Methods of enzymatically separating stereoisomers of a racemic mixture of a reactive ester

Methods of separating stereoisomers of a racemic mixtures of a compound employ porcine pancreatic lipase which selectively transesterfies one enantiomer of an ester function which is proximate to a chiral isomeric site are presented. The compound is a reactive ester of the formula: ##STR1## The method comprises obtaining a racemic mixture of the reactive ester, reacting the reactive ester with a poly(ethylene glycol) of average molecular weight of about 100 to about 10,000 Daltons and the enzyme. The reactive ester, the poly(ethylene glycol) and the enzyme are reacted in a medium in proportions other conditions effective to form a chiral site proximate poly(ethylene glycol) ester. The chiral site approximate poly(ethylene glycol) ester is then separated from the reaction medium, the reactive ester, the poly(ethylene glycol) and the enzyme.

TECHNICAL FIELD 
This invention relates to the resolution of enantiomers by stereospecific 
enzymatic transeseterification of an ester function placed up to 2 C-atoms 
away from a chiral center and separating the two stereoisomers based on 
their different physicochemical properties. 
BACKGROUND ART 
The resolution of enantiomers by enantioselective hydrolysis with an enzyme 
is known. However, the recent discovery that many hydrolytic enzymes work 
well in low polarity organic solvents (Cambou, B. and Kilbanov, A. M. J. 
Am. Chem. Soc., 1984, 106, 2687; Zaks, A., and Kilbanov, A. M., Science, 
1984, 224, 1249) has allowed for enzymatic resolution to be extended to 
new reactions such as esterification (Cambou, B. and Kilbanov, A. M., 
Biotechnology and Bioengineering, 1984, XXVI, 1449), and 
transesterification (Cambou, B. and Kilbanov, A. M., J. Am. Chem. Soc., 
1984, 106, 2687; Cambou, B. and Kilbanov, A. M., Biotechnology and 
Bioengineering, 1984, XXVI, 1449). 
Resolution by enzymatic transesterification solves some of the problems 
associated with enzymatic hydrolyses. Among these are the low solubility 
of many organic compounds in water, the difficulty of recovering the 
enzyme for reuse, and the requirement for adjusting the ph as the reaction 
progresses. Moreover, an increase in enzyme stability has been reported 
when it is used in an organic solvent and higher reaction temperatures are 
tolerated (Zaks, A. and Kilbanov, A. M., Science, 1984, 224, 1249). 
The resolution of a racemic mixture of esters may be carried out by 
transesterifying one enantiomer of a racemic ester with an achiral alcohol 
(Cambou, B. and Kilbanov, A. M. Biotechnology and Bioengineering, 1984, 
XXVI, 1449). More commonly, however, one enantiomer of a racemic alcohol 
transesterifiers and achiral ester. (Cambou, B. and Kilbanov, A. M., J. 
Am. Chem. Soc., 1984, 106, 2687; Cambou, B. and Kilbanov, A. M., 
Biotechnology and Bioengineering, 1984, XXVI, 1449). The latter case, 
which is useful for resolving alcohols, leads to the necessity for 
separating an ester from an alcohol. The first case, which is useful for 
resolving racemic mixtures of esters, leaves both enantiomers in the form 
of esters. The separation of the isomers in most cases requires tedious 
chromatography or careful distillation. (Cambou, B. and Klibanov, A. M., 
Biotechnology and Bioengineering, 1984, XXVI, 1449). This has been a 
substantial drawback which accounts for the limited application of this 
technology up to the present time. 
The resolution of 3,4'-epoxybutyrate by stereoselective enzymatic 
hydrolysis of its methyl or other alkyl esters was recently reported by 
Mohr et al. (Mohr et al., Helv. Chim. Acta., 1987, 70, 142; Mohr et al., 
Tetrahedron Letters, 1989, 30 (19(, 2513) and by Bianchi et al. (Bianchi 
et al., J. Org. Chem., 1988, 53, 104)). Mohr et al. hydrolyzed a methyl 
ester with pig liver esterase and obtained the unchanged (R) ester and the 
(S) acid. However, the enantiomeric excesses of each compound after 
separation was not very high. Bianchi et al., supra, conducted the 
reaction with 13 different enzymes and concluded that porcine pancreatic 
lipase (PPL) provides the best stereoselectivity when alkyl, e.g., butyl, 
isobutyl and octyl, esters are hydrolyzed. 
DISCLOSURE OF THE INVENTION 
This invention relates to a compound of the formula 
EQU R.sup.1 OC--R--CH(--R.sup.2)(--R.sup.3) 
wherein 
R is (C.sub.0 -C.sub.2)alkyl or --O(C.sub.0 -C.sub.2)alkyl, all of which 
may be further substituted with (C.sub.1 -C.sub.4)alkyl; 
R.sup.1 is selected from the group consisting of --OH, --O(C.sub.1 
-C.sub.15)alkyl or alkenyl, --O--(C.sub.3 -C.sub.15)cycloalkyl, 
cycloalkenyl, cycloalkynyl and --O--(C.sub.4 -C.sub.22)aryl, all of which 
may contain N, S or O in the chain or ring structure and be further 
substituted with one or more (C.sub.1 -C.sub.4)alkyl, OH, NO.sub.2 or 
halogen, and R.sup.1 is preferably CF.sub.3 CH.sub.2 O, CCl.sub.3 CH.sub.2 
O, ClCH.sub.2 CH.sub.2 O or 
##STR2## 
wherein X is Cl, NO.sub.2 or F; R.sup.2 is selected from the group 
consisting of (C.sub.1 -C.sub.12)alkyl, alkenyl or alkynyl, (C.sub.3 
-C.sub.15)cycloalkyl, cycloalkenyl, cycloalkynyl, (C.sub.4 -C.sub.22)aryl 
and (C.sub.5 -C.sub.23)alkylaryl or arylalkyl, all of which may be 
substituted in the ring or chain with O, N or S; and 
R.sup.3 is selected from the group consisting of R.sup.2, OH, NH.sub.2, 
OR.sup.2, NHR.sup.2, NR.sup.2, SR.sup.2, O(C.sub.1 -C.sub.6)acyl or aroyl, 
NH(C.sub.1 -C.sub.6)acyl or aroyl, --CH(R.sup.2)(R--COR.sup.1) and 
halogen, wherein R.sup.2 is different from R.sup.3 ; 
or R.sup.2 and R.sup.3 form a ring selected from the group consisting of 
(C.sub.3 -C.sub.15)cycloalkyl, cycloalkenyl or cycloalkynyl, (C.sub.4 
-C.sub.22)aryl, and (C.sub.5 -C.sub.23)alkylaryl or arylalkyl, all of 
which may be further substituted in the ring or chain with N, S or O, said 
compound being selected from the group consisting of the stereoisomers 
thereof in substantially stereochemically pure form. 
Also disclosed herein are polymers of the formula 
##STR3## 
wherein R is (C.sub.0 -C.sub.2)alkyl or --O(C.sub.0 -C.sub.2)alkyl, all of 
which may further be substituted with (C.sub.1 -C.sub.4)alkyl; 
P is a poly(alkylene glycol) of the formula --O--(--(CH.sub.2).sub.n 
--O).sub.x --, wherein n is 1 to 10 and x is 1 to 1,000; and 
R.sup.2, R.sup.3 and R are as disclosed above. 
Also disclosed herein are polymers of the formula 
##STR4## 
wherein R is (C.sub.0 -C.sub.2)alkyl; 
R.sup.2 is H or is as described above; and 
R.sup.3 is as described above but different from R.sup.2, and 
P is a poly alkylene glycol of the formula --O--(--(CH.sub.2).sub.n 
--O).sub.x --, wherein n is 1 to 10 x is 1 to 10,000 and z is 1 to 10,000. 
This invention also relates to a method of separating the isomers of a 
racemic mixture of the compound of the invention, which method comprises 
obtaining a racemic mixture of an ester of the formula R.sup.4 
--OC--R--CH(--R.sup.2)(--R.sup.3), wherein R, R.sup.2, and R.sup.3 are as 
described above, and R.sup.4 is O(C.sub.1 -C.sub.6)alkyl or O(C.sub.4 
-C.sub.10)aryl, and preferably --OCH.sub.2 CX.sub.3 or O(C.sub.4 -C.sub.10 
aryl, all of which may be substituted with O or S and/or in the ring with 
halogen NO.sub.2 or OCH.sub.2 CX.sub.3, wherein X is halogen; 
reacting the reactive ester with a poly(ethyleneglycol) of average 
molecular weight about 150 to 20,000 daltons and an enzyme capable of 
enantioselectively transesterifying an ester function which is proximate 
to an (S) (or (R))chiral isomeric site; the reactive ester, the 
poly(ethylene glycol) and the enzyme being reacted in a medium, in 
proportions and under conditions effective to form a chiral site proximate 
poly(alkyleneglycol) ester of one enantiomer of the ester; and 
separating the chiral site proximate poly(alkyleneglycol) ester(s) from the 
reaction medium, the unchanged enantiomer of the reactive ester, the 
enzyme and, subsequently, the poly(alkyleneglycol). 
A method of preparing polymers of the formula 
##STR5## 
comprising obtaining a reactive diester of the formula 
##STR6## 
wherein R, R.sup.1, R.sup.2, and R.sup.3 are as defined above; reacting 
the reactive diester with a poly(alkylene glycol) of the formula 
HO((CH.sub.2).sub.n --O--)H.sub.x H of average molecular weight about 150 
to 20,000 daltons and an enzyme capable of enantioselectively 
transesterfying an ester function which is proximate to a pair of chiral 
isomeric sites; the reactive ester, poly(alkylene glycol) and the enzyme 
being reacted in a medium, in proportions and under conditions effective 
to form a bis chiral site proximate polyester between the diol and one 
stereoisomer of the diester. 
A more complete appreciation of the invention and many of the attended 
advantages thereof will be readily perceived as the same becomes better 
understood by reference to the following detailed description. Other 
objects, advantages and features of the present invention will become 
apparent to those skilled in the art from the following discussion. 
BEST MODE FOR CARRYING OUT THE INVENTION 
This invention arose from a desire to improve the ease of separating two 
stereoisomeric esters while retaining advantages associated with the known 
enzyme-catalyzed transesterification in an organic medium. The present 
invention is an improvement over the method disclosed by Mohr, et al, 
supra, Bianchi et al., supra, Klibanov et al, supra, and Mohr et al, 
supra, and it relies, inter alia, on a combination of stereospecific 
enzyme transesterification with a polymeric alcohol of predetermined 
characteristics and the separation of the stereoisomers by relying on 
different physicochemical characteristics of the compounds. 
The alcohol which is utilized herein is of a polymeric nature and has a 
molecular weight range suitable for imparting to the transesterified 
isomer properties which are substantially different from those of the 
untransesterified stereoisomer. The polymeric alcohol utilized for the 
transesterification must have low solubility in the reaction solvent. In 
this manner, the transesterified stereoisomer, by taking on the 
characteristics of the polymeric alcohol, becomes insoluble and easily 
separable from the unchanged isomer by known separation techniques. After 
the stereoisomer transesterified with the insoluble polymer alcohol is 
separated from the reaction mixture, the unmodified stereoisomer may be 
isolated by evaporating the solvent or other known separation techniques 
such as distillation, extraction into a solvent, and the like. The 
insoluble transesterified ester may be hydrolyzed or again transesterified 
by chemical or enzymatic catalysis to thereby obtain the free acid or a 
low molecular weight alcoholic ester thereof. 
This invention therefore provides stereochemically pure esters containing 
at least one chiral center, where the chiral center is positioned up to 2 
C-atoms away from the carboxyl moiety. The compounds of the invention have 
the formula 
EQU R.sup.1 OC--R--C(--R.sup.2)(--R.sup.3), 
wherein 
R is (C.sub.0 -C.sub.2)alkyl or --O(C.sub.0 -C.sub.2)alkyl, which may be 
further substituted with (C.sub.1 -C.sub.4)alkyl; 
R.sup.1 is selected from the group consisting of --OH, --O(C.sub.1 
-C.sub.15)alkyl or alkenyl, and O(C.sub.3 -C.sub.15)cycloalkyl, 
cycloalkenyl, cycloalkynyl, O(C.sub.4 -C.sub.22)aryl and O(C.sub.5 
-C.sub.23)alkylaryl or arylalkyl, all of which may contain N, S, or O in 
the chain or ring structure and be further substituted with one or more 
(C.sub.1 -C.sub.4)alkyl, OH, NO.sub.2 or halogen; 
R.sup.2 is selected from the group consisting of (C.sub.1 -C.sub.12)alkyl, 
alkenyl or alkynyl, (C.sub.3 -C.sub.15)cycloalkyl, cycloalkenyl or 
cycloalkynyl, (C.sub.4 -C.sub.22)aryl, and (C.sub.5 -C.sub.23)alkylaryl or 
arylalkyl, all of which may be substituted in the ring or chain with O, N 
or S; and 
R.sup.3 is selected from the group consisting of R.sup.2, OH, NH.sub.2, 
OR.sup.2, NHR.sup.2, SR.sup.2, O--(C.sub.1 -C.sub.6)aryl, NH--(C.sub.1 
-C.sub.6) acyl, --CH(R.sup.2)(--R--COR.sup.1) and halogen, wherein R.sup.2 
is different from R.sup.3 ; 
or R.sup.2 and R.sup.3 form a ring selected from the group consisting of 
(C.sub.3 -C.sub.15)cycloalkyl, alkenyl or alkynyl, (C.sub.4 -C.sub.22)aryl 
and (C.sub.5 -C.sub.23)alkylaryl or arylalkyl, all of which may be further 
substituted in the ring or chain with N, S or O, said compound being 
selected from the group consisting of the isomers thereof in substantially 
stereochemically pure form. 
R.sup.1 is preferably CF.sub.3 CH.sub.2 O, CClCH.sub.2 O, ClCH.sub.2 
CH.sub.2 O.sub.2 or 
##STR7## 
wherein X is Cl, NO.sub.2 or F. 
The substantially stereospecifically pure compounds of the invention have 
about 95% or greater purity, and in some cases up to about 97% or greater 
purity. These stereoisomers are, in a most preferred embodiment, 
substantially devoid of detectable amounts of other isomers of the same 
compound. 
A particularly preferred group of stereoisomers is that having the (R) 
configuration. Another particularly preferred group is that having the (S) 
stereochemical configuration. Other preferred groups are those where R is 
held by a single bond, and where R is C.sub.1 -alkyl. Preferred groups are 
also those wherein R.sup.1 is OH or a poly(alkylene glycol) having a 
molecular weight of about 100 to 20,000 daltons, and more preferably about 
1000 to 5,000 daltons. Also preferred are those wherein R.sup.2 is H or an 
aliphatic hydrocarbon. Other preferred groups are those wherein R.sup.2 is 
aryl or a cycloaliphatic residue. 
Still other preferred groups of compounds are those where R.sup.3 is 
R.sup.2, OR.sup.2, or --CH(--OR.sup.2)(--R--COOR.sup.1) and the like as 
described above, as long as R.sup.2 is different from R.sup.3. Another 
preferred group is that where R.sup.2 and R.sup.3 form a ring structure 
such as a cycloaliphatic or aromatic ring, optionally containing N, S or 
O. 
The group of compounds where R.sup.3 is --CH)--OR.sup.2)(--R--COOR.sup.1) 
have at least two chiral centers and the method of the invention may be 
applied to separate all stereoisomers from one another by itself or in 
combination with the method of the co-filed application by the present 
inventors entitled "Substantially Pure Stereoisomers and Method of 
Preparation", the entire contents of which are incorporated herein by 
reference. 
A particularly preferred group of stereochemically pure compounds is that 
having more than one chiral center in the molecule. These compounds are 
obtained as racemic mixtures of the (R,R), (R,S), and (S,S) stereoisomers 
thereof. These stereoisomers may be separated from one another by a 
repetitive application of the method of the invention. 
The compounds of the invention may be prepared by the method disclosed 
herein or by any other known method. The present method, however, permits 
the large-scale high stereochemical purity preparation of these compounds. 
The stereochemically pure compounds of the invention are suitable for the 
preparation of polymers which have non-linear optical properties, as 
components of catalysts for making optically active compounds or as media 
for the separation of optically active compounds. Examples of such 
polymers are helical polymers having a single screw sense. The pitch and 
period of the helical polymers can be adjusted by varying the structure of 
the alcoholic stereoisomer utilized. The nitrogen-containing compounds 
also permit binding of metals to the helix to make chiral catalysts. 
The polymers of the invention may be any polymer formed by random, block or 
alternate binding of the present chiral ester stereoisomers of the 
invention with other bridging structures. By means of example, the polymer 
of the invention may have the general formula 
##STR8## 
wherein R is (C.sub.0 -C.sub.2)alkyl or --O(C.sub.0 -C.sub.2)alkyl, which 
may further be substituted with (C.sub.1 -C.sub.4)alkyl; 
P is a poly(alkylene glycol) of the formula --O--((CH.sub.2).sub.n).sub.x, 
wherein n is 1 to 10 and x is 1 to 10,000; and 
R, R.sup.2 and R.sup.3 are as disclosed above. 
Also disclosed herein are are polymers of the formula 
##STR9## 
wherein R is (C.sub.0 -C.sub.2)alkyl; 
R.sup.2 is H or is as described above; 
R.sup.3 is as described above but different from R.sup.2 ; and 
P is poly(alkylene glycol) of the formula --O--(--(CH.sub.2).sub.n 
--O).sub.x, wherein n is 1 to 10, x is 1 to 10,000, and z is 1 to 10,000. 
The polymers of the invention may be prepared by the method of the 
invention by reaction of an excess of a racemic mixture of the 
stereoisomeric chiral compound of the invention described above with a 
diol of the formula HO((CH.sub.2).sub.n --O--).sub.x H in the presence of 
an enzyme capable of stereoselectively transesterifying one enantiomer of 
the chiral compound with the diol at each end, two molecules of the chiral 
compound being required to completely transesterify both alcoholic 
functions of each diol molecule. The reaction is conducted in a medium, in 
proportions of the reactants and under conditions effective to form a bis 
site proximate polyester between the diol and one stereoisomer of the 
diester. 
Also part of this invention is a method of separating the stereoisomers of 
a racemic mixture of the compounds of the invention comprising 
obtaining a racemic mixture of a reactive ester of the formula R.sup.1 
--OC--R--CH--(R.sup.2)(--R.sup.3), wherein R, R.sup.2, and R.sup.3 are as 
described above, and R.sup.1 is O(C.sub.1 -C.sub.6)alkyl, --O--Ar, 
--OCH.sub.2 CH.sub.2 X or --OCH.sub.2 CX.sub.3, wherein X is halogen and 
the Ar may further contain N, S or O in the ring structure and; 
reacting the reactive ester with a poly(ethylene glycol) or diol terminated 
polyether of average molecular weight about 100 to 20,000 daltons under 
conditions effective to form the poly(ethyleneglycol) or poly ether 
alcohol ester of one enantiomer of the ester; and 
separating the chiral site proximate poly(ethylene glycol) ester(s) from 
the reaction medium, the reactive ester, and the enzyme and subsequently 
from the poly(ethylene glycol). 
The racemic mixtures of a reactive ester as described above are either 
commercially available or may be prepared by methods known in the art. For 
example, this may involve taking a mixture of a chiral acid and treating 
it with excess alcohol in the presence of an acid catalyst or with a 
dehydrating agent such as dicyclohexyl carbodiimide (DCC) in the presence 
of a catalyst such as 4-(N,N dimethylamino)pyridine (DMAP) as described by 
Hassner and Alexanian (Hassner and Alexanian, Tetrahedron Lett., 1978, 
4475). 
The racemic mixture of the reactive ester is reacted with a poly(ethylene 
glycol) of average molecular weight about 100 to 20,000 daltons, more 
preferably about 500 to 10,000, and still more preferably about 1,000 to 
5,000 daltons. This reaction is conducted in the presence of an enzyme 
capable of stereoselectively transesterifying one enantiomer of an ester 
function which is proximate to a chiral site. The reactants are provided 
in a liquid medium, preferably an anhydrous organic compound which acts as 
a solvent for the ester substrate but not the poly(ethylene glycol) and 
also preferably under e.g., a non-oxidizing atmosphere. The non-aqueous 
conditions under which the transesterification step is conducted are 
advantagous because most organic esters are far more soluble in organic 
solvents than in water. The solubility of the substrate ester is, 
therefore, ensured while the transesterified poly(ethylene glycol) ester 
resulting from the transesterification is insoluble and their separation 
is easily obtained. 
The enzyme may also be recovered or separated from an organic solvent in a 
far more simple manner than it is from an aqueous medium. In addition, the 
non-aqueous enzymatic reaction has the advantage that no acidic product is 
formed and therefore, the pH of the reaction need not be adjusted as the 
reaction progresses. 
The enzyme transesterification reaction is conducted at a temperature of 
about 10.degree. to 75.degree. C., more preferably about 25.degree. to 
60.degree. C., and more preferably 35.degree. to 50.degree. C. This is a 
range of temperatures within which the reaction proceeds well and the 
poly(ethylene glycol) stays in the liquid state. 
The method of the invention thus permits the resolution of one enantiomer 
of a racemic mixtures of esters by transesterication of one enantiomer of 
the mixture with an achiral alcohol followed by cooling to cause 
separation of the transesterified enantiomer from the reaction mixture. 
Prior art methods for separation of racemic ester mixtures by 
enzyme-catalyzed transesterification required the subsequent separation of 
one stereoisomer from another by somewhat cumbersome methods such as 
distillation or chromatography. This probably accounts for their limited 
application, particularly, their lack of suitable adaptation to 
large-scale preparation. 
The alcohol utilized as a substrate for the transesterification reaction is 
sufficiently large and its properties thus dominate those of the chiral 
acyl residue to which it is bound. The alcohol is insoluble in the 
reaction solvent and the ester product (the transesterified product) also 
has low solubility in the reaction solvent. This allows for an easy 
removal of the transesterified ester enantiomer from the unchanged ester 
enantiomer by simple filtration, decantation or centrifugation. 
Particularly preferred organic solvents are symmetrical and unsymmetrical 
aliphatic ethers, aliphatic hydrocarbons, aromatic hydrocarbons, alkyl 
halides, arylalkyl ethers and aryl halides, among others. However, any 
other organic solvents which act as such for the reactive ester isomer 
while permitting that the transesterified esters precipitate or otherwise 
separate are suitable. 
The reaction of the reactive ester substrate with the polyalkylene glycol 
and the enzyme is preferably conducted in proportions of about 1 mmol:10 
mmol:2.5 g to 1 mmol:0.5 mmol:0.05 g, more preferably about 1 mmol:5 
mmol:1.0 to 1 mmol:0.6 mmol:0.1 g, and still more preferably about 1 
mmol:1 mmol:0.5 g to 1 mmol:0.7:0.2 g. 
The separation step is conducted at a temperature where the poly(ethylene 
glycol) and the poly(ethylene glycol) ester(s) are in solid form or in 
liquid form, but having low solubility in the reaction solvent. A 
particularly appropriate temperature range may be found for the separation 
of each specific poly(ethylene glycol)/solvent combination utilized and is 
in general lower than the temperature utilized for the enzymatic reaction. 
Typically, the separation temperature is less than about 30.degree. C., 
and in some instances less than about 20.degree. C., and for lower 
molecular weight poly(alkylene glycols), it may be less than about 
5.degree. C. An artisan would know how to find appropriate temperature 
ranges for various molecular weight PEGs without undue experimentation. 
This separation step is suitable for separating the reactive ester and the 
reaction medium from the enzyme, the poly(ethylene glycol) and the 
poly(ethylene glycol) ester(s). What remains in the organic medium is the 
reactive ester of the unchanged enantiomer while the enzyme, the 
poly(ethylene glycol) and the poly(ethylene glycol) ester(s) precipitate 
out of the medium. 
Thereafter, the poly(ethylene glycol) and the poly(ethylene glycol) 
ester(s) are separated from the enzyme by extraction with a solvent for 
the poly(ethylene glycol) and the poly(ethylene glycol) ester(s). 
Typically, solvents that may be utilized herein are dichloromethane, 
chloroform, and tetrahydrofuran. However, any other solvent for the 
poly(ethylene glycol) and the poly(ethylene glycol) ester(s) that is not a 
solvent for the enzyme is suitable. 
After separating the enzyme from the solution, the poly(ethylene glycol) 
may be separated from the ester(s) being resolved by converting the latter 
to the corresponding soluble alkyl ester(s), placing the mixture under 
conditions effective for the poly(ethylene glycol) to form a separate, 
preferably solid phase, and separating the poly(ethylene glycol) from the 
alkyl ester(s) which is soluble in the extraction solvent. 
Suitable conditions for converting the poly(ethylene glycol) ester(s) to 
the corresponding (C.sub.1 -C.sub.4) alkyl ester(s) are the reaction of 
the poly(ethylene glycol) ester(s) with an alkyl alcohol in a proportion 
of about 1 mmol:200 mmol to 1 mmol:1 mmol, more preferably about 1 
mmol:100 mmol to 1 mmol:5 mmol, and still more preferably about 1 mmol:30 
mmol to 1 mmol:15 mmol in a alkaline or acidic medium at a acidity 
equivalent to about pH 2.0 and a temperature of about 25.degree. C. to 
60.degree. C. 
The poly(ethylene glycol) ester may be mixed with stirring in a suitable 
alkyl alcohol such as methanol in the proportions specified above. A 
catalytic amount of base such as sodium methoxide or an acid such as 
concentrated sulfuric may be added to the solution and the mixture 
stirred, e.g., at room temperature until no further reaction is observed. 
The alkyl ester product may be isolated by reducing the reaction mixture 
to almost dryness and extracting the residue with an appropriate organic 
solvent such as isopropyl ether. The ether phase may then be evaporated to 
yield the alkyl ester. 
The conditions for the separation of the poly(ethylene glycol) from the 
ester(s) remaining in solution encompass lowering the temperature, 
permitting the poly(ethylene glycol) to precipitate or separate from the 
solution and, without varying the temperature, centrifuging and, if 
necessary, then filtering or decanting the solution away from the 
separated poly(ethylene glycol) phase. These are all techniques known in 
the art which need not be further described herein. 
In a particularly preferred embodiment of the method, the poly(ethylene 
glycol) ester(s) is converted to the corresponding soluble (C.sub.1 
-C.sub.4)alkyl ester(s) by adding thereto a (C.sub.1 -C.sub.4)alkanol and 
a transesterifying enzyme in a medium under conditions effective to form 
the corresponding alkyl ester(s). 
In another preferred embodiment of the invention, the method further 
comprises subjecting a partially stereo-chemically pure reactive ester(s) 
separated from the chiral site proximate poly(ethylene glycol) ester(s) to 
another reaction step to form further chiral site proximate poly(ethylene 
glycol) ester(s) by transesterification with poly(ethylene glycol) of any 
previously unreacted chiral site proximate reactive ester having the 
stereochemistry preferred by the enzyme, and then separating the new 
chiral site proximate poly(ethylene glycol) ester(s) from the reaction 
medium, the reactive ester(s), the enzyme, and, subsequently, the 
poly(ethylene glycol). 
The conditions for conducting these steps are similar to the ones described 
above. However, an artisan would know how to vary different parameters in 
order to attain further conversion of one enontiomer of the reactive 
substrates into the products if necessary. 
In still another preferred embodiment of the invention, the method further 
comprises separating the reactive ester and the reaction medium from the 
enzyme, the poly(ethylene glycol) and the poly(ethylene glycol) ester(s) 
at a temperature where the poly(ethylene glycol) and the poly(ethylene 
glycol) ester(s) are in solid form and then separating the reactive 
ester(s) from the reaction medium. The reactive ester recovered in this 
manner is substantially in the form of one enantiomer of the reactive 
ester(s) which is substantially free from the other enantiomer. 
The temperature at which the unchanged reactive ester and the reaction 
medium are separated from the enzyme, the poly(ethylene glycol) and the 
poly(ethylene glycol) ester(s) is as described above. Typically, the 
temperature is about -70.degree. to 100.degree. C., and more preferably 
about -10.degree. to 30.degree. C. Other conditions for the separation 
steps are generally as described above. 
The reactive ester may be separated from the reaction medium by methods 
known in the art such as evaporation of the medium, distillation and the 
like. This technology is substantially known and will not be described in 
further detail herein. 
In a most preferred embodiment of the method, the enzyme utilized for the 
transesterification step is porcine pancreatic lipase. This enzyme has 
been found, in many instances, to provide the best stereospecificity for 
the transesterification reaction. 
As already indicated, the racemic mixture of the reactive ester may be 
subjected more than one time to the present method. 
The method of the invention will be exemplified by reference to 
2,2,2-trichloroethyl-3,4-epoxybutanoate and its transesterification with 
poly(ethylene glycol). This reaction is shown in the following Scheme. 
##STR10##

Having now generally described this invention, the same will be better 
understood by reference to certain specific examples, which are included 
herein for purposes of illustration only and are not intended to be 
limiting of the invention or any embodiment thereof unless so specified. 
EXAMPLES 
EXAMPLE 1 
Selective transesterification of 2,2,2-trichloroethyl 
(S)-3,4-epoxybutanoate. 
The (S)-enantiomer from racemic 2,2,2-trichloroethyl 
(R,S)-3,4-epoxybutanoate, ((R,S) compound 1), was selectively 
transesterified by low molecular weight (about 1500 Daltons) polyethylene 
glycols (PEG) using porcine pancreatic lipase (PPL) as the catalyst. The 
reaction required about 5 hours to consume one-half of the racemic ester 
at 45.degree. C., a temperature capable of keeping the PEG as a molten 
phase in diisopropyl ether. 
##STR11## 
The separation of the PEG (S)-ester, (compound (S)-2), from the unchanged 
compound (R) 1 was achieved by cooling the reaction mixture to 0.degree. 
C. and filtering off the enzyme and the solidified compound 2. The 
(R)-enantiomer was isolated in high yield and high enatiomeric excess by 
evaporating the solvent from the filtrate. The PEG ester of the 
(S)-enantiomer (compound(S)-2), was recovered by extracting it into 
methylene chloride and filtering off the catalyst. 
The structure and enantiomeric purity of the (R) 1 compound was proven by 
its conversion to the corresponding (-)-carnitine chloride (compound 3). 
This was done by enzymatic hydrolysis of the trichloroethyl ester followed 
by treatment with trimethylamine and acidification with HCl as described 
by Bianchi, et al. (Bianchi, D. et al, supra) 
The carnitine displayed a rotation [.alpha.].sub.D -22.9.degree. 
corresponding to an enantiomeric excess of &gt;96% by comparison with the 
literature value of [.alpha.].sub.D -23.7.degree. for the natural product. 
(Strack, E., Lorenz, J., Z. Physiol. Chem, 1960 318, 129). 
##STR12## 
Resolution of a racemic mixture of 3,4-epoxybutyrates by enantioselective, 
enzymatic hydrolysis of the methyl or other alkyl esters has recently been 
reported by Mohr et al. (Mohr, P.; Rosslein, L.; Tamm, C., Helv. Chim. 
Acta., 1987, 70, 142) and by Bianchi et al. (Bianchi et al., supra). In 
the former, the methyl ester was hydrolyzed with pig liver esterase to 
give a 40% recovery of the unchanged (R)-ester and a 30% yield of the 
(S)-acid. An ee of 97% was found for the derivative prepared from the acid 
though it may have been improved by recrystallization. The derivative 
prepared from the unchanged (R)-ester indicates an ee of 82%. Though 
supported by theory (Chen, C. S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J., 
J. Am. Chem. Soc., 1982 104, 7294) the author's statement that a high ee 
for either enantiomer can be achieved by stopping the reaction after 40% 
reaction or after 60% reaction was not proven experimentally. 
A survey of 13 different enzyme preparations reported by Bianchi, et al., 
supra, led to the conclusion that PPL (steapsin) provided the best 
enantioselectivity when alkyl (particularly butyl, isobutyl, and octyl) 
esters were hydrolyzed. However, to achieve an ee of &gt;95% for the 
unchanged (R)-ester, it was necessary to hydrolyze 60-70% of the starting 
material. 
The (S)-enantiomer compound (S)-2 was further converted to the 
corresponding methyl ester by PPL catalyzed transesterification with 
methanol. Based on the rotation of +10.6.degree. reported (Mohr, P.; 
Rosslein, L.; Tamm, C.; Helv. Chim. Acta. 1987, 70, 142) for 
methyl-(R)-3,4-epoxybutanoate corresponding to an ee of 82%, the rotation 
of -11.61.degree. observed herein for the (S)methyl ester that is isolated 
from the PEG by transesterification would correspond to an ee of 89%. 
EXAMPLE 2 
Preparation of 2,2,2-trichloroethyl 3-butenoate 
8.130 g (95.0 mmol) of vinylacetic acid were dissolved in 50 mL of 
methylene chloride and treated successively with 14.34 g (96.0 mmol) of 
2,2,2-trichloroethanol and 0.40 g (3.3 mmol) of 4-(dimethylamino)pyridine 
(DMAP) following the general method of Hassner and Alexanian (Hassner, A.; 
and Alexanian, V., Tetrahedron Lett., 1978, 4475). 
The mixture was then cooled to 0.degree. C. After 15 min., 19.81 g (96.0 
mmol) of dicyclohexylcarbodiimide (DCC) and 25 mL of methylene chloride 
were added with stirring. A white precipitate was observed to form 
immediately. 
When the DCC addition was complete, the mixture was allowed to warm to 
ambient temperature and remain there for two days. The precipitate was 
then filtered off and the filtrate washed successively with three portions 
of a saturated solution of citric acid, two portions of saturated aqueous 
sodium bicarbonate, and one portion of saturated brine. 
After drying over magnesium sulfate, the solvent was evaporated to give a 
light yellow oil. The oil was passed through a 4-inch column of silica 
(Merck Grade 60/60 Angstrom) and eluted with methylene chloride to obtain 
18.45 g (89.3%) of a colorless liquid that was shown by VPC to be &gt;95% 
pure. 
.sup.1 H NMR (CDCl.sub.3): .delta.3.25 (d of t,J=6.9, 1.4 Hz, 2H), 4.76 (s, 
2H), 5.25 (m, 2H), 5.95 (m, 1H); 
.sup.13 C NMR (CDCl.sub.3): .delta.169.8, 129.0, 119.3, 95.0, 73.9, 38.5. 
EXAMPLE 3 
Preparation of 2,2,2-trichloroethyl (R,S)-3,4-epoxybutanoate ((R,S) (1)) 
10.0 g of 2,2,2-trichloroethyl-3-butenoate were dissolved in 50 mL of 
methylene chloride and treated with 11.1 g (.about.1.4 equiv.) of 
m-chloroperoxybenzoic acid in 100 mL of methylene chloride following the 
general method of Dahill et al. (Dahill, R. T.; Dorsky, J.; Easter, W., J. 
Org. Chem. 1970, 35, 251). The reaction was allowed to continue for 3 days 
at ambient temperature after which time VPC analysis showed the starting 
alkene to have been consumed. 
The mixture was then cooled in an ice bath and a saturated sodium sulfite 
solution added with stirring until a KI starch test was negative. The 
m-chlorobenzoic acid which precipitated during the reaction was filtered 
off and the filtrate washed with three portions of the aqueous sodium 
sulfite solution, two portions of saturated aqueous sodium bicarbonate, 
and one portion of saturated brine. 
After drying over magnesium sulfate, the solvent was evaporated and the 
oily product purified by distillation in vacuo. 
B.P. 65.degree.-70.degree. C. 0.1 mm. 
.sup.1 H NMR (CDCl.sub.3): .delta.2.59 (d of d, J=4.8, 2.6 Hz, 2H), 2.72 
(d, J=5.79 Hz, 2H), 2.86 (t, J=4.4 Hz, 1H), 3.35 (m, 1H), 4.78 (s, 2H). 
.sup.13 C NMR (CDCl.sub.3): .delta..sub.c 168.4, 94.6, 73.9, 47.3, 46.2, 
37.5 
Anal. Calc. for C.sub.6 H.sub.7 Cl.sub.3 O.sub.3 ; C 30.87; H, 3.02. Found: 
C, 31.53; H, 3.15. 
EXAMPLE 4 
Preparation of the Poly(ethyleneglycol) (PEG) Substrate. 
PEG of molecular weight 1300-1600 (Aldrich) was chosen because of its 
solubility and melting point characteristics. 
Very low molecular weight oligomers were removed by the following 
procedure. To 200 mL of anhydrous isopropyl ether under a dry nitrogen 
atmosphere were added 25 g of PEG. The mixture was stirred and warmed to 
about 45.degree. C. 
The mixture was held at this temperature for two hours. During this time 
the polymer melted but formed a separate liquid phase at the bottom of the 
reaction vessel. Upon cooling to 0.degree. C. in an ice/water bath, the 
PEG solidified and some dissolved polymer precipitated. The solid PEG was 
fractured into a relatively fine powder by rapid stirring. 
The powder was recovered by filtration, washed with additional cold 
isopropyl ether, and dried under vacuum at room temperature. The procedure 
yielded 24.2 g of PEG. 
EXAMPLE 5 
Resolution of 2,2,2-trichloroethyl (R,S)-3,4-epoxybutanoate (Compound (R,S) 
1) 
100 mL of anhydrous isopropyl ether were placed in a 300 mL three-necked 
round bottom flask equipped with a magnetic stirrer and a dry nitrogen 
inlet. 16 g (11.0 mmol, assuming a molecular weight of 1500) of the 
previously prepared PEG were added to the flask. The mixture was heated to 
about 45.degree. C. with stirring. In rapid succession thereafter were 
added 4.0 g (17.1 mmol) of (R,S) 1 compound and 4.3 g of PPL (35% protein, 
activity=35-70 units per mg, Sigma) which had been dried for three days in 
vacuo over phosphorus pentoxide as previously described (Wallace, J. S.; 
and Morrow, C. J., J. Polym. Sci. Part A: Polym. Chem., 1989, 27, 2553). 
After 4.5 hrs, VPC analysis indicated that 50% of the starting ester had 
been consumed and the reaction was stopped by rapid cooling in an 
ice/water bath. The PPL and esterified PEG mixture were filtered from the 
cold mixture and washed with cold isopropyl ether. The filtrate was 
concentrated by evaporation to yield 1.72 g (86%) of 2,2,2-trichloroethyl 
(R) (+)-3,4-epoxybutanoate (compound (R)-1). 
[.alpha.].sub.D.sup.23 +5.05.degree. (C=4, CHCl.sub.3). 
The .sup.1 H and .sup.13 C NMR spectra were identical with those described 
above for the racemic material. 
The recovered PEG and esterified PEG was dissolved in CH.sub.2 Cl.sub.2 and 
freed of the insoluble PPL enzyme by suction filtration using a fitted 
glass funnel. The recovery of solid material was 15.82 g (98.9%). 
.sup.1 H NMR (CDCl.sub.3): .delta.3.62 (s) and 2.6 (br s) were strong 
absorptions arising from the PEG. 
.delta.2.54 (d of d of d), 2.80 (t), 3.25 (m) comprise a weak set of 
absorptions from the 3,4-epoxybutyrate. 
There is no absorption near .delta.4.8 for the methylene of a 
trichloroethyl ester. 
EXAMPLE 6 
Isolation of (S)-3,4-epoxybutanoate as a Methyl Ester 
To 100 mL of anhydrous isopropyl ether were added 14.5 g (9.4 mmol) of the 
recovered PEG and the mixture was stirred and heated to 50.degree. C. In 
rapid succession, were added to the mixture 8.25 g (0.25 mmol) of 
anhydrous methanol and 2.2 g of PPL. The reaction was stopped after 
approximately 30 hours by cooling the reaction vessel in an ice/water 
bath. The solidified PEG was then collected by filtration and washed with 
cold isopropyl ether. 
The filtrate was concentrated by evaporation to yield 0.91 g (92%) of 
methyl (S) (-)-3,4-epoxybutanoate. 
[.alpha.].sub.D.sup.20.degree. -11.61.degree. (c=1.8, CHCl.sub.3) (Lit. 
[.alpha.9 .sub.D.sup.r.t. : -10.67.degree. (c=1.8, CHCl.sub. for the 
unchanged enantiomer from the enzymatic hydrolysis (Mohr, P.; Rosslein, 
L.; Tamm, C., Helv. Chim. Alta, 1987, 70,142). 
The protein NMR spectrum was identical with that described previously 
(Mohr, P.; Rosslein, L.; Tamm, C., Helv. Chim. Alta, 1987, 70,142). 
The TLC and VPC behaviors were identical with those of an authentic racemic 
sample. 
EXAMPLE 7 
Conversion of 2,2,2-Trichloroethyl-(R)-3,4-Epoxybutanoate (Compound (R)-1) 
to (R) (-)-Carnitine Chloride (Compound 3) 
1.5 g, (6.42 mmol) of 2,2,2-Trichloroethyl (R)-3,4-epoxybutanoate (compound 
(R)-1) were suspended in 15 mL of 0.1M phosphate buffer that had been 
adjusted to pH 7.8. To this mixture were added 200 mg of the lipoprotein 
lipase Amano P from Pseudomonas sp. (AMANO Int'l Enzyme Co., Troy, Va.) 
The mixture was stirred at ambient temperature while maintaining the pH 
near 7.5 by slowly adding 1M aqueous NaOH. After about 4 hours the 
consumption of base ceased and the reaction mixture was extracted with 
2.times.10 mL of methylene chloride to remove the trichloroethanol that 
had been freed. 
The aqueous solution of (R)-3,4-epoxybutanoate was converted to (R) 
(-)-carnitine chloride (compound 3) in 72% yield following the method 
described by Bianchi et al., supra). 
[.alpha.].sub.D.sup.25.degree. -22.9.degree. (lit. 
[.alpha.].sub.D.sup.25.degree. -23.7.degree. 
M. P.: 146.degree. C., decomp. (Lit. 142.degree. C., decomp., (Strack, E.; 
Lorenz, J. Z., Physiol. Chem., 1960, 318, 129) 
.sup.1 H NMR (D.sub.2 O): .delta.2.51 (Strack, E.; Lorenz, J. Z., Physiol. 
Chem., 1960, 318, 129) (two d of d, 2H), 3.06 (s, 9H), 3.34 (m, 2H), 4.52 
(m, 1H). 
The enantiomeric excess as determined by comparison of the optical rotation 
with the literature value was 96.6%. 
The carnitine chloride displayed a rotation [.alpha.].sub.D -22.8.degree. 
corresponding to an enantiomeric excess of 96% by comparison with the 
literature value of [.alpha.].sub.D -23.7% for the natural product. 
EXAMPLE 8 
Proof of the Structure and Enantiomeric Purity of the 2,2,2-Trichloroethyl 
(R)-3,4-Epoxybutanoate by Its conversion to (R)-(-)-Carnitine Chloride 
The unchanged 2.2.2-trichloroethyl (R)-3,4-epoxybutanoate was converted to 
the well known (R) (-)-carnitine chloride by enzymatic hydrolysis of the 
trichloroethyl ester with Amano P enzyme, a nonspecific lipoprotein lipase 
from Pseudomonas species, followed by treatment with trimethylamine and 
acidification with HCl as described by Bianchi et al., supra. 
##STR13## 
EXAMPLE 9 
Evidence Against Improvement in the Enantiomeric Purity of the Unchanged 
2,2,2-Trichloroethyl (R)-3,4-Epoxybutanoate during its Enzymatic 
Hydrolysis Using Amano P Enzyme 
A survey of 13 different enzyme preparations by Bianchi et al., supra, led 
to the conclusion that PPL provides the best enantioselectivity during the 
hydrolysis of 3,4-epoxybutanoates if alkyl (particularly butyl, isobutyl, 
and octyl) esters are hydrolyzed. An ee of &gt;95% can be achieved for the 
unchanged (R)-ester if 60-70% of the starting material is hydrolyzed. 
Bianchi et al, supra, have shown that the hydrolysis of isobutyl 
3,4-epoxybutanoate using Amano P enzyme as the catalyst occurs without 
stereoselectivity. We have found that the hydrolysis of 
2,2,2-trichloroethyl (R,S)-3,4-epoxy butanoate in the presence of Amano P 
also proceeds without stereoselectivity. 
##STR14## 
The complete hydrolysis of both enantiomers requires only four hours, the 
same time as was required for Amano P to hydrolyze the pure (R) enantiomer 
during its conversion to carnitine chloride. Thus, the high enantiomeric 
purity of this enantiomer is attributable entirely to the selectivity of 
the PPL during the transesterification by PEG, and not to a double 
resolution process involving the Amano P. 
EXAMPLE 10 
Comparison of Stereochemical Purity of Isomers Obtained by the Method of 
the Invention and a Prior Art Method. 
(a) Previously Reported Enzyme-Catalyzed Resolution of 3,4-Epoxybutyrates 
The resolution of 3,4-epoxybutyrate by enantioselective enzymatic 
hydrolysis of the methyl or other alkyl ester was reported by Mohr et al., 
(Mohr, P.; Rosslein, L.; Tamm, C., Helv. Chim. Acta, 1987, 70, 142; Mohr, 
P.; Rosslein, L.; Tamm, C., Tetrahedron Lett., 1989, 30, 2513) and by 
Bianchi et al. (Bianchi, D.; Cabri, W.; Cesti, P.; Francalanci, F.; Ricci, 
M., J. Org., Chem., 1988, 53, 104). 
In the former report, the methyl ester was hydrolyzed with pig liver 
esterase to give 40% recovery of unchanged (R)-ester and a 30% yield of 
the (S) acid. An enantiomeric excess of 97% was reported for the (S) acid 
based on its conversion to (+)-gamma-amino-.beta.-hydroxybutyric acid. 
The authors established an ee of 82% for the unchanged methyl 
(R)-3,4-epoxybutyrate ([.alpha.].sub.D.sup.22: +10.67.degree. (c=1.8, 
CHCl.sub.3)) by converting it to methyl (S)-3-hydroxypentanoate and 
comparing the specific rotation found with that reported for a sample of 
the opposite enatiomer of the same material. The latter material had been 
shown optically pure by HPLC analysis of the 
(S)-.alpha.-methoxy-.alpha.-trifluoromethylphenylacetate derivative (Mori, 
K.; and Ikunaka, M., Tetrahedron, 1984, 40, 3471). 
(b) Optical Purity of the Transesterified Enantiomer by the Method of the 
Invention 
The poly(ethylene glycol) ester of (S)-3,4-epoxybutanoate was converted to 
methyl (S)-3,4-epoxybutanoate by transesterification with methanol using 
porcine pancrease lipase as the catalyst. The conditions were as follows. 
To 100 mL of anhydrous isopropyl ether was added 14.5 g (9.4 mmol) of the 
recovered PEG ester and the mixture stirred and heated to 50.degree. C. 
8.25 g (0.25 mmol) of anhydrous methanol and 2.2 g of PPL were added in 
rapid succession to the mixture. After approximately 30 hrs, the reaction 
was stopped by cooling in an ice/water bath and then the solidified PEG 
was collected by filtration and washed with cold isopropyl ether. The 
filtrate was concentrated by evaporation to yield 0.91 g (92%) of methyl 
(S)-(-)-3,4-epoxybutanoate. 
Based on a specific rotation of -10.67.degree. indicating an ee of 82% for 
methyl (S)-3,4-epoxybutyrate (Mohr, P.; Rosslein, L.; Tamm, C., 
Tetrahedron Lett., 1989, 30, 2513) the rotation of -11.61.degree. observed 
for the methyl (S)-3,4-epoxybutanoate isolated from the PEG corresponds to 
an ee of 89% for that compound. 
##STR15## 
The invention now being fully described, it will be apparent to one of 
ordinary skill in the art that many changes and modifications can be made 
thereto without departing from the spirit or scope of the invention as set 
forth herein.