration 25 mM) and GlcNAc-Gln.
-200 nmol (5 mM) of Fmoc was dissolved in 24 µl of 0.1 M phosphate buffer (pH 6.25), 16 µl of enzyme solution containing 160 µU of endo-M was added, and 37
The reaction was performed at a temperature of 3 ° C. for 3 hours. After stopping the reaction, the reaction solution is distilled water to 1 m.
Diluted to 1 and analyzed the reaction product by HPLC. When the transfer reaction product is TF-SGP, 5.0%, TF-AS
In the case of GP, the yield was 13.7%. The transfer reaction product was isolated by HPLC fractionation, and as a result of mass spectrometry, TF
-SGP transfer reaction product has an ion peak corresponding to a molecular weight of 2574, and TF-ASGP transfer reaction product has an ion peak corresponding to a molecular weight of 1992 (m / z).
[MH] 1992) was observed, and the compound in which the disialo double-chain complex sugar chain was transferred to GlcNAc-Gln-Fmoc and the asialo double-chain complex sugar chain were GlcN, respectively.
It was confirmed that the compound was a compound transferred to Ac-Gln-Fmoc.

[0050]

Example 9 Human Transferrin-Derived Asialo Sugar Chain GlcNAc
-Gln-DNS transglycosylation reaction: A dansylated (DNS) derivative of GlcNAc-Gln (GlcNAc-Gln-DNS) (molecular weight 585) as a sugar chain receptor is a compound in which the α amino group of Gln is protected by a DNS group. Was synthesized as. Asialoglycopeptide (TF) as a sugar chain donor
-ASGP) was reacted for 3 hours under the same reaction conditions as in Example 4. When the reaction product was collected by HPLC and subjected to mass spectrometry, an ion peak (m / z) corresponding to a molecular weight of 2004 was observed, and the asialo double-chain complex type sugar chain was Gl.
It was confirmed that the compound was transferred to cNAc-Gln-DNS.

[0051]

INDUSTRIAL APPLICABILITY According to the present invention, a novel complex glycopeptide can be easily synthesized by enzymatically adding a sugar chain to a synthetic substrate having a GlcNAc residue. The peptide of the sugar chain receptor is G if there is asparagine (Asn) or glutamine (Gln) in the amino acid sequence of the peptide chain.
An N-linked sugar chain can be added via the lcNAc residue, and a completely new complex glycopeptide that is not found in nature can be synthesized. The sugar chain to be added may be either a high-mannose type sugar chain or a complex type sugar chain containing sialic acid, and a desired complex glycopeptide can be synthesized. Naturally, not only non-natural but also natural ones can be synthesized. The present invention provides a research technique for elucidating the physiological role played by the sugar chains of glycoconjugates, together with application to medicine.

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Claims (9)

[Claims] 1. The method according to claim 1, wherein a sugar chain of a glycoconjugate (sugar chain donor) is converted to N-acetylglucosamine (G) in the presence of endoglycosidase.
A method for producing a complex glycopeptide by transferring to a synthetic substrate (sugar chain receptor) having an lcNAc) residue. 2. The method according to claim 1, wherein the synthetic substrate having a GlcNAc residue is a compound represented by the following formula (Formula 1). Embedded image (In the formula, R ¹ represents H, an amino-protecting group, an amino acid, a peptide or a peptide in which an α-amino group of an N-terminal amino acid is protected. R ² represents an OH, a carboxyl-protecting group, an amino acid, a peptide or a carboxyl group of a C-terminal amino acid. Represents a protected peptide, where n is 1 or 2.). However, substances prepared by enzymatic decomposition of naturally occurring glycoconjugates are excluded. 3. The method according to claim 1, wherein the amino acid to which GlcNAc binds is asparagine (Asn). 4. The method according to claim 1, wherein the amino acid to which GlcNAc binds is glutamine (Gln). 5. The protecting group for the α-amino group of the N-terminal amino acid is 9
-Fluorenylmethyloxycarbonyl (Fmoc)
Group, tert-butyloxycarbonyl (Boc) group, 3-nitro-2-pyridinesulfenyl (Npys) group, benzyloxycarbonyl (Z) group or dansyl (DN
The method according to claim 2, which is a S) group. 6. C protecting group of the carboxyl group of the terminal amino acid tert-butyl (Bu ^t), The method of claim 2 is benzyl (Bzl) or methyl (Me) group. 7. The method according to claim 7, wherein the endoglycosidase is endo-β-N-.
Acetylglucosaminidase (EC 3.2.1.96)
The method of claim 1, wherein 8. The method according to claim 1, wherein the reaction is carried out under enzyme rate limiting conditions. 9. A sugar chain of a complex sugar (sugar chain donor) is converted to N-acetylglucosamine (G) in the presence of endoglycosidase.
A complex glycopeptide produced by transferring to a synthetic substrate (sugar chain receptor) having an lcNAc) residue.