Enantioselective preparation of s-2R.sub.1,2R.sub.2 -1,3-dioxolane-4-methanol and derivatives thereof

Process for the enantioselective conversion of an enantiomer mixture of 2R.sub.1,2R.sub.2 -1,3-dioxolane-4-methanol including the following steps: combining the enantioselective enzyme, PQQ-dependent alcohol dehydrogenase with an enantiomer mixture of 2R.sub.1, 2R.sub.2 -1,3-dioxolane-4-methanol of the formula: ##STR1## where R.sub.1 and R.sub.2 independently are selected from the group consisting of hydrogen, an optionally branched alkyl group, an aryl, and R.sub.1 and R.sub.2 with the carbon atom to which they are attached being an optionally substituted carbocyclic ring, the combination of ingredients producing 2R.sub.1,2R.sub.2 -3-dioxolane-4-methanol enriched in the S-enantiomer i.e. S-2R.sub.1,2R.sub.2 -1,3-dioxolane-4-methanol.

BACKGROUND AND SUMMARY OF THE INVENTION 
This invention relates to a process for the enantioselective conversion of 
an enantiomer mixture of 2R.sub.1, 2R.sub.2 -1,3-dioxolane-4-methanol of 
the formula: 
##STR2## 
where R.sub.1 and R.sub.2 independently represent hydrogen or an 
optionally branched alkyl group, an aryl, or R.sub.1 and R.sub.2, together 
with the carbon atom to which they are attached, represent an optionally 
substituted carbocyclic ring, the product of said conversion being 
enriched in one of the enantiomers, which is achieved by subjecting this 
enantiomer mixture to the action of an enantioselective enzyme. 
Such a process is known from EP-A-244,912. According to this published 
patent application, a product enriched in R-enantiomer can be obtained 
from an enantiomer mixture of an above-disclosed compound by subjecting 
such a mixture to the action of an enzyme that may originate from a great 
number of micro-organisms. According to the prior method it is mainly the 
S-enantiomer of the relevant compounds that is converted into the 
R-2R.sub.1, 2R.sub.2 -1,3-dioxolane-4-carboxylic acid, so that a 2R.sub.1, 
2R.sub.2 -1,3-dioxolane-4-methanol enriched in R-enantiomer also remains. 
In said prior application it is also stated that the oxidation product, 
which contains an excess of R-2R.sub.1, 2R.sub.2 
-1,3-dioxolane-4-carboxylic acid, can be reduced to the corresponding 
alcohol, in which the amount of S-2R.sub.1,2R.sub.2 
-1,3-dioxolane-4-methanol is predominating. 
For many applications, in particular for the use as starting compounds for 
the preparation of pharmaceutical compounds, it is desirable to have 
S-2R.sub.1, 2R.sub.2 -1,3-dioxolane-4-methanol at one's disposal. For this 
purpose it is desirable to be able to prepare a product enriched in 
S-enantiomer directly from an enantiomer mixture of 2R.sub.1, 2R.sub.2 
-1,3-dioxolane-4-methanol. This can, for example, be achieved by means of 
an enantioselective enzymatic process. 
DETAILED DESCRIPTION OF THE INVENTION 
A process has now been found for the enantioselective conversion of an 
enantiomer mixture of 2R.sub.1, 2R.sub.2 -1,3-dioxolane-4-methanol of the 
formula: 
##STR3## 
where R.sub.1 and R.sub.2 independently represent hydrogen or an optically 
branched alkyl group, an aryl, or R.sub.1 and R.sub.2, together with the 
carbon atom to which they are attached, represent an optionally 
substituted carbocyclic ring, the product of said conversion being 
enriched in one of the enantiomers, which is achieved by subjecting the 
enantiomer mixture to the action of an enantioselective enzyme by, and 
this characterizes the invention, preparing 2R.sub.1, 2R.sub.2 
-1,3-dioxolane-4-methanol enriched in S-enantiomer while applying a 
suitable pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase as 
enantioselective enzyme. 
R.sub.1, R.sub.2 mostly represent H, or an C.sub.1 -C.sub.10 (cyclo) alkyl, 
a C.sub.5 -C.sub.10, preferably C.sub.6, aryl or R.sub.1 and R.sub.2 form 
together with the C-atom to which they are attached a carbocyclic ring 
with 4-10, preferably 5 or 6, C-atoms. R.sub.1, R.sub.2 and/or the 
carbocyclic ring they form together with the C-atom to which they are 
attached may be substituted with halogen atoms, lower alkyl (C.sub.1 
-C.sub.4) or lower alkoxy (C.sub.1 -C.sub.4). 
The so-called PQQ-dependent alcohol dehydrogenase to be used in the present 
invention is known in itself from, for instance, the dissertation 
"Quinoprotein (PQQ-containing) Alcohol Dehydrogenase" by J. A. Duine (PhD 
thesis, Delft, 19 Sep. 1985) and from B. W. Groen et al. in Biochem. J. 
234 (1986) p. 611 ff. PQQ stands for pyrrolo-quinoline-quinone, i.e., 
2,7,9-tricarboxy-1H-pyrrolo(2,3f)quinoline-4,5-dione. Suitable 
PQQ-dependent alcohol dehydrogenases are e.g. Pseudomonas testosteroni or 
Gluconobacter suboxydans. The PQQ-dependent alcohol dehydrogenase 
originating from or obtained from Pseudomonas testosteroni is eminently 
suitable for the subject process. The Pseudomonas testosteroni 
micro-organism can be cultured by the method described by B. W. Groen et 
al., loc., cit., but also by any other suitable cultivation method. Such 
cultivation methods are so commonly known and have been described so 
frequently in patent specifications and scientific publications as to 
render it superfluous to describe these cultivation methods in this 
context. The enzyme preparation as used in the subject invention is not 
restricted by purity and the like and may be both a crude enzyme solution 
and purified enzyme, but it may also consist of (permeabilized and/or 
immobilized) microbial cells that possess the desired activity, or of a 
homogenate of cells or whole cells possessing such activity. The enzyme 
may also be used in immobilized form or in chemically modified form. When 
some undesired opposite enzyme activity is present in the enzyme 
preparation used, it is recommended that this undesired activity be 
eliminated or be suppressed in order to obtain maximum enantioselectivity. 
The invention is in no way restricted by the form in which the enzyme for 
the subject invention is used. Within the scope of the invention use may 
also be made of a PQQ-dependent alcohol dehydrogenase originating from a 
mutant of Pseudomonas testosteroni or from genetically modified 
micro-organisms. Preferably use is made of a PQQ-dependent alcohol 
dehydrogenase originating from or obtained from Pseudomonas testosteroni 
ATCC 15666, ATCC 15667, NCIB 8893, NCIB 8955 or NCIB 10808. According to 
Jin Tamaoko et al. in Int. J. Syst. Bact. 37 (1987) p. 52 ff, Pseudomonas 
testosteroni should be classified as Comamonas testosteroni. The organisms 
are referred to as Pseudomonas testosteroni in this application. 
As is apparent from the publication by B. W. Groen et al. (loc. cit.), the 
PQQ-dependent alcohol dehydrogenase, such as can be obtained, for 
instance, from Pseudomonas testosteroni, is not active in all cases with 
regard to the oxidation of some alcohols: for activity a sufficient amount 
of PQQ must be present. If enough PQQ is not present in the cultivation of 
the micro-organism, then it is desirable for oxidation purposes to add it 
in sufficient quantities. Instead of PQQ alone, PQQ analogues may also be 
used, such as monoesters of PQQ (at the 2-position), 4-hydroxy-PQ (PQ 
standing for pyrroloquinoline) or 5-hydroxy-PQ after oxidation, 
3-methyl-PQQ, 3-ethyl-PQQ, 3-propyl-PQQ, N-methyl-PQQ, N-ethyl-PQQ, 
8-methyl-PQQ or the PQQ-acetone adduct and other aldehyde and ketone 
adducts, as described by J. A. Jongejan, B. W. Groen and J. A. Duine in 
"PQQ and Quinoproteins" (J. A. Jongejans and J. A. Duine, eds.), Kluwer 
Academic Publishers, Dordrecht, 1989, pp. 205-216. 
Enantioselective enzymatic conversions are known by themselves. The 
enantioselectivity resides in a difference in conversion rate between the 
enantiomers in question. Reaction products are obtained that are enriched 
in one of the enantiomers. For practical purposes it generally is 
desirable for one of the enantiomers to be obtained in a large excess. 
This is achieved by terminating the conversion at a certain degree of 
conversion. For enantioselective enzymatic hydrolyses this is described by 
Qu-Ming Gu et al. in Tetrahedron Letters 27 (1986), 5203 ff., and more in 
general by Ghing-Shih Chen et al. in J. Am. Chem. Soc. 104 (1982), 7294 
ff. The general doctrine of enantioselective enzymatic conversions 
described in these publications also applies to the subject process. 
According to the subject process the PQQ-dependent alcohol dehydrogenase 
oxidizes the R-enantiomer of 2R.sub.1, 2R.sub.2 -1,3-dioxolane-4-methanol 
at a more rapid rate to form the S-enantiomer of the corresponding 
2R.sub.1, 2R.sub.2 -1,3-dioxolane-4-carboxylic acid, as a result of which 
a reaction product is obtained, enriched in S-2R.sub.1, 2R.sub.2 
-1,3-dioxolane-4-methanol. The conversion rates of the enantiomers appear 
to differ considerably, as a result of which at 50% conversion of a 
racemic mixture an enantiomer excess of more than 90% of the 2R.sub.1, 
2R.sub.2 -1,3-dioxolane-4-methanol can be obtained, while an enantiomer 
excess of more than 95% can be reached at a conversion between 50 and 55%. 
The range of starting proportion of PQQ dependent alcohol dehydrogenase 
relative to the enantiomer mixture is 1 mg-200 mg cells (dry weight) per g 
R,S-substrate. 
Generally, the amount of PQQ is 0.5-2 equivalents with respect to the 
enzyme. For optimal results a minimum quantity of 1 equivalent is 
required. 
The cells used in the reaction may be separated from the reaction mixture 
and reused in a subsequent conversion, without considerable loss of 
activity. 
According to the subject process, preferably, the S-enantiomer is prepared 
from 2,2-dimethyl-1,3-dioxolane-4-methanol. An enantiomer excess of 
S-2,2-dimethyl-1,3-dioxolane-4-methanol of at least 95% can be obtained. 
For example, in one embodiment of the present invention, conversion to 
S-2,2-dimethyl-1,3-dioxolane-4-methanol is by suspending 10-200 g of wet 
cells in 0.2-10.0 l potassium phosphate buffer and saturated with 0.5-60 
.mu.mol of PQQ to which is added 10-10.00 g R,S-2,2-dimethyl-1,3- 
dioxolane-4-methanol whereafter the solution is kept at 
15.degree.-50.degree. C. temperature and 5-9 pH by titration for 6-72 hr 
amount of time. 
This S-2,2-dimethyl-1,3-dioxolane-4-methanol is an important starting 
product for the preparation of pharmaceuticals, crop protection and/or 
agricultural pest control agents, as is also reported in a publication by 
J. Jurczak et al. in Tetrahedron 42 (1986) 447 ff. The R- and 
S-enantiomers of 2,2-dimethyl-1,3-dioxolane-4-methanol are important 
chiral C.sub.3 synthons in organic synthesis. Examples of syntheses of 
more complex structures from such C.sub.3 synthons are .beta.-receptor 
blocking agents and sn-glycerylphosphoryl choline (GPC). 
Racemic 2,2-dimethyl-1,3-dioxolane-4-methanol can be prepared in the way 
known for the preparation of acetals by acid-catalyzed coupling of 
glycerol and acetone. For compounds of the formula presented in the above, 
in which R.sub.1 and R.sub.2 do not both represent a methyl group, 
glycerol is to be coupled with the aldehyde or ketone in question.

EXAMPLES 
The invention will be further elucidated by the following examples, without 
being restricted by these examples. In these examples the analyses of the 
reaction products, notably with regard to the determination of the 
enantiomeric excess (ee), were performed using the method described below 
(TAGIT method). It is possible, if so desired, to calculate the 
selectivity value for the reaction with the aid of the formulae of Chen et 
al. (J. Am. Chem. Soc. 104, 7294 ff. (1982)). For conversion determination 
use was made of column separation over an Aminex HPX-87H HPLC column with 
0.01N H.sub.2 SO.sub.4 as mobile solvent and glycerol detection on the 
basis of the refractive index. 
All analyses were always performed using samples taken, at different points 
of time, from the reaction mixtures of the experiments, the amounts being 
10-50 ml for conversion determination and 10-50 ml for ee determination, 
depending on the conversion. 
In ee determination the samples are extracted with two times 20 ml 
dichloromethane, after which 200 .mu.l of the residue obtained after 
extract evaporation is dissolved in 5 ml diethylether. Subsequently 0.35 g 
tosylchloride and about 0.5 g potassium hydroxide powder are added to the 
ether solution, and the resulting solution is stirred at room temperature 
for 15 minutes. The tosyl addition product of the 2R.sub.1, 2R.sub.2 
-1,3-dioxolane-4-methanol is then isolated by extraction with diethylether 
and water, after which the ether fraction is washed with water and 
evaporated. The tosyl-2R.sub.1, 2R.sub.2 -1,3-dioxolane-4-methanol 
obtained is then dissolved in 2.0 ml n-butylamine and heated for 1 hour at 
100.degree. C. The reaction product is subsequently isolated by extraction 
with diethylether and water, after which the ether fraction is washed four 
times with water and evaporated. This residue is dissolved in 1.0 ml 
acetonitrile, after which 2.5 .mu.l of the resulting solution is mixed 
with 50 .mu.l acetonitrile and 1 mg 
2,3,4,6-tetra-0-acetyl-.beta.-glucopyranosyl-isothiocyanate (TAGIT). Five 
minutes after mixing, the ee is determined using a reversed phase 
(C.sub.18) column with 60/40 methanol/water as eluent solvent. 
This method for determination of the enantiomer excess has been found to be 
very suitable. It gives an excellent separation of the R- and 
S-enantiomers of 2R.sub.1, 2R.sub.2 -1,3-dioxolane-4-methanol (in 
derivatized form), as is illustrated in FIGS. 1, 2 and 3, which are added 
as specimen chromatograms of this separation. FIG. 1 relates to the 
racemate, for FIG. 2 use has been made of the commercially available 
S-enantiomer (Janssen Chimica), and FIG. 3 relates to the product obtained 
according to the invention after 56% conversion. 
EXAMPLE I 
pseudomonas testosteroni (American Type Culture Collection No. 15667) was 
cultured in a fermentor containing 20 l of mineral salts medium 
(composition per liter of demineralized water: 15.4 g K.sub.2 
HPO.sub.4.3H.sub.2 O, 4.52 g KH.sub.2 PO.sub.4, 0.5 g MgCl.sub.2. 6H.sub.2 
O, 3 g (NH.sub.4).sub.2 SO.sub.4,15 mg CACl.sub.2, 15 mg 
FeSO.sub.4.7H.sub.2 O and the trace elements Cu, B, Mn, Zn, Mo), to which, 
after sterilization, 100 ml ethanol had been added. The pH was 7.0. The 
fermentor contents were kept at 28.degree. C. and stirred at a speed of 
350 rpm. The aeration rate was 3.5 l per minute. After 80 hours of 
cultivation the cells were harvested by centrifuging. A portion of these 
cells (wet weight 11 g) was suspended in 100 ml tris/HCl buffer (50 mM, pH 
7.5), to which 5.3 .mu.mol PQQ had been added. After the cells had been 
saturated with PQQ, 1.8 l of the same buffer and 20 g R,S-2 
2-dimethyl-1,3-dioxolane-4-methanol were added. During incubation the cell 
suspension was aerated (1.0 l air per minute) and stirred (400 rpm), while 
the pH was kept at 7.5 by titration with 0.2N NaOH. The temperature was 
kept at 28.degree. C. 
During this oxidation, at regular intervals of four hours, samples were 
taken, which were analyzed in the way described above. Sampling occurred 
at 4 hr. (Sample 1), 8 hr. (Sample 2), 12 hr. (Sample 3) and 16 hr. 
(Sample 4). The results are summarized in Table 1. FIG. 3 refers to the 
results of this Example, for below Sample No. 4 at 56% conversion. 
TABLE 1 
______________________________________ 
Conversion 
ee (S-enantiomer) 
Sample (%) (%) 
______________________________________ 
1 15 17 
2 32 45 
3 45 75 
4 56 99 
______________________________________ 
Thus with respect to Sample 4, there was 56% conversion of 
2,2-dimethyl-1,3-dioxolane-4-methanol and the enantiomeric excess (ee) of 
the S-enantiomer of 2,2-dimethyl-1,3-dioxolane-4-methanol was 99%. 
EXAMPLE II 
Using the purification method described in Biochem. J. (1986) 234, 611 ff., 
PQQ-dependent alcohol dehydrogenase was recovered from Pseudomonas 
testosteroni ATCC 15667, cultured as described in Example I. The isolated 
enzyme was dissolved in 20 ml potassium phosphate buffer (pH 8.0; 50 mM) 
and saturated with 100 nmol PQQ. Subsequently 1.01 mmol potassium 
ferricyanide and 0.5 mmol R,S-2,2-dimethyl-1,3-dioxolane-4-methanol were 
added. The pH of the solution was kept at 8.0 by titration with 0.1N NaOH. 
After reduction of ferricyanide the reaction mixture was analyzed. There 
was 51% conversion of 2,2-dimethyl-3-dioxolane-4-methanol yielding the 
S-2,2-dimethyl-1,3-dioxolane-4-methanol with ee value of 99%. 
EXAMPLE III 
The alcohol dehydrogenase recovered from Pseudomonas testosteroni ATCC 
15667 (see Example II) was dissolved in 20 ml potassium phosphate buffer 
(pH 8.0; 50 mM) and saturated with 100 nmol PQQ, and subsequently excess 
potassium ferricyanide and 0.5 mmol 2,2-pentylene-1,3-dioxolane-4-methanol 
were added. After oxidation of about 50% of the substrate the reaction was 
stopped and the reaction mixture analyzed. Conversion appeared to be 54% 
and the enantiomer excess of the S-enantiomer was 99%. 
EXAMPLE IV 
Example III was repeated with 2,2-butylene-1,3-dioxolane-4-methanol instead 
of 2,2-pentylene-1,3-dioxolane-4-methanol. The conversion was 53% and the 
enantiomer excess of the S-enantiomer 99%. 
EXAMPLE V 
A 2.0 l Erlenmeyer flask, filled with 500 ml mineral salts medium (see 
Example I), 2.5 ml ethanol and 2 .mu.mol PQQ, was inoculated with 
Pseudomonas testosteroni ATCC 15667. Both PQQ (filter sterilized) and 
ethanol had been added after sterilization by heating of the medium. The 
cultivation temperature was 28.degree. C., and shaking took place at 200 
rpm. After 36 hours the cells were harvested by centrifuging, after which 
they were suspended in 100 ml potassium phosphate buffer (pH 7.0; 50 mM). 
After addition of 1.0 g R,S-2,2-dimethyl-1,3-dioxolane-4-methanol, the 
reaction medium was incubated at 28.degree. C. while being shaken (200 
rpm). After 24 hours the reaction medium was analyzed. Conversion was 54%, 
and the ee value for the S-enantiomer 96%. 
EXAMPLE VI 
Using the method of Example V, Pseudomonas testosteroni ATCC 15666, NCIB 
8893 and NCIB 10808 were respectively cultured, and each time the 
PQQ-dependent alcohol dehydrogenase obtained was used for enantiospecific 
oxidation of R,S-2,2-dimethyl-1,3-dioxolane-4-methanol as described in 
example IV, with similar results as regards the enantioselectivity. 
EXAMPLE VII 
Using the purification method described in Agric. Biol. Chem. 42 (1978) 
2045-2056, purified, membrane bound, PQQ-dependent alcohol dehydrogenase 
recovered from Gluconobacter suboxydans ATCC 621, was dissolved in 20 ml 
potassium phosphate buffer (pH=7.0; 50 mM) and saturated with 100 nmol 
PQQ. Subsequently excess potassium ferricyanide and 0.5 mmol 
R,S-2,2-dimethyl-1,3-dioxolane-4-methanol were added. After oxidation of 
about 60% of the substrate, the reaction was stopped. There was 64% 
conversion to S-2,2-dimethyl-1,3-dioxolane-4-methanol with an enantiomeric 
excess of 96%. 
EXAMPLE VIII 
Pseudomonas testosteroni ATCC 15667 was cultured in a fermentor containing 
5 l of a medium containing mineral salts as described in Example I (pH 
=7), to which, after sterilization, 25 ml ethanol, as a carbon source, was 
added. At the end of the exponential growth phase, the pH was increased to 
7.5. Subsequently 150 mmol R,S-2,2-dimethyl-1,3-dioxolane-4-methanol and 
2.5 mg PQQ were added while the pH was kept at 7.5 by titration with 0.2N 
NaOH. After oxidation of 57% of the substrate added, the cells were 
separated off with a centrifuge, and the remaining substrate was isolated 
by extraction. The enantiomer excess of the S-enantiomer of 
2,2-dimethyl-1,3-dioxolane-4-methanol dioxolane was .gtoreq.99.5%. 
EXAMPLE IX 
The cells remaining from example VIII were reused. To this end the cells 
were suspended in 1.0 l tris/HCl buffer (50 mM, pH 7.5), to which 20 g 
R,S-2,2-dimethyl-1,3-dioxolane-4-methanol were added. After oxidation of 
54% of the racemate (54% conversion), the S-enantiomer of 
R,S-2,2-dimethyl-1,3-dioxolane-4-methanol remained with an enantiomeric 
excess of 97%. 
While only a few exemplary embodiments of this invention have been 
described in detail, those skilled in the art will recognize that there 
are many possible variations and modifications which may be made in the 
exemplary embodiments while yet retaining many of the novel and 
advantageous features of this invention. Accordingly, it is intended that 
the following claims cover all such modifications and variations.