Enzymatic process for the preparation of (S)-cyanohydrins

Details Enzymatic process for the preparation of (S)-cyanohydrins Enzymatic process for the preparation of (S)-cyanohydrins

1998-12-29

2001-05-01

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing nitrogen-containing organic compound

C435S232000

Reexamination Certificate

active

06225095

ABSTRACT:

Cyanohydrins are of importance, for example, for the synthesis of alpha-hydroxy acids, alpha-hydroxy ketones, and beta-aminoalcohols, which are used for obtaining biologically active substances, e.g. pharmaceutical active compounds, vitamins or alternatively pyrethroid compounds.
The preparation of a cyanohydrin can be carried out by addition of hydrocyanic acid (HCN) to the carbonyl group of an aldehyde or of an unsymmetrical ketone, enantiomer mixtures of unsymmetrical cyanohydrins resulting.
Since normally only one of the two enantiomers in a biologically active enantiomer mixture is biologically active, there has not been a lack of attempts to find a process for the preparation of the (S)-enantiomer of an optically active cyanohydrin in as high as possible optical purity.
Thus, for example, in Makromol. Chem. 186, (1985), 1755-62 a process for obtaining (S)-cyanohydrins by reaction of aldehydes with hydrocyanic acid in the presence of benzyloxycarbonyl-(R)-phenylalanine-(R) histidine methyl ester as a catalyst is described. The optical purity of the (S)-cyanohydrins obtained, however, is extremely unsatisfactory.
EP-A-0 326 063 describes an enzymatic process for the preparation of optically active (R)- or (S)-cyanohydrins by reaction of aliphatic, aromatic or heteroaromatic aldehydes or ketones with hydrocyanic acid in the presence of (R)-oxynitrilase (EC 4.1.2.10) from
Prunus amygdalis
or oxynitrilase (EC 4.1.2.11) from Sorghum bicolor. Examples of the stereospecific preparation of aliphatic (S)-cyanohydrins are not indicated. This is not surprising, since in Angew. Chemie 102 (1990), No. 4, pp 423-425 it is stated by inventors who are mentioned in EP-A-0 326 063 that no aliphatic (S)-cyanohydrins can be prepared using hydrocyanic acid in the presence of the (S)-oxynitrilase from Sorghum bicolor. This finding is also confirmed by F. Effenberger et al. in Tetrahedron Letters Vol. 31 No. 9 (1990), pp. 1249-1252.
EP 0 632 130 further describes a process in which aliphatic aldehydes or unsymmetrical aliphatic ketones are reacted stereospecifically with hydrocyanic acid and oxynitrilase from
Hevea brasiliensis
to give (S)-cyanohydrins. The reaction is carried out according to EP 0 632 130, preferably in an aqueous diluent without addition of organic solvents, since these, as described in EP 0 632 130, rapidly inhibit the activity of the enzyme.
The processes known up to now were mostly carried out under rather dilute conditions, either in an aqueous or organic system or in a two-phase system. This procedure, however, has disadvantages for many starting materials. Thus, for example, 3-phenoxybenzaldehyde or 4-fluoro-3-phenoxybenzaldehyde or various ketones are poor substrates, so that a high use of enzyme is necessary in order to obtain an acceptable yield of cyanohydrins in good optical purity.
It has now unexpectedly been found that the reaction of a large number of carbonyl compounds, such as, for example, aliphatic, alicyclic, unsaturated, aromatically substituted aliphatic, aromatic, and also heteroaromatic aldehydes and ketones, to give the corresponding cyanohydrins in high yield and high optical purity is possible in a process which is more concentrated in relation to the prior art and with lower use of enzyme if the reaction is carried out in an emulsion. Unexpectedly, the enzyme activity under the conditions of an emulsion such as, for example, high stirring energy, which in the case of many proteins lead to deactivation, remains stable.
The present application therefore relates to a process for the preparation of the (S)-enantiomers of optically active cyanohydrins by reaction of an aldehyde or of a ketone with a cyanide group donor in the presence of a native or of a recombinant (S)-hydroxynitrile lyase, which comprises stirring a reaction mixture of
a) an aldehyde or ketone dissolved in an organic water-immiscible or slightly miscible diluent,
b) an aqueous (S)-hydroxynitrile lyase solution and
c) a cyanide group donor
in such a way that an emulsion is formed, which is maintained up to the end of the reaction by stirring, after which the corresponding (S)-cyanohydrin is isolated from the reaction mixture by phase separation after reaction is complete
The starting materials employed in the process according to the invention are an aldehyde or a ketone, a cyanide group donor, an aqueous solution of a native or recombinant hydroxynitrile lyase (Hnl) and an organic diluent which is immiscible or slightly miscible with water.
Aldehydes in this case are understood as meaning aliphatic, aromatic or heteroaromatic aldehydes. Aliphatic aldehydes in this case are understood as meaning saturated or unsaturated aliphatic, straight-chain, branched or cyclic aldehydes. Preferred aliphatic aldehydes are straight-chain aldehydes, in particular having 2 to 18 C atoms, preferably from 2 to 12, which are saturated or mono- or polyunsaturated. The aldehyde can in this case have both C—C double bonds and C—C triple bonds. The aldehyde can be unsubstituted or substituted by groups which are inert under the reaction conditions, for example by optionally substituted aryl or heteroaryl groups such as phenyl or indolyl groups, or by halogen, ether, alcohol, acyl, carboxylic acid, carboxylic acid ester, nitro or azido groups.
Examples of aromatic or heteroaromatic aldehydes are benzaldehyde and variously substituted benzaldehydes such as, for example, 3,4-difluorobenzaldehyde, 3-phenoxybenzaldehyde, 4-fluoro-3-phenoxybenzaldehyde, and further furfural, anthracene-9-carbaldehyde, furan-3-carbaldehyde, indole-3-carbaldehyde, naphthalene-1-carbaldehyde, phthalaldehyde, pyrazole-3-carbaldehyde, pyrrole-2-carbaldehyde, thiophene-2-carbaldehyde, isophthalaldehyde or pyridine aldehydes etc.
Ketones are aliphatic, aromatic or heteroaromatic ketones in which the carbonyl carbon atom is unequally substituted. Aliphatic ketones are understood as meaning saturated or unsaturated, straight-chain, branched or cyclic ketones. The ketones can be saturated or mono- or polyunsaturated. They can be unsubstituted, or substituted by groups which are inert under the reaction conditions, for example by optionally substituted aryl or heteroaryl groups such as phenyl or indolyl groups, or by halogen, ether, alcohol, acyl, carboxylic acid, carboxylic acid ester, nitro or azido groups.
Examples of aromatic or heteroaromatic ketones are acetophenone, indolylacetone etc.
Aldehydes and ketones which are suitable for the process according to the invention are known or can be prepared in the customary manner.
Hydrocyanic acid is added as a cyanide group donor. The hydrocyanic acid can in this case also be released from one of its salts such as, for example, NaCN or KCN only shortly before the reaction and added to the reaction mixture in undiluted form or in dissolved form.
As a hydroxynitrile lyase (Hnl), native (S)-hydroxynitrile lyases, e.g. from cassava and
Hevea brasiliensis,
and also recombinant (S)-Hnl. Preferably, the native Hnl used is Hnl from
Hevea brasiliensis
or
Manihot esculenta.
Suitable recombinant (S)-Hnl is obtained, for example, from genetically modified microorganisms such as, for example,
Pichia pastoris
or
Saccharomyces cerevisiae.
Preferably, recombinant (S)-Hnl from
Pichia pastoris
is employed.
As a result of the functional overexpression in the methylotrophic yeast
Pichia pastoris,
this Hnl can be obtained in any desired amount (M. Hasslacher et al., J. Biol. Chem. 1996, 271, 5884). This expression system is particularly suitable for fermentations at a high cell density. Thus it is possible to obtain approximately 20 g of pure enzyme per liter of fermentation medium. The specific activities of the purified recombinant protein achievable are approximately twice as high as those of the natural enzyme, which was isolated from the leaves of the tree
Hevea brasiliensis.
After cell destruction, the cytosolic fraction can be used without further purification, whereby the expenditure of labor is minimized. The enzyme is not glycosylated and also has no prosthetic group which would lead

Affiliated with

Also associated with

Rating

Enzymatic process for the preparation of (S)-cyanohydrins does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Enzymatic process for the preparation of (S)-cyanohydrins, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Enzymatic process for the preparation of (S)-cyanohydrins will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2537633