JP2003212891A - N-acetylglucosaminyl-cellooligosaccharide derivative and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a non-natural type new cellooligosaccharide derivative, and to provide a method for producing the same. <P>SOLUTION: This method for producing the new cellooligosaccharide derivative, an N-acetylglucosaminly-cellooligosaccharide derivative, comprises the following process: using a cellooligosaccharide or a derivative thereof as a saccharide receptor and an oxazoline derivative of N-acetyllactosamine as a saccharide donor, a chitinase is subjected to them under alkaline conditions to obtain a non-natural type N-acetyllactosaminyl-cellooligosaccharide derivative where N-acetyllactosamine is transferred via a β-1,4-linkage to the non-reduced terminal of the cellooligosaccharide or the derivative thereof; subsequently, β-D galactosidase is subjected to the non-natural type N-acetyllactosaminyl- cellooligosaccharide derivative to obtain the objective N-acetyl glucosaminyl- cellooligosaccharide derivative where N-acetylglucosamine is transferred via a β-1,4-linkage to the non-reduced terminal of the cellooligosaccharide or the derivative thereof; wherein it is preferable that the chitinase (preferably, Bacillus-derived one) be subjected to both the saccharide receptor and donor under the condition of pH 8-10. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel N-acetylglucosaminyl-cellooligosaccharide derivative and a method for producing the same.

[0002]

2. Description of the Related Art Advances in separation / purification technology and analysis technology are elucidating the structure and function of complex sugar chains.
Sugar chains are involved in various life phenomena, and development of new drugs and the like using sugar chains is expected.

The amount of information that a sugar chain has depends on the type, number, bonding position, steric structure, etc. of the sugar as a structural unit, and is extremely enormous. Therefore, it has an unknown sequence or bond. The possibility that sugar chains exist cannot be denied.

[0004] In developing and researching new drugs using sugar chains, constructing a non-natural sugar chain has the potential to make new discoveries and improve the function of sugar chains. There is also the possibility of adding new functions.

Up to now, as a method for synthesizing sugar chains,
Organic synthetic chemical methods and methods using enzymatic transferases are known.

[0006]

However, the synthetic method of synthetic organic chemistry requires a multi-step reaction process by repeated protection / deprotection of functional groups and a plurality of purification operations, and therefore is industrial scale. Was extremely difficult to implement.

On the other hand, the method of synthesizing a sugar chain using transferase is much simpler than the above-mentioned chemical synthesis method, and since it does not involve the production of isomers, a sugar chain having a clear structure can be efficiently produced. Although it can be obtained well, it is difficult to obtain a large amount of transferase, and the sugar nucleotide as a substrate is unstable and expensive,
Implementation on an industrial scale was costly. In addition, transferase has a weak point that sugar recognition is extremely strict and only certain sugar chains can be synthesized, and there is a problem that it is not suitable for the synthesis of modified sugar chains and non-natural sugar chains.

Therefore, an object of the present invention is to provide a novel non-natural cellooligosaccharide derivative and a method for producing the same.

[0009]

[Means for Solving the Problems] In the course of research for constructing a sugar chain using a sugar hydrolase, the present inventors have found that the receptor specificity of chitinase, which is an enzyme that decomposes chitin under alkaline conditions. However, they have found that they are relatively tolerant and recognize cellobiose and cellotriose that do not have an acetamide group at the 2-position as a sugar acceptor, and have completed the present invention based on this finding.

That is, one of the present invention is an N-acetylglucosaminyl-cellooligosaccharide derivative represented by the following general formula (1).

[0011]

[Chemical 2]

Another aspect of the present invention is that a cellinooligosaccharide or cellooligosaccharide derivative serving as a sugar acceptor and an oxazoline derivative of N-acetyllactosamine serving as a sugar donor are treated with chitinase under alkaline conditions. After that, the method for producing an N-acetylglucosaminyl-cellooligosaccharide derivative is characterized by further reacting β-D-galactosidase.

In the above-mentioned production method, it is preferable that chitinase is allowed to act on the sugar acceptor and the sugar donor under the conditions of pH 8 to 10. In addition, the chitinase,
It is preferably a chitinase from the genus Bacillus .

According to the present invention, a cellooligosaccharide or a cellooligosaccharide derivative is used as the sugar acceptor, and an oxazoline derivative of N-acetyllactosamine is used as the sugar donor, and a chitinase is allowed to act on these to react with the cellooligosaccharide or A non-natural N-acetyllactosaminyl-cellooligosaccharide derivative in which N-acetyllactosamine is transferred to the non-reducing end of the cellooligosaccharide derivative through a β-1,4 bond is obtained, and β-D-galactosidase is allowed to act on the derivative. Thus, a non-natural cellooligosaccharide derivative (N-acetylglucosaminyl-cellooligosaccharide derivative) in which N-acetylglucosamine is transferred to the non-reducing end of a cellooligosaccharide or a cellooligosaccharide derivative through a β-1,4 bond can be prepared easily and easily. It can be prepared inexpensively. The N-acetylglucosaminyl-cellooligosaccharide derivative is a novel physiologically active substance,
It can be expected to be used as medicines, diagnostics, cosmetics, foods, agricultural chemicals, fertilizers, etc. Further, the reducing end of the cellooligosaccharide derivative can be provided with a polymerizing group for immobilization or a functional group for detection, and therefore, for example, as a substrate for cellulase activity measurement or a column packing material. Expected to be used.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The basis of the method for synthesizing an N-acetylglucosaminyl-cellooligosaccharide derivative of the present invention is to allow a chitinase to act on an oxazoline derivative of N-acetyllactosamine, which is a sugar donor, under alkaline conditions. As a result, after transfer to the non-reducing terminal side of the cellooligosaccharide or cellooligosaccharide derivative serving as a sugar acceptor by β-1,4 bond, β-D-galactosidase is further allowed to act to form a galactose residue at the non-reducing terminal. Is to disconnect.

In the present invention, the oxazoline derivative of N-acetyllactosamine (hereinafter, simply referred to as oxazoline derivative) means a compound having a structure represented by the following formula (2), which is derived from N-acetyllactosamine.

[0017]

[Chemical 3]

The above oxazoline derivative, for example, acts acetyl chloride on N-acetyllactosamine to chlorinate the anomeric position and protects the hydroxyl group, forms an oxazoline ring with tetraethylammonium chloride and sodium hydrogen carbonate, and finally sodium. It can be obtained by carrying out deacetylation with methoxide (K. Sas
aki, S. Ahlfors, T. Frejd, J. Kihlberg, G. Magnuss
on, J. Org. Chem., 53, 5629 (1988), S. Nishimura,
H. Kuzuhara, Y. Takiguchi, K. Shimahara, Carbohyd
r. Res., 194, 223 (1989)).

As cellooligosaccharides or cellooligosaccharide derivatives serving as sugar acceptors, cellooligosaccharides of disaccharides to hexasaccharides and those obtained by modifying their reducing terminals with a methyl group, an acrylamide group, a paranitrophenyl group or the like are preferably used. To be

As the chitinase, a classification of carbohydrate hydrolase by Henrissat et al. (Henrissat, B., Biochem.
J., 280, 309 (1991), Henrysat, B., A. Bairoch., B
iochem.J., 293, 781 (1993)), family 1
A chitinase belonging to Group 8 is used, and a chitinase derived from the genus Bacillus is particularly preferably used.

Chitinase is a bacterium of the genus Bacillus (for example,
It can be prepared by preparing a crude enzyme from a culture solution of Bacillus circulans (IFO 13627, etc.) by, for example, ammonium sulfate precipitation, and further purifying it using a chitin column or the like if necessary (T. Watanabe, K. Suzuki, W. Oyanagi, K. Oh
nishi, H. Tanaka, J. Biol. Chem., 265, 15659 (199
0)). The chitinase may be a purified one or a crude enzyme. In addition, a commercially available chitinase may be used, and for example, a trade name “chitinase” ( Bacill
us sp. Origin, manufactured by Wako Pure Chemical Industries, Ltd., etc. can be used.

In the present invention, Bacillus sp. A chitinase derived from is particularly preferably used.

[0023] The β-D-galactosidase is not particularly limited, but for example, Streptococcus pneumoniae (ATCC630).
5)), Bacillus circulans (IFO 13627) and the like can be used.

Β-D-galactosidase can be extracted from the culture broth of these cells by, for example, ammonium sulfate precipitation (Green, AA et a.
l .: Methods in Enzymology, vol. 1, p76, 1955)
Gel filtration chromatography (Miyazaki et al.
Agric Bio Chem, 52 , 625-631, 1988). Alternatively, a commercially available β-D-galactosidase may be used, and for example, the trade name "lactose-degrading enzyme preparation Biol
acta FN5 "(from Bacillus circulans , manufactured by Daiwa Kasei)
Etc. can be used. In addition, it is preferable that β-D-galactosidase have N-acetylhexosaminidase activity removed.

In the present invention, Streptococcus
Β- from Pneumonia ( Streptococcus pneumoniae )
D-galactosidase is particularly preferably used.

In the present invention, N represented by the general formula (1)
The -acetylglucosaminyl-cellooligosaccharide derivative can be obtained, for example, as follows.

That is, the above-mentioned oxazoline derivative and cellooligosaccharide or cellooligosaccharide derivative are alkaline (preferably pH 7 to 11, more preferably pH 8 to 10).
It is dissolved in the buffer solution prepared as described above, chitinase is added thereto, and the reaction is performed at 20 to 40 ° C. During the reaction, the reaction solution was sampled with time to confirm the disappearance of the peak of the oxazoline derivative by high performance liquid chromatography, and then the enzyme was inactivated, and the target product (N-acetyllactosaminyl was analyzed by high performance liquid chromatography or the like. -Cellooligosaccharide derivative).

The obtained N-acetyllactosaminyl-cellooligosaccharide derivative is added to a buffer solution (preferably pH).
5-7, more preferably pH 6-6.5) in β-D-
To obtain an N-acetylglucosaminyl-cellooligosaccharide derivative represented by the general formula (1) by allowing galactosidase to act, cleaving the galactose residue at the non-reducing end, and purifying by high performance liquid chromatography or the like. You can

In the present invention, the enzymatic reaction using chitinase is carried out under the alkaline conditions as described above to give a sugar donor to the non-reducing terminal side of the cellooligosaccharide or cellooligosaccharide derivative serving as a sugar acceptor. The oxazoline derivative of N-acetyllactosamine can be efficiently transferred by β-1,4 bond.

In the present invention, it is preferable to use the oxazoline derivative and the cellooligosaccharide or cellooligosaccharide derivative in a molar ratio of 0.3 to 3: 1.

The amount of chitinase added is not particularly limited as long as the transfer reaction proceeds sufficiently. For example, in the reaction system containing 0.1 mol / L of the above oxazoline derivative, the amount of chitinase added is 50. ~ 500 mU / m
L is preferable, and 70 to 150 mU / mL is more preferable. In the present invention, chitinase 1U (unit) means 1 μmol of N-acetyl chitosan per minute from N-acetyl chitosan when the enzyme is allowed to act on N-acetyl chitosan under the conditions of pH 6.8 and 37 ° C. It means the amount of enzyme that produces a reducing sugar corresponding to glucosamine.

The amount of β-D-galactosidase added is not particularly limited as long as the reaction proceeds sufficiently. For example, 5 to 10 mmol of the substrate (N-acetyllactosaminyl-cellooligosaccharide derivative) is added. In the reaction system containing / L, the addition amount of β-D-galactosidase is 5 to 2
50 mU / mL is preferable, and 25-125 mU / mL is more preferable. In the present invention, βU-galactosidase 1U (unit) means pH 6.0, 37 ° C.
When the enzyme is allowed to act on para-nitrophenyl-β-D-galactoside under the conditions of 1), it means the amount of enzyme that liberates 1 μmol of para-nitrophenol from para-nitrophenyl-β-D-galactoside in 1 minute.

In the case of the enzyme reaction using chitinase, examples of the buffer solution used when carrying out the above-mentioned enzyme reactions include Tris buffer solution, phosphate buffer solution, carbonate buffer solution and the like. In the case of an enzymatic reaction using β-D-galactosidase, for example, Sodium cacodylate buffer solution, phosphate buffer solution and the like can be mentioned.

The conditions for high performance liquid chromatography are, for example, a column such as "TSK-gel Amide-80" (trade name, manufactured by Tosoh Corporation), "Asahipak NH2P-50 4E" (trade name, manufactured by Showa Denko KK). Acetonitrile / water mixed solvent (acetonitrile / water (v / v) = 80/20)
~ 60/40) and the like.

[0035]

EXAMPLES The present invention will be described more specifically below with reference to examples. Production Example 4.0 g of N-acetyllactosamine was mixed with 20 g of acetyl chloride.
mL was added, and the reaction was carried out at room temperature for 4 days while stirring. During the reaction, the reaction was followed by TLC (ethyl acetate / hexane (v / v) = 4/1).

The reaction solution was concentrated by evaporation, diluted with an excess amount of methylene chloride, and then partitioned with cold water and saturated sodium hydrogen carbonate solution until the pH became neutral. The obtained organic solvent phase was dried over anhydrous sodium sulfate. After drying in, it was filtered to remove sodium sulfate.

The obtained filtrate was concentrated by evaporation and further dried under reduced pressure to completely remove the solvent, and N-chloride was added.
Acetyl-3,6,2 ', 3', 4 ', 6'-hexa- O -acetyl-α
-Lactosaminyl (hereinafter referred to as compound (I)) 6.8
0 g was obtained. The following reaction was carried out as it was without purification.

3.01 g of the above compound (I) was dissolved in 25 mL of acetonitrile, and this solution was added to 1.0 g of tetraethylammonium chloride and 1.0 g of sodium hydrogencarbonate dried with calcium chloride for one and a half hours.
Refluxed at 0 ° C for 1 hour and 40 minutes. After confirming the completion of the reaction by TLC (ethyl acetate / hexane (v / v) = 4/1), a glass filter G4 (trade name, manufactured by Shibata Scientific Co., Ltd.) was used to remove solids, and the obtained filtrate was decompressed. After concentrating with, diluted with an excess amount of methylene chloride, partitioned with cold water and saturated sodium hydrogen carbonate solution until the pH of the aqueous phase becomes neutral, and the obtained organic solvent phase is dried over anhydrous sodium sulfate, The sodium sulfate was removed by filtration.

After the obtained solution was concentrated by evaporation, silica gel flash column chromatography (Gel: trade name "Silica Gel 60", manufactured by Merck, parti
clesize: 0.04-0.063mm, developing solvent: ethyl acetate / hexane (v / v) = 4/1)
-Methyl {3,6 di- O -acetyl-4- O- (2,3,4,6-tetra- O -acetyl-β-D-galactopyranosyl) 1,2-dideoxy-α-glucopyrano} 1.40 g of [2,1-d] -2-oxazoline (hereinafter referred to as compound (II)) was obtained.

The above compound (II) under an argon atmosphere.
1.40 g was dissolved in anhydrous methanol 270 mL,
A methanol solution of sodium methoxide was added so as to have a concentration of 0.01 M, and the mixture was reacted at room temperature for 2 hours. T
After confirming the completion of the reaction by LC (ethyl acetate / hexane (v / v) = 4/1), the ion exchange resin Amberlite IR-120 (H ⁺ ) (trade name, manufactured by Organo Corporation) was added until the reaction solution became neutral. ) Was added and then filtered to remove the ion exchange resin.

The obtained filtrate was concentrated by evaporation and further dried under reduced pressure to give 2-methyl {4- O- (β-D-galactopyranosyl) 1,2-dideoxy-α-D-glucopyrano} [ 0.80 g of 2,1-d] -2-oxazoline (hereinafter referred to as oxazoline derivative (I)) was obtained. The structure of the oxazoline derivative (I) was confirmed by NMR.

Example 1 36.5 mg of the above oxazoline derivative (I) was placed in an Eppendorf tube and (N-acrylamidomethyl) was added.
-Aminocarbonylethyl-1-thio-β-D-cellobioside 16.9 mg and 1 mL of 50 mM Tris buffer (pH 9.0) in which 325 mU of commercial chitinase were dissolved were added, and the mixture was placed in a constant temperature bath at 40 ° C to carry out the reaction. .

During the reaction, the reaction was traced by high performance liquid chromatography, and after confirming that the peak of the oxazoline derivative had completely disappeared, heat deactivation was carried out at 90 ° C. for 20 minutes. The high performance liquid chromatography was performed under the following condition (1).

Conditions (1) Column: "TSK-gel Amide-80 (4.6 x 25 mm)" (trade name,
Solvent: Acetonitrile / water (v / v) = 75/25 Temperature: 40 ° C. Flow rate: 1.0 mL / min Detection: RI Then, using the obtained reaction liquid, the following conditions (2) are used. High-performance liquid chromatography was performed to collect the target fraction.

-Condition (2) Column: "Inertsil ODS-3 (10.0 x 25 mm)" (trade name, G
Solvent: water / methanol (v / v) = 900/70 temperature: room temperature flow rate: 3.0 mL / min detection: UV (210 nm) Then, the collected fractions are concentrated by evaporation and freeze-dried. , N-acetyllactosaminyl represented by the following formula:
4.7 mg of a cellooligosaccharide derivative was obtained.

[0046]

[Chemical 4]

4.5 mg of the obtained N-acetyllactosaminyl-cellooligosaccharide derivative was added to 730 μL of 20 mM phosphate buffer (pH 6.0) and commercially available β-D-galactosidase (derived from Streptococcus pneumoniae , trade name “β1”). , 4-galactosidase ", manufactured by CALBIOCHEM) 1
0.7 mU was added and the reaction was carried out in a constant temperature bath at 37 ° C.

During the reaction, the reaction was traced by high performance liquid chromatography, and after confirming that the peak of the N-acetyllactosaminyl oligosaccharide derivative had completely disappeared, 90
Heat deactivation was performed at 20 ° C. for 20 minutes. The high performance liquid chromatography was performed under the above condition (1).

Then, the obtained reaction solution was subjected to high performance liquid chromatography under the following condition (3) to collect a fraction of the desired product.

Condition (3) Column: "Inertsil ODS-3" (trade name, manufactured by GL Sciences) Solvent: water / methanol (v / v) = 900/70 Temperature: room temperature Flow rate: 5.0 mL / min Detection : UV (210 nm) Then, the collected fraction is concentrated by evaporation and freeze-dried to obtain N-acetylglucosaminyl represented by the following formula.
2.3 mg of a cellooligosaccharide derivative was obtained.

[0051]

[Chemical 5]

The above-mentioned N-acetyllactosaminyl-cellooligosaccharide derivative and N-acetylglucosaminyl-cellooligosaccharide derivative were analyzed by ¹ H, ¹³ CNMR and MALDI.
The structure was confirmed by TOFMASS.

[0053]

As described above, according to the present invention,
A cellooligosaccharide or a cellooligosaccharide derivative as a sugar acceptor,
An oxazoline derivative of N-acetyllactosamine was used as a sugar donor, and a chitinase was allowed to act on these derivatives under alkaline conditions to give N-acetyllactosamine β-1,4 at the non-reducing end of a cellooligosaccharide or a cellooligosaccharide derivative.
A bound non-natural type N-acetyllactosaminyl-cellooligosaccharide derivative is obtained, and β-D-galactosidase is allowed to act on this to give N-acetylglucosamine at the non-reducing end of the cellooligosaccharide or the cellooligosaccharide derivative.
A non-natural type N-acetylglucosaminyl-cellooligosaccharide derivative having -1,4 linkage can be prepared easily and inexpensively. This N-acetylglucosaminyl-cellooligosaccharide derivative can be expected to be used as a novel physiologically active substance as a drug, a diagnostic agent, a cosmetic, a food, a pesticide, a fertilizer and the like. Further, the reducing end of the cellooligosaccharide derivative,
Since a polymerizing group for immobilization, a functional group for detection and the like can be added, it can be expected to be used as a substrate for cellulase activity measurement or a column packing material, for example.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Mao Kuriyama             07 Aoba, Aramaki, Aoba-ku, Sendai-shi, Miyagi Tohoku University             On campus (72) Inventor Masaya Fujita             07 Aoba, Aramaki, Aoba-ku, Sendai-shi, Miyagi Tohoku University             On campus (72) Inventor Takeshi Watanabe             Niigata Prefecture Niigata City Igarashi Ninomachi 8050 Niigata             Inside the university F-term (reference) 4B064 AF21 CA21 CB07 CC03 CD09                       DA01                 4C057 CC03 CC05 DD01 DD03 JJ14

Claims (4)

[Claims] 1. An N-acetylglucosaminyl-cellooligosaccharide derivative represented by the following general formula (1). [Chemical 1] 2. A cellooligosaccharide or cellooligosaccharide derivative which serves as a sugar acceptor, and an oxazoline derivative of N-acetyllactosamine which serves as a sugar donor are allowed to act with chitinase under alkaline conditions, and then β-D-galactosidase is added. A method for producing an N-acetylglucosaminyl-cellooligosaccharide derivative, characterized in that: 3. The pH of the sugar acceptor and the sugar donor is 8
The method for producing an N-acetylglucosaminyl-cellooligosaccharide derivative according to claim 2, wherein a chitinase is allowed to act under the conditions of 10 to 10. 4. The method for producing an N-acetylglucosaminyl-cellooligosaccharide derivative according to claim 2, wherein the chitinase is derived from the genus Bacillus .