IE47954B1 - Process for the preparation of maltoheptaoside derivatives - Google Patents

Description

The present invention is concerned with a process for the preparation of compounds which are useful as substrates for the determination of a-amylase.

The determination of α-amylase in serum is an impor5 ' tant clinical parameter for the function of the pancreas.

The commercially available reagents for the determination of α-amylase are preponderantly based upon a system in which starch is broken down by α-amylase and the fragments formed are determined in the visible range of light or in 1C the UV range, depending upon whether coloured starch or native starch is used in the test as the substrate for the amylase. An important disadvantage of these processes and reagents is that starch, which is a macromolecule, cannot be sufficiently standardised and characterised so that the rate of reaction of individual batches can vary very greatly and, when carrying out measurements, a standard must always be included. For better results, a more uniform substrate would be necessary which provides dependable results in the case of fission.

A step forwards in the direction of a more uniform substrate occurred with the use of maltopentaose. This is split by α-amylase into maltotriose and maltose which are converted by α-glucosidase into glucose which can then be determined by any desired method, for example by the known hexokinase method. 47854 Besides maltopentose, it has also already been proposed truse maltotetraose and maltohexaose as substrate (see V.S. Patent Specification Nos. 3,879,263 and 4,000,042). However, in this case, the results obtained with the tetraose were markedly poorer than those obtained with the pentaose and with hexaose even worse results were obtained than with the tetraose. Thus, in the case of maltotetraose and -pentaose, it is still possible to obtain a stoichiometric reaction whereas in the case of the hexaose, just still tolerable deviations from the stoichiometric reaction were ascertained.

A disadvantage of maltopentaose, which was also found in the case of the tetraose, is, however, that a considerable reagent blank occurs, i.e. the measurement reaction already starts before the sample to be determined is added. Furthermore, this reagent blank is not constant in the case of comparatively high substrate concentrations. Indeed, it changes for more than 25 minutes before a constancy of this side reaction is achieved.

It has also been ascertained that the assumed different fission of maltopentaose by pancrease α-amylase and saliva α-amylase, which would have enabled a differentiation, does not actually exist (see J. BC., 1970, 245, 39173927; J.Biochem., 51, p.XVIII, 1952).

Therefore, it is an object of the present invention to provide a process for the preparation of compounds which are useful as substrates for the determination of a-amylase, these compounds having a higher degree of purity and uniformity than the kncwn substrates, being readily obtainable and satisfying the requirements with regard to the blank •17 9 5 4 - 4 value without serum, the length of the lag phase and the achievable maximum activity.

Thus, according to the present invention, there is provided a process for the preparation of compounds of the general formula:- in which R is a phenylglucoside, mononitrophenylglucoside, dimtropher.ylglucoside, sorbitol or gluconic acid group, wherein peracetylated maltoheptaose is reacted with phenol, nitrophenol or dinitrophenol in the presence of a FriedelCrafts catalyst to give a compound (I), in which R is phenylglucoside, mononitrophenylglucoside or dinitrophenylglucoside group; or wherein maltoheptaose is either reduced with sodium borohydride to give a compound (I), in which R is a sorbitol group, or is oxidised with bromine to give a compound (I), in which R is a gluconic acid group.

In the compounds of general formula (I), the phenyl groups can be in the a- or β-position. In the case of the dinitrophenyl groups, the two nitro groups can be present in any desired position, for example as 2,4-, 2,6- or 3,5substituents.

The maltoheptaose derivatives (Γ) can be prepared in various ways. In the case of the phenylated derivatives, there can be used not only chemical but also enzymatic methods. The chemical syntheses are, in principle, based 9 5 4 - 5 upon the reaction of peracetylated maltoheptaose with the appropriate phenol in the presence of a Friedel-Crafts catalyst. This method can be used not only for phenol itself but also for mononitrophenol and dinitrophenol.

On the other hand, it is also possible first to prepare the phenyl derivative and subsequently to nitrate it, for example with the use of the process described in Bull. Chem. Soc. Japan, 34, 718/1961. This method is particularly suitable for the mononitro derivative. Under certain circumstances, a separation of the resultant o and p-nitrophenyl derivatives can also be carried out.

This reaction is preferably carried out by melting or boiling under reflux in non-polar solvents with zinc chloride, stannic chloride or titanium tetrachloride as Friedel-Crafts catalyst. After the introduction of the phenol or nitrophenol, the protective groups are split off in known manner, for example with sodium methoxide, ammonia, potassium hydroxide or barium methoxide, in each case in methanolic solution, or with aqueous barium hydroxide solution.

The enzymatic preparation of the phenyl derivatives can take place by transglucosidatxon of the phenyl glucoside or of the corresponding nitrated phenyl glucoside with acyclodextrin, amylose or soluble starch in the presence of a specific microbial transferase. For this purpose, use is made of a transferase from Bacillus macerans. In this case, for this transglucosidation, use is made of the kncwn amylase from Bacillus macerans (E.C. 2.4.1.' 19. DSM 24; isolation see J. A. de Pinto, L. L. Campbell, Biochemistry, 7, 114/1968? transfer reaction see Methods in Carbohydrate 7 9 5 4 - 6 Chemistry, Vol. 11, 347 (1963)) which, besides its hydrolytic and cyclising action, clearly also displays a glucosyltransferring effectiveness.

Those compounds of general formula (1) in which R is a sorbitol residue can be obtained from maltoheptaose by reduction with sodium borohydride under mild conditions and those compounds of general formula (1) in which R is a gluconic acid group can be obtained from maltoheptaose by oxidation with bromine.

Specifi cation Pa tent/No. 47953 describes and claims a process and a reagent for the determination of α-amylase which makes use of the compounds (1) obtained by the process of the present invention.

The compounds prepared by the process according to the present invention are readily obtainable in a high state of purity. A simple process for the preparation of maltoheptaose is described in German Patent Specification No. P 27 41 191.2.

The following Examples are given for the purpose of illustrating the present invention:Example 1 Preparation of a-phenylmaltoheptaoside a) Tridecosacetyl-p-D-maltoheptaose 57.6 g. (50 mM) maltoheptaose and 41 g. (500 mM) anhydrous sodium acetate are suspended in 543 ml. (5.75 mole) acetic anhydride and vigorously stirred at 100°C. for 4 hours, with the exclusion Of moisture. After cooling to about 60°C., the reaction mixture is stirred into about 1 litre ice water and further stirred overnight at 4°C., a viscous, semi-cyrstalline mass thereby precipitating out. - 7 After pouring off the supernatant, the residue is again stirred with ice water, the product thereby crystallising through. It is separated off, washed and dried. There are obtained 80.7 g. (76% of theory) tridecosacetyl-p-DRT maltoheptaose in the form of colourless crystals; =+ 137.5° (c.=1.15 chloroform); m.p. 150°C. (not sharp) (RT means ambient temperature).

The mother liquor (the decantate) is evaporated to dryness in a vacuum and the residue triturated with water and brought to crystallisation. There are obtained a RT o further 22.3 g. of product; [o] =136 (c,=l, chloroform); m.p. 150°C.

This, the total yield of product is 103 g. (=97% of theory).

The product can be recrystallised from ethanol until, after repeated recrystallisations,the melting point and optical rotation do not change. Therefore, the product is, on the atom, of optically pure configuration (β). b) Dodecosacetyl-a-phenyl-D-maltoheptaoside 9.54 g. (4.5 mM) Tridecosacetyl-3-D-maltoheptaose, 0.61 g. (4.5 mM) freshly melted zinc chloride and 4.23 g. (45 mM) distilled phenol are stirred for 2.5 hours at 100°C. with the exclusion of moisture. While still warm, the reaction mixture is dissolved in ethyl acetate and washed twice with 100 ml. amounts of water, three times with 100 ml. IN aqueous sodium hydroxide solution, once with 100 ml. IN acetic acid and once with 100 ml. saturated aqueous sodium chloride solution, the initially brown solution thereby becoming pale yellow. After drying over anhydrous magnesium sulphate, the solution is evaporated to dryness and taken 7 9 5 4 - 8 up in 30 ml. warm methanol. After standing overnight, syrupy material separates out which is crystallised from ethanol. There are obtained 8.7 g. dodecosacetyl-a-phenylD-maltoheptaoside (70% of theory): m.p. 155—165°C. (decomp.)? RT o [a] =+147 (c=8 in chloroform). c) Phenyl-a-D-maltoheptaoside .8 g. (5 mM) Peracetyl-l-phenyl-a-D-maltoheptaoside are dissolved in 200 ml. warm anhydrous methanol and mixed at ambient temperature, with the addition of some dioxan, while stirring with 30 ml. 0.IN sodium methoxide solution and then stirred at ambient temperature for 16 hours. After 20 minutes, the product begins to separate out in semicrystalline form. Finally, it is mixed with 200 ml. acetone, cooled to 4°C. and filtered off with suction. The product is dissolved in 160 ml. water, decolorised with active charcoal and desalinated with a cation exchange resin (Dowex 50 H+) There are obtained 5.6 g. (91% of theory) phenyl-a-D-maltoRT heptaoside in the form of a colourless lyophilisate: [a] & =+ 176° (c=10 in water).

The product still contains some free maltoheptaose and, according to HPLC analysis (detection at 254 nm), also 2 UVpositive impurities. These are removed by chromatography on cross-linked dextran (Sephadex LH 20), using water as elution agent. (Sephadex is a Registered Trade Mark).

Example 2 Preparation of p-nitrophenyl-a-D-maltoheptaoside a) Peracetyl-p-nitrophenyl-a-D-maltoheptaoside 13,6 g. (20 mM) Peracetyl-maltoheptaose, prepared in the manner described in Example la), are dissolved in 90 ml. anhydrous benzene, together with 11.9 g. (100 mM) p-nitro47954 - 9 phenol and mixed, while stirring and with the exclusion of moisture, with 10.4 g. (4.5 ml.; 40 mM) SnCl,. A voluminous mass thereby precipitates out which, however, dissolves again upon heating. The reaction mixture is boiled under reflux for 1 hour. Upon cooling, a viscous mass precipitates out which, after the addition of 00 ml. ethyl acetate, again goes into solution. Upon stirring the solution into 180 ml. 2N aqueous sodium carbonate solution, SnOfOH)^ pre- cipitates out. This is separated off, with the addition of some active charcoal. The aqueous phase is separated off and the organic phase well washed, finally with a saturated aqueous solution of sodium chloride. After drying and evaporating the solution, there is obtained a resinous product which is crystallised from ethanol. There are obtained 6.2 g. (41% of theory) peracetyl-p-nitrophenyl-a-D-maltoheptaoside; m.p. 100°C. (decomp.); fa]^T=+131° (c=l in chloroform).

The same product is also obtained when the reaction is carried out in chloroform or when the reaction is carried out with titanium tetrachloride instead of with stannic chloride. b) p-Nitrophenyl-a-D-maltoheptaoside .5 g. (7 mM) Peracetyl-p-nitrophenyl-a-D-maltoheptaoside is slurried in 100 ml. anhydrous methanol and mixed with ml. of an approximately IN sodium methoxide solution. The starting material, becomes honey-like and dissolves slowly.

After some time, the crystalline deposition of the de-acetylated product commences which, after stirring o\ernight, is complete. It is filtered off with suction, wished with methanol and dried. There are obtained 2.8 g. (86% of RT O theory) p-nitrophenyl-a-D-maltoheptaoside; [a & =+124 (c= 0.6 in water). - 10 The product is purified on cross-linked dextran (Sephadex LH 20), using water as elution agent. There are obtained 1.2 g. (45% of theory) of the above-described compound which is active in the enzymatic test.

Furthermore, the compound is also obtainable by the nitration of α-phenylmaltoheptaoside, obtained according to Example 1, with nitrating acid in the manner described in Bull. Chem. Soc. Japan, 34, 7Π (1961).

When using dinitrophenol instead of mononitrophenol, the corresponding dinitrophenyl compound is obtained.

Example 3 p-Nitrophenyl-o-maltooligosaccharides by Enzymatic Synthesis with Bacillus Maeerans Amylase (E.C.2.4.1.19 from Bac. Mac. DSM 24) In addition to a hydrolytic and cyclising action.

Bacillus maeerans amylase also has glyeosyl-transferring properties which can be utilised for the synthesis of oligosaccharides and derivatives thereof (see Methods in Carbohydrate Chemistry, 11, 347 (1963)). For the synthesis of p20 nitrophenyloligosaccharides, this process was optimised in the following manner; 680 mg. Bacillus maeerans amylase (E.C. 2.4.1.19 from Bac. mac. DSM 24) (lyophilisate) (0.46 U/mg. weighed amount, protein content of the weighed amount 28.5%, free from p25 nitrophenyl-a-D-glucoside-splitting activities), 400 mg. aD-p-nitrophenylglucoside, 3.5 g. a-cyclodextrin and 70 ml.

Soerensen phosphate buffer (pH 6.2; 0.01 M) are mixed. The o batch is incubated for 24 hours at 37 C. For purification, α-cyclodextrin and formed β-cyclodextrin are first separa30 ted off by means of the tetrachloroethylene inclusion com479 54 pound. After chromatography on cross-linked dextran (Sephadex LH 20), there are obtained 50 mg. of lyophilisate of p-nitrophenylmaltoheptaoside, which is highly active in the amylase assay.

Example 4 Preparation of Maltoheptaitol g. Maltoheptaose are dissolved in 50 ml. water, 2 g. sodium borohydride are added thereto portionwise and the reaction mixture is stirred for 75 minutes at ambient temperature until the test for reducing sugar is negative. Sodium ions are removed by chromatography on a cation exchange resin (Dowex 50 H ) (pH value of the solution after passage through the exchange resin=3.5). (Dowex is a Registered Trade Mark). The eluates from the exchange resin are evaporated in a vacuum and taken up and evaporated several times with methanol/water (addition of water for dissolving the product) for the removal of boric acid as methyl ester. The solution obtained, which is finally neutral, is lyophilised. There are obtained 9 g. (90% of theory) maltoheptaitol, which is free of reducing sugars.

Content (determined enzymatically) by glucose by sorbitol water 86.1% 89.0% 8.5% Example 5 Preparation of Maltoheptagluconic Acid 11.5 g. (0.01 mole) Maltoheptaose and 6 g. barium benzoate are dissolved in 150 ml. water. While stirring and cooling, 1 ml. bromine is added thereto and stirring continued for 36 hours. After driving off excess bromine by means of - 12 nitrogen, the reaction mixture is mixed with 4 ml. 4N sulphuric acid and some active charcoal, filtered and the filtrate extracted with chloroform in order to remove benzoic acid. The aqueous solution is mixed with 3.2 g. silver carbonate and stirred (pH: neutral). The insoluble salts are filtered off and the clear solution passed over 20 ml. Amberiite” JR H . (Amberiite is a Registered Trade Mark). The acidic eluate is immediately neutralised with sodium hydroxide and lyophilised. There are obtained 8 g. (72% of theory) maltoheptagluconic acid.

Content (determined enzymatically) by glucose by gluconic acid

Claims (6)

1. CLAIMS: 1. Process for the preparation of compounds of the general formula:ch 2 oh ch 2 oh H< OH OH (I) 5 in which R is a phenylglucoside, mononitrophenylglucoside, dinitrophenylglucoside, sorbitol or gluconic acid group, wherein peracetylated maltoheptaose is reacted with phenol, nitrophenol or dinitrophenol in the presence of a FriedelCrafts catalyst, followed by removal of the acetyl protective 10 groups, to give a compound (I), in which R is phenylglucoside, mononitrophenylglucoside or dinitrophenylglucoside group; or wherein maltoheptaose is either reduced with sodium borohydride to give a compound (I), in which R is a sorbitol group, or is oxidised with bromine to give a compound (1), in which R is a 15 gluconic acid group.

2. Modification of the process according to claim 1 for the preparation of a compound (I) in which R is a nitrophenylglucoside group, wherein a maltoheptaose derivative (I), in which R is a phenylglucoside group, is nitrated 20 with a nitrating acid.

3. A process according to claim 1 or 2, wherein the acyl groups are split off with sodium methoxide,ammonia, potassium hydroxide or barium methoxide, all in methanolic solution or with aqueous barium hydroxide solution. 4. 79 54

4. Modification of the process according to claim 1 for the preparation of a compound (I), in which R is a phenyl glucoside, mononitrophenylglucoside or dinitrophenylglucoside group, wherein the phenylglucoside or nitrated phenylglucoside 5. Is transglucosidated with α-cyclodextrin, amylose or soluble starch in the presence of Bacillus macerans amylase.

5. Process according to claim 1 for the preparation of compounds (1), substantially as hereinbefore described and exemplified.

6. 10 6. Compounds (I) as claimed in claim 1, whenever prepared by the process according to any of claims 1 to 5.