FI68418C - FRAMEWORK FOR MALTOHEPTAOS DERIVATIVES - Google Patents

Description

6841 8

Method for preparing maltoheptaose derivatives

The invention relates to a process for the preparation of maltoheptaose derivatives of the formula

CH „OH | CHo0H

J-0 / -% OH OH 5 wherein R is a phenylglucoside, mononitrophenylglucoside or dinitrophenylglucoside group.

Determination of serum α-amylase levels is an important clinical parameter in pancreatic function. Commercially available reagents for the determination of α-Amylase are mainly based on a system in which α-amylase cleaves starch and the cleaved parts formed are determined in the visible or UV range, depending on whether colored starch or natural starch was used as the amylase substrate in the experiment. An essential disadvantage of this method or reagents is that the starch as a macromolecule cannot be characterized and standardized well enough, so that the rates of change of private batches become very different and standard experiments must always be used in the measurements. To achieve better results, a more uniform substrate would be needed that would give reliable results when digested.

Master application 782789 (FI-C 61916) describes a method for the determination of α-amylase in body fluids by enzymatic digestion of a maltoheptose derivative of the above formula I and subsequent determination of the cleavage products in a manner known per se.

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The assay for cleavage products is preferably performed with α-Glu cosidase. In this case, both enzymes, α-Amylase and α-glucosidase, cleave the phenyl group, the mononitrophenyl group or the dinitrophenyl group, which is easily determined. The phenyl groups may be in the α- or 3-position. In the case of the α-position, only the cleavage by α-amylase and α-glucosidase occurs and the cleaved substituted or unsubstituted phenols can be easily determined by known color reactions. However, substituents in the β-position can also be used. In the latter case, in addition to α-glucosidase, 3-glucosidase is also used.

In the case of dinitrophenyl groups, both nitro groups may be in the desired position, for example as 2,4-, 2,6- or 3,5-substituents.

The nitrophenol or dinitrophenol released during the cleavage of the nitro-containing substituent is itself a colored compound which is of course optically detectable. If the phenol itself is cleaved, this can be determined by known methods, for example by reacting it with a nucleophilic substance such as 3-methyl-6-sulfonyl-benzthiazolone-hydrazone- (2) (HSK) in the presence of monophenol oxidase. In this reaction, a red dye is formed which is measurable.

It is an object of the present invention to provide a process for the preparation of maltoheptaose derivatives of the above formula I and this is carried out by a process characterized in that said phenylglucoside or nitrated phenylglucoside is reacted with α-cyclodextrin in the presence of cyclomaltodextransine dextransin

Thus, in this enzymatic preparation method, transglucosidation of phenylglucoside or similar nitrated phenylglucosides is performed with 68 α 8 -cyclodextrin in the presence of a known cyclonal dextrin glucanotransferase.4. (1968) 114; transfer reaction: Methods in Carbohydr. Chemistry, Vol. II 347), which, in addition to its hydrolytic and cyclizing effect, apparently also has a glucosyl transfer effect.

The following example illustrates the invention.

Example Preparation of p-nitrophenyl-α-maltoheptaoside by enzymatic synthesis of cyclomaltodextrin using glucanotransferase (Bacillus macerans amylase from E.C.2.4.1.19 Bac. Mac. DSM 24).

In addition to its hydrolytic and cyclizing effect, Bacillus macerans amyPase also has glycosyl-transferring properties that can be exploited in the synthesis of oligosaccharides and their derivatives (Methods in Carbohydrate Chemistry II, 347). For the synthesis of p-nitrophenyl oligosaccharides, this method was optimized as follows: 680 mg Bacillus macerans amylase E.C.2.4.1.19 Bac. mac. DSM 24) (lyophilisate) (0.46 U / mg weighed batch, protein content in the weighed batch 28.5%, no p-nitrophenyl-α-D-glucoside cleavage activity), 400 mg α-Dp-nitrophenyl glucoside, 3, 5 g α-cyclodextrin, and 70 ml Soerensen's phosphate buffer, pH 6.2; 0.01 moles, mixed together. The batch is incubated for 24 hours at 37 ° C. For purification, the α- and the resulting 8-cyclo-dextrin are then first separated via a tetrachlorethylene closure compound. After chromatography with cross-linked dextran (Sephadex LH20), 50 mg of p-nitrophenyl-maltoheptaoside lyophilisate, which has proved to be very active in the amylase test, are obtained.