JP3002113B2 - Method for producing carbohydrate or complex carbohydrate - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION This invention utilizes the transglycosylation activity of glycosidases, relates to the production how the carbohydrate or glycoconjugate was remodeling.

[0002]

2. Description of the Related Art The sugar chains of glycoproteins play an important role in various biological activities such as the second messenger system of immunity. Therefore, modification or substitution of a sugar chain of a glycoprotein is useful in the pharmaceutical industry. The reason is that this technique makes it possible to create a new type of drug that can reach the target cells or maintain its efficacy. There are chemical methods and enzymatic methods for modifying or substituting the sugar chains of this glycoprotein. Since chemical reactions usually require organic solvents or harsh conditions, they can cause glycoprotein denaturation. On the other hand, since a method using an enzyme is usually carried out in an aqueous solution such as a buffer, the protein is not denatured. Another advantage of the enzymatic method is that the enzymes act very specifically. A method of sugar chain remodeling by an enzymatic method that is currently widely used is described in H. DH Joziasse et al. [European Journal of Biochemistry (Eur. J. Bioche
m.), Vol. 191, pp. 75-83 (1990)], which uses exoglycosidase or glycosyltransferase, and is a sequential conversion from a non-reducing end of a sugar chain. A report of a glycosyltransfer reaction using endoglycosidase is disclosed in B. RB Trimble et al. [Journal of Biological Chemistry, Vol. 261,
12000 to 12005 (1986)], Flavobacterium meningosepticum.
ingosepticum) and those relating to endo-β-N-acetylglucosaminidase F (endo-F).
M. RM Bardales et al. [Journal of Biological Chemistry, Volume 264,
198993-19897 (1989)], which relates to endo-α-N-acetylgalactosaminidase derived from Diplococcus pneumoniae. The former is a report that glycerol becomes an acceptor, and the latter is a report that glycerol, tris, p-nitrophenol, serine, and threonine become acceptors. On the other hand, as a report on a remodeling mechanism of a sugar chain in which a carbohydrate or a complex carbohydrate directly acts as an acceptor, there is a report on Arthrobacter protoformie described in JP-A-5-64594.
phormiae), and mucols described in Biochemistry, Vol. 65, p. 1014 (1993), as well as those relating to endo-β-N-acetylglucosaminidase A (endo-A).
Endo-β-N- from Mucor hiemalis
There is one related to acetylglucosaminidase M (Endo-M).

[0003] As described above, if the stability and biological activity of glycoconjugates obtained by remodeling sugar chains are enhanced as compared with natural glycoconjugates, it is very useful when applied to pharmaceuticals.
However, in the conventional remodeling method using exoglycosidase or glycosyltransferase, an enzymatic reaction must be performed for each sugar residue.
The number of reaction steps increases, which is very complicated. Also, in the case of the above-described glycosyltransfer reaction using endo-F or endo-α-N-acetylgalactosaminidase derived from Diprococcus pneumoniae, the carbohydrate or complex carbohydrate does not serve as an acceptor. On the other hand, a transglycosylation reaction using endo-A or endo-M is a remodeling reaction of a sugar chain in which a carbohydrate or a complex carbohydrate directly serves as an acceptor, and is currently the simplest and practical remodeling of a sugar chain. Is the way. Endo-A used for sugar chain remodeling is Arthrobacter protoformier AKU 0647 (FERM BP)
-4948), and the enzyme decomposes the portion of the oligomannose-type sugar chain indicated by the arrow in the following formula (Formula 1).

[0004]

Embedded image

This Endo-A undergoes a transfer reaction of the following formula (Formula 2).

[0006]

Embedded image X-GlcNAc-Y + Z → X-
GlcNAc-Z + Y

In this transfer reaction, X represents a saccharide,
When endo-A is used as the enzyme, X acts well on a homo-oligomer composed of mannose, glucose and the like, and a hetero-oligomer composed of two or more components such as mannose, glucose and N-acetylglucosamine.

Y is a carbohydrate or a complex carbohydrate, and when endo-A of the present invention is used, glucose, mannose, N-
It acts well on carbohydrates such as monosaccharides such as acetylglucosamine, homo-oligomers of these two or more sugars, and hetero-oligomers consisting of these two or more components.
Or, a glycoconjugate in which a polypeptide is bound through Asn, a glycoconjugate in which a polypeptide is bound through Thr or Ser, or a Thr or Ser at the end thereof may be used.

As the acceptor Z for the transfer reaction using endo-A, the reaction is well performed when a saccharide or complex saccharide having a free sugar at the C-4 position at its non-reducing terminal is used as the acceptor. Among the free sugars at the C-4 position of the acceptor, those having the same conformation at the C-4 and C-5 positions as glucose are particularly preferable, and glucose, glucosamine, N-acetylglucosamine, mannose, mannosamine, It works well on monosaccharides such as N-acetylmannosamine and allose, or on saccharides having these saccharides as non-reducing terminals, or on complex saccharides. In addition, α- and β-
Methyl glycosides, or α- and β-p-nitrophenyl glycosides, glucose-1-phosphate, mannose-
It also acts on carbohydrates such as 1-phosphate. A sugar chain transfer reaction using Endo-A is usually performed by adding a saccharide or complex saccharide of an acceptor to a starting solution containing a raw material saccharide or complex saccharide, an endoglycosidase, and a buffer. In this transfer reaction, an organic solvent, an inorganic salt, or the like is added to the starting solution, and the reaction may be converted to a hydrophobic state.If the organic solvent is, for example, methanol, a poorly water-soluble saccharide or complex saccharide may be used. it can. In the case of endo-A, it reacts well in the presence of 40% methanol and shows a relative activity of about 80% even in the presence of 50% methanol. Also, DMSO
(Dimethylsulfoxide) and DMF (N, N-dimethylformamide) can also be used.

[0010]

As described above, the remodeling of a sugar chain by the enzymatic method requires the mild reaction conditions,
The utility is high in that the specificity of the enzyme can be utilized. However, in a sugar chain remodeling reaction using exoglycosidase or endoglycosidase, the resulting remodeled sugar chain is hydrolyzed by glycosidase, and a hydrolysis product that is not desirable for the intended purpose is found. An object of the present invention is not to generate a hydrolysis product of the remodeled sugar chain, it is to provide a manufacturing how the carbohydrate or glycoconjugate.

[0011]

SUMMARY OF THE INVENTION In general, the present invention relates to a method for producing a saccharide or a complex saccharide, comprising End-A and Endo-A.
The method of manufacturing a carbohydrate or a glycoconjugate using the remodeling reaction represented by the following formula by endoglycosidase selected from fine end -M (1), comprising the step of performing the remodeling reactive water soluble solvent And that
Aqueous solvent, wherein the aqueous solvent der Rukoto containing a water-soluble ketone and / or dioxane. AB + C → AC + B (1) (where A is a saccharide containing GlcNAc at the terminal, and B is a saccharide
Carbohydrate or complex carbohydrate, C indicates carbohydrate or complex carbohydrate)

The present inventors have conducted a remodeling reaction of a saccharide or glycoconjugate using glycosidase in the presence of a specific water-soluble organic solvent, so that the reaction proceeds efficiently and the remodeling It has been found that hydrolysis of the product by glycosidase is suppressed, and the desired remodeling product can be produced in good yield, and the present invention has been completed.

The glycosidase used in the present invention includes, for example, exoglycosidase described in the above-mentioned European Journal of Biochemistry.
Examples of the endoglycosidase include endo-F described above and endo-α-N- derived from Diprococcus pneumoniae.
Acetylgalactosaminidase, endo-A, endo-
M etc. In the presence of a glycosidase selected from these glycosidases, the present invention provides the following formula (Formula 3):

[0014]

Embedded image AB + C → AC + B

(A is a carbohydrate containing GlcNAc at the terminal , B is a carbohydrate or complex carbohydrate, and C is a carbohydrate or complex carbohydrate). In the present invention, the reaction is carried out under conditions in which hydrolysis of AC, which is a product of the formula (Formula 3), is suppressed. Examples of the saccharide represented by the group A in the above formula (Formula 3) other than the terminal GlcNAc include monosaccharides, disaccharide or higher oligomers, or Glc at the terminal thereof.
Those bound by NAc are indicated. For example, when endo-A is used as an enzyme, A is X-GlcNA
In this case, X is a homo-oligomer composed of mannose, glucose and the like, and a hetero-oligomer composed of two or more components such as mannose, glucose and N-acetylglucosamine. When endo-M is used as the enzyme, A is X'-GlcN
Ac. In this case, X ′ is exemplified by the above-mentioned homo-oligomer, or a hetero-oligomer composed of two or more components such as mannose, glucose, N-acetylglucosamine, and galactose. In addition, the above formula (Formula 3)
Examples of the saccharide or glycoconjugate represented by the group B or C in the
Monosaccharide, disaccharide or higher oligomer, or those in which a specific amino acid is bonded to their terminal, or those in which a polypeptide is bonded via the amino acid, and further, a specific amino acid or a polypeptide in which the polypeptide is bonded to the amino acid There is something. For example, when the endo-A or endo-M of the present invention is used, examples of B or C include glucose, mannose, N-acetylglucosamine, galactose, fucose, fructose, xylose, p-nitrophenyl glycoside derivative, methyl glycoside There are carbohydrates such as monosaccharides such as derivatives, homo-oligomers of these two or more sugars, hetero-oligomers consisting of two or more of these components, and Asn or complex carbohydrates to which a polypeptide is bound via Asn, Thr or Ser, Thr or S
It may be a glycoconjugate or the like to which a polypeptide is bound via er.

This condition is achieved by carrying out the remodeling reaction represented by the above formula (Formula 3) in the presence of a selected water-soluble organic solvent. As the water-soluble organic solvent, water-soluble ketones and / or dioxane may be used, and the remodeling reaction is performed in an aqueous solvent containing these solvents. As the water-soluble ketones, acetone, methyl ethyl ketone and diethyl ketone can be used.

The amount of the water-soluble ketone or dioxane added to the aqueous solvent for the remodeling reaction is such that the hydrolysis of the remodeling product by glycosidase is suppressed and the remodeling product can be obtained efficiently. Good.
For example, in the case of acetone, it is better to add and use 50 V / V% or less in view of the enzyme stability in an aqueous solvent containing acetone and the efficiency of the remodeling reaction. Also in the case of dioxane, it is preferable to add and use 50 V / V% or less for the same reason.

The transfer reaction of the present invention is usually carried out by adding a saccharide or complex saccharide of an acceptor to a starting solution containing a raw material saccharide or complex saccharide, glycosidase, and a buffer. The amount of raw materials or acceptors used is not particularly limited,
Although it can be used up to its saturation amount, a state in which the acceptor is present in excess is preferable. Usually, an acceptor of 200 mM or more is used. However, in the present invention, 1
Even at an acceptor concentration of about 0 mM, the reaction proceeds and has a remarkable effect in a remodeling reaction using an acceptor having poor solubility or an acceptor which is difficult to use quantitatively. Next, the amount of glycosidase to be used is not particularly limited and can be appropriately selected from a wide range, but is usually 0.1 mU or more, more preferably 3 mU to 10 U per 1 ml of the starting solution.
You can use it to the extent. As the buffer, the pH is 5 to 11
Any suitable buffer may be used, and the reaction is usually performed in an acetate buffer having a pH of about 6.

In the following, endo-A is used as a glycosidase.
Man ₉ GlcNAcGlcNA as the starting glycoconjugate
c-Asn [Biocarb Chemicals
als): M ₉ GN ₂ Asn], Man ₆ G
The present invention will be described in further detail using lcNAcGlcNAc-Asn (manufactured by BioCurb Chemicals, Inc .; hereinafter, abbreviated as M ₆ GN ₂ Asn) and GlcNAc (manufactured by Wako Pure Chemical Industries, Ltd.) as an acceptor.

[0020]

TABLE 1 Endo-A 3 mUM ₉ GN ₂ Asn 3 nmol GlcNAc 10 μmol 25 mM acetic acid Buffer solution (pH 6.0) 20 µl (containing 25% of solvent) Reaction temperature 37 ° C Reaction time 10 minutes ──────────────────────────── ──

Using various solvents under the reaction conditions shown in Table 1,
Table 2 shows the results of examining the effects of various organic solvents on the endo-A remodeling reaction. As shown in Table 2,
In the presence of acetone, the substrate was Man ₉ G by endo-A.
lcNAc (hereinafter abbreviated as M ₉ GN) and GlcNAc-A
sn (hereinafter abbreviated as Asn-Gn) and its M ₉
The GN is transferred to the acceptor GlcNAc, and the remodeling sugar chain Man ₉ GlcNAcGlcNAc
(Hereinafter abbreviated as M ₉ GN ₂ ), and acetone is an organic solvent for a remodeling reaction which is superior to conventionally known DMF and DMSO. Table 2
In the formula, THF indicates tetrahydrofuran.

[0022]

[Table 2] Table 2 {Solvent M ₉ Gn ₂ (%)} {Water 37 DMF 53 DMSO 70 acetone 100 CH ₃ CN 9 THF 13} ──────────

Next, the stability of Endo-A in an aqueous solvent containing acetone is shown in FIG. That is, FIG. 1 shows a 25 mM acetate buffer (pH 6.0) 20 at each acetone concentration.
After preincubation of 3 mU of End-A in μl at 37 ° C. for each time, 1 μl of the preincubation solution was added to 20 μl of 25 mM acetate buffer containing ₃ nmol of M ₉ GN ₂ Asn, and 37 ° C.
And the activity of endo-A was measured. The vertical axis represents the relative activity (%) of each treated enzyme when the activity of untreated endo-A was defined as 100%, and the horizontal axis represents the preincubation time. (Minutes). As shown in FIG. 1, Endo-A is extremely stable in aqueous solvents containing 30% acetone.

Next, it is usually preferable that an excess of the acceptor is present in the sugar chain remodeling reaction. FIG. 2 shows the relationship between the remodeling reaction and the acceptor concentration in the present invention. That is, FIG. 2 shows that the reaction was carried out in the presence of 30% acetone using GlcNAc at each concentration according to the conditions described in Table 1, and the amount of M ₉ GN _{2 and} the amount of M ₉ GN were determined by HPAEC / PAD. The system was measured with a system (manufactured by Dionex), and the vertical axis is PAD
The response and the abscissa indicate the GlcNAc concentration (M). As shown in FIG. 2, GlcNA in the presence of 30% acetone
Even at a c concentration of 25 mM, M ₉ GN ₂ was sufficiently produced. Further, at a GlcNAc concentration of 200 mM or more,
Hydrolysis of remodeling sugar chains is sufficiently suppressed, and Glc
At an NAc concentration of 500 mM, the production of the product M ₉ GN by the hydrolysis is completely suppressed.

Next, the formation of remodeled sugar chains by endo-A in an aqueous solvent containing acetone and an aqueous solvent not containing acetone is shown in FIG. That is, FIG.
%, 30%, 40% and 50%, the reaction was carried out according to the conditions shown in Table 1, and M ₉ GN ₂ Asn, M ₉
The amounts of GN ₂ , M ₉ GN, and Asn-GN were measured. The vertical axis indicates% of each substance, and the horizontal axis indicates reaction time (minutes). As shown in FIG. 3, no M ₉ GN was formed in the presence of 30 to 50% of each acetone. To Also generation of M ₉ GN ₂ in the presence of acetone non about 50% at 30 min, M ₉ in the presence of 30% acetone
The amount of generated GN ₂ has reached 100%. In order to further study the acceptor in the reaction solution containing acetone,
In the presence of acetone (3 for L-Fuc and L-Gal)
5%, others 30%), 3 nmol of M ₉ GN ₂ Asn,
20 μl of a 25 mM acetate buffer (pH 6.0) containing 4 μmol of the acceptor and 1 mU of Endo-A was added to 37
° C, 15 minutes for L-Fuc and L-Gal, 1 for others
After reacting for 0 minutes, the remodeling sugar chain generation rate was measured. Table 3 shows the results. As shown in Table 3, Man, L-
Fuc, L-Gal, Fru, Xyl, 2-deoxy-Glc (manufactured by Sigma), 6-deoxy-Gl
c (manufactured by Sigma), methyl (methyl) α-GlcNAc
(Manufactured by Sigma), 3-O-Me-Glc (manufactured by Sigma)
It can be seen that a sugar transfer reaction has occurred.

[0026]

[Table 3] Table 3 {Acceptor remodeling sugar chain formation rate (%)} ────────────────────────── GlcNAc 96.5 Man 86.7 L-Fuc 25.6 L-Gal 19.3 Fru 6.3 Xyl 24.2 2-deoxy-Glc 90.9 3-deoxy-Glc 0.0 6-deoxy-Glc 77.3 methyl α-GlcNAc 94.3 3-O-Me-Glc 36.1 ─────────────────────────

FIG. 4 shows the formation of remodeled sugar chains by endo-A in an aqueous solvent containing dioxane and an aqueous solvent not containing dioxane. That is, FIG. 4 shows the remodeling reaction in the presence of 0%, 20%, and 30% of dioxane measured over time, the vertical axis represents the generation rate (%) of the remodeling sugar chain, and the horizontal axis represents the reaction time. (Min). FIG.
As shown in the figure, the remodeling reaction proceeded efficiently in the presence of 30% dioxane, and the hydrolysis of the remodeling sugar chain was suppressed.

FIG. 5 is a graph showing the relationship between the acceptor concentration and the generated remodeling sugar chain. The vertical axis indicates the remodeling sugar chain generation rate (%), and the horizontal axis indicates the acceptor concentration (M). . As shown in FIG.
Even in mM, the remodeling reaction progressed, and about 50%
3 shows the production rate of remodeling sugar chains of the present invention. Furthermore, at an acceptor concentration of 100 mM or more, the remodeling sugar chain production rate is 1
In this case, the hydrolysis of the remodeling sugar chain was completely suppressed.

As described above, in the transfer reaction of a saccharide by glycosidase, the remodeling efficiency is remarkably improved by adding a selected water-soluble organic solvent, acetone and dioxane to the reaction solution. The above acetone and dioxane may be used alone, but may be used in combination with acetone and dioxane.

The remodeling reaction in the presence of acetone and / or dioxane is usually performed at room temperature to about 60 ° C., preferably at a temperature of about 30 to 40 ° C. and at a pH of about 5 to 11.
The transfer reaction is completed usually in about 5 minutes to 5 hours, preferably in about 10 to 60 minutes, depending on the reaction conditions. The resulting remodeled carbohydrate or complex carbohydrate contains no by-products of the reaction, and can be easily separated and purified from the reaction end solution according to known means. For example,
The remodeled saccharide or complex saccharide may be separated from the reaction completed solution by gel filtration column chromatography, ion exchange resin column chromatography, or the like, and then concentrated, desalted, freeze-dried, or the like.

According to the present invention, the remodeling reaction proceeds even at a low acceptor concentration, and a remodeling product can be efficiently obtained even when an acceptor having low solubility or an acceptor having a limited amount is used. Acetone and dioxane have a higher remodeling reaction rate than DMSO and DMF, and have a low boiling point, so that they can be easily distilled off from the remodeling reaction solution, and are extremely useful as a substance for promoting the remodeling reaction.

[0032]

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.

Example 1 Examination of Organic Solvent to be Added in Endo-A Transglycosylation Reaction In a 25 mM acetate buffer (pH 6.0), 25
% Organic solvents (DMF, DMSO, acetone, CH ₃ C
N, THF) and 3 nmol of M ₉ GN ₂ Asn, and 5
Prepare a reaction solution containing 00 mM GlcNAc,
Put 3 mU of End-A in each (total volume 20 μ
l) After incubation at 37 ° C for 10 minutes,
The reaction solution was evaporated to dryness under reduced pressure. The dried product was redissolved in water and analyzed by the HPAEC / PAD system. The results are shown in Table 2. As shown in Table 2, the production rate of M ₉ GN ₂ was 100% in the presence of acetone.

Example 2 Investigation of the stability of Endo-A in a reaction solution containing various concentrations of acetone Each of the solutions was prepared in a 25 mM acetate buffer (pH 6.0).
%, 30%, 40%, 45%, and 50% acetone, prepare 3 mU of End-A in each (total volume 20 μl), 0 min at 37 ° C., 5 min.
Minutes, 10, 20, and 30 minutes. 2 μl each of the reaction solution was taken, put into a separately prepared 25 mM acetate buffer (pH 6.0) containing ₃ nmol of M ₉ GN ₂ Asn, and incubated at 37 ° C. for 10 minutes. After a heat treatment at 100 ° C. for 3 minutes, a quarter of the reaction solution was analyzed by an HPAEC / PAD system to check the residual activity. FIG.
Fig. 4 is a diagram showing the relationship between the preincubation time (minutes, horizontal axis) and the relative activity (%, vertical axis) in an acetone-containing reaction solution, and shows that almost no inactivation occurs in 30% acetone.

Example 3 Examination of Acceptor Concentration in Endo-A Transglycosylation Reaction The transglycosylation reaction was performed using ₃ nmol of M ₉ GN ₂ Asn as a donor, and 3 mU of endo-A was 25 mM containing 30% acetone. In acetate buffer (pH 6.0), the concentration of GlcNAc as an acceptor was 25 mM, 50 mM
M, 100 mM, 200 mM, 300 mM, and 500
Each reaction changed to mM was performed at 37 ° C. for 10 minutes. The reaction was stopped by heat treatment at 100 ° C. for 3 minutes, and the hydrolysis product M ₉ GN and the remodeling product M ₉ GN ₂ were subjected to HPAEC / PAD system.
Was measured, and the results are shown in FIG. In FIG. 2, the ordinate represents the PAD response and the abscissa represents the concentration of GlcNAc (M).
It can be seen that when the concentration is 200 mM or more, the transfer reaction almost completely precedes the hydrolysis reaction.

Example 4 Time-dependent change of the product of the sugar chain transfer reaction 3 nmol of M ₉ GN ₂ Asn and 50 mM of GlcN
Ac and 3 mU of Endo-A in 20 μl of 25 mM acetate buffer (pH 6.0) in the presence of 0-50% acetone
Incubate at 37 ° C for 2 minutes, 4 minutes, 8 minutes, 1 minute
After 5 minutes and 30 minutes, respectively,
Analysis was performed by the EC / PAD system, and the results are shown in FIG. In FIG. 3, the vertical axis represents the generation rate (%) of the remodeling sugar chain, and the horizontal axis represents the reaction time (minute). As shown in FIG. 3, the sugar chain donor (M ₉ GN ₂ As
n; *) is reduced and the remodeling product M ₉ GN
₂ (Δ) was found to increase, and after 30 minutes in the presence of 30% acetone, 100% of the remodeling product was obtained.

Example 5 Investigation of sugar chain transfer reaction to various acceptors in an acetone-containing reaction solution 3 nmol of M ₉ GN ₂ Asn as a donor, 4 μmo
1 acceptor, 1 mU of endo-A and acetone (35% for L-Fuc and L-Gal, 30 for others)
% Of 25 mM acetate buffer (pH 6.
0) at 37 ° C. and 15 for L-Fuc and L-Gal.
Minutes, the others were incubated for 10 minutes. The reaction was stopped by heat treatment at 100 ° C. for 3 minutes, and the reaction solution was evaporated to dryness under reduced pressure. The dried product was redissolved in 1/4 of the water of the reaction solution, and analyzed by an HPAEC / PAD system. Table 3 shows the results. However, 2-
When deoxy-Glc and 3-O-Me-Glc were used as acceptors, the reaction solution was subjected to a graphitized carbon column [Shondon Scientific (Shondon Scientific).
(manufactured by ientific). Table 3 shows GlcNAc, Man, L-Fuc, L-Gal, and Fr.
u, Xyl, 2-deoxy-Glc, 3-deoxy-G
lc, 6-deoxy-Glc, methyl α-GlcNA
It shows the remodeling sugar chain production rate (%) when c, 3-O-Me-Glc was used as the acceptor. M
an, L-Fuc, L-Gal, Fru, Xyl, 2-
Deoxy-Glc, 6-deoxy-Glc, methyl α-
It can be seen that a sugar chain transfer reaction has also occurred for GlcNAc and 3-O-Me-Glc.

Example 6 Examination of Acetone Concentration in Endo-A Sugar Chain Transfer Reaction 3 μmol of M ₉ GN ₂ Asn as a donor, 4 μmol of GlcNAc as an acceptor, 20 μl of 25 mM acetic acid containing 1 mU of endo-A and acetone The buffer (pH 6.0) was incubated at 37 ° C. for 15 minutes. The reaction was stopped by heat treatment at 100 ° C. for 3 minutes, and the reaction solution was evaporated to dryness under reduced pressure.
This dried product was redissolved in 1/4 of water of the reaction solution,
Analysis was performed using the EC / PAD system, and the results are shown in FIG. That is, FIG. 6 shows the acetone concentration (%, abscissa) in the acetone-containing reaction solution and M ₉ GN ₂ which is a donor (open circle), a hydrolysis product (closed circle), and a remodeling product (open square mark). FIG. 4 is a diagram showing the relationship between the respective quantitative ratios (%, vertical axis). It can be seen that the proportion of the remodeling product at the acetone concentration of 20% is the highest.

Example 7 Remodeling of Glycopeptide Sugar Chain A glycopeptide having a two-chain complex type sugar chain was prepared from bovine fibrinogen by digestion with a mixture of chymotrypsin and trypsin. The glycopeptide was purified by HPLC and its N-terminus was modified with a naphthylacetyl group. Next is Mucor Himaris (FERM BP-4991)
Endo-M derived from Agricultural and Biologi
cal Chemistry), vol. 54, pp. 97-106 (199)
Purified by the method described in 0). The glycopeptide was thoroughly digested with the enzyme. 1.3 ml of 0.1 M ammonium acetate buffer (pH 6.0) containing 1.25 mmol of glycopeptide and 1.77 mU of Endo-M was incubated at 37 ° C overnight. Glycopeptides before and after digestion with Endo-M were analyzed by HPLC, and the results are shown in FIG.
That is, FIG. 7 is a diagram showing the results of HPLC analysis before (upper) and after (lower) digestion of the glycopeptide with Endo-M, in which the vertical axis shows the fluorescence intensity and the horizontal axis shows the retention time (min). . HPLC column RP-C ₃ (4.6 × 250m
m), Solution A: 50 mM ammonium acetate solution, Solution B: 50 mM containing 50% acetonitrile
Using an ammonium acetate solution, elution was carried out at a flow rate of 1 ml / min at room temperature and 45% B solution. The glycopeptide was detected using a fluorescence detector having an excitation wavelength of 320 nm and a fluorescence wavelength of 380 nm. As shown in FIG. 7, it can be seen that the glycopeptide was almost completely digested and the double-stranded complex type sugar chain was removed. After the deglycosylated glycopeptide [GlcNAc-pentapeptide] was purified by HPLC, its amino acid composition was examined. FIG. 8 shows the result. That is, FIG. 8 is a diagram showing the results of amino acid composition analysis of GlcNAc-pentapeptide.
The UV absorption at 4 nm is shown, and the retention time (min) is shown on the horizontal axis. As shown in FIG. 8, the amino acid composition of GlcNAc-pentapeptide is Asp: Glu: Val: Lys =
1: 2: 1: 1. Based on the previously reported amino acid sequence of fibrinogen, this glycopeptide has the following formula (Formula 4):

[0040]

[Image Omitted] Gln-Val-Glu-Asn (CHO)-
Lys (SEQ ID NO: 1 in Sequence Listing)

(However, CHO indicates a sugar residue bonded to the amide group of Asn). Next, 14 nmol of GlcNAc-pentapeptide was used as an acceptor, and 3 nmol of M ₉ GN ₂ As was used as a donor.
n, 1 containing 3 mU of Endo-A and 35% acetone
0 μl of 50 mM ammonium acetate buffer (pH 6.
0) was incubated at 37 ° C. for 15 minutes. The reaction solution was subjected to HPLC analysis under the same conditions as in FIG. FIG. 9 shows the result. That is, FIG. 9 is a diagram showing the results of HPLC analysis of the glycopeptide before the remodeling reaction (upper) and the glycopeptide after the remodeling reaction (lower), with the vertical axis representing the fluorescence intensity and the horizontal axis representing the retention time (minutes). ). As shown in FIG. 9, a peak considered to be a remodeling product could be confirmed. The remodeling product GlcNAc-
The yield from the pentapeptide was about 5% considering the size of the peak in the chromatogram.

Example 8 Study of endo-A transglycosylation reaction in reaction solutions containing various concentrations of dioxane 4 mU of endo-A and 100 mM of GlcNAc were added to 25 mM acetate buffer (pH 6.0), respectively. Put in, 3
Preincubation was performed at 7 ° C. for 10 minutes. Then, 0%, 10%, 20%, 30%
%, 40%, and 50% of dioxane, and 4 nmol of M ₆ GN ₂ Asn was added as a sugar chain donor (total volume: 40 μl), incubated at 37 ° C. for 10 minutes, and subjected to chain transfer. The reaction was performed. 100 ℃
The reaction was stopped by heat treatment for 3 minutes.
Analyzed by PLC. As shown in Table 4, when the concentration of dioxane was 30% or more, it was found that the transfer reaction almost completely took precedence over the hydrolysis reaction. The PA conversion was carried out according to the method described in Japanese Patent Publication No. 5-65108, and M ₆ G
PA compound of N ₂ (hereinafter abbreviated as M ₆ GN ₂ -PA), M
PA compounds of ₆ GN (hereinafter abbreviated as M ₆ GN-PA) were separately quantified. The remodeling sugar chain generation rate in Table 4 was determined by the following equation (Equation 1).

[0043]

## EQU1 ## Remodeling sugar chain formation rate = [(M ₆ GN ₂ -P
A production amount) / (M ₆ GN-PA production amount + M ₆ GN ₂
-Amount of PA)] x 100

[0044]

Table 4 ──────────────────────────────── Dioxane concentration (%) Remodeling sugar chain generation rate ( %) {0 60 10 90 20 94 30 100 100 50 100} ──────────────────────────

Example 9 Time-dependent change of the transglycosylation product Endo-A 4 mU and 100 mM GlcNAc were added to 25
Three reaction vials pre-incubated at 37 ° C. in a mM acetate buffer (pH 6.0) were prepared. 1
After 0 minutes, dioxane was added to the final concentrations of 0%, 20%, and 30%, and then a sugar chain donor, M ₆ GN ₂ As.
The reaction was carried out at 37 ° C. after adding 6 μg of n. 10 minutes, 3
After 0 minute, 60 minutes, 90 minutes, 180 minutes, and 300 minutes, sampling was performed, and PA treatment was performed after heat treatment at 100 ° C. for 3 minutes. The product was analyzed by HPLC, and the amount of M ₆ GN ₂ -PA produced was measured and shown in FIG. In FIG. 4, the vertical axis represents the generation rate of M ₆ GN ₂ -PA with respect to the sugar chain donor, that is, the remodeling sugar chain generation rate (%), and the horizontal axis represents the reaction time (minute).
Is shown. As shown in FIG. 4, the dioxane concentration was 20%,
Or at 0%, the remodeling product M ₆ GN
₂ is 60 with respect to the sugar chain donor M ₆ GN ₂ Asn.
% Production yield is not exceeded. In the presence of 30% dioxane, a production yield of 100% M ₆ GN ₂ was obtained after 60 minutes. Further, it was found that the transfer product was not hydrolyzed even if the reaction was continued for 240 minutes thereafter.

Example 10 Examination of Acceptor Concentration in 30% Dioxane in Endo-A Sugar Chain Transfer Reaction Using 4 nmol of M ₆ GN ₂ Asn as a sugar chain donor, 4 mU of Endo-A was converted to 30% dioxane. Including 2
In a 5 mM acetate buffer (pH 6.0), the concentration of GlcNAc as an acceptor was 0, 10, 50, 10
Reaction vials with 0, 200, 300, and 500 mM were prepared, and incubated at 37 ° C. for 10 minutes to perform a sugar chain transfer reaction. The reaction was stopped by heating at 100 ° C. for 3 minutes, and the generated M ₆ GN ₂ was
After the conversion, the product was analyzed by HPLC. The result is shown in FIG.
In FIG. 5, the vertical axis represents M ₆ GN ₂ -P for the sugar chain donor.
A generation rate, ie, remodeling sugar chain generation rate (%)
, And the horizontal axis shows the GlcNAc concentration (M). As shown in FIG. 5, it was found that when GlcNAc was 100 mM or more, the transfer reaction almost completely took precedence over the hydrolysis reaction.

[0047]

According to the present invention, there is provided a method for producing a remodeled sugar chain which does not produce a reaction by-product. The method of the present invention is particularly useful in the production of a substance obtained by remodeling the sugar chain of a physiologically active substance that plays an important role in a living body.

[0048]

[Sequence list]

SEQ ID NO: 1 Sequence length: 5 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: peptide Sequence characteristics: The fourth Xaa is an amide group of Asn It is an Asn derivative with a sugar residue attached.

[Brief description of the drawings]

FIG. 1 is a diagram showing the stability of an enzyme under each acetone concentration.

FIG. 2 is a diagram showing a relationship between a donor concentration and a reaction product yield.

FIG. 3 is a diagram showing the relationship between reaction product yield and time under each acetone concentration.

FIG. 4 is a diagram showing the relationship between the remodeling generation rate and time under each dioxane concentration.

FIG. 5 is a diagram showing a relationship between a donor concentration and a remodeling generation rate.

FIG. 6 is a diagram showing the relationship between acetone concentration and donors, hydrolysates, and remodeling products.

FIG. 7 shows HPLC analysis results before and after digestion of a glycopeptide with endo-M.

FIG. 8 shows the results of amino acid composition analysis of GlcNAc-pentapeptide.

FIG. 9: H before and after remodeling reaction of glycopeptide
It is a figure which shows a PLC analysis result.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Shojiro Iwahara 3325-7 Ikedo, Miki-cho, Kida-gun, Kagawa Prefecture (72) Inventor Akihiro Kondo 3-4-1 Seta, Otsu-shi, Shiga Prefecture Takara Shuzo Co., Ltd. In-house (72) Inventor Ikunoyuki Kato 3-4-1, Seta, Otsu-shi, Shiga Takara Shuzo Co., Ltd. Central Research Laboratory (58) Field surveyed (Int. Cl. ⁷ , DB name) C12P 21/00- 21/06 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A method selected from End-A and End-M.
A method for producing a saccharide or glycoconjugate using a remodeling reaction represented by the following formula (1) with an endoglycosidase, which comprises a step of performing the remodeling reaction in an aqueous solvent ; method for producing a carbohydrate or a glycoconjugate said aqueous solvent and said aqueous solvent der Rukoto containing a water-soluble ketone and / or dioxane. AB + C → AC + B (1) (where A is a saccharide containing GlcNAc at the terminal, and B is a saccharide
Carbohydrate or complex carbohydrate, C indicates carbohydrate or complex carbohydrate) 2. The method according to claim 1, wherein the water-soluble ketones are acetone, methyl ethyl ketone and diethyl ketone.