JPH0638784A - Production of sugar chain containing sialic acid as constituent element - Google Patents

Abstract

PURPOSE:To obtain a sialic acid useful for the investigation of the relationship between sugar chain and physiological activity, etc., on an industrial scale at a low cost from an inexpensive raw material by the rearrangement reaction of a sialyl group donor with a sialyl group acceptor in the presence of a sialidase. CONSTITUTION:Lactose monohydrate is used as a sialyl group acceptor and dissolved in 0.2M acetic acid buffer solution (pH5.0) containing 30% dimethylformamide. A sialic acid dimer is added as a sialyl group donor together with a sialidase to the solution and mixed at 37 deg.C for 1hr. After 1hr of mixing, the reaction liquid is heated at 100 deg.C to inactivate the sialidase and terminate the reaction. The denaturated enzyme is removed by filtration, the reaction liquid is fractionated by high-performance liquid chromatography and the fraction eluted at the retention time equal to that of an alpha 2-6 sialyllactose specimen is collected and concentrated to obtain the sugar chain containing sialic acid bonded to sialic acid galactose through alpha-glycoside bond as a constituent element.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing a sugar chain having sialic acid as a constituent element.

[0002] The present invention provides a novel method for producing a sugar chain having sialic acid as a constituent element, which is very useful for research on sialic acid and sugar chains containing the same.

[0003]

BACKGROUND OF THE INVENTION Sialic acid is a general term for acyl derivatives of neuraminic acid, and is widely present in the living world other than plants as a constituent of various glycoproteins and glycolipids. .

Sialic acid rarely exists in free form in nature, is bound by an α-glycoside bond, and generally occupies the non-reducing end of a heterooligosaccharide of glycoprotein or ganglioside. It also exists in the natural world as an oligosaccharide or a polysaccharide in the form of colominic acid or the like. Sialic acid,
It is present in glycoproteins in the secretions of the digestive tract, urethra, vagina, etc., and imparts high viscosity. Further, sialic acid is known to play an important role in the residence time of glycoproteins in the living body, since the glycoconjugate inhibits the degradation by degrading enzymes. Therefore, in order to control the blood life of a glycoprotein, a technique for freely binding a sialic acid residue to a sugar chain of a target glycoprotein is required. More recently, it has been found that the ligand on the lymphocyte side that binds to the adhesion protein (secretin) of vascular endothelial cells stimulated by inflammation is a sugar chain called sialyl Lewis X. In pursuit of this, attempts have been made to synthesize sugar chains containing sialic acid. Glycolipids are said to be involved in cell recognition, and there is a demand for a technique for binding sialic acid to glycolipids that also do not have sialic acid.

However, in the present situation, the synthesis of sugar chains containing sialic acid is only partially performed by a chemical synthesis method or an enzymatic method.

[0006] That is, first, in the chemical synthesis method, although a technique itself for binding sialic acid to other sugar chains has been reported, it is difficult to bind a sialyl group to another sugar by an α bond. , It requires repeated binding and elimination of protecting groups, which results in extremely long steps, and therefore the chemical synthesis method is disadvantageous especially for industrial mass production, and already in industrial mass production. No method has yet been reported for production.

On the other hand, two types of synthetic methods using enzymes are known. One is a method using a transferase, which is a reaction in which CMP-neuraminic acid is used as a substrate and a sialyltransferase (sialyltransferase) transfers a sialyl group to another sugar chain. However, transferase is contained in animal organs, and it is necessary to express it in microorganisms or animal cells by genetic engineering techniques in order to obtain it in large quantities. Also,
The substrate in this reaction, CMP-neuraminic acid, is
Although it is produced by a chemical synthesis method or an enzymatic synthesis method, none of them is an easy reaction, so that it is expensive.

[0008] Another reaction of the enzymatic method is an attempt to synthesize a sialic acid-containing sugar chain by a dehydration condensation reaction which is a reverse reaction of sialidase which is a hydrolase, and is currently announced. The reaction yield of sialyllactose synthesis by the dehydration condensation reaction of sialic acid and lactose is 1% or less, which is far from practical use. That is, in this method, since the selectivity is low, a large amount of substances other than the target binding compound are produced as a by-product.

As described above, the sialic acid residue plays an important role in physiological activity in sugar chains in glycoproteins and glycolipids in vivo, and for the study of the relationship between sugar chains and physiological activity. In spite of the demand for a simple synthetic method, there is still no established method.

[0010]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies in view of the present situation, and as a result, by utilizing the transfer activity of sialidase which is a hydrolase, sialic acid 2
It was found that a sialic acid-bonded sugar chain can be synthesized by transferring the sialic acid at the non-reducing end of the monomer to another sugar chain, and the present invention was completed based on this new finding.

The dehydration condensation reaction, which is the reverse reaction of sialidase hydrolysis that has been conventionally carried out, involves the reaction of sialic acid (X) and a sialyl group acceptor (Y) as substrates in the form of X + Y → X-Y + H ₂ O. In the present invention, a sugar chain having an acid is produced, whereas in the present invention, a sialidase transfer reaction from a sialyl group donor (X-Z) and a sialyl group acceptor, that is, in the form of X-Z + Y → X-Y + Z. This is a reaction which is completely different from the former because it produces a sugar chain having sialic acid. As can be seen from this, the method for producing a sugar chain having sialic acid by the method of the present invention is novel.

It should be noted that 15 or more kinds of sialic acid are naturally known, and in the present invention, a reaction for transferring N-acetylneuraminic acid, which is the most widely found among them in the living world, is described. However, sialic acid other than N-acetylneuraminic acid can be transferred as long as it is a sialidase substrate.

As the donor of the sialyl group, sialic acid 2
Polymers, colamic acid, which is a polymer of sialic acid, or sialic acid glycosides such as other synthetic substrates, neuraminic acid derivatives are used, but are not limited to these, and other things such as hydrolysis of polysialic acid Substances or other sugar chains containing sialic acid, for example, glycolipids of erythrocyte membrane may be used.

As the sialyl group acceptor, sugar chains having galactose at the non-reducing end, such as lactose, galactose and N-acetyllactosamine, are used in the examples. This is because the acid is always bound to galactose. However, in order to synthesize a non-naturally occurring sugar chain, a sugar chain having no galactose at the non-reducing end can also be used as a sialyl group acceptor.

Further, the origin of sialidase which is a hydrolase is not particularly limited. Clostridium perfringen as described in this example.
s origin and Arthrobacter ureafac
When an enzyme derived from iens is used, the sialyl group binds to the 6-position of the sugar residue of the receptor by an α2-6 bond, but by appropriately selecting and using a sialidase derived from another organism, the sialyl group can be obtained. It is also possible to synthesize a sugar chain having α2-3 linked to the 3-position of the sugar residue of the receptor.

That is, when an enzymatic reaction is carried out using, for example, a virus-derived sialidase (a specific example is Newcastle disease virus-derived sialidase), α2-3 linked sialyl oligosaccharides are obtained site-selectively. be able to. Moreover, at that time, no matter what kind of sialyl group donor is used, α2-
The regioselectivity of the triple bond is not compromised.

Therefore, according to the present invention, it is possible to regioselectively and efficiently synthesize α2-6 linked sugar chains and α2-3 linked sugar chains simply by changing the enzyme used. Become. That is, according to the present invention, as described above, for example, by using an enzyme of bacterial origin, among sugar chains containing sialic acid that naturally exist and have useful physiological properties, α2-6-linked sugar chains are used. It has become possible to freely combine and position-selectively. However, naturally, α2-
There are sialic acid-containing sugar chains that are linked by α2-3 bonds in addition to 6-bonds, and glycoproteins or glycolipids containing such sialic acid are known to play an important role in vivo. There is. Therefore, the establishment of a selective synthesis method of not only α2-6-linked sialyl oligosaccharides but also α2-3-linked sialyl oligosaccharides has been desired.
It has become possible as described above.

Therefore, according to the present invention, α2-3 linked sialyl oligosaccharides and α2-6 linked sialyl oligosaccharides can be freely synthesized. Moreover, according to the present invention, various sialic acid-containing oligosaccharides can be synthesized from sialic acid dimer, which is an inexpensive sialyl group donor and sialidase, which is an inexpensive hydrolase, and the process is simple and simple. Together, the method according to the present invention is suitable as an industrial method.

The transfer reaction is carried out under the optimum temperature and pH of the sialidase used while monitoring the components of the reaction solution by HPLC etc., and when the amount of the desired reaction product reaches the maximum. The reaction is stopped by heating or the like. After stopping the reaction, the enzyme is removed and the target substance is collected by HPLC or the like.

It should be noted that the addition of an organic solvent is preferable because it improves the yield when the reaction solution is mixed with an organic solvent such as dimethylformamide or acetonitrile, but it is not an essential requirement of the present invention.

Examples of the present invention will be described below.

[0022]

Example 1 Clostridium perfri
Synthesis of sialyllactose by sialidase derived from ngens Lactose monohydrate 36 mg as sialyl group acceptor
Was dissolved in 100 μl of 0.2 M acetate buffer (pH 5.0) containing 30% dimethylformamide. 10 mg of sialic acid dimer and sialidase (Clostridium perf) as a sialyl group donor were added to this solution.
derived from Ringens, manufactured by Sigma) 10 mg (3 uni)
t) was added and mixed at 37 ° C. for 1 hour. After 1 hour, the reaction solution was heated to 100 ° C. to inactivate the sialidase and stop the reaction. After removing the denatured enzyme by filtration, the reaction solution was used as a Mono Q column (Pharmacia).
It was fractionated by HPLC connected to. The results are shown in Fig. 1.

Fractions (peak A) eluted at the same retention time (8.82 minutes) as the α2-6 sialyllactose standard were collected and concentrated to give 3.1 mg of solid. This substance was hydrolyzed by a sialidase derived from Salmonella (manufactured by Sigma) to release sialic acid and lactose, which revealed that the sialic acid was α-glycoside-linked to lactose. Further, the 400 MHz ¹ H NMR spectrum of this substance was measured (FIG. 2), and 400 of the α2-6 sialyllactose standard was measured.
It was in agreement when compared with the MHz ¹ H NMR spectrum. From the above results, it was identified that the substance obtained by the method of the present invention is α2-6-bonded sialyllactose.

The ¹ H NMR spectrum is for the sample dissolved in heavy water, XL-40 manufactured by Varian.
It is measured using a 0 NMR spectrometer.

[0025]

Example 2 Arthrobacter ureaf
Synthesis of sialyllactose by sialidase derived from aciens Lactose 36 mg as a sialyl group acceptor, 30%
0.2 M phosphate buffer (pH 7.
0) Dissolved in 100 μl. To this solution was added 10 mg of sialic acid dimer and sialidase (Arthrobacter ureafaciens) as a sialyl group donor.
(Origin, manufactured by Sigma) 0.1 ml (1 unit) was added and mixed at 37 ° C. After 3 hours, the reaction solution was heated to 100 ° C. to inactivate the sialidase and stop the reaction. The reaction solution was measured by HPLC connected to a Mono Q column (Pharmacia). The same elution position as the α2-6 sialyllactose standard (peak A; 5.54 min)
At the peak of free sialic acid (peak B; 9.05).
The peak of the product having an area of about 3% of the area of (min) was observed (FIG. 3). From this result, it was revealed that α2-6 sialyllactose in which sialic acid was α-glycosidically linked to lactose was synthesized.

[0026]

Example 3 Synthesis of Sialylgalactose 72 mg of galactose as a sialyl group acceptor and 10 mg of sialic acid dimer as a sialyl group donor
Dissolve in 0 μl of 0.1 M acetate buffer (pH 5.0),
To this was added 30 μl of acetonitrile. To this solution, sialidase (Clostridium perfr)
Ingens-derived, Sigma) 7mg added and 37 ° C
And reacted for 2 hours. After completion of the reaction, the reaction solution was heated to 100 ° C. to inactivate the sialidase to stop the reaction, and the denatured enzyme was removed by filtration. After that, the reaction solution is
When fractionated by HPLC with an oQ column connected, a product peak corresponding to an area intensity of 2.1% of the area intensity of the sialic acid dimer was observed. The fraction corresponding to this peak was taken and concentrated, and the solid content of 400 MHz ¹ H
When the NMR spectrum was measured (FIG. 4; measurement conditions and equipment were the same as in Example 1), a peak peculiar to the α2-6 bond appeared. From this result, it was confirmed that α2-6-bonded sialylgalactose was produced.

[0027]

Example 4 Synthesis of Sialyl N-Acetyllactosamine 110 mg of N-acetyllactosamine as a sialyl group acceptor and 50 mg of sialic acid dimer as a sialyl group donor were mixed with 150 μl of 0.1 M acetate buffer ( pH 5.
It was dissolved in 0) and 50 μl of acetonitrile was added thereto. To this solution, sialidase (Clostridiu)
derived from m perfringens, manufactured by Sigma) 20 m
g was added and reacted at 37 ° C. for 2 hours. After completion of the reaction, the reaction solution was heated to 100 ° C. to inactivate the sialidase to stop the reaction, and the denatured enzyme was removed by filtration. After that, the reaction solution was fractionated by HPLC with a Mono Q column connected, and a peak of the product corresponding to an area intensity of 7.7% of the area intensity of the sialic acid dimer was observed. A 400 MHz ¹ H NMR spectrum of a solid obtained by collecting and concentrating a fraction corresponding to this peak was measured (FIG. 5; measurement conditions and equipment were the same as in Example 1).
A peak peculiar to -6 bond appeared. From this result, α2
It was confirmed that -6-bonded sialyl N-acetyllactosamine was produced.

[0028]

Example 5 Synthesis of α2-3 linked sialyllactose using lactose as an acceptor 100 mg of sialic acid dimer and 250 mg of lactose
It was dissolved in 500 μl of 0.1 M phosphate buffer (pH 7.0), and the sialidase (Oxford Glyco System) derived from Newcastle disease virus was added to this solution.
0.1 unit was added, and the mixture was mixed by rotation at 37 ° C. and reacted for 3 days. After the reaction solution was placed in boiling water for 5 minutes to inactivate the enzyme, it was filtered through a 45 μm membrane and fractionated by HPLC connected to a HiLoad Q column (Pharmacia) to obtain the desired product. The HPLC conditions were 0 to 20 mM saline flowing in a 240-minute linear gradient. The α2-3 linked sialyllactose was eluted in the 125-135 min portion. The obtained trisaccharide component was desalted by gel filtration, lyophilized and weighed to find that it was 1.5 mg. This is 9% of the theoretical yield calculated based on the reacted sialic acid dimer.
Met. The 500 MHz ¹ H NMR spectrum of this compound is shown in FIG.

[0029]

Example 6 Reaction Example Using N-Acetyllactosamine as Receptor 100 mg of sialic acid dimer and 350 mg of N-acetyllactosamine were mixed with 400 μl of 0.1 M phosphate buffer (pH).
7.0), and sialidase (Oxford Glyco) derived from Newcastle disease virus was added to this solution.
(System Co., Ltd.) 0.1 unit was added, and the mixture was rotatively mixed at 37 ° C. and reacted for 3 days. After the reaction solution was placed in boiling water for 5 minutes to inactivate the enzyme, it was filtered through a 45 μm membrane and separated by HPLC with a HiLoad Q column (Pharmacia) to obtain the desired product. HPLC conditions are 0 to 20 mM saline solution 240
Run with a linear gradient of minutes. The α2-3 linked sialyllactose was eluted in the portion of 130 to 140 minutes.
The obtained trisaccharide component was desalted by gel filtration, lyophilized and weighed to find 1.2 mg. This was 6% of the theoretical yield calculated based on the reacted sialic acid dimer. 500 MHz ¹ H of this compound
The NMR spectrum is shown in FIG.

[0030]

[Example 7] Example of reaction using colominic acid as a donor 100 mg of colominic acid 20 mg and lactose 50 mg
20 ml of Newcastle disease virus-derived sialidase (Oxford Glyco System) was dissolved in 0.1 M phosphate buffer (pH 7.0), and the mixture was mixed by rotation at 37 ° C. for 3 days to carry out the reaction. went. The reaction solution is placed in boiling water for 5 minutes to inactivate the enzyme, and then filtered through a 45 μm membrane to obtain Mo.
HPLC connected to no Q column (Pharmacia)
Was analyzed by. The HPLC conditions were 0 to 20 mM saline flowing in a 20-minute linear gradient. Standard α
Although 2-3-bonded sialyllactose was eluted around 7.0 minutes, a peak was similarly detected at 7.0 minutes when the reaction solution was measured (FIG. 8). 3% of theoretical yield calculated based on the area of sialic acid produced by hydrolysis
Met.

[0031]

Example 8 Reaction Example Using Synthetic Substrate as Donor 2-O- (p-nitrophenyl) -N-acetyl-α-
D-neuraminic acid sodium salt (manufactured by Seikagaku Corporation)
10 mg and N-acetyllactosamine 30 mg, dimethyl sulfoxide and 0.2 M phosphate buffer (pH 6.5)
Of the sialidase (Oxfo) derived from Newcastle disease virus.
rd Glyco System) (40 mm units) was added, and the mixture was rotatively mixed at 37 ° C. and reacted for 48 hours.
Place the reaction mixture in boiling water for 5 minutes to inactivate the enzyme,
Filter through a 45 μm membrane and use Mono Q
It was analyzed by HPLC connected to a column (Pharmacia). HPLC conditions are 0 to 10 mM saline 2
Flowed with a 0 minute linear gradient. Peaks were detected at 10.67 minutes (Fig. 9), ¹ and separated the peak H
By measuring NMR, α2-3 linked sialyl
It was identified as N-acetyllactosamine. It was 5% of the theoretical yield calculated based on the area of sialic acid produced by hydrolysis.

[0032]

INDUSTRIAL APPLICABILITY The present invention is capable of synthesizing a sialic acid-containing sugar chain by transferring a sialyl group of a substrate containing a sialyl group to another sugar chain using sialidase which is a hydrolase. is there. That is, the present invention has established a simple method for synthesizing oligosaccharides containing sialic acid, which plays an important role in glycoproteins in living organisms and sugar chains in glycolipids.

According to the present invention, not only α2-6-linked sialyl oligosaccharides but also α2-3 linked sialyl oligosaccharides, which are said to play an important role in vivo, are regioselectively and other. It has become industrially possible to synthesize easily and inexpensively as compared with the method.

[Brief description of drawings]

FIG. 1 Clostridium perfringe
The HPLC chart by the Mono Q column of the reaction product after performing reaction using sialidase derived from ns is shown. The measurement conditions are 0 mM NaCl concentration from 0 to 3 minutes, 3 to 12 minutes NaCl concentration 10 mM
Then, after that, elution was performed at a NaCl concentration. Detection is 2
The absorbance was 20 nm. Peak A is α2-6 sialyllactose.

[Fig. 2] Clostridium perfringe
After the reaction was performed using sialidase derived from ns, Mo
The substance obtained by concentrating and solidifying the fraction eluted at the same retention time as the α2-6 sialyllactose standard by HPLC using a No Q column was 400 MHz ¹ H.
The spectrum when subjected to NMR is shown. The sample was dissolved in heavy water and measured. Chemical shift is TSP as internal standard
(Sodium3- (timetylsilyl))
It was measured using a tetradeutero propionate).

FIG. 3 Arthrobacter ureafaci
The HPLC chart of the reaction product after performing a reaction using ens sialidase is shown. 8 mM NaCl concentration from 0 to 18 minutes, 50 mM Na concentration thereafter
Elution was performed with Cl concentration. The detection method is the same as in the case of FIG. Peak A is α2-6 sialyllactose and peak B is free sialic acid.

[Fig. 4] Fig. 4 shows a reaction using galactose as a sialyl group acceptor and sialic acid dimer as a sialyl group donor, followed by concentration and solidification of a product fraction eluted by HPLC using a Mono Q column. 400 substances obtained by
The spectrum when applied to MHz ¹ H NMR is shown.

FIG. 5 shows a reaction using N-acetyllactosamine as a sialyl group acceptor and a sialic acid dimer as a sialyl group donor, followed by HPLC using a Mono Q column.
The spectrum obtained by subjecting a substance obtained by concentrating and solidifying the product fraction eluted in (4) to 400 MHz ¹ H NMR is shown.

[Fig. 6] 500 MHz of a standard product (manufactured by Sigma, containing about 10% α2-6-linked sialyllactose) (upper) and enzymatically synthesized α2-3 sialyllactose (lower).
¹ H NMR spectrum is shown. Each sample was dissolved in heavy water and measured. Chemical shift is TSP (Sodium
3- (trimethylsilyl) tetrad
eutero propionate) was used as a standard.
The next peak was eliminated by the presaturation method.

FIG. 7 shows a 500 MHz ¹ H NMR spectrum of enzymatically synthesized α2-3 linked sialyl N-acetyllactosamine. The measurement conditions are UNITY manufactured by Varian.
It is the same as FIG. 1 except that a -500 NMR apparatus was used.

FIG. 8: Mono of the reaction solution of colominic acid and lactose
An HPLC chart using a Q column is shown. The measurement conditions were such that the salt concentration was changed from 0 to 10 mM with a gradient from 0 to 20 minutes. The detection was performed by the absorbance at 220 nm. The peak at 7.00 is the product, 10.3
The peak of 5 is sialic acid.

FIG. 9 shows an HPLC chart of a reaction solution of a synthetic substrate and N-acetyllactosamine. The measurement conditions are the same as in FIG.
The peak at 10.67 is the product and the peak at 14.96 is sialic acid.

Claims (4)

[Claims] 1. A method for producing a sugar chain comprising sialic acid as a constituent, which comprises subjecting a sialyl group donor and a sialyl group acceptor to a transfer reaction in the presence of sialidase. 2. A sialylic group donor is a sialic acid dimer,
The method for producing a sugar chain comprising sialic acid according to claim 1, which is a colominic acid, a sialic acid glycoside, and / or a neuraminic acid derivative. 3. The method for producing a sugar chain comprising sialic acid as a constituent element according to claim 1 or 2, wherein the sialidase is an enzyme derived from a microorganism or a virus. 4. The sugar chain which is a sugar chain comprising α2-6-bonded or α2-3-bonded sialic acid as a constituent element.
A method for producing a sugar chain comprising the sialic acid according to claim 3 as a constituent element.