JPH1060006A - Production of nonreduced-terminal azidized acetylmaltooligosyl bromide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily produce the subject compound suitable as a substrate for the determination of α-amylase activity in high reaction yield without accompanying side reactions by reacting a specific oligosaccharide with titanium tetrabromide in the presence of a nonpolar organic solvent. SOLUTION: The objective compound of the formula II such as 6<3> -azido-6<3> - deoxynonaacetylmaltotriosyl bromide can be produced by reacting (A) a nonreduced-terminal azidized acetylmaltooligosaccharide of the formula I (Ac is acetyl; (n) is 0-5) with (B) titanium tetrabromide in the presence of (C) a non-polar organic solvent. Preferably, the component C is a mixture of dichloromethane and ethyl acetate, the amount of the component B is usually 0.5-3.0 times mol-equivalent based on the component A and the reaction is carried out at 20-35 deg.C for 1-3 days.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to the following formula (II):

[0002]

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(Wherein Ac represents an acetyl group, and n represents an integer of 0 to 5), and relates to a method for producing a non-reducing terminal azidated acetylmaltooligosyl bromide. More specifically, the present invention provides 4-nitrophenol, 2-nitrophenol, 2-nitrophenol at the reducing end of non-reducing end azido maltooligosaccharides useful as a substrate for measuring α-amylase activity contained in serum or other biological fluids. In order to efficiently produce a non-reducing terminal azido acetylmaltooligosyl bromide as an intermediate material when producing a non-reducing terminal azido maltooligoside derivative into which an aromatic chromophoric group such as halogeno-4-nitrophenol is introduced, Method.

[0004]

2. Description of the Related Art The present inventors have previously described a method for producing acetylmaltooligosyl bromide by reacting acetylmaltooligosaccharide with phosphorus tribromide in a nonpolar organic solvent in the presence of water. (Japanese Patent Publication No. 5-37156). In addition, a method of reacting hydrogen bromide in acetic acid with acetylmaltooligosaccharide [“Methods o
f Carbohydrate Chemistry "
2, 226 (1963)]. However, in the latter method, since a side reaction occurs in which a glycosyl bond between sugars is cut, the reaction yield is remarkably low particularly in acetylmaltooligosaccharide having a glucose polymerization degree of 3 or more. On the other hand, the former method has a polymerization degree of 3
Even with the above acetylmaltooligosaccharides, the desired acetylmaltooligosyl bromide is provided in high yield. However, when an azide group is present at the non-reducing end of acetylmaltooligosaccharide, a reduction reaction of the azide group or a substitution reaction from the azide group to a bromo group occurs as a side reaction.
It has the disadvantage of greatly reducing the reaction yield.
The present inventors have previously found that a non-reducing terminal azido-maltooligoside having an aromatic chromophoric group introduced at the 1-position of the reducing terminal glucose is extremely useful in α-amylase activity measurement. (JP-A-5-1091
No.).

[0005]

DISCLOSURE OF THE INVENTION The present invention overcomes the above-mentioned drawbacks of the conventionally used or reported methods for producing acetylmaltooligosyl bromide derivatives and provides a non-reducing terminal at the reducing end of an azido maltooligosaccharide. An object of the present invention is to provide a method for efficiently producing a non-reducing terminal azidoacetylmaltooligosyl bromide, which is an intermediate raw material when producing a non-reducing terminal azidomaltooligoside derivative having an aromatic chromophoric group introduced therein. It is a thing.

[0006]

Means for Solving the Problems As a result of various studies for achieving the above object, the present inventors have found that non-reducing terminal azidoacetylmaltooligosaccharides can be added to titanium tetrabromide (non-polar organic solvent). When TiBr ₄ ) is acted on, a bromine atom is efficiently introduced into position 1 of the reducing terminal glucose,
It has been found that non-reducing terminal azidated acetylmaltooligosyl bromide can be efficiently obtained without causing side reactions such as sugar-sugar-glycosyl bond cleavage side reaction, azide group reduction reaction or substitution reaction with bromine, Based on this finding, the present invention has been completed. That is,
The present invention provides the following general formula (I)

[0007]

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In the presence of a non-polar organic solvent, titanium tetrabromide acts on a non-reducing terminal azidoacetylmaltooligosaccharide represented by the formula: Characterized by the following general formula (II):

[0009]

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(Wherein Ac and n have the same meanings as described above), which is a method for producing a non-reducing terminal azidated acetylmaltooligosyl bromide.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
First, the non-reducing terminal azido-acetylmaltooligosaccharide represented by the general formula (I), which is a starting material in the present invention, may be obtained by any method. For example, as previously reported by the present inventors, commercially available 6 -O-tosyl-β-cyclodextrin is reacted with N ₃ Na to give 6-azido-6.
-Deoxy-β-cyclodextrin, and simultaneously with or after the action of cyclodextrinase having specific properties, exo-type saccharifying enzymes are reacted to form a non-reducing terminal azido maltooligosaccharide. (Japanese Unexamined Patent Publication No. 5-262784), and then obtained by reacting acetic anhydride in pyridine, for example. The non-reducing terminal azido-malto-oligosaccharide represented by the general formula (I), which is a starting material in the present invention, has a non-reducing terminal azido-malto-oligoside having an aromatic chromophoric group introduced therein, which is an α-form. Since it is used as a substrate for amylase activity measurement, the action of α-amylase is likely to occur, and the degree of glucose polymerization is 3 to 7, that is, n is 1 to 5 in the general formula (I) (a specific example is 6 ³ - azido -6 ³ - deoxy dec acetyl maltotriose, 6 ⁴ - azido -6 ⁴ - deoxy tridecafluoro acetyl maltotetraose, 6 ⁵ - azido -6 ⁵ - deoxy hexadecanol acetyl maltopentaose, 6 ⁶ - azido -6 ⁶ - deoxy nonadecamethylene acetyl maltohexaose, 6 ⁷ - azido -6 ⁷ - deoxy docosapentaenoic acetyl maltoheptaose) It is preferable to use

In order to carry out the present invention, titanium tetrabromide is allowed to act on the non-reducing terminal azidoacetylmaltooligosaccharide represented by the general formula (I) in the presence of a nonpolar organic solvent. The amount of titanium used is usually 0.5 to 3.0 times the molar equivalent of the non-reducing terminal azidoacetylmaltooligosaccharide, preferably 1.0 to 2.0 times the molar equivalent.

The nonpolar organic solvent used in the present invention may be any solvent which is inert to the bromination reaction and does not promote a side reaction. Dichloroethane, chloroform,
Carbon tetrachloride, ethyl acetate, diethyl ether, 1,4-
Examples thereof include dioxane and tetrahydrofuran. Among them, a mixed solution of dichloromethane-ethyl acetate is preferable because of a high reaction yield and easy availability. For example, a mixing ratio of dichloromethane: ethyl acetate is 50: A ratio of 1 to 2: 1, preferably 10: 1 is used. The reaction temperature of the bromination reaction in the present invention is usually 0 to 50 ° C (or the boiling point of the solvent used), but is preferably 20 to 35 ° C to avoid side reactions. The reaction time is affected by the concentration, temperature and the like.
~ 3 days.

The thus obtained general formula (I)
Specific examples of the non-reducing terminal azide acetyl maltooligosyl bromide represented by I) is 6 ³ - azido -6 ³
-Deoxynonaacetylmaltotriosyl bromide,
6 ⁴ - azido -6 ⁴ - deoxy dodeca acetyl maltotetraosyl bromide, 6 ⁵ - azido -6 ⁵ - deoxy pentadecanoyl acetyl maltopentaose O Sil bromide, 6 ⁶ -
Azido -6 ⁶ - deoxy octadecanols acetyl maltohexaose O Sil bromide, 6 ⁷ - azido -6 ⁷ - like deoxy hen eicosapentaenoic acetyl maltoheptaose Oh sill bromide and the like. The reaction solution containing the obtained non-reducing terminal azidated acetylmaltooligosyl bromide, if necessary,
Post-treatments such as neutralization with a base and liquid-liquid extraction, and purification operations such as column chromatography and recrystallization can yield the target product with high purity (content).

The obtained non-reducing terminal azidated acetylmaltooligosyl bromide is reacted with 4-nitrophenol, 2-halogeno-4-nitrophenol or the like in the presence of, for example, silver oxide or silver perchlorate. If the following formula (III)

[0016]

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(Wherein Ac and n have the same meanings as described above, and X represents a hydrogen atom or a halogen atom), wherein an aromatic color-forming group is introduced at the 1-position of the reducing terminal glucose. It is possible to obtain a reducing terminal azidoacetylmaltooligoside (Koenigs-Knorr reaction). Then, by reacting potassium carbonate, ammonia and the like in methanol, for example, the acetyl group can be removed, and the following formula (IV)

[0018]

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(Ac, n and X in the formula have the same meanings as described above).

Hereinafter, the present invention will be described in more detail with reference to Reference Examples, Examples and Comparative Examples, but the present invention is not limited to these examples. The high performance liquid chromatography in each example was performed by using an ODSAQ-312 column (6.0 mm ID × 150 mm) manufactured by YMC (hereinafter referred to as “OD
S ") or TSKgel Ami manufactured by Tosoh Corporation
A de-80 column (4.6 mm ID × 250 mm) (hereinafter abbreviated as “amide”) was used. The chromatography was performed using a mixture of acetonitrile / water (v / v) at a flow rate of 1.0 ml / min. The following examples show the columns used, the detection method, the mixing ratio of the eluent, and the retention time (t _R ).

[0021] <Reference Example> 6 ⁵ - azido -6 ⁵ - preparation of deoxy-hexadecanol acetyl maltopentaose (1) 6-azido-6-deoxy -β- cyclodextrin preparation commercial 6-O-tosyl -β -35 g of cyclodextrin
(27 mmol) was dissolved in 100 ml of DMF, 5.3 g (81 mmol) of sodium azide was added, and the mixture was reacted at 95 ° C. for 1.5 hours. Then, the reaction solution was concentrated to dryness under reduced pressure, and the obtained residue was dissolved by adding 30 ml of water, and a small amount of crystal seeds were added. The crystals were separated by filtration with a glass filter, washed with 5 ml of cold water and 50 ml of cold ethanol, and dried to give 6-azido-
27 g of 6-deoxy-β-cyclodextrin (23
mmol, yield 86%). Infrared absorption spectrum (cm ⁻¹ ): 3390, 2920,
2120, 1642, 1414, 1370, 1340,
1304, 1156, 1080, 1030 High-performance liquid chromatography [amide column, RI detection,
Eluent 3: 2]: t _R = 6.6 min Elemental analysis: C N as C ₄₂ H ₆₉ N ₃ O ₃₄ Theoretical value (%) 43.49 6.00 3.62 Actual value (%) 43. 28 6.11 3.53

(2) Production of 6 ^m -azidated maltooctaose 5.0 g (4.31 mmol) of 6-azido-6-deoxy-β-cyclodextrin obtained in the above (1) was previously heated to 40 ° C. The solution was poured into 200 ml of a warmed 10 mM phosphate buffer (pH 7.5) with stirring, and completely dissolved. There cyclodextrinase 23U
And 10.0 g (55.5 mmol) of glucose were added, and the mixture was reacted at 40 ° C. with stirring for 7 hours. After completion of the reaction, the reaction solution was heated with stirring at 90 ° C. for 15 minutes. Subsequently, the reaction solution was cooled to room temperature, filtered with a standard supercell (manufactured by Celite), washed with water, and the filtrate and the washing solution were combined and purified by ODS column chromatography, and an acetonitrile-water mixture (volume) 0% → 5
% Gradient) and concentrated to dryness to give 2.6 g of 6 ^m -azide maltooctaose (m is an integer of 1 to 8).
(1.94 mmol, 45.0%). High performance liquid chromatography [amide column, RI detection,
Eluent 3: 2]: t _R = 12.4 min The cyclodextrinase described above was obtained from Bacillus sphaericus.
us) E-244 strain [Microtechnical Laboratories No. 2458 (FER)
MBP-2458) deposited in a culture medium, and cyclodextrinase (EC 3.2.
1.54) (Appl. Microbial Biot)
echnol. 39, pp. 714-719, 19
1993).

[0023] (3) 6 ⁵ - azido -6 ⁵ - 6 was obtained in the deoxy maltopentaose of manufacturing said (2) ^m - the azide maltooctaose ose 600mg (0.448mmol), 5mMPIPE
Dissolve in 30 ml of S buffer (pH 6.0) with stirring, and add thereto 300 U of cyclodextrin glucanotransferase (enzyme solution manufactured by Amano Pharmaceutical Co., Ltd.) and 1.8 g (5.26 mmol) of maltose. The reaction was carried out while stirring at 40 ° C. for 4.5 hours.
After completion of the reaction, the reaction solution was heated with stirring at 90 ° C. for 15 minutes. Subsequently, the reaction solution was cooled to room temperature, filtered through a membrane filter (0.45 μm), 1500 U of glucoamylase was added, and the reaction was performed with stirring at 40 ° C. for 12 hours. After the completion of the reaction, the reaction solution was cooled at 90 ° C. for 15 minutes.
Heat with stirring for minutes. Subsequently, the reaction solution was cooled to room temperature, filtered through a membrane filter (0.45 μm), and the obtained filtrate was subjected to ODS column chromatography, purified, and mixed with acetonitrile-water (volume ratio 0% →
5% and eluted with gradient), the eluate fractions of 1.5% acetonitrile and lyophilized 6 ⁵ - azido -6 ⁵ - deoxy maltopentaose 220 mg (0.258 mmol,
57.6%). 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): 2.80~4.00 (m)} , 4.64 (0.5
H, d, J = 8.0 Hz), 5.23 (0.5 H, d,
J = 3.5 Hz), 5.35 (4H, d, J = 3.5H)
z) High performance liquid chromatography [amide column, RI detection,
Eluent 3: 2]: t _R = 6.9 min

[0024] (4) 6 ⁵ - azido -6 ⁵ - deoxy hexadecanol acetyl maltopentaose of manufacturing the above (3) 6 was obtained in the same manner as ⁵ - azido -6 ⁵ - deoxy maltopentaose 5.0g (5.86mmo
l) was dissolved in 100 ml of pyridine, and 50 ml of acetic anhydride was dissolved.
(529 mol) was added and reacted at room temperature for 2 days. Next, pyridine, acetic anhydride, and acetic acid in the reaction solution were distilled off.
The resulting residue was purified by subjecting to silica gel column chromatography, dichloromethane - ethyl acetate - methanol mixture (volume ratio 99: 50: 1) was evaporated to eluted fractions of the solvent of the eluted purpose, 6 ⁵ - azido -6 ⁵ - deoxy hexadecanol acetyl maltopentaose O - scan 8.10 g (5.3
(1 mmol, 90.6%). Infrared absorption spectrum (cm ⁻¹ ): 2106, 1750,
1374, 1250 High-performance liquid chromatography [ODS column, RI detection,
Eluent 7: 3]: t _R = 8.6 min

[0025]

EXAMPLES 6 ⁵ - azido -6 ⁵ - deoxy - 6 was obtained as a prepared in Reference Example of pentadecanoyl- acetyl maltopentaose O Sil bromide ⁵ - azido -6 ⁵ - deoxy hexadecanol acetyl maltopentaose 5. 0g
(3.28 mmol) was dissolved in 25 ml of dichloromethane, and 2.5 ml of ethyl acetate and 1.93 g of titanium tetrabromide were dissolved.
(5.25 mmol) was added and reacted at 30 ° C. for 1 day. Next, 6.90 g (50 mm) of potassium carbonate was added to the reaction solution.
ol), and the mixture was stirred at room temperature for 30 minutes. Then, insolubles were filtered off with a glass filter and washed with 25 ml of dichloromethane. After distilling off dichloromethane and ethyl acetate in the mixed solution obtained by combining the filtrate and the washing solution, the obtained residue was purified by silica gel column chromatography, and a dichloromethane-ethyl acetate-methanol mixed solution (volume ratio: 99: 5
The solvent of the target elution fraction eluted in 0: 1) was distilled off,
6 ⁵ - azido -6 ⁵ - deoxy pentadecanoyl acetyl maltopentaose O Sil bromide 4.82g (3.12mmo
1, 95.1%). Melting point (° C.): 119.0-121.0 Infrared absorption spectrum (cm ⁻¹ ): 2108, 1740,
1350, 1250, 1040, 760 High-performance liquid chromatography [ODS column, RI detection,
Eluent 7: 3]: t _R = 10.3 min Elemental analysis: C ₆₀ H ₈₀ BrN ₃ O ₃₉ CH N Theoretical value (%) 46.58 5.21 2.72 Actual value (%) 46. 39 5.41 2.53

[0026] <Comparative Example> phosphorus tribromide 6 using ⁵ - azido -6 ⁵ - deoxy pentadecanoyl acetyl maltopentaose O sill 6 produced the obtained analogously to Example bromide ⁵ - azido -6 ⁵ - Deoxyhexadecaacetyl maltopentaose 5.0 g
(3.28 mmol) was dissolved in 25 ml of dichloromethane, and 0.94 ml of water and 0.305 ml of phosphorus tribromide (3.
21 mmol) and reacted at 30 ° C. for 1 day. Next, 4.14 g (30 mmol) of potassium carbonate was added to the reaction solution.
After stirring at room temperature for 30 minutes, insolubles were filtered off with a glass filter and washed with 25 ml of dichloromethane. After distilling off dichloromethane and ethyl acetate in the mixed solution obtained by combining the filtrate and the washing solution, the obtained residue was purified by silica gel column chromatography, and a dichloromethane-ethyl acetate-methanol mixed solution (volume ratio: 99: 5
The solvent of the target elution fraction eluted in 0: 1) was distilled off,
6 ⁵ - azido -6 ⁵ - deoxy pentadecanoyl acetyl maltopentaose O Sil bromide 3.62g (2.34mmo
1, 71.3%). Its physicochemical properties and elemental analysis were completely consistent with those obtained in Example 1. In this column chromatography, 6 ⁵ -
Azide-6 ⁵ -deoxypentadecaacetylmaltopentaosyl bromide other than 6
0.840 g (0.538 g) of ⁵ -N-acetyl-6 ⁵ -deoxypentadecaacetylmaltopentaosyl bromide
mmol, 16.4%) and 6 ⁵ - bromo-6 ⁵ - deoxy pentadecanoyl acetyl maltopentaose O Sil bromide 0.458g (0.289mmol, 8.8%) was obtained. 6 ^5-N-acetyl -6 ⁵ - deoxy pentadecanoyl acetyl maltopentaose O Sil bromide; HPLC [ODS column, RI detection,
Eluant _{7: 3]: t R =} 4.7min 6 5 - bromo-6 ⁵ - deoxy pentadecanoyl acetyl maltopentaose O Sil bromide; HPLC [ODS column, RI detection,
Eluent 7: 3]: t _R = 10.9 min

[0027]

According to the present invention, an aromatic chromogenic group is introduced as a glycoside at position 1 of the reducing terminal glucose of the non-reducing terminal azido-maltooligosaccharide, which is extremely suitable as a substrate for measuring α-amylase activity. Formula (IV)
The above general formula (I), which is extremely useful as an intermediate in the production of a non-reducing terminal azido-malto-oligoside represented by
The non-reducing terminal azidated malto-oligosyl bromide represented by I) can be obtained simply and with high reaction yield without causing side reactions.

Claims (2)

[Claims] 1. The following general formula (I): (Wherein Ac represents an acetyl group, and n represents an integer of 0 to 5), and reacting titanium tetrabromide with the non-reducing terminal azidated acetylmaltooligosaccharide in the presence of a nonpolar organic solvent. The following general formula (II): (Wherein Ac and n have the same meanings as described above), a process for producing a non-reducing terminal azidated acetylmaltooligosyl bromide. 2. The non-reducing terminal azide according to claim 1, wherein n of the non-reducing terminal azido acetylmaltooligosaccharide according to claim 1 is 3, and the non-polar organic solvent is a mixture of dichloromethane and ethyl acetate. For producing acetylmaltooligosyl bromide bromide.