Non-homogeneous systems for the resolution of enantiomeric mixtures
The present invention relates to a process for the biocatalyst-mediated enantioselective conversion of enantiomeric mixtures of hydrophobic esters uing a biphasic solvent system. More particularly, the present invention relates to the enzyme-mediated enantioselective synthesis of anti-viral compounds, such as 2-hydroxymethyl-5-(5-flurocytosin-1-yl)-1,3-oxathiolane (FTC) and its analogues, in a non-homogenous reaction system.
1. A process for the resolution of a compound of Formula A:
wherein:
X=F;
Y=S;
wherein the process comprises the steps of:
(a) dispersing an enantiomeric mixture of a compound of Formula B
wherein:
R is C 1 –C 8 alkyl, alkenyl, or alkynyl
X=F;
Y=S
at a concentration of between about 5% and about 45% (weight/volume of a non-homogeneous system), in an organic solvent system to produce an organic component;
(b) providing an aqueous solvent system to produce an aqueous component; and
(c) contacting the organic component and the aqueous component to form a non-homogeneous system, under conditions which permit the resolution of the mixture with a hydrolase enzyme to produce a chiral non-racemic ester of Formula B and a non-racemic alcohol of Formula A
wherein said hydrolase enzyme is dispersed in either said organic component, said aqueous component or said non-homogeneous system.
2. The process of claim 1 , wherein the compound of Formula B is 2-butyryloxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane.
3. The process of claim 2 , wherein the organic component comprises between about 5 and about 90% of the non-homogeneous system; and the non-homogeneous system also comprises between about 1 and about 20% of surfactant.
4. The process according to any one of claim 1 , 2 or 3 , wherein the hydrolase enzyme is selected form the group consisting of porcine liver esterase, porcine pancreatic lipase, Pseudomonas species lipase, Aspergillus niger lipase and subtilisin.
5. The process according to claim 4 , wherein the hydrolase enzyme is a crosslinked enzyme crystal.
6. The process according to claim 5 , wherein the crosslinked enzyme crystal is crosslinked with glutaraldehyde.
7. The process according to claim 4 , wherein the hydrolase enzyme is an immobilized enzyme.
8. The process according to claim 4 , wherein the hydrolase enzyme is a soluble enzyme.
9. The process according to claim 4 , wherein the hydrolase enzyme is porcine liver esterase.
10. The process according to any one of claim 1 , 2 or 3 , wherein the chiral non-racemic ester is isolated from the organic component.
11. The process according to any one of claim 1 , 2 or 3 , wherein the chiral non-racemic alcohol is isolated from the aqueous component.
12. The process according to any one of claim 1 , 2 or 3 , wherein the enantiomeric mixture is dispersed in the organic component to a concentration of between about 5% to about 15%.
13. The process according to any one of claim 1 or 2 , wherein the enantiomeric mixture is dispersed in the organic component to a concentration of between about 10% to about 20%.
14. The process according to any one of claim 1 , 2 or 3 , wherein the organic component comprises not more than about 50% water miscible organic solvent.
15. The process according to claim 14 , wherein the organic component comprises one or more solvents selected from the group consisting of C 4 –C 8 alcohols, nitromethane, dichloromethane, toluene, methyl isobutyl ketone, tert-butyl acetate and alkanes.
16. The process according to claim 15 , wherein the organic component comprises one or both of n-amyl alcohol and 3-methyl-3-pentanol.
17. The process according to claim 3 , wherein the surfactant is selected from the group consisting of cationic surfactants, anionic surfactants and nonionic surfactants.
18. The process according to claim 17 , wherein the surfactant is selected from the group consisting of Tween 20™, Tween 80™, Prionex™, Teepol HB7™, Tergitol TMN-6™, Tergitol TMN-10™, Tergitol NP-4™, Tergitol 15-S-3™, Igepal CA-630™, Tyloxapol™, Glucode-oxycholic acid, octyl β-gluco-pyranoside, dioctyl sulfosuccinate, and deoxycholic acid.
19. The process according to claim 18 , wherein the surfactant is Tween-80™.
20. The process according to claim 18 , wherein the surfactant is dioctyl sulfosuccinate.
21. The process according to claim 3 , wherein the surfactant is added to the organic component.
22. The process according to claim 3 , wherein the surfactant is added to the aqueous component.
23. The process according to claim 3 , wherein the surfactant is added to said non-homogeneous system.
24. The process according to claim 3 , wherein the surfactant is formulated with the hydrolase enzyme.
25. The process according to any one of claim 1 , 2 or 3 , wherein the aqueous solvent system comprises water and excipients selected from the group consisting of buffering salts, alkalizing agents, anti-microbial preservatives, stabilizers, filtering aids, co-enzymes, excipients that facilitate dispersion and excipients that facilitate function of the enzyme.
26. The process according to claim 25 , wherein the aqueous solvent system comprises water buffered with phosphate buffer at a pH of greater than about 7.
27. The process according to claim 25 , wherein the aqueous solvent system comprises water buffered with 2-amino-2-(hydroxymethyl)-1,3-propanediol or TRIS™.
28. The process according to any one of claim 1 , 2 or 3 , wherein the conditions which permit the resolution comprise a temperature of between about 5° C. and about 45° C.