Enzymatic processes for preparing (S)-cyanohydrins

Details Enzymatic processes for preparing (S)-cyanohydrins Enzymatic processes for preparing (S)-cyanohydrins

1999-06-25

2002-01-08

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Preparing nitrogen-containing organic compound

C435S280000

Reexamination Certificate

active

06337196

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.
A cyanohydrin can be prepared by addition of a cyanide group to the carbonyl carbon of an aldehyde or of an unsymmetrical ketone, mixtures of enantiomers of optically active cyanohydrins resulting.
Since in a biologically active mixture of enantiomers only one of the two enantiomers is biologically active, there has been no lack of attempts to find a process for the preparation of the (S)-enantiomer of an optically active cyanohydrin in as high an optical purity as possible.
Thus, in Makromol. Chem. 186, (1985), 1755-62, for example, 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 highly unsatisfactory.
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 is described in EP-A-0 326 063. Examples of the stereo-specific 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 with hydrocyanic acid from the (S)-oxynitrilase from Sorghum
EP 0 632 130 additionally 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 rapidly inhibit the activity of the enzyme, as described in EP 0 632 130.
EP 0 539 767 describes a similar process for the preparation of (S)-cyanohydrins, using specific cyanide group donors instead of hydrocyanic acid. EP 0 539 767 also indicates that organic solvents rapidly inhibit the activity of the enzyme.
The use of recombinant hydroxynitrile lyase from Hevea brasiliensis in an aqueous buffer system is described in Tetrahedron Letters Vol. 52, No. 23, 1996, pp. 7833-7840.
It has now unexpectedly been found that the use of recombinant hydroxynitrile lyase (Hnl) from
Hevea brasiliensis
makes possible 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, the recombinant Hnl being distinguished by a high resistance to organic solvents.
The invention therefore relates to a process for the preparation of the (S)-enantiomer of an optically active cyanohydrin by reaction of an aldehyde or of a ketone with a cyanide group donor, which comprises reacting the aldehyde or the ketone with a cyanide group donor in an organic diluent in the presence of a recombinant (S)-hydroxynitrile lyase from
Hevea brasiliensis
and isolating the (S)-cyanohydrin formed from the reaction mixture.
Starting materials employed in the process according to the invention are an aldehyde or a ketone, a cyanide group donor, a recombinant hydroxynitrile lyase and a diluent.
Aldehydes are in this case understood as meaning aliphatic, aromatic or heteroaromatic aldehydes. Aliphatic aldehydes are in this case 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 or variously substituted benzaldehydes such as, for example, 3-phenoxybenzaldehyde, additionally furfural, anthracene-9-carbaldehyde, furan-3-carbaldehyde, indole-3-carbaldehyde, naphthalene-l-carbaldehyde, phthalaldehydes, 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 identically or unidentically 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 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, benzophenone etc. Aldehydes and unsymmetrical ketones are preferably reacted.
Aldehydes and ketones which are suitable for the process according to the invention are known or can be prepared in the customary manner.
A possible cyanide group donor is hydrocyanic acid or a cyanohydrin of the general formula R
1
R
2
C(OH) (CN). In the formula I, R
1
and R
2
independently of one another are hydrogen or a hydrocarbon group which is unsubstituted or substituted by groups which are inert under the reaction conditions, or R
1
and R
2
together are an alkylene group having 4 or 5 C atoms, where R
1
and R
2
are not simultaneously hydrogen. The hydrocarbon groups are aliphatic or aromatic, preferably aliphatic groups. R
1
and R
2
are preferably alkyl groups having 1 to 6 C atoms, the cyanide group donor is very preferably acetone cyanohydrin.
The cyanide group donor can be prepared according to known processes. Cyanohydrins, in particular acetone cyanohydrin, are also commercially available.
Preferably, hydrocyanic acid or acetone cyanohydrin is employed as the cyanide group donor. The hydroxynitrile lyase employed is recombinant (S)-Hnl from
Hevea brasiliensis
. Suitable recombinant (S)-Hnl is obtained, for example, from genetically modified microorganisms such as, for example,
Pichia pastoris
or
Saccharomyces cerevisiae.
Recombinant (S)-Hnl from
Pichia pastoris
is preferably employed. By 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 having a high cell density. Thus, it is possible to obtain approximately 20 g of pure enzyme per liter of fermentation medium. The achievable specific activities of the purified recombinant protein are approximately twice as high as those of the natural enzyme, which was isolated from the leaves of the tree
Hevea brasiliensis.
After cell disruption, the cytosolic fraction can be used without further purification, by means of which the expenditure of work is minimized. The enzyme is not glycosylated and also has no prosthetic group which would lead to inactivation during removal of the protein moiety. The Hnl can be employed at room temperature for a number of days without significant loss of activity, and is adequately stable at −20° C. in the long term. As a result, the possibility results of using the same enzyme batch a number of times. The enzyme is also distinguis

Affiliated with

Also associated with

Rating

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

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2818283