Acylation of alcohols with Pseudomonas lipase immobilized on a polystyrene resin

The O-acylation of alcohols is carried out by reacting a vinyl ester or a carboxylic ester with Pseudomonas lipase immobilized on a hydrophobic carrier such as a polystyrene-based carrier. The polystyrene carrier preferably has a surface area of 100-1000 m.sup.2 /g, a pore volume of 25 to 75% and a pore diameter of 25-1200 .ANG.. Crosslinking may be carried out after the lipase is immobilized on the carrier.

Optically active alcohols are often important chiral precursors of 
biologically active substances such as, for example, of pharmaceuticals, 
natural products, crop protection agents or else of liquid crystal 
components. 
An economic preparation process which ensures enzymatic racemate resolution 
and thus the preparation of the optically active alcohols is therefore of 
great importance. The same applies to enzymatic stereodifferentiation of 
prochiral compounds such as, for example, the esterification of 
enantiotopic hydroxyl groups of 2-substituted 1,3-propanediols. In 
addition, acylation with enzymatic catalysis is of importance, in contrast 
to chemical acylation, for particularly sensitive substrates such as, for 
example, certain primary or secondary alcohols. 
Some pharmacological agents whose preparation is facilitated and made more 
economical by the process according to the invention are products such as 
NSAIDs (nonsteroidal antiinflammatory drugs), beta-blockers, 
bronchospasmolytics, antimycotics, pyrethroids, tetramisole, 
tetrahydrozoline, (R)-(-)-tomoxetine and (S)-(+)-fluoxetine, and 
prostaglandins and carbohydrates. Chiral building blocks for the synthesis 
of protease inhibitors, for example of renin, can be obtained considerably 
more straightforwardly by using enzymatic processes. 
It is already known that vinyl esters can be transesterified with enzymatic 
catalysis and with the addition of alcohols in the presence of solvents 
such as, for example, tetrahydrofuran (M. Degueil-Castaing et al., 
Tetrahedron Letters, Vol. 28, No. 9, pages 953-954, 1987). Pig pancreatic 
lipase was used as enzyme. No stereoselectivity was observed. 
Also known is the enzymatic separation of racemic alcohols based on a 
selective enzyme-catalyzed transesterification reaction with vinyl esters 
in the absence of solvents. The enzymes used are immobilized lipases from 
pig liver and pancreas and from the microorganisms Pseudomonas, Candida, 
Mucor, Rhizopus and Penicillium (EP 032 19 18). 
It is furthermore known that it is possible to employ carboxylic esters for 
the transesterification (G. Carpani, F. Orsini, M. Sisti, L. Verotta, 
Gazz. Chim. Ital. 119, p. 463-465 (1989)) and cyclic carboxylic anhydrides 
for the acylation (Y. Terao et al., Chem. Pharm. Bull. 37, p. 1653-1655 
(1989)). 
In European Patent Application EP 0 25 42 43, chiral compounds are prepared 
optically pure from prochiral diols by reaction with vinyl acetate in the 
presence of hydrolases. This is achieved by selective esterification of 
only one of the two enantiotopic primary OH groups. 
It is also known that immobilized lipases can be employed for the 
hydrolysis and transesterification of fats, oils and similar compounds (M. 
Mittelbach, J. Am. Oil. Chem. Soc. 67, 168-170 (1990)). 
Hsu et al. (Tetrahedron Letters, Vol. 31, No. 44, p. 6403-6406 (1990)) 
describe the reaction of secondary alcohols using XAD-8 immobilized lipase 
from Pseudomonas and find an increased rate of reaction of the substrate. 
However, there are no statements in the publication about the useful lives 
(stability) or the thermal stability of the immobilized enzyme with 
negligible loss of activity. 
It has now been found, surprisingly, that the O-acylation of alcohols using 
immobilized Pseudomonas lipase can be carried out particularly efficiently 
by immobilizing the enzyme by binding to hydrophobic carriers such as 
polystyrene-based adsorber resins. 
Hence the invention relates to: 
A process for the acylation of alcohols, where a vinyl ester of the formula 
I 
##STR1## 
in which R.sup.1 is hydrogen, C.sub.1 -C.sub.18 -alkyl which is optionally 
substituted by halogen, or is phenyl or (C.sub.1 -C.sub.3) 
-alkoxy-(C.sub.1 -C.sub.4)-alkyl and 
R.sup.2 is hydrogen or methyl, or 
a carboxylic ester of the formula II 
##STR2## 
in which R.sup.1 has the abovementioned meaning, and R.sup.7 is either 
fluorine, chlorine or hydrogen, where all R.sup.7 must be identical, or 
R.sup.7 is fluorine, chlorine, bromine or cyano and hydrogen, where two 
R.sup.7 must be hydrogen, or R.sup.7 is fluorine or chlorine and hydrogen, 
where only one R.sup.7 is hydrogen and the two other substituents are 
identical, or 
one of the cyclic carboxylic anhydrides succinic or glutaric anhydride is 
reacted with an alcohol in the presence of immobilized Pseudomonas lipase, 
which comprises employing Pseudomonas lipases which are immobilized on 
polystyrene-based adsorber resins. 
The invention is described in detail hereinafter, especially in its 
preferred embodiments. The invention is furthermore defined by the 
contents of the claims. 
In the process according to the invention, the vinyl or methylvinyl ester 
or the carboxylic ester of the formula II or the cyclic carboxylic 
anhydride, which acts as solvent or is dissolved in another organic 
solvent, is cleaved into a ketone, aldehyde or alcohol and an acyl 
radical, the latter undergoing enzymatic acylation with the added alcohol 
(substrate). 
Suitable carrier materials are polystyrene-based adsorber resins. All 
carriers can be obtained commercially. 
The polystyrene-based carrier materials which are used have a pore volume 
of 25-70, but preferably 35-55%, a surface area of 100-1000 m.sup.2 /g, 
but preferably 200-750 m.sup.2 /g, and a pore diameter of 25-1300 .ANG., 
preferably 50-250 .ANG.. 
Employed as enzyme are Pseudomonas lipases [lipase P from Pseudomonas 
cepacia (also called FP or PS) Amano Pharmaceuticals, Nagoya, Japan]. 
To immobilize the enzyme, 0.01 to 2 g, but preferably 0.1-1.5 g, of enzyme 
per 10 ml of carrier are stirred in 0,005-1M potassiumphosphate buffer, pH 
5-9, but preferably pH 6-8, for 1-20 h. After the reaction time, the 
buffer is removed by filtration with suction through a frit, and the 
enzyme/carrier mixture is washed with large quantities of water, acetone 
and vinyl acetate. The carrier is ready for use in this state and can be 
stored in the dry state. 
The quantity of carrier to be loaded with enzyme is chosen freely depending 
on the size of the batch, on the reactivity of the alcohol, on the 
expected reaction time and on the required level of conversion. It can 
easily be determined by preliminary tests. 
The vinyl and methylvinyl esters of the formula I which cannot be bought 
can be prepared in a straightforward manner, for example by noble 
metal-catalyzed transesterification of vinyl acetate with the appropriate 
carboxylic acids. The transesterification is preferably catalyzed by 
Pd.sup.2+. 
The vinyl esters can also be synthesized by an Hg.sup.2+ -catalyzed 
addition of acetylene. 
The carboxylic esters of the formula II, just like the cyclic carboxylic 
anhydrides (succinic and glutaric anhydride), can be bought or prepared by 
standard processes. 
The alcohols which cannot be bought are obtained, for example, by reduction 
of the corresponding ketones, most of which can be bought, or by 
a-halogenation of corresponding ketones with subsequent reduction to the 
alcohol. Other alcohols or ketones which cannot be bought can be prepared 
straightforwardly by processes known from the literature, for example by 
Grignard or other conventional addition reactions. 
By alcohols are meant an alcohol of the formula III 
##STR3## 
in which R.sup.3 is C.sub.1 -C.sub.18 -alkyl or C.sub.3 -C.sub.10 
-cycloalkyl, it also being possible for these radicals to be 
halogen-substituted, and 
R.sup.4 is epoxy-C.sub.1 -C.sub.5 -alkyl, where the epoxy group is in the 
position .beta. to the OH group in the radical of the formula II or 
R.sup.4 is C.sub.1 -C.sub.10 -alkyl, C.sub.2 -C.sub.10 -alkenyl, C.sub.2 
-C.sub.10 -alkynyl, C.sub.3 -C.sub.8 -cycloalkenyl, where the alkyl, 
alkenyl, alkynyl and cycloalkenyl radicals are optionally substituted by 
COOH, halogen, NO.sub.2, CN, C.sub.1 -C.sub.4 -alkoxycarbonyl or phenyl, 
it being possible in turn for the phenyl radical to be substituted by 
halogen, NO.sub.2, CN or C.sub.1 -C.sub.4 -alkoxy, or R.sup.4 is aryl or 
heteroaryl, where the aryl or heteroaryl radicals are optionally 
substituted by C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -alkoxy, halogen, 
NO.sub.2, CN or N-PG, where PG is an amino-protective group, 
or in which 
R.sup.3 and R.sup.4 together are an alkylene or alkenylene radical of the 
formula IVa, b 
##STR4## 
in which n is 1, 2 or 3, and 
R.sup.5 and R.sup.6 are identical or different and are hydrogen, C.sub.2 
-C.sub.4 -alkenyl, or C.sub.1 -C.sub.4 -alkyl or 
R.sup.5 and R.sup.6 together are fused-on phenyl or fused-on naphthyl, 
where the phenyl or naphthyl radical is optionally substituted by C.sub.1 
-C.sub.4 -alkyl, C.sub.1 -C.sub.4 -alkoxy, NO.sub.2, CN or halogen, it 
also being possible for a methylene unit in the alkenylene chain to be 
replaced by a carbonyl group, or 
an alcohol of the formula V 
##STR5## 
in which R.sup.8 is hydrogen or an alkyl group and 
R.sup.9 is alkyl, aralkyl, aryl, benzyl or a naphthylmethyl group. 
It is also possible to use all polyhydric alcohols as substrate. 
By halogens in the alcohol of the formula III are meant fluorine, chlorine, 
bromine and iodine, especially chlorine and bromine. By "aryl" are meant, 
for example, phenyl, naphthyl, phenanthryl, anthryl and fluorenyl, 
especially phenyl, naphthyl and phenanthryl. By "heteroaryl" are meant, 
for example, furyl, thienyl, pyrrolyl, pyridyl, pyrimidyl, pyrazinyl, 
pyridazinyl, pyrazolyl, isoxazolyl, imidazolyl, oxazolyl, thiazolyl and 
indolyl, especially furyl, thienyl, pyrrolyl and pyridyl. By the 
amino-protective group "PG" are meant the amino-protective groups 
customarily employed in peptide chemistry, for example benzyloxycarbonyl 
(Z), benzoyl, benzyl, butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl 
(Fmoc), benzhydryl, allyloxycarbonyl (Aloc), tosyl, methoxymethyl (MOM), 
tetrahydropyranyl (THP), acetyl, but also alkyl or cycloalkyl groups, such 
as for example, N-methyl, N,N-dimethyl. By "fused-on phenyl" or "fused-on 
naphthyl" is meant a phenyl or naphthyl radical in which the C--C double 
bond of the radical of the formula III is part of the phenyl or naphthyl 
radical. The optionally substituted radicals R.sup.1, R.sup.3, R.sup.4, 
R.sup.5 and R.sup.6 are preferably monosubstituted. 
Alkyl and alkenyl radicals with 3 and more carbon atoms, and alkynyl 
radicals with 4 and more carbon atoms, can be both straight-chain and 
branched. 
The alcohol to be acylated is employed in a concentration of 0.05-200%, 
preferably 0.5-10%, based on the volume of the vinyl ester. 
At least 0.5 mole equivalents of the vinyl radical must be employed for the 
acylation of the alcohol. 
The reaction of the alcohols is carried out batchwise or in a continuous 
process. 
The racemate resolution of the alcohols can be carried out in the process 
according to the invention with an increase in activity of at least 90% 
compared with conventional processes. 
The enzyme immobilized on a carrier shows scarcely any loss of activity in 
the continuous process even after some months of use, in which there is 
alternation of reaction runs and phases of non-use of the immobilized 
enzyme. This even applies when the reaction is carried out at elevated 
temperatures. 
For batchwise reaction with immobilized Pseudomonas lipase, the vinyl ester 
of the formula I or the carboxylic ester of the formula II or the cyclic 
carboxylic anhydride, but preferably vinyl acetate or a solution of the 
vinyl ester, is introduced into a (non-polar) organic solvent, and the 
alcohol to be reacted is added. Suitable and preferred solvents are 
ethers, but very particularly symmetrical and unsymmetrical, branched and 
unbranched dialkyl ethers. Also suitable and preferred are hydrocarbons, 
very particularly linear, branched or cyclic hydrocarbons of C.sub.4 
-C.sub.8. Pseudomonas lipase immobilized on a carrier is added to the 
suspension, and is stirred or shaken at constant temperature. The 
completion of the reaction is checked by TLC, GC or HPLC. The immobilized 
enzyme is subsequently removed by filtration and thoroughly washed with a 
solvent (see above) or vinyl acetate, and the solution is concentrated in 
vacuo. The alcohol/ester mixture which remains as residue in the case of 
racemate resolution is separated by column chromatography on silica gel or 
by extraction, crystallization or distillation. Other acylation products 
frequently result in sufficient purity so that purification is 
unnecessary. 
To carry out the reaction in the continuous process, the Pseudomonas lipase 
P/FP/PS which is immobilized on a carrier is packed into a glass column 
and washed with the solvent in which the reaction is carried out, i.e. 
with vinyl acetate, another vinyl ester or another organic solvent. 
The substrate solution is subsequently allowed to run through at a constant 
rate at constant temperature. 
The ratio of carrier [ml] to immobilized lipase [g] to vinyl acetate [ml] 
to the concentration of the substrate [% by volume] is in the range 
5-100:1:5-10,000:0.5-200 when vinyl acetate is employed (i.e. without 
further addition of solvent). 
If the reaction is carried out in a vinyl ester, the alcohol to be acylated 
is employed in concentrations of 0.05-200% by volume. 
The level of the conversion can be controlled virtually as required by 
adjusting the dropping rate and can easily be determined by preliminary 
tests. 
The space-time yields depend directly on the absolute values of the 
abovementioned parameters, but especially on the column dimensions, i.e. 
the quantity of enzyme which is immobilized on the carrier in the column. 
The column dimensions can be chosen freely but, on the laboratory scale, 
are preferably of the order of 10-500 ml. With a preferred column packing 
with 50 ml of enzyme immobilized on a carrier, with a 1% strength 
substrate solution and with a flow rate of 10 drops/min, space-time yields 
of about 0.5-300 g/l/h are achieved. 
The reaction temperature during the process is (-)10.degree. to 
(+)100.degree. C., preferably (+)0.degree.-60.degree. C. 
The reaction times depend on the nature of the alcohol to be reacted, the 
concentration thereof and the quantity of the enzyme immobilized on the 
carrier and vary between 1 h and 4 weeks. They are preferably between 3 h 
and 3 days. 
The products acetaldehyde or acetone resulting from the process according 
to the invention, and the alcohols liberated for the acylation, as well as 
the enantiomeric alcohols (substrates) resulting in the case of selective 
acylation, i.e. carboxylic esters and unreacted alcohol, can be separated 
in a known manner by using all customary methods, which have, however, to 
be tested in the individual case, preferably by chromatography on silica 
gel or one of the other abovementioned processes.

EXAMPLE 
General procedure 
To immobilize the enzyme, the carrier material is either 
a) employed untreated, 
b) subsequently crosslinked with glutaraldehyde after the enzyme 
immobilization (Tab. 1) 
re a) To immobilize the lipase on the carrier, 50 ml of the carrier are 
suspended in 100 ml of potassium phosphate buffer, pH 7.0, and 500 mg of 
lipase P are added. The mixture is stirred at RT for 3 h, filtered and 
washed thoroughly with water. 
re b) Subsequent crosslinking For the subsequent crosslinking, the process 
is carried out as described in a) but, after the stated immobilization 
time, crosslinking is carried out with 4 ml of glutaraldehyde solution 
(25% strength). After 1 hour, the enzyme immobilized on the carrier is 
filtered off and washed with water. 
The preferred carriers may be characterized as follows: 
______________________________________ 
.RTM.XAD-2 
.RTM.XAD-4 
______________________________________ 
Pore volume [%] 42 51 
Density 1.02 1.02 
Surface area [m.sup.2 /g] 
330 750 
Pore diameter [.ANG.] 
90 50 
______________________________________ 
The carrier is stored in water or dry. 
500 mg of the alcohol to be reacted are suspended in 20 ml of vinyl 
acetate. To this is added the immobilized lipase, and the mixture is 
stirred at constant temperature. 
After the reaction is complete, the immobilized enzyme is removed by 
filtration. The remaining solution is completely evaporated in vacuo. 
The acylation products present in the residue can be separated by standard 
processes, for example by silica gel chromatography. 
Table 2 indicates the starting materials and resulting products, the 
variable process parameters (quantity of enzyme, quantity of carrier, 
quantity of alcohol, quantities of vinyl esters, reaction temperature, 
reaction time) and the product characteristics and chemical yield. 
For accurate determination of the activity it is necessary to determine 
accurately the quantity of enzyme bound to the carrier. 
The basic assumption in the tests described here for the process according 
to the invention is that Pseudomonas lipase is a single protein with a 
salt content of 37% or 63% of the protein (% by weight). This salt content 
is determined by dialysis. 
To determine the immobilization yield, the test which has already been 
described is carried out with a batch of 20 ml of XAD-2 carrier and 2 g of 
Pseudomonas lipase. The immobilization and washing solutions are 
collected, combined and lyophilized. 1.77 g of residues composed of enzyme 
and buffer salts are obtained from this pool. It is known from the weight 
obtained after lyophilization of pure buffer that the quantity of salt is 
0.38 g in the quantity of buffer solution employed. 
Thus, from the 1.77 g of residue it is necessary to subtract, on the one 
hand, 0.38 g of buffer salts and, on the other, 0.74 g of salts of the 
enzyme (=37%; see above). 
The remaining quantity of 0.65 g ought to correspond to the quantity of 
non-immobilized enzyme. 
Subsequent dialysis of the residue shows that a small quantity of salt is 
still present, so that the quantity of enzyme remaining is 0.58 g instead 
of the theoretical value of 0.65 g. Thus the quantity of immobilized 
enzyme is between 0.61 and 0.68 g. This means that the immobilization 
yield is 51%. 
The calculation is carried out once more by way of example for Example 1 
(phenylethanol) mentioned in Table 2. 
______________________________________ 
50 mg of lipase P (obtained commercially) .times. 
0.63 actual content of lipase P enzyme .times. 
0.51 immobilization yield .fwdarw. 
16 mg of lipase immobilized on the carrier 
50 mg of free lipase P .times. 
0.63 actual content of lipase P enzyme .fwdarw. 
31.5 mg of actual content of lipase P enzyme 
16 mg of immobilized enzyme provide a conversion of 33.4% 
31.5 mg of free enzyme provide a conversion of 20.7% .fwdarw. 
activity: 320%. 
______________________________________ 
The enzymatic racemate resolution can be carried out not only as described 
above in the presence of vinyl acetate but also in the presence of other 
vinyl esters such as, for example, of chloroacetic acid, lauric acid and 
phenylacetic acid. For this, lipase P immobilized on the carrier is packed 
into a glass column and washed with 150 ml of t-butyl methyl ether, which 
is used as solvent for the following reaction. 
The column is subsequently charged with a solution of substrate and vinyl 
ester, each of which are dissolved in 250 ml of t-butyl methyl ether. This 
solution is allowed to run slowly through the column, and the composition 
after the solutions have completely passed through is determined by gas 
chromatography. The quantities to be employed, and the flow-through and 
reaction times and the results are to be found in Table 3. 
Example of transesterification with ethyl acetate: 
0.5 ml of ethyl acetate is stirred with 2.5% strength phenylethanol in 
t-butyl methyl ether (10 ml) and 5 ml of enzyme immobilized on XAD-2 
(theoretical quantity of enzyme: 50 mg) at room temperature. The reaction 
showed 50% conversion of the phenylethanol after 5 days. 
The control with non-immobilized enzyme showed a distinctly lower 
conversion after 7 days. 
The conversion was determined by TLC. 
Example of the esterification of primary OH groups: 
Geraniol is dissolved 2.5% strength in 10 ml of vinyl acetate and stirred 
with the addition of 5 ml of enzyme immobilized on XAD-2 (theoretical 
quantity of enzyme: 50 mg) at room temperature. Quantitative acetylation 
was carried out with immobilized enzyme after only 1 h, whereas the free 
enzyme required 50% more time. 
Example of the acetylation of dl-pantolactone - test of the long-term 
stability at elevated temperature in a continuous process: 
400 ml of lipase P immobilized on XAD-2 are packed into a 
temperature-controllable jacketed glass column. A 0.1% strength solution 
of dl-pantolactone (in vinyl acetate/t-butyl methyl ether 1:9; total 
volume 2 l) is passed at a flow rate of 0.11 ml/min through the column at 
50.degree. C. 
The column volume is compensated by subsequent washing with t-butyl methyl 
ether at the same rate. GC analysis of the reaction solution shows 71.6% 
conversion to pantolactone acetate in this first run. The washed column is 
stored in the dry state at RT and temperature-equilibrated for 5 h before 
each new start of another run. During the next 5 months a further 11 
continuous reactions are carried out in this manner. The 12th run (under 
identical conditions apart from: flow rate 0.13 ml/min), carried out 5 
months later, shows 70.7% conversion to pantolactone acetate. 
TABLE 1 
__________________________________________________________________________ 
The reaction time is 6 h in each case 
Previously 
Subsequently 
functionalized 
crosslinked 
% conversion to acetate 
Ex. with chloro- 
with 1st Quantity 
No. 
Carrier 
acetic acid 
glutaraldehyde 
Run 2nd 
3rd 
4th 
used 
__________________________________________________________________________ 
Control 15-17 
4-6 in each case 
1 .RTM.Amberlite 
-- -- 33.4 
38.4 
38 33.5 
(X) 
XAD-2 
2 XAD-4 -- -- 19.35 
11.94 
12.31 
8.08 
XAD-16 -- -- 17.20 
14.11 
14.91 
10.06 
XAD-1180 
-- -- 17.61 
13.03 
12.71 
11.19 
3 XAD-12 -- -- 19 4.45 
-- -- 
4 XAD-2 -- yes 18.45 
24.93 
17.31 
13.46 
5 XAD-4 -- yes 15.66 
16.74 
14.63 
11.91 
6 XAD-12 -- yes 26.06 
9.7 
-- -- 
__________________________________________________________________________ 
(X) The following were used in Example 1: 250 mg of phenylethanol, 10 ml 
of vinyl acetate, 5 ml of carrier. 
TABLE 2 
__________________________________________________________________________ 
(Amberlite XAD-2 was used as carrier in each case) 
__________________________________________________________________________ 
Quantity Quantity 
Racemo: 
Precursor 
A: B: of vinyl 
Reaction 
Conver- 
Ex. 
prochir. 
Quantity 
Conc. 
carrier 
enzyme 
Temp. 
ester 
time sion 
No. 
alcohol 
[g] in % 
[ml] 
[g] [.degree.C.] 
[ml] [h] [%] 
__________________________________________________________________________ 
1 1-phenyl- 
A: 0.25 
2.5 5 0.05 RT 10 6 33.4 
ethanol 
B: 0.25 
2.5 0.05 RT 10 6 20.7 
C: 0.5 
1 50 5 RT 50 -- 50 
2 1-phenyl- 
A: 0.50 
2.5 10 1 RT 20 30 46.1 
propanol 
B: 0.52 
2.5 1 RT 20 30 37.1 
C: -- 
-- -- -- RT -- -- -- 
3 2-chloro- 
A: 0.51 
2.5 10 1 RT 20 48 51.7 
1-phenyl- 
B: 0.51 
2.5 1 RT 20 48 32.3 
ethanol 
C: 0.5 
0.2 50 5 RT 50 -- 34 
4 panto- A: 0.5 
2.5 10 1 RT 20 48 32.2 
lactone 
B: 0.49 
2.5 1 RT 20 48 29.7 
C: 0.5 
0.2 50 5 50.degree. C. 
250 -- 42 
5 alleth- 
A: 0.5 
2.5 10 1 RT 20 4 45 
rolone B: 0.51 
2.5 1 RT 20 4 42 
C: 0.5 
1 50 5 RT 500 -- 64 
6 1-(6-acetoxy- 
A: -- 
-- -- -- -- -- -- 
naphthyl)- 
B: -- 
-- -- -- -- -- -- 
ethanol 
C: 14 
1 50 5 RT 1400 -- 52 
7 2-(1-naphthyl- 
A: 1 2 2.5 0.25 0.degree. C. 
50* 16 98 
methyl)-pro- 
B: 1 2 0.25 0.degree. C. 
50* 16 95 
pane-1,3-diol 
C: 1 0.5 50 5 15.degree. C. 
200* 6 n.d. 
__________________________________________________________________________ 
Racemo: Space-time 
Ex. 
prochir. 
Act. 
yield Alcohol 
chem. Acetate 
chem. 
No. 
alcohol 
[%] [g/l/h] 
ee [.alpha.].sub.D.sup.25 
yield 
ee [.alpha.].sub.D.sup.25 
yield 
__________________________________________________________________________ 
1 1-phenyl- 
320 -- n.d. 
n.d. n.d. 
&gt;95% 
+105.6 
n.d. 
ethanol 
-- -- " " " &gt;95% 
+105.1 
n.d. 
-- 3 &gt;95% 
-44 43% &gt;95% 
104.1 
44% 
2 1-phenyl- 
250 -- 77% -38 45% &gt;95% 
102.7 
36% 
propanol 
-- -- 55% -27.4 54% &gt;95% 
+100.1 
33% 
-- -- -- -- -- -- -- -- 
3 2-chloro- 
320 -- n.d. 
n.d. 35% &gt;95 +75.6 
39% 
1-phenyl- 
-- -- n.d. 
n.d. 62% &gt;95% 
-- 25% 
ethanol 
-- 0.5 n.d. 
n.d. n.d. 
n.d. 
n.d. n.d. 
4 panto- 220 -- n.d. 
n.d. 38% &gt;95% 
+12.7 
22% 
lactone 
-- -- n.d. 
n.d. 63% &gt;95% 
+12.7 
28% 
-- 0.5 n.d. 
n.d. n.d. 
&gt;95% 
+11.4 
37% 
5 alleth- 
210 -- &gt;95% 
+14.5 42% &gt;84% 
-27.6 
44% 
rolone -- -- &gt;95% 
+14.8 43% 92% -29.9 
41% 
-- 6 &gt;95% 
+14.6 28% 52% -15.87 
63% 
6 1-(6-acetoxy- 
-- -- -- -- -- -- -- -- 
naphthyl)- 
-- -- -- -- -- -- -- -- 
ethanol 
-- 3 &gt;95% 
-30 45% 92% +82.35 
48% 
7 2-(1-naphthyl- 
190 -- -- -- -- 95% +39.5 
94% 
methyl)-pro- 
-- -- -- -- -- 97% +40.3 
91% 
pane-1,3-diol 
-- 30 -- -- -- 88% +36.5 
n.d. 
__________________________________________________________________________ 
A = batch test with immob. enzyme 
B = comparison batch with free enzyme 
C = test in continuous process 
RT = room temperature 
n.d. = not determined 
*= a vinyl acetate/dimethoxyethane/diethyl ether mixture was used 
ee: measured on the basis of the optical rotation 
TABLE 3 
______________________________________ 
Acylation of phenylethanol in various vinyl esters 
Rac. Vinyl ester Quantity 
Reaction 
Con- 
Ex. alcohol.sup.+ 
Type/Quantity 
enzyme/ 
time version 
No. [g] [ml] carrier* 
[h] [%] 
______________________________________ 
1 2.5 g chloroacetic 5/50 5 67.6 
acid/12.5 
2 2.5 g lauric acid/5 
5/50 5 41.7 
3 2.5 g phenylacetic 5/50 16 24.4 
acid/5 
______________________________________ 
.sup.+ phenylethanol was employed as rac. alcohol in each case. 
* .RTM.Amberlite XAD2 was employed as carrier in each case. 
The process according to the invention has the following advantages 
compared with conventional processes for racemate resolution of alcohols: 
A) The space-time yields are distinctly increased owing to increased enzyme 
activity. 
B) Very long useful lives of the immobilized enzyme permit particularly 
economic use of the biocatalyst. 
C) The activity of the enzyme is durable (Tab. 1; see below (a)). 
D) High thermal stability of the immobilized enzyme. 10 ml of 0.5% strength 
phenylethanol solution in toluene are mixed with 100 mg of free lipase or 
1 ml of immobilized enzyme and subsequently with 0.1 ml of vinyl 
phenylacetate in each case. The free lipase shows 3.2% conversion after 
stirring at RT for 5 hours, and no longer shows activity after the same 
time has elapsed under reflux at 110.degree. C., whereas the immobilized 
lipase still shows 1-2% conversion at 110.degree. C. The conversion is 
determined by GC test (Reoplex on .RTM.Chromosorb). 
(a) 250 mg of racemic phenylethanol in 20 ml of vinyl acetate are shaken 
with 2 ml of immobilized lipase at 50.degree. C. for 6 h. The conversion 
is subsequently determined by GC, and the immobilized enzyme is removed by 
filtration and thoroughly washed with vinyl acetate. Renewed reaction 
under identical conditions is carried out the next day. 
(b) The process is carried out in the same way using 0.2 g of the free 
enzyme. 
(a) A conversion of 48.7% is measured in the 1st run after 6 h. 
A conversion of 21.2% is measured in the 10th run after 6 h. 
(b) In the 1st run, the conversion after 6 h is 46.1%. Product no longer 
detectable after the 7th run. 
The activity of the enzyme is ensured in the long term.