JPH07289280A - Production of nonreducing terminal 6-azido-maltopentaose - Google Patents

Abstract

PURPOSE:To efficiently. obtain the subject compound useful for a substrate, etc., for measuring the activities of an alpha-amylase by enzymolyzing a 6-azido- cyclodextrin in the presence of glucose and then reacting the resultant compound with a converting enzyme and a saccharifying enzyme in the presence of maltose. CONSTITUTION:This method for producing a nonreducing terminal 6-azido- maltopentaose expressed by formula III is to react a 6-azido-cyclodextrin expressed by formula I [(n) is 5 or 6] with a cyclodextrinase having functions to cleave the cyclodextrin and produce a maltooligosaccharide in accordance with the glucose polymerization degree of the cyclodextrin and the higher substrate specificity in the hydrolytic rate or affinity for the cyclodextrin than those of polysaccharides or a straight-chain oligosaccharide having the same polymerization degree as that of the cyclodextrin in the presence of glucose, produce the 6-azido-maltooligosaccharide expressed by formula II [(m) is 3-6 when (n) is 5 and 3-7 when (n) is 6], subsequently react the resultant azido- maltooligosaccharide with a cyclodextrin glucanotransferase and exo type saccharifying enzymes.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel method for producing non-reducing terminal 6-azido maltopentaose. More specifically, the present invention is itself a substrate for measuring α-amylase activity, and in particular, it is an aromatic chromophore as a glycoside at the 1-position of reducing terminal glucose, which is extremely useful as a substrate for measuring α-amylase activity. The present invention relates to a method for efficiently producing a non-reducing terminal 6-azido maltopentaose used as an intermediate for a non-reducing terminal 6-azido maltopentaoside derivative having a group introduced therein.

[0002]

2. Description of the Related Art As a method for producing a non-reducing end-modified oligosaccharide, cyclomaltodextrin glucanotransferase is allowed to act on modified cyclodextrin in the presence of an acceptor, followed by glucoamylase or α.
-A method for acting a glucosidase is known (Japanese Patent Laid-Open No. 63-170393), but this method is unavoidable because the proportion of by-products that cannot be reused is relatively large, and therefore it is based on the raw materials used. The yield could not be increased, and it was not always satisfactory as an industrial method.

Further, the present inventors first produced a non-reducing terminal 6-azido maltoperse by reacting 6-azido cyclodextrin with cyclodextrinase and then with exo type saccharifying enzymes. A method has been proposed (Japanese Patent Laid-Open No. 5-262784). However, this method is also practically due to the cleavage site when cleaving cyclodextrin with cyclodextrinase,
The production of by-products that cannot have the desired structure cannot be suppressed, and the yield is still unsatisfactory.

[0004]

The present invention is particularly applicable to α-
A non-reducing terminal 6 which is extremely useful as a substrate for measuring amylase activity. A non-reducing terminal 6 having an aromatic chromophoric group as a glycoside introduced at the 1-position of glucose is used as an intermediate of an azido maltopentaoside derivative. -Provided for the purpose of providing a method for efficiently producing azido maltopentaose in a large amount.

[0005]

The inventors of the present invention have conducted various studies to achieve the above-mentioned object, and as a result, a cyclodextrinase, which is a hydrolase having a specific physicochemical property, cleaves cyclodextrin. And 6-azidocyclodextrin obtained by reacting 6-azidated cyclodextrin with glucose in the presence of glucose. When a sugar transferase, cyclodextrin glucanotransferase, is allowed to act on azidated maltooligosaccharides in the presence of maltose, maltose causes the second and third glucose residues from the 6-azidated glucose residue toward the reducing end. 6-azido maltopentaose which is the non-reducing end of the target substance in the reaction solution Content of malto-oligosaccharides on the reducing end side of Escherichia coli is increased, and exo-type saccharifying enzymes are allowed to act after or before the enzymatic action of the latter to hydrolyze and remove glucose residues on the non-reducing end side. As a result, it was found that non-reducing terminal 6-azido maltopentaose can be efficiently and easily produced, and the present invention has been completed based on these findings.

That is, the present invention has the general formula

[Chemical 4] In the presence of glucose, 6-azidated cyclodextrin represented by the formula (n is 5 or 6)
The action of cleaving cyclodextrin to form malto-oligosaccharide derived from the degree of glucose polymerization of cyclodextrin and (b) the hydrolysis rate or affinity for cyclodextrin is higher than that of polysaccharide or linear oligosaccharide having the same degree of polymerization as cyclodextrin. By reacting cyclodextrinase with a large substrate specificity, the general formula

[Chemical 5] (Wherein n has the same meaning as described above, m is an integer of 3 to 6 when n is 5, and an integer of 3 to 7 when n is 6) to produce a 6-azido maltooligosaccharide. And then allowing the cyclodextrin glucanotransferase and exo-type saccharifying enzymes to act in any order in the presence of maltose, the formula

[Chemical 6] The present invention provides a method for producing non-reducing terminal 6-azido maltopentaose represented by:

The present invention will be described in detail below. The 6-azidated cyclodextrin represented by the general formula (I), which is the starting material in the present invention, may be produced by any method. For example, commercially available α- or β-cyclodextrin (the degree of glucose polymerization is 6 , 7), and preferably can be produced from β-cyclodextrin by a known method because it is inexpensive.

A preferred embodiment of this production method will be described. First, cyclodextrin is dissolved in a solvent such as pyridine, and the cyclodextrin is mixed with 2 to 7 parts thereof.
Double molar amount of tosyl chloride is added, usually 15 ~ 30 ℃
By reacting at a temperature in the range of about 4 to 6 hours for tosylation of only one of the 6-position hydroxyl groups, and if necessary purification by a conventional method, 6-O-tosylcyclodextrin is obtained. Then, 2 to 50 times molar amount of sodium azide is added to 6-O-tosylcyclodextrin in a polar solvent, and usually 3 to 3 at a temperature in the range of 70 to 90 ° C.
The reaction is carried out for about 10 hours to replace the tosyloxy group with an azido group. As the polar solvent used at this time, for example, water,
Acetone, 1,4-dioxane, acetonitrile, dimethylformamide and the like can be mentioned, and these may be used alone or in admixture of two or more. And if necessary,
6-Azidocyclodextrin can be obtained by purification according to a conventional method such as a method of filtering tetrachloroethane inclusion complex from an aqueous solution or a method of filtering toluene inclusion complex [Carbohydrate Research (Carbohydrate
r. Res. ), Vol. 18, pp. 29-37 (19
71)]].

Thus, the 6-azidated α-cyclodextrin (n = 5) or 6-azidated β-cyclodextrin (n = 6) as the starting material represented by the general formula (I) can be easily prepared. However, 6-azido β-cyclodextrin is preferable as a raw material in view of the enzymatic reaction rate in the subsequent step.

FIG. 1 shows that 6-azido β-
It is explanatory drawing which shows the production | generation process of the target substance in the manufacturing method of this invention taking the case where cyclodextrin is used as an example.

In FIG. 1, G and G ^N3 are glucose residues, glucose residues in which the hydroxyl group at the 6-position is replaced with an azide group, CDase and CGTa, respectively.
se means cyclodextrinase and cyclodextrin glucanotransferase, which will be described later, respectively.

Next, 6-represented by the above general formula (I)
Regarding cyclodextrinase acting on azidated cyclodextrin, (a) it has an action of cleaving cyclodextrin to produce maltooligosaccharide derived from the glucose polymerization degree of cyclodextrin, and (b)
Hydrolysis rate or affinity for cyclodextrin is not particularly limited as long as it has a larger substrate specificity than a linear oligosaccharide having the same degree of polymerization as a polysaccharide or cyclodextrin, and is not particularly limited. There is no particular restriction on the origin.

As such an enzyme, a known cyclodextrinase having the following physicochemical properties can be mentioned as a suitable one. In addition, this cyclodextrinase (hereinafter referred to as C)
Dase) is a hydrolase and is a cyclomaltodextrinase.
also referred to as "inase" and belongs to EC 3.2.154 [[Appl Microbiol
Biotechnol ", Vol. 39, pp. 714-719 (1993)].

Physicochemical properties (a) Action: It has the action of cleaving cyclodextrin to form maltooligosaccharide derived from the glucose polymerization degree of cyclodextrin.

(B) Substrate specificity: Hydrolyzation rate or affinity for cyclodextrin is higher than that of polysaccharide or linear oligosaccharide having the same degree of polymerization as cyclodextrin.

(C) Optimum pH and stable pH range: When β-cyclodextrin is used as a substrate, it has an optimum pH in the vicinity of pH 8.0 and has a stable pH range of 5.5 to 9.5. . (D) Optimum temperature of action: Has a proper temperature of action around 40 ° C. (E) Deactivation: It has a property of being almost deactivated by treatment at a temperature of 50 ° C. or higher for 15 minutes.

(F) Inhibition and activation: Hg ²⁺ , Cu ²⁺ ,
90% or more inhibition by Zn ²⁺ , Ni ²⁺ and Fe ²⁺ ,
It has the property of being activated by Ca ²⁺ and Mg ²⁺ by 10 to 30%.

(G) Molecular weight: The molecular weight by gel filtration is 144,000 and the molecular weight by SDS PAGE is 72,000. That is, this enzyme has a molecular weight of 7
It is a dimer composed of 2,000 subunits.

The titer of this enzyme is 2% (w / v).
Concentration of β-cyclodextrin solution (500 μl) and appropriate amount of enzyme in 100 mM phosphate buffer (p
H7.5) (500 μl) was mixed and allowed to react at a temperature of 40 ° C. for an appropriate time, then the reaction was stopped by boiling for 10 minutes, and the produced maltoheptaose was quantified by high performance liquid chromatography. When the amount of enzyme was small, it was determined by measuring the reducing power by the Somogene Nelson method using glucose as a standard.

Regarding the enzyme unit of this enzyme, the amount of the enzyme that produces 1 micromol of maltoheptaose per minute was 1 unit. An enzyme having such a property belongs to the genus Bacillus, for example, and a microorganism producing the enzyme, for example, Bacillus sphicus (Bacillus sph).
aericus) E-244 strain [Institute of Industrial Science and Technology, Institute of Microbial Engineering (currently Institute of Biotechnology, Institute of Industrial Science and Technology), Micro Engineering Lab. No. 2458 (FERM BP-245)
8) has been deposited] and the like are cultivated in a medium and collected from this culture.

The physicochemical properties of the above-mentioned enzyme and its producing bacterium Bacillus sphaericus E-244 strain (FERM
The mycological properties of BP-2458) and the method for producing an enzyme using the same are known (JP-A-3-15384 and JP-A-3-86701).

The 6-azido cyclodextrin represented by the above general formula (I) is added to the above C in the presence of glucose.
When Dase is allowed to act, glucose is transferred only once to an unspecified position of the cyclic sugar chain due to the transglycosylation activity of the enzyme, and 6-positions at various positions represented by the general formula (II) are obtained.
A reaction solution containing a mixture of 6-position azido-malto-oligosaccharides having 2-position azido-glucose residues as a main component is obtained. In this reaction, the presence of glucose increases the theoretical production of a substance that can be the target compound by about 30% as compared with the absence thereof. When maltose was used instead of glucose, this transglycosylation reaction was hardly observed.

The concentration of 6-azidated cyclodextrin (starting substance) in this enzymatic reaction is not particularly limited, but it is preferable to adjust it so that the concentration is not less than the Km value for the substrate of CDase. The amount of glucose to coexist is not particularly limited, and is usually selected from the range of 3 to 20 times the molar amount of 6-azidated cyclodextrin.
The amount of enzyme is not particularly limited, but it may be added in an appropriate amount so that the amount of product is maximized within the reaction time, and is usually in the range of 0.5 to 10 units per 1 g of the starting material. To be elected.

The enzyme reaction conditions are not particularly limited as long as they are within the range of action pH and action temperature of CDase, but the reaction is usually carried out under the conditions of pH 6.5 to 9.0 and temperature 35 to 45 ° C. A reaction time of 30 minutes to 48 hours is suitable. Furthermore, in this reaction, if necessary, 1,
4-dioxane, acetone, dimethyl sulfoxide (D
A water-soluble organic solvent such as MSO) or N, N-dimethylformamide (DMF) may be added.

After completion of the enzymatic reaction by CDase, acid treatment with, for example, hydrochloric acid, acetic acid, etc., 75-1
It is preferable to deactivate the enzyme by heat treatment at 00 ° C. for 30 to 180 minutes.

In this way, a reaction solution containing a mixture of various 6-azidated maltooligosaccharides represented by the general formula (II) as a main component is obtained.
For example, when the starting material is 6-azido-β-cyclodextrin (n = 6), a compound mainly composed of a mixture of 6 ^p -azido-maltooctaose (p = 2 to 8) is obtained. , in the case of 6-azide α- cyclodextrin (n = 5) is similarly 6 ^q - is obtained as a main component a mixture of azide maltoheptaose (q = 2 to 7 integer) .

Symbols such as 6 ⁸ -and 6 ⁷ -are, for example,
It is shown that the hydroxyl group at the 6-position of the glucose residues 8th and 7th from the reducing end side of glucose constituting malto-oligosaccharide is substituted.

Next, a cyclodextrin glucanotransferase is added to a reaction solution containing a mixture of various 6-azidated maltooligosaccharides represented by the general formula (II) thus obtained as a main component in the presence of maltose. And by reacting exo-type saccharifying enzymes in any order, that is, after allowing cyclodextrin glucanotransferase to act in the presence of maltose, the exo-type saccharifying enzymes are allowed to act, or the exo-type saccharifying enzymes are reacted. After acting, the cyclodextrin glucanotransferase acts in the presence of maltose,
A non-reducing terminal 6-azido maltopentaose represented by the above general formula (III) is obtained as a target substance.

That is, by reacting cyclodextrin glucanotransferase in the presence of maltose on a reaction solution containing a mixture of various 6-azidated maltooligosaccharides represented by the general formula (II) as a main component, maltose Selective transfer from the 6-azido-glucose residue to the second and third glucose residues toward the reducing end, and 4 glucose residues were polymerized from the 6-azido-glucose residue toward the reducing end. A general formula whose main component is a maltooligosaccharide having a structure

[Chemical 7] A reaction solution containing a mixture of various 6-azidated maltooligosaccharides represented by the formula (n and m have the same meaning as described above) is obtained.

[0030] The enzymatic reaction in the presence of maltose, as shown in FIG. 1, in the reaction solution, the reducing end in the non-reducing ends 6 azide maltopentaose and molecular 6 ⁵ - azide malto 6-with pentaose part
It has the significance of increasing the content of azidated maltooligosaccharides. That is, 6-azido maltopentaose in the reaction solution before the enzymatic reaction and 6 ⁵ on the reducing end side in the molecule
The theoretical content of 6-azido-malto-oligosaccharide having an azido-maltopentaose moiety is as follows when the starting material is 6-azido-α-cyclodextrin (n = 5) represented by the general formula (I). In the case of 1/7, 6-azido-β-cyclodextrin (n = 6), it is 1/6, but due to the enzymatic reaction, their theoretical contents in the reaction solution are 5/7 and 4 respectively. It can be increased to 5 or 4 times as much as / 6, and as a result, high yield of the non-reducing terminal 6-azido maltopentaose of the target substance can be obtained by acting exo-type saccharifying enzymes in the next step. You can get at a rate.

The reaction solution obtained by the enzymatic reaction of the previous step may be directly used for the enzymatic reaction of this step. However, in this step, the glucose transfer of glucose and various unsubstituted maltooligosaccharides contained in the reaction solution is carried out. Since they occur as side reactions, it is preferable to remove them beforehand by, for example, octadecyl silica gel (ODS) or activated carbon column chromatography in order to improve the reaction yield.

Further, as shown in FIG. 1, the reaction solution obtained in the previous step, 6 ³ , 6 ² -azidated maltooctaose or 6 ³ , 6 ² -azidated maltoheptaose,
Since the glycosyl transfer in this step does not occur and is present in the reaction solution as it is, even if these 6-azido maltooligosaccharides are removed in advance by an appropriate purification method such as activated carbon column chromatography or ODS column chromatography, Good.

Cyclodextrin glucanotransferase used in the present invention
Rin glucanotransferase (hereinafter referred to as CGTase) is a known transferase, and is a cyclomaltodextrin glucanotransferase (c
ychromaltodextrin glucanano
EC).
It belongs to 4.1.19. The origin of CGTase is not particularly limited, and includes, for example, the genus Bacillus [eg, Bacillus maceran].
s), Bacillus megaterium
etc.] derived from the genus Klebsiella [eg Klebsiella pneumoniae (Klebs)
iellapneumoniae) etc.] and the like are used.

The reaction conditions for the action of this CGTase are the same as those used in ordinary enzyme reactions and are not particularly limited, but the pH is 5.5 to 8.0 and the temperature is 35 to 5.
The reaction time at 0 ° C. is preferably about 0.5 to 48 hours. C
The amount of GTase used is not particularly limited as long as it is used in the case of a normal enzyme reaction, but it is 50 to 50 g with respect to the total weight of 1 g of the mixture of various 6-azidated maltooligosaccharides represented by the general formula (II). It is good to choose in the range of 1000 units. Further, the amount of maltose to be coexisted is not particularly limited, and is selected from the range of 3 to 20 times the molar amount of the mixture of various 6-azidated maltooligosaccharides obtained in the previous step.

In this reaction, 1,4-
A water-soluble organic solvent such as dioxane, acetone, DMSO, DMF or isopropanol may be added. For the purpose of removing glucose by-produced in the reaction system, enzymes that do not react with maltose or maltooligosaccharides, but react only with glucose, such as glucose oxidase,
Glucose dehydrogenase and the like can also be added.

After completion of the enzymatic reaction with CGTase, acid treatment using, for example, hydrochloric acid or acetic acid,
The enzyme is inactivated by heat treatment at 100 ° C. for 30 to 180 minutes.

In this way, a reaction solution containing a mixture of various 6-azidated maltooligosaccharides represented by the general formula (IV) as a main component can be obtained. As the reaction solution, for example, the starting material is 6-azido. Β-cyclodextrin (n = 6), 6 ⁵ -azido maltononaose to 65 ⁵ -azido maltopentases (having a degree of polymerization of glucose of 9, 8, 7, 6 and 5) , And in the case of 6-azidated α-cyclodextrin (n = 5), 6
⁵ -Azido maltooctaose to 65 ⁵ -Azido maltopentose (having a degree of polymerization of glucose of 8, 7, 6 and 5) as a main component can be mentioned.

Then, in this reaction solution, the
Unreacted 6 noted³, 6²-Azido maltooctaose
Or 6³, 6²-C in addition to azido maltoheptaose
Generated from maltose coexisted by the action of GTase
Glucose, maltotriose, maltotetraose
6 caused by further glycosyl transfer^Four-Azido Maruto
Kutaose ~ 6^Four-Azido maltotetraose (glu
Degree of polymerization of course is 8, 7, 6, 5 and 4), 6⁶
-Azido maltodecaneose ~ 6⁶-Azido maltohe
Xaose (the degree of polymerization of glucose is 10, 9, 8, 7 and
6 and 6), 6 ⁷-Azido-maltoundecanose-
6⁷-Azido maltoheptaose (polymerization of glucose
Degree 11, 10, 9, 8 and 7) etc.
It

It should be noted that the enzyme 6-azidated cyclodextrin, which is a starting material, is not directly subjected to the enzymatic reaction by CDase in the presence of glucose as in the method of the present invention, but the enzyme by CGTase is directly present in the presence of maltose. the reaction for 6 be ⁵ - reducing end side of the azide maltopentaose and molecular 6 ⁵ - reaction solution containing 6-azido-malto oligosaccharides with azide maltopentaose unit is obtained, but in this case Has a very low reaction yield and cannot achieve the object of the present invention. As a cause of this, it is considered that CGTase easily causes a cleavage reaction or a rearrangement reaction in addition to the transfer reaction of maltose.

Next, the exo-type saccharifying enzyme is allowed to act on the reaction solution thus obtained, so that the 6-azido-glucose residue does not exist at the non-reducing end side so that the 6-azido glucose residue becomes the non-reducing end. nonreducing end 6 azide maltopentaose represented substituted glucose residue at the a is hydrolyzed formula (III) (6 ⁵ - azide maltopentaose)
The main component of is obtained. The general formula (II
OH in I) indicates that the OH group is the α-anomer, β-anomer or a mixture of both.

In addition, as a secondary component, 6 ²
-Azido-maltose, 6 ³ -Azido-maltotriose, 6 ⁴ -Azido-malto tetraose, 6 ⁶ -Azido-malto hexaose, 6 ⁷ -Azido-malto heptaose and the like are contained.

Examples of the exo-type saccharifying enzymes used in this case include known glucoamylase and α-glucosidase. These may be used alone or in combination. Further, the origin thereof is not particularly limited, and examples thereof include glucoamylase derived from Rhizopus and α-glucosidase derived from yeast.

The reaction conditions when the exo-type saccharifying enzymes are allowed to act may be appropriately selected depending on the working pH and working temperature range of the enzyme used, but usually at pH 4.0 to 8.0 and temperature 35 to 50 ° C., The reaction is performed for about 0.5 to 48 hours.

The amount of the exo-type saccharifying enzyme used is not particularly limited as long as it is used in a usual enzymatic reaction, but it is usually 6 kinds represented by the general formula (IV).
-Selected in the range of 50 to 1000 units for a total weight of 1 g of the mixture of azido maltooligosaccharides. Further, this enzymatic reaction can be stopped by acid treatment, heat treatment or the like as in the case of CDase or CGTase.

Next, the case where CGTase is allowed to act after the exo-type saccharifying enzyme is allowed to act on the reaction solution containing various 6-azido maltooligosaccharides represented by the general formula (II) as the main components will be described.

That is, in this case, the glucose residue on the non-reducing end side is hydrolyzed and removed by the action of exo-type saccharifying enzymes, and the starting material is 6
-In the case of azido-β-cyclodextrin, the non-reducing terminal 6-azido maltooctaose to the non-reducing terminal 6-
Azido maltose (the degree of polymerization of glucose is 8, 7,
6, 5, 4, 3 and 2), and the starting material is 6
In the case of azido-α-cyclodextrin, the non-reducing end 6-azido maltoheptaose to the non-reducing end 6-
Azido maltose (the degree of polymerization of glucose is 7, 6,
A reaction solution containing a mixture of 5, 4, 3 and 2) as a main component is obtained.

The reaction conditions with the exo-type saccharifying enzymes in this case may be appropriately selected depending on the working pH and working temperature range of the enzyme to be used, but usually pH 4.0 to 8.0 and temperature 35.
The reaction is carried out at -50 ° C for about 0.5-48 hours. Further, the amount of exo-saccharifying enzymes used is not particularly limited, but is usually in the range of 50 to 1000 units per 1 g of the total weight of the mixture of various 6-azidated maltooligosaccharides represented by the general formula (II). Is selected in.

Then, this enzymatic reaction is stopped by acid treatment or heat treatment in the same manner as described above, and then CGTase is allowed to act in the presence of maltose as described above to give the compound represented by the above general formula (IV). The resulting 6-azido-malto-oligosaccharide has no non-reducing terminal glucose residue. At this time, regarding the reaction conditions and the like for allowing CGTase to act in the presence of maltose,
The same as the above is adopted.

In a particularly preferred embodiment of the present invention,
For example, by reacting 6-azidated cyclodextrin represented by the general formula (I) with CDase in the presence of glucose, when the product becomes maximum, the reaction is temporarily stopped by acid treatment or heat treatment. After that, for example, the reaction solution is subjected to ODS column chromatography and the like to remove glucose and various unsubstituted maltooligosaccharides, and then CGTase is allowed to act in the presence of maltose, and when the product becomes maximum, Deactivate CGTase by acid treatment, heat treatment, or the like. Next, after adjusting the liquidity to an appropriate pH, let exo-type saccharifying enzymes act,
A method in which the reaction is stopped by acid treatment, heat treatment, or the like when the product becomes maximum.

Next, the desired non-reducing terminal 6-azido maltopentaose represented by the general formula (III) is separated from the thus obtained reaction solution containing non-reducing terminal 6-azido malto-oligosaccharide. Purification is carried out, but there is no particular limitation on this separation and purification method, and a method conventionally used for separation and purification of oligosaccharides can be used. For example, activated carbon column chromatography, ODS column chromatography, silica gel column chromatography,
A method of collecting fractions by using thin layer chromatography or the like can be adopted.

The non-reducing terminal 6-azido maltopentaose represented by the general formula (III) thus obtained can itself be used as a substrate for measuring α-amylase activity. Which can be used very suitably as a substrate for use, for example, a general formula in which an aromatic chromophore is introduced as a glycoside at the 1-position of the reducing terminal glucose of the compound,

[Chemical 8] It is useful as an intermediate for a non-reducing terminal 6-azido maltopentaoside derivative represented by (X in the formula is an aromatic color-forming group).

In the non-reducing terminal 6-azido maltopentaoside derivative represented by the general formula (V), the aromatic chromophoric group of X may be any as long as it can be spectroscopically detected. , And the derivative is α-anomer (α-glycoside) or β-anomer (β-glycoside)
Either of them may be used.

Represented by the above general formula (V)
Examples of the compound include 2-chloro-4-nitrophenyl
Le = 6^Five-Azido-6^Five-Deoxy-β-maltopentao
Sid, 2-fluoro-4-nitrophenyl = 6^Five-Horse mackerel
Do-6^Five-Deoxy-α-maltopentaoside, 4-ni
Trophenyl = 6^Five-Azido-6^Five-Deoxy-β-mal
Topentaoside, phenol indo-3'-chlorophe
Nil = 6^Five-Azido-6^Five-Deoxy-β-maltopenta
Osid, resazurinyl = 6^Five-Azido-6^Five-Deoxy-
β-maltopentaoside, 4-aminophenyl = 6^Five-
Azide-6^Five-Deoxy-β-maltopentaoside, 4
-Methylumbelliferonyl = 6^Five-Azido-6^Five-Deo
Xy-α-maltopentaoside, 2-chloro-4-nit
Rophenyl = 6^Five-Azido-6^Five-Deoxy-α-malto
Pentaoside, luciferinyl = 6 ^Five-Azido-6^Five-De
Oxy-β-maltopentaoside and the like can be mentioned.

Incidentally, the non-reducing terminal 6-azido maltopentaose represented by the general formula (III) is α-
Reagent for measuring α-amylase activity when used as a substrate for measuring amylase activity, method for measuring α-amylase activity, and further non-reducing terminal 6-azid maltopentaoside derivative represented by the general formula (V) , A reagent for measuring α-amylase activity when the derivative is used as a substrate for measuring α-amylase activity, and a method for measuring α-amylase activity are already known (for example, JP-A-5-262784). .

[0055]

According to the method of the present invention, the above-mentioned general formula (II
The non-reducing terminal 6-azido maltopentaose represented by I) can be mass-produced efficiently and unprecedented by a short reaction process and a simple operation using inexpensive cyclodextrin as a starting material.

[0056]

EXAMPLES Next, the present invention will be described in more detail by way of examples. The specific optical rotation in each example is a value measured by the D line of sodium at 25 ° C.

Example 1 Preparation of 6 ⁵ -azido-6 ⁵ -deoxymaltopentaose (non-reducing terminal 6-azidomaltopentaose) (1) 6-azido-6-deoxy-β-cyclodextrin (6- (A) Manufacture of 6-O-tosyl-β-cyclodextrin Commercially available β-cyclodextrin [manufactured by Shimizu Minato Sugar Co., Ltd.]
100 g (88.2 mmol) was dissolved in 200 ml of pyridine, and tosyl chloride 22 g, 22 g, was added at intervals of 30 minutes.
After adding 23 g (total 67 g, 350 mmol) and adding the last tosyl chloride, the reaction was carried out at room temperature for 1.5 hours while stirring. Then add 10 ml of water to this reaction solution.
After adding, the solvent was distilled off under reduced pressure, 100 ml of water was added to the obtained concentrated liquid, and the solvent was distilled off again under reduced pressure.
After 1000 ml of water was added to the obtained residue and the mixture was stirred, a small amount of crystal seeds were added and the mixture was allowed to stand at room temperature for crystallization. The crystals are filtered off with a glass filter and water 200m
3, washed with 300 ml of methyl ethyl ketone and 300 ml of methyl ethyl ketone and then dried to give 37.2 g (28.9 mmol, yield 32.60%) of 6-O-tosyl-β-cyclodextrin.
8%) was obtained.

Melting point (° C): 172.0 to 174.0 (decomposition) Infrared absorption spectrum (cm- ¹ ): 3400, 293
0,1642,1632,1600,1424,136
0,1300,1178,1156,1078,102
8 Nuclear magnetic resonance spectrum (200 MHz) ppm: (DM
SO-d ₆ ) 2.44 (3H, s), 3.15 to 4.4
5 (m), 4.76 (2H, br.s), 4.85 (5
H, br. s), 7.44 (1H, d, J = 8.8H
z), 7.75 (1H, d, J = 8.8Hz)

High performance liquid chromatography [TSK gel Amide-80 column (4.
6 mm ID x 250 mm), RI detection, eluent: acetonitrile / water = 3: 2 (v / v), flow rate: 1.0 ml /
min]: t _R = 5.5 min

(B) Preparation of 6-azido-6-deoxy-β-cyclodextrin 35 g (27 mmol) of 6-O-tosyl-β-cyclodextrin obtained in (a) was dissolved in 100 ml of DMF to prepare sodium azide. 5.3 g (81 mmol) was added and reacted at 95 ° C. for 1.5 hours. Then, the reaction solution was concentrated to dryness under reduced pressure, 30 ml of water was added to the obtained residue to dissolve it, a small amount of crystal seed was added, and the mixture was left to cool to 5 ° C. for crystallization. The crystals were filtered off with a glass filter, washed with 5 ml of cold water and 50 ml of cold ethanol, and then dried to obtain 27 g (23 mmol, yield 86%) of 6-azido-6-deoxy-β-cyclodextrin.

Melting point (° C.): 215.0 to 218.0 (decomposition) Infrared absorption spectrum (cm ⁻¹ ): 3390, 2920,
2120, 1642, 1414, 1370, 1340,
1304, 1156, 1080, 1030 High performance liquid chromatography [TSKg manufactured by Tosoh Corporation]
el Amide-80 column (4.6 mm ID x 25
0 mm), RI detection, eluent: acetonitrile / water =
3: 2 (v / v), flow rate: 1.0 ml / min]: t _R
= 6.6 min Specific rotation [α]: (c 0.510,1,4-dioxane / H ₂ O = 1: 1 (v / v); + 145 °

Elemental analysis value: C H N theoretical value (%) 43.49 6.00 3.62 as C ₄₂ H ₆₉ N ₃ O ₃₄ Actual value (%) 43.28 6.11 3.53

(2) Preparation of CDase 1% (w / v) β-cyclodextrin, 1% (w /
v) peptone, 0.5% (w / v) NaCl and 0.1
100 ml of a liquid medium (using tap water, pH 7.0) consisting of% (w / v) yeast extract was placed in a 500 ml Sakaguchi flask and sterilized at 120 ° C. for 20 minutes.
In addition, Bacillus sphaericus E-244 (FERM
One platinum loop was inoculated from the stored slant of BP-2458), and cultured at 30 ° C. for 1 day with shaking. 50m of this culture
1 ml was inoculated into a 3000 ml mini jar containing 2000 ml of medium prepared by the same medium composition and sterilization conditions as above, and aerated and agitated at 30 ° C., 1 vvm and 350 rpm for 2 days. The cells were separated from the culture solution by centrifugation at 8000 rpm for 20 minutes, and 500 ml of 10 mM phosphate buffer (pH 7.0) containing 2% (w / v) Triton X-100.
The microbial cells were suspended and stirred at 25 ° C for 1 day. After the bacterial cell residue was removed from the suspension by centrifugation at 12000 rpm for 20 minutes, the supernatant was dialyzed against 10 mM phosphate buffer (pH 7.0) for 16 hours. The dialyzed product obtained was centrifuged at 12000 rpm for 20 minutes to remove insoluble materials, and the supernatant was used as a crude enzyme solution (1).

Then, about 500 ml of this crude enzyme solution (1)
(Total activity 200 units, protein amount 2083 mg, specific activity 0.1, pH 7.0) in 10 mM phosphate buffer (pH
It was applied to a DEAE sepharose packed column (φ34 × 170 mm) equilibrated with 7.0) to adsorb the enzyme, and then elution was performed with a gradient gradient of 0 to 1.5 M NaCl. The active fractions thus obtained were collected to obtain 105 ml of crude enzyme solution (2) (total activity 145 units, specific activity 0.58, yield 72.5%). Then, 20 ml of this crude enzyme solution (2) (total activity 31 units, protein amount 29 mg) was added to 100 m containing 1M sodium sulfate.
Ether 5 equilibrated with M phosphate buffer (pH 7.0)
After applying to a PW packed column (φ21.5 × 150 mm) to adsorb the enzyme, elution was performed with a gradient of 1M to 0 sodium sulfate. 50 ml of the crude enzyme solution (3) was collected by collecting the active fractions thus obtained.
(Total activity 72 units, specific activity 2.93, yield 36%) was obtained.

(3) Preparation of 6 ^p -azido maltooctaose (p = 2-8 integer) 5.0 g (4.31 mmol) of 6-azido-6-deoxy-β-cyclodextrin obtained in (1) ) Was preheated to 40 ° C. in 10 mM phosphate buffer (pH
= 7.5) It was added to 200 ml while stirring and completely dissolved. 23 units of the crude enzyme solution (3) of CDase obtained in the above (2) and 10.0 g of glucose (55.
5 mmol) was added and the reaction was carried out while stirring at 40 ° C. for 7 hours. After completion of the reaction, the reaction solution was heated at 90 ° C. for 15 minutes while stirring. Then, the reaction solution was cooled to room temperature, filtered through Standard Super Cell (manufactured by Celite), washed with water, and the filtrate and the washing solution were combined and subjected to ODS column chromatography for purification, and an acetonitrile-water mixed solution was added. Elution with (volume ratio 0% → 5% gradient), concentration to dryness, and 6 ^p -azido maltooctaose (p = integer of 2 to 8) 2.6 g (1.94 mmol, 4
5.0%) was obtained.

High Performance Liquid Chromatography [TSK gel Amide-80 column (4.
6 mm ID x 250 mm), RI detection, eluent: acetonitrile / water = 3: 2 (v / v), flow rate: 1.0 ml /
min]: t _R = 12.4 min

(4) Preparation of 6 ⁵ -azido-6 ⁵ -deoxymaltopentaose 6 ^p -azido maltooctaose 600 obtained in (3)
mg (0.448 mmol) was dissolved in 30 ml of 5 mM PIPES buffer solution (pH = 6.0) with stirring, and there, Contizyme [enzyme solution manufactured by Amano Pharmaceutical Co., Ltd.] equivalent to 300 units of CGTase and 1.8 g of maltose were dissolved.
(5.26 mmol) was added, and the reaction was carried out while stirring at 40 ° C. for 4.5 hours. After the reaction is completed, the reaction solution is heated to 90 ° C.
The mixture was heated for 15 minutes while stirring. Subsequently, the reaction solution was cooled to room temperature, and a membrane filter (0.45 μm) was used.
After filtering with 1., 1500 units of glucoamylase [manufactured by Toyobo Co., Ltd.] was added, and the reaction was performed while stirring at 40 ° C. for 12 hours. After the reaction is completed, the reaction solution is heated to 90 ° C.
The mixture was heated for 15 minutes while stirring. Subsequently, the reaction solution was cooled to room temperature, and a membrane filter (0.45 μm) was used.
The resulting filtrate was subjected to ODS column chromatography for purification and eluted with an acetonitrile-water mixture (volume ratio 0% → 5% gradient), and the eluted fraction of 1.5% acetonitrile was frozen. Dried 6 ⁵ -Azido-6 ⁵
-Deoxy-maltopentaose 220 mg (0.25
8 mmol, 57.6%) was obtained.

Melting point (° C): 176.0-179.0 Infrared absorption spectrum (cm ^-1 ): 3400, 2920,
2110, 1628, 1406, 1360, 1278,
1240, 1144, 1076, 1022 Nuclear magnetic resonance spectrum (200 MHz) ppm (D
₂ O): 2.80 to 4.00 (m), 4.64 (0.5
H, d, J = 8.0 Hz), 5.23 (0.5 H, d,
J = 3.5Hz), 5.35 (4H, d, J = 3.5H
z) High performance liquid chromatography [TSKg manufactured by Tosoh Corporation]
el Amide-80 column (4.6 mm ID x 25
0 mm), RI detection, eluent: acetonitrile / water =
3: 2 (v / v), flow rate: 1.0 ml / min]: t _R
= 6.9 min Specific rotation [α]: (c 0.544, H ₂ O); +16
9 ° Elemental analysis value: C ₃₀ H ₅₁ N ₃ O ₂₅ C H N theoretical value (%) 42.21 6.02 4.92 Measured value (%) 42.03 6.13 4.69

Example 2 Production of 6 ⁵ -azido-6 ⁵ -deoxymaltopentaose (method of allowing exo-type saccharifying enzymes to act before CGTase action) (1) Non-reducing end 6 ^s -Azido malto-oligosaccharide (s =
5.0 g (4.31 m) of 6-azido-6-deoxy-β-cyclodextrin obtained in the same manner as in (1) of Example 1.
mol) was heated to 40 ° C. in advance to 10 m
The mixture was poured into 200 ml of M phosphate buffer (pH = 7.5) while stirring and completely dissolved. To this, 23 units of CDase obtained in the same manner as in (2) of Example 1 and 10.0 g (55.5 mmol) of glucose were added, and the reaction was carried out while stirring at 40 ° C. for 7 hours. After completion of the reaction, the reaction solution was heated at 90 ° C. for 15 minutes while stirring. Then, the reaction solution was cooled to room temperature, and a membrane filter (0.
(45 μm) and then the reaction solution was adjusted to pH = 6.0 by adding 1N hydrochloric acid. To this, add 1500 units of glucoamylase [manufactured by Toyobo Co., Ltd.], and add 1 unit at 40 ° C.
The reaction was performed while stirring for 2 hours. After completion of the reaction, the reaction solution was heated at 90 ° C. for 15 minutes while stirring. The obtained reaction solution was cooled to room temperature, filtered with a membrane filter (0.45 μm), and the filtrate was subjected to ODS column chromatography for purification, and the mixture was mixed with acetonitrile-water (volume ratio 0% → 5% gradient). ), Concentrated to dryness, and the non-reducing terminal 6 ^s -azid maltooligosaccharide (s
About 3 g of a mixture of = 2 to 8).

(2) Preparation of 6 ⁵ -azido-6 ⁵ -deoxymaltopentaose 6 ^s -azidated maltooligosaccharide (s = 2 to 2 obtained in (1)
1/5 volume of a mixture of 8 integers) (about 600 mg) except for using as the starting material the same procedure as in Example 1 (4), 6 ⁵ - azido -6 ⁵ - deoxy maltopentaose 1
84 mg (0.216 mmol, 2 steps total yield 25.
1%) was obtained. The physicochemical properties of this product were completely in agreement with those obtained in (1) of Example 1.

Example 3 Preparation of 6 ⁵ -azido-6 ⁵ -deoxymaltopentaose (1) Preparation of 6-azido-6-deoxy-α-cyclodextrin (a) 6-O-tosyl-α-cyclodextrin Manufacture of commercially available α-cyclodextrin [manufactured by Shimizu Port Refinery]
The same operation as in Example 1 (1) (a) was performed except that 100 g (103 mmol) was used as the starting material, and 34.2 g (30.4 g) of 6-O-tosyl-α-cyclodextrin was obtained.
mmol, yield 30%).

Melting point (° C.): 160.0 to 162.0 (decomposition) Infrared absorption spectrum (cm ⁻¹ ): 3450, 2930,
1644, 1632, 1600, 1422, 1358,
1300, 1176, 1154, 1078, 1030

Nuclear magnetic resonance spectrum (200 MHz) p
pm: (DMSO-d ₆ ) 2.43 (3H, s), 3.
20-4.44 (m), 4.76 (2H, br.s),
4.84 (5H, br.s), 7.44 (1H, d, J
= 8.8 Hz), 7.75 (1H, d, J = 8.8H
z)

High performance liquid chromatography [TSK gel Amide-80 column (4.
6 mm ID x 250 mm), RI detection, eluent: acetonitrile / water = 3: 2 (v / v), flow rate: 1.0 ml /
min]: t _R = 4.8 min

(B) Preparation of 6-azido-6-deoxy-α-cyclodextrin 34.2 g (30.4 mmol) of 6-O-tosyl-α-cyclodextrin obtained in (a) was used as a starting material. The same operation as in Example 1 (1) (b) was performed except that
-Azido-6-deoxy-α-cyclodextrin 2
7.8 g (27.8 mmol, yield 92%) was obtained.

Melting point (° C.): 216.0 to 217.0 (decomposition) Infrared absorption spectrum (cm ⁻¹ ): 3390, 2930,
2120, 1644, 1416, 1368, 1342,
1304, 1158, 1082, 1032 High performance liquid chromatography [TSKg manufactured by Tosoh Corporation]
el Amide-80 column (4.6 mm ID x 25
0 mm), RI detection, eluent: acetonitrile / water =
3: 2 (v / v), flow rate: 1.0 ml / min]: t _R
= 5.8 min Specific rotation [α]: (c 0.400, H ₂ O); +12
9 ° Elemental analysis value: as C ₃₆ H ₆₅ N ₃ O ₂₉ C H N theoretical value (%) 43.33 5.96 4.21 actual measurement value (%) 43.13 5.88 4.12

(2) Preparation of 6 ⁵ -azido-6 ⁵ -deoxymaltopentaose 5.0 g (5.02 mmol) of 6-azido-6-deoxy-α-cyclodextrin obtained in (1) was used as a starting material. The same operation as in Example 1 (3) and (4) was performed except that it was used, and 1.04 g (1.22 mmol, total yield of 24 steps) of 6 ⁵ -azido-6 ⁵ -deoxy-maltopentaose was obtained.
%) Was obtained. The physicochemical properties of this product were completely consistent with those obtained in (4) of Example 1.

Comparative Example The yields of the method of the present invention and a known method were compared. The result is shown below as a comparative example. (1) Method of the present invention 5.0 g (4.31 mmo) of 6-azido-6-deoxy-β-cyclodextrin obtained in the same manner as in Example (1)
Using l) as a starting material and performing an activated carbon treatment (removal of unreacted glucose not adsorbed on activated carbon) instead of ODS column chromatography, the same operation as in Example 1 (3) was performed, and then Example 1 ( 6 ⁵ in the same way as 4)
Azido-6 ⁵ -deoxymaltopentaose 1.41g
(1.65 mmol) was obtained. The yield at this time is 38%
Met.

(2) Known method 1 [Method of reacting 6-azido-6-deoxy-β-cyclodextrin with CDase and then reacting exo-type saccharifying enzymes (JP-A-5-262784)] 6 g of 6-azido-6-deoxy-β-cyclodextrin obtained in the same manner as in Example (1) (4.31 mmol)
Was dissolved in 1.0 liter of 10 mM phosphate buffer (pH = 7.8), and CDas obtained in the same manner as in Example 1 (2)
e490 units were added, and the mixture was reacted at 40 ° C. for 2 hours while stirring. Next, the same operation as in Example 1 (3) was carried out except that purification by ODS column chromatography was not performed, and the concentrated dried product [6 ^r -azido maltoheptaose (r = integer of 1 to 7) was contained. ] Then, this was dissolved in 250 ml of 20 mM acetate buffer (pH 4.5), 2500 units of glucoamylase [manufactured by Toyobo Co., Ltd.] was added, and the mixture was reacted at 40 ° C. for 40 hours while stirring. After completion of the reaction, the reaction solution was heated with stirring at 90 ° C. for 15 minutes, cooled to room temperature, and then filtered with a membrane filter (0.45 μm).
Purified by DS column chromatography, eluted with acetonitrile-water mixture (volume ratio 0% → 5% gradient), and the eluted fraction of 1.5% acetonitrile was freeze-dried to give 6 ⁵ -azido-6 ^5. 0.48 g (0.563 mmol) of -deoxymaltopentaose was obtained. The total yield of the two steps at this time was 13%.

(3) Known method 2 [6-Azido-6-deoxy-β-cyclodextrin is allowed to act on CGTase in the presence of maltose and then acted on by exo-type saccharifying enzymes (Japanese Patent Application Laid-Open No. S60-18753). No. 63-170393)] 6-azido-6-deoxy-β-cyclodextrin 519 mg (0.448 m) obtained in the same manner as in Example (1).
except that the mol) as a starting material was conducted in the same manner as in Example 1 (4), 6 ⁵ - to give a deoxy maltopentaose 107 mg (0.125 mmol) - azido -6 ^5. The yield at this time was 28%. From the comparison of the yields of (1) to (3), it can be seen that the method of the present invention is remarkably excellent.

[Brief description of drawings]

FIG. 1 6-azido-6-deoxy-as a starting material
Explanatory drawing of the manufacturing method of this invention using (beta)-cyclodextrin.

Claims (2)

[Claims] 1. A general formula: In the presence of glucose, 6-azidated cyclodextrin represented by the formula (n is 5 or 6)
The action of cleaving cyclodextrin to form malto-oligosaccharide derived from the degree of glucose polymerization of cyclodextrin and (b) the hydrolysis rate or affinity for cyclodextrin is higher than that of polysaccharide or linear oligosaccharide having the same degree of polymerization as cyclodextrin. By reacting cyclodextrinase with a large substrate specificity, the general formula: (Wherein n has the same meaning as described above, m is an integer of 3 to 6 when n is 5, and an integer of 3 to 7 when n is 6) to produce a 6-azido maltooligosaccharide. And then allowing the cyclodextrin glucanotransferase and the exo-type saccharifying enzymes to act in any order in the presence of maltose. A method for producing non-reducing terminal 6-azido maltopentaose represented by: 2. The 6-azidated cyclodextrin is α
The production method according to claim 1, which is obtained from -cyclodextrin or β-cyclodextrin.