AU615318B2 - Process for the preparation of s-cyanohydrins - Google Patents

Abstract

The preparation of (S)-cyanohydrins by reaction of the corresponding oxo compounds with hydrocyanic acid can be achieved enzymatically with the aid of oxynitrilase (4.1.2.11) from Sorghum bicolor. This is preferably carried out in aqueous medium at pH values below 6.0, in particular below 4.5, in order to suppress competing chemical reactions and racemisations. The reaction temperatures are, in particular, between -5 DEG C and +50 DEG C. Of particular interest is the reaction of aromatic aldehydes, specifically benzaldehyde and its derivatives, and the generation of aldononitriles by reaction of aldoses, especially of sugars with 3 to 9 carbon atoms, with hydrocyanic acid.

Description

COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMLET SPECIFCATION NAME ADDRESS OF APPLICANT: Degussa Aktiengesellschaft Weissfrauenstrasse 9 D-6000 Frankfurt am Main 1 Federal Republic of Germany Kemforschungsanlage Julich Gesellschaft mit beschrankter Haftung Postfach 1913 D-5170 Julich Federal Republic of Germany NAME(S) OF INVENTOR(S): Maria-Regina KULA Uwe NIEDERMEYER Ingeborg Maria STURTZ S ADDRESS FOR SERVICE: DAVIES COLLISON Patent Attorneys 1 Little Collins Street, Melbourne, 3000.

COMPLETE SPECIFICATION FOR THE INVENTION ENTITLED: Process for the preparation of S-cyanohydrins The following statement is a full description of this invention, including the best method of performing it known to me/us:- Background of the Invention The present invention relates to a process for the preparation of S-cyanohydrins by reaction of the corresponding oxo compounds with hydrocyanic acid.

Optically-active cyanohydrins have great importance for obtaining optically-active a-amino alcohols, a-hydroxy carboxylic acids, heterocycles and pyrethroid insecticides.

The preparation of R-cyanohydrins with the aid of chiral catalysts from the corresponding aldehydes with hydrocyanic acid, especially using Roxynitrilase (4.1.2.10) from Prunus amygdalus, has been disclosed.

This enzymatic reaction with R-oxynitrilase which has been known for a long time (since 1908), was investigated in detail in the 1960s by Becker (JACS 88:4299 (1966)), who described the preparation of optically-active cyanohydrins with oxynitrilase bound to ion exchangers at pH 5.4 n German Patent 1,300,111.

la According to Effenberger et al. (Ancew. Chem.

99:491 (1987)), the enzymatic reaction should be carried out in organic solvents which are immiscible with water in order to suppress the competing chemical reaction that leads to racemates.

By contrast, the enzymatic preparation of optically-pure S-cyanohydrins has not hitherto been disclosed.

Summary of the Invention It is therefore an object of the present invention to produce optically-pure S-cyanohydrins.

These and other objects according to the invention are achieved by a process for preparation of an S-cyanohydrin, comprising the steps of combining an oxo compound with hydrocyanic acid and oxynitrilase (4.1.2.11) from Sorghum bicolor to form a reaction mixture, and enzymatically reacting the oxo compound with the hydrocyanic acid to produce an S-cyanohydrin.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

-2- Detailed Description of the Preferred Embodiments It has now been found that the oxynitrilase (4.1.2.11) obtainable from Sorghum bicolor can be used to obtain optically-pure S-cyanohydrins enzymatically.

The process according to the invention for the preparation of S-cyanohydrins by reaction of oxo compounds aldehydes or ketones) with hydrocyanic acid comprises carrying out the reaction enzymatically with the aid of oxynitrilase (4.1.2.11) from Sorghum bicolor.

It is true that the oxynitrilase (4.1.2.11) has been known for some time Bove et al., J.

Biol. Chem. 236:207 (1961)) and its reactivity in terms of the enzymatic cleavage of cyanohydrins has been investigated Mao et al., Phytochemistry 6:473 (1967)). However, it has not hitherto been disclosed that it is possible with this oxynitrilase to obtain S-cyanohydrins having high optical purity.

The enzymatic reaction of aromatic aldehydes, especially of benzaldehyde and its derivatives, with hydrocyanic acid has been particularly investigated by the present inventors.

Furthermore, the enzymatic S-cyanohydrin formation can be utilized with advantage for the production of optically-active nitriles of aldonic acids by reaction of aldoses, in particular aldehyde sugars with 3 to 9 carbon atoms, with hydrocyanic acid in the presence of S-oxynitrilase, resulting in interesting nitriles of aldonic acids which are increased by one carbon atom and which can easily be -3hydrolyzed to the aldonic acid or hydrogenated to the polyhydroxy amine.

To obtain S-cyanohydrins with particularly high optical purity enzymatically it is necessary to suppress the purely chemical reaction of the aldehydes with hydrocyanic acid, which leads to racemates, adversely affecting the optical purity of the products. In addition, chiral cyanohydrins are prone to racemization, which further reduces the optical purity of the product.

Thus, the pH values and temperatures used are preferably those at which the competing react ons and racemizations can be suppressed. In this cor ection, the reactivity of the aldehyde and of the cyanohydrin determines how low the pH must be reduced for these concurrent reactions to be negligible.

In this respect it has been established that it is possible to use the oxynitrilase (4.1.2.11) from Sorghum bicolor in a pH range of such acidity that the enzymatic reaction predominates by far.

This is because the reaction optimum for the enzyme is at pH 4.9, and it still has 40% of the maximum activity at pH 3.25. Thus, this pH tolerance of the oxynitrilase (4.1.2.11) makes it possible to obtain products with high optical purity.

In this connection, alteration of the substrate concentrations and/or the reaction temperature provides additional opportunities for inhibiting the chemical concurrent reaction relative to the enzymatic reaction so that there is a certain range of very favorable conditions that are particularly preferred for practice of the invention.

pH values below about 6.0, in particular below about 4.5, are preferably used.

The reaction temperature is expediently in the range from about to The reaction is t.lfl.

1 carried out in aqueous medium, which ought to contain a maximum of about 40%, in particular not more than about 25%, of organic co-solvent.

It has to be borne in mind in this connection that the oxynitrilases have sufficient activity only if the lowering of pH is accompanied by a reduction in the co-solvent content. However, because the enzyme activity is considerably diminished by the presence of even small amounts of organic solvents ethanol), it is advisable and preferable not to add any solvent.

The reactions can be carried out with the natural enzyme as well as with the immobilized enzyme. The enzyme is stable over time in both forms so that it can also be used in a continuous procedure.

It is possible and expedient to carry out a continuous reaction, particularly by using an enzyme membrane reactor. The progress of the reaction can, in view of the different optical ratations of the precursor monosaccharide) and product nitrile of aldonic acid), be followed particularly straight- t forwardly with a polarimeter.

The precursor and product are separated by i thin-layer chromatography using polar mobile phases such as, for example, butanol:pyridine:water in the ratio 6:4:3 Quantitative separation and ;I i N T 'y determination of the diastereomers with the aid of HPLC are likewise possible.

The invention is further described by means of the following illustrative examples.

Example 1 p-Hydroxybenzaldehyde (52 mg 0.5 mmol) is dissolved in 9.4 ml of 50 mM citrate buffer of pH 3.75 and equilibrated at 20°C. To this is added 500 l of (S)-oxynitrilase solution and 800 p1 of 4.2 M aqueous HCN solution. The (S)-oxynitrilase solution employed has an activity of 84 U/ml, where 1 U catalyzes the formation of one pmol/min phydroxymandelonitrile at 20'C and pH 3.75. The reaction is followed by polarimetry (A 578 nm). A constant optical rotation is obtained after 15 to min, and the reaction is complete. Subsequently the reaction mixture is extracted four times with 10 ml of diethyl ether. The combined organic phases are dried over sodium sulfate, and the solvent is stripped off in a rotary evaporator. The residue is subsequently washed three times with 10 ml of pentane each time, and the product is dried in vacuo.

Chem. yield: 64.8 mg (87 of theory) Optical purity: 99 ee The optical purity is determined as the N,Obis(pentafluoropropionyl)-2-amino-l-(phydroxyphenyl)-ethanol derivative of the mandelonitrile by capillary gas chromatography by the method of H. Frank et al. Chromatogr. 146:197- 206 (1987)) on a chiral separating phase (FS- Chirasil-Val, 25 m x 0.32 mm) The procedure for the derivatization is as follows: 1-2 mg of the mandelonitrile are reduced with 250 1l of a 1 M diborane solution (in tetrahydrofuran) in chloroform at room temperature in min. After the excess diborane is hydrolyzed with a few drops of ethanol and the solvent is stripped off, the resulting amino alcohol is directly acylated with 20 pl of pentafluoropropionic anhydride in methylene chloride at room temperature in 15 min.

Finally, excess anhydride is stripped off in a rotary evaporator, and the residue is taken up in methylene chloride and analyzed by gas chromatography.

Example 2 m-Hydroxybenzaldehyde (61 mg reacted at pH 3.25 with 450 pM of 4.2 solution in accordance with Example 1.

is complete after 30 to 40 minutes.

hydroxy-mandelonitrile is isolated as Example 1.

0.5 mmol) is M aqueous HCN The reaction The described in Chem. yield: Optical purity: 67 mg (90 of theory) 98 ee The optical purity is determined via the N,Obis-pentafluoropropionyl-2-amino-l-(mhydroxyphenyl)-ethanol as in Example 1.

Example 3 m-Methylbenzaldehyde (60 mg 0.5 mmol) is reacted with 475 il of 4.2 aqueous HCN solution and 500 i1 of oxynitrilase solution at pH 3.25 in accordance with Example 1. The reaction is complete after about 45 min. The isolation is carried out in analogy to Example 1, (S)-3-methylmandelonitrile being obtained.

Chem. yield: 59 mg (80 of theory) Opzical purity: 96 ee The optical purity is determined via the N,Obis-pentafluoropropionyl ester amide of the corresponding amino alcohol as in Example 1.

Example 4 Benzaldehyde (53 mg 0.5 mmol) is reacted with 475 pl of 4.2 M aqueous HCN solution and 1,500 1l of (S)-oxynitrilase solution at pH 3.25 in accordance with Example 1. The reaction is complete after about 45 min. The isolation is carried out in analogy to Example 1.

Chem. yield: 48 mg (80 of theory) Optical purity: 96 ee The optical purity is determined via the N,Obis-pentafluoropropionyl ester amide as in Example 1.

The S-oxynitrilase solution employed has an activity of 84 U/ml, where 1 U catalyzes the formation of one pM/min p-hydroxymandelonitrile at 20"C and pH 3.75.

-8- Example Continuous production of (S)-p-hydroxymandelonitrile in a stirred reactor with immobilized oxynitrilase The reaction principle is applied to the formation of larger amounts of product in a continuous procedure using (S)-oxynitrilase immobilized on Eupergit® C (Rohm, Darmstadt).

In a continuous procedure in a 10 ml reactor, (S)-p-hydroxymandelonitrile is produced with a spacetime yield of 57.9 g/(l d) over 72 h.

Characteristic reaction data: p-Hydroxybenzaldehyde

HCN

Na citrate Hold-up time Catalyst concentration Running time Enzyme inactivation Conversion in reaction Enantiomeric excess Temperature 21 400 45 3,960 0.15 72 about 3 98 mM mM mM sec g/ml pH 3.75 Example 6 Preparation of a nitrile of an aldonic acid Reaction mixture: 150 mg of D(-)-arabinose (100 mmol); optical rotation: [a]20 -103.01 ml/g dm] (c

D

-9-

I

3.75/citrate buffer) 4 ml of sodium citrate buffer mM; pH 3.75) 100 U of enzyme stock solution oxynitrilase) 50 p1 of hydrocyanic acid The arabinose is dissolved in 4 ml of sodium citrate buffer, and to this solution are added the enzyme solution and the hydrocyanic acid, and the reaction mixture is shaken at room temperature for days.

The reaction is followed by means of the optical rotation and by thin-layer chromatography.

The Rf values of the precursor and product separated from one another on silica gel plates using the above-mentioned mobile phase are 0.68 and 0.81 respectively.

Optical rotation of the product solution: [a] 2 0 -45.54 ml/g dm] (c 3.75/citrate

D

buffer) .1

Claims (14)

1. A process for preparation of an S- cyanohydrin, comprising the steps of: combining an oxo compound with hydrocyanic acid and oxynitrilase (4.1.2.11) from Sorghum bicolor to form a reaction mixture; and enzymatically reacting the oxo compound with the hydrocyanic acid to produce an S- cyanohydrin.

2. The process as claimed in claim 1, wherein the process is carried out in aqueous medium at pH values below abaut

3. The process as claimed in claim 1, wherein the oxo compound is an aldehyde.

4. The process as claimed in claim 3, wherein the aldehyde is benzaldehyde or a d" vs ive Lhr1s.

The process as claimed in claim 1, wherein the oxo compound is an aldose, and the S-cyanohydrin produced is a nitrile of an aldonic acid, wherein the aldonic acid has one more carbon atom than the aldose.

6. The process as claimed in claim 5, wherein an aldose of the general formula R-CHO is reacted, in which R is a polyol residue of the formula -[CHOH]n- CH 2 0H with n 1 to 7. -11- -12

7. The process as claimed in claim 1, wherein the process is carried out in aqueous medium at Ph values below

8. The process as claimed in claim 3, wherein the aldehyde is an aromatic aldehyde.

9. The process claimed in claim 6, wherein the aldose is a su iaving from 3 to 9 carbon atoms.

The proc as claimed in claim 1, wherein the reaction temperatures is between -5 0 C and +50 0 C.

11. The process as claimed in claim 2, wherein .he reaction temperatures is between -5 0 C and

12. The process as claimed in claim 7, wherein the reaction temperatures is between -5 0 C and +50 0 C.

13. An S-cyanohydrin produced by the process of claim 1.

14. An S-cyanohydrin produced by the process of claim 12. A process as claimed in claim 1, or an S-cyanohydrin produced thereby, substantially as hereinbefore described with reference to the Examples. DATED this 8th day of July, 1991. DEGUSSA AKTIENGESELLSCHAFT and FORSCHUNGSZENTRUM JULICH GMBH By Their Patent Attorneys DAVIES COLLISON 7 v c 910708,inmdaL20a:\381 O4deg.res,12