JPH08280397A - Production of gal-glcnac-man - Google Patents

Abstract

PURPOSE: To produce the subject glucide useful as a synthetic intermediate, etc., for a complex glucide saccharide chain such as a glycoprotein or a glycolipid by carrying out the saccharide transfer reaction of a solution containing GlcNAc-Man and a galactose donor with a glycosidase. CONSTITUTION: An N-acetylhexosaminidase is reacted with a solution containing mannose (Man) and N-acetylglucosamine (GlcNAc) to prepare GlcNAc-Man. The resultant GlcNAc-Mann and a galactose donor (e.g. lactose) are then dissolved in a solution comprising dimethyl sulfoxide and 0.2M potassium phosphate buffer solution (pH 6.0) and a glycosidase (e.g. β-galactosidase) is added thereto. Reaction is carried out at 37 deg.C for 5hr under shaking and the reactional solution is then treated at 100 deg.C for 5min to inactivate the enzyme. The resultant solution is subsequently treated with an active carbon column, fractionated and purified at a linear concentration gradient of water-ethanol to afford the objective Gal- GlcNAc-Man useful as a synthetic intermediate, etc., for synthesizing a complex glucide saccharide chain such as a glycoprotein or a glycolipid.

Description

Detailed Description of the Invention

[0001]

The present invention relates to Ga, which is an important synthetic intermediate in the synthesis of glycoconjugate sugar chains such as glycoproteins and glycolipids.
The present invention relates to a method for producing l-GlcNAc-Man using glycosidase.

[0002]

2. Description of the Related Art Gal-GlcNAc-Man is a common structure existing at the non-reducing side chain end of an asparagine-linked sugar chain among glycoprotein sugar chains, and is an important synthetic intermediate in the total synthesis of sugar chains. Is.

Conventionally, as a method for producing Gal-GlcNAc-Man, an organic chemical production method and a method using an enzyme are known.

When manufactured by an organic chemical method, 1
It is produced by the condensation reaction of N-phthaloyllactosamine in which all the other hydroxyl groups have been activated and mannose in which only the hydroxyl group at the 2-position has been released (T. O.
gawa et al., Carbohydrate Res., 123 (1983) C8).

In the case of using an enzyme, GlcNAc
-Man as a sugar acceptor, UDP-Gal as a sugar donor, and glycosyltransferase (galactosyltransferase)
A possible method is to transfer Gal to cNAc-Man.

[0006]

However, in order to produce Gal-GlcNAc-Man by an organic chemical method, a multistep reaction of 14 steps is required, and an expensive reagent such as a silver catalyst is used in the condensation reaction. Had to use.

On the other hand, when it is produced using a glycosyltransferase, there is a problem that the sugar donor UDP-Gal is expensive and the galactosyltransferase is also expensive and unstable.

[0008]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have found that GlcNAc-M
In a solution containing an and a galactose donor, β
-Gal-GlcNAc-Man was successfully produced by carrying out the transfer reaction of galactosidase. According to the method of the present invention, by appropriately selecting β-galactosidase, the G-linked G-linked GlcNAc-Man G
It is possible to produce al-GlcNAc-Man.

Since the method of the present invention is produced using an enzyme, it is possible to carry out the reaction under mild conditions as compared with the organic chemical production method. Further, by combining the method of the present invention and the method for producing GlcNAc-Man described in Japanese Patent Application No. 6-185198,
Gal-Gl in only two steps from raw sugar (galactose donor, N-acetylethylglucosamine and mannose)
It is also a feature of the present invention that cNAc-Man can be produced.

In the method of the present invention, inexpensive lactose or para-nitrophenyl-β-galactoside (pNp-β-Gal) can be used as the galactose donor, and β-galactosidase which catalyzes the glycosyl transfer reaction is also inexpensive. Moreover, it is stable.

The contents of the present invention will be described in more detail below.

The origin of β-galactosidase [EC 3.2.1.23] used in the present invention is not particularly limited, and GlcNA
β- which selectively transfers galactose to a desired position of c
Choose galactosidase. That is, when selectively transferring galactose to the 4-position of N-acetylglucosamine, for example, using p-galactosidase from pneumococcus (Diplococcus pneumoniae) or Bacillus circulans (Bacillus circulans), and the like, N-acetylglucosamine In the case of selectively transferring galactose to the 6-position of, for example, using β-galactosidase derived from E. coli, when selectively transferring the galactose to the 3-position of N-acetylglucosamine, for example, bovine testis The β-galactosidase derived from may be used.

GlcNAc-Man is produced by a known organic chemical method or a method using an enzyme. Examples of the method using an enzyme include production by dehydration condensation reaction with N-acetylhexosaminidase in a solution containing mannose and N-acetylglucosamine, which is disclosed in Japanese Patent Application No. 6-185198.

As the galactose donor, a sugar having a galactose reducing end, such as lactose, or a synthetic substrate pNp-β-Gal can be used.

Galactose donor and GlcNAc in the reaction
The molar ratio with -Man is preferably 1: 0.1 to 1: 5.

The present invention will be described in more detail with reference to the following examples.

[0017]

【Example】

Example 1 (Production of Galβ1-4GlcNAcβ1-2Man) 210 mg of pNp-β-Gal and 267 mg of GlcNAcβ1-2Man were combined with 42
It was dissolved in a solution consisting of 0 μl of dimethylsulfoxide and 2.1 ml of 0.2 M potassium phosphate buffer (pH 6.0), and 1 unit of β-galactosidase (derived from Streptococcus pneumoniae, manufactured by Boehringer Mannheim) was added to this 37 Shake at ° C. After 5 hours, the reaction was heated at 100 ° C. for 5 minutes to heat inactivate the enzyme. The HPLC chromatograph of the reaction solution is shown in FIG. The reaction solution was applied to an activated carbon column to purify the product by a linear concentration gradient of water-12% ethanol. The eluate was fractionated in 20 ml portions, and after confirming the eluted fraction of the product by HPLC, the fraction number 86-98 was concentrated.
6.1 mg of Galβ1-4GlcNAcβ1-2Man was obtained. Product
^{The 13} C-NMR spectrum is shown in FIG.

Example 2 (Production of Galβ1-4GlcNAcβ1-2Man) 159 mg of pNp-β-Gal and 214 mg of GlcNAcβ1-2Man were mixed with 71
It was dissolved in a solution consisting of 7 μl of acetonitrile and 1.63 ml of 0.2 M potassium phosphate buffer (pH 6.0), to which 20 units of β-galactosidase (derived from Bacillus circulans, manufactured by Daiwa Kasei Co., Ltd.) was added. Shake at ° C. After 2 hours, the reaction solution was heated at 100 ° C. for 5 minutes to inactivate the enzyme by heat. The reaction solution was applied to an activated carbon column to purify the product by a linear concentration gradient of water-12% ethanol. 20
The eluate was fractionated by ml, the eluted fraction of the product was confirmed by HPLC, and the fraction number 66-88 was concentrated to give 27.6 mg of Galβ1-4GlcNAcβ1-2Man.

Example 3 (Production of Galβ1-4GlcNAcβ1-6Man) 120 mg of pNp-β-Gal and 150 mg of GlcNAcβ1-6Man were added to 54
It was dissolved in a solution consisting of 0 μl of acetonitrile and 1.2 ml of 0.2 M potassium phosphate buffer (pH 6.0), and 32 units of β-galactosidase (derived from Bacillus circulans, manufactured by Daiwa Kasei Co., Ltd.) was added to this 37 Shake at ° C. After 2 hours, the reaction solution was heated at 100 ° C. for 5 minutes to inactivate the enzyme by heat. The reaction solution was applied to an activated carbon column to purify the product by a linear concentration gradient of water-12% ethanol. 20
The eluate was fractionated by ml, and after confirming the eluted fraction of the product by HPLC, the fraction No. 72-84 was concentrated to give 36.9 mg of Galβ1-4GlcNAcβ1-6Man.
The ¹³ C-NMR spectrum of the product is shown in FIG.

[0020]

According to the present invention, Gal-GlcNAc-Man is added to Gl
The binding of Gal to cNAc-Man can be regioselectively controlled, and it can be produced more easily and cheaply than the organic chemical production method. Gal-GlcNAc-Man thus obtained
Is useful as a synthetic intermediate in the total synthesis of sugar chains.

[Brief description of drawings]

FIG. 1 is an HPLC chromatograph of a reaction solution obtained by reacting pNp-β-Gal and GlcNAcβ1-2Man with β-galactosidase derived from Streptococcus pneumoniae.

FIG. 2 is a ¹³ C-NMR spectrum of Galβ1-4GlcNAcβ1-2Man produced by a transfer reaction of β-galactosidase derived from pneumococcus using pNp-β-Gal and GlcNAcβ1-2Man as raw materials.

[Fig. 3] Galβ1-4GlcNAcβ1-6M produced by a transfer reaction of β-galactosidase derived from Bacillus circulans using pNp-β-Gal and GlcNAcβ1-6Man as raw materials.
It is a ¹³ C-NMR spectrum of an.

Claims (2)

[Claims] 1. A method for producing Gal-GlcNAc-Man, which comprises performing a transglycosylation reaction with glycosidase in a solution containing GlcNAc-Man and a galactose donor. 2. The method for producing Gal-GlcNAc-Man according to claim 1, wherein the glycosidase is β-galactosidase.