Method of converting racemic hydantoins into optically active aminoacids

A method is disclosed for converting racemic hydantoins into optically active aminoacids, wherein microorganisms of the Pseudomonas genus are used to hydrolyze the hydantoins into derivatives of D,L-aminoacids. Comparatively high temperatures and a definitely basic pH are favorable factors.

This invention relates to enzymatic complexes which are capable of 
converting racemic hydantoins into optically active aminoacids, and more 
particularly it relates to the hydrolysis of hydantoins into aminoacid 
derivatives having the "D" configuration, said hydrolysis resorting to the 
use of particular microorganisms which use hydantoin as a nitrogen source. 
A few aminoacids, especially phenyl-glycine and p-hydroxyphenylglycine are 
important intermediates for the preparation of compounds which are widely 
used in the pharmaceutical industry. 
A number of attempts have been made in the past to obtain D-aminoacids, but 
none of such methods could be carried out on an industrial scale. 
The chemical methods as used hitherto for the separation of optical isomers 
are very expensive and give low yields. They are based on the use of 
camphorsulphonic acid. 
Another method provides for a selective hydrolysis of the D-acyl-aminoacid 
with the acylase enzyme. However, the D-acylases are comparatively rare 
and always impure because of the presence of L-acylase, which makes the 
procedure difficult and is conducive to the obtainment of a product having 
poor optical purity. 
The enzymatic hydrolysis which is the subject-matter of the present 
invention, conversely, permits the preparation of a single stereo-isomeric 
form of an aminoacid, or a derivative thereof, from a racemic compound. 
A method for the enzymatic resolution of D,L-aminoacids or of their 
derivatives has already been suggested in U.S. Pat. No. 3,964,970. This 
method essentially comprises the step of subjecting to an enzymatic 
hydrolysis by hydropyrimidine hydrolase (E.C.3.5.2.2); extracted from 
calf's liver, the racemic form of compounds having the following general 
formula: 
##STR1## 
The hydrolysis takes place according to the pattern: 
##STR2## 
It has now been found that the enzymatic resolution of racemic compounds 
having the general formula (1) according to the pattern (2) can be carried 
out also with hydrolases which are supplied by microorganisms of the 
Pseudomonas genus, which have never been employed in such a kind of 
reaction. 
The method, the subject of the present invention, comprises the steps of 
producing the specific enzyme which is formed during the growth of 
microorganisms of the Pseudomonas class and hydrolyzing hydantoins into 
derivatives of D,L-aminoacids with this enzyme, wherein the enzyme can be 
used directly as a bacterial suspension or culture medium, or it can be 
extracted from the cells and the culture medium. 
The test for the selective hydrolysis of hydantoin derivatives of 
D,L-aminoacids through microorganisms was carried out as follows : the 
bacterial strains, isolated from earth, plants, debris of various kinds, 
etc., as well as strains from bacterial collections were inoculated from 
slant into 250-mls flasks containing 50 mls of the following culture 
medium: 
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Meat peptone 10 grams per liter 
Yeast extract 10 grams per liter 
Glucose 5 grams per liter 
NaCl 3 grams per liter 
5-(D,L)-methylhydantoin 
1 gram per liter 
pH = 7.2 
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Sterilization 30 mins. at 110.degree. C. 
After 20 to 24 hours of incubation (orbital stirring) at 30.degree. C., 
500-ml flasks, containing 100 mls of the same medium, were incubated with 
5 mls of the above pre-culture. 
After 18 to 22 hours of additional incubation, the enzymatic reaction with 
the resting cells was carried out : in test tubes with 10 mls of phosphate 
buffer, 0.07 M, pH = 8.5, with 20 micromol/ml of 5-(D,L)-phenylhydantoin, 
there was added 1 ml of the bacterial suspensions (dry weight about 50 
milligrams per ml). After 30 minutes of incubation at 30.degree. C., the 
reaction with p-dimethylaminobenzaldehyde was carried out for the 
quantitative determination of the as-formed carbamyl derivative (J. Biol. 
Chem., 238, 3325 (1963). The strains which have been employed are 
tabulated in Table 1. Those of them which are identified by the numbers 
942 and 945 have been deposited with the Centralbureau voor 
Schimmelcultures where they have been assigned the symbols CBS 145.75 and 
146.75, respectively. 
TABLE 1 
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N-carbamylphenylglycine as a 
Strain % of the theoretical yield 
______________________________________ 
Pseudomonas sp. 
940 41 
941 50 
942 64 
943 50 
944 50 
945 57 
946 50 
947 15 
948 15 
949 20 
950 20 
951 20 
952 20 
953 20 
954 20 
955 20 
Pseudomonas 
fluorescens 956 10 
Pseudomonas sp. 
957 41 
Pseudomonas 
ATCC 11299 9 
Pseudomonas 
oleovorans CL 
59 17 
Pseudomonas 
desmolyticum 
NCIB 8859 25 
Pseudomonas 
fluoroscens 
ATCC 11250 25 
Pseudomonas 
putida 
ATCC 12633 14 
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The bacterial strains listed in TABLE 1 grow well in all the common 
laboratory media. They are all capable of using hydantoin as a single 
nitrogen source. The growth takes place at a temperature ranging from 
5.degree. C. to 40.degree. C., the optimum being between 25.degree. C. and 
30.degree. C. 
As regards the classification by genera, the scheme of the Bergey's Manual, 
7th Edition was followed together with the recommendations by Lysenko (J. 
Gen. Microbiol. 25, 379 (1961) and by Stanier, Palleroni, Doudoroff (J. 
Gen. Microbiol. 43, 159-271 (1966). 
It has been found that during the enzymatic hydrolysis of D-hydantoin at an 
alkaline pH (pH 7-10) a nonenzymatic racemization of the remaining 
L-hydantoin takes place. For this reason, and as a result of the 
continuous removal of D-hydantoin by enzymatic hydrolysis, all the 
carbamyl derivative obtained at the end of the reaction is of the D-form. 
The identity of D(-)-N-carbamylphenylglycine was evidenced after 
crystallization of the reaction product, on the basis of the IR, NMR, and 
mass spectra and the elemental analysis. 
The specific rotation is [.alpha.].sub.D.sup.25 = - 137.degree. (c= 1% in 
1N NH.sub.4 OH) corresponding to the one as reported in the literature. 
The racemization rate of L-hydantoin is a function of the temperature and 
of the pH and is the faster, the higher is the temperature and the more 
basic is the pH. However, by working at a pH in the vicinity of 7.5, the 
racemization rate is sufficiently high so as not to limit the reaction 
speed. 
The temperature of the enzymatic reaction can be maintained between 
10.degree. C. and 60.degree. C. For practical reasons, however, 
temperatures comprised between 25.degree. C. and 40.degree. C. are 
preferred. 
According to the present invention, the hydrolysis of the hydantoins takes 
place not only in the presence of microorganisms which are being grown or 
in the presence of intact cells of them, but also in extracts of the above 
enumerated microorganisms. The microorganisms can be cultured, for 
example, in a liquid nutrient medium so as to obtain an accumulation of 
hydrolase in the cells and the DL-hydantoins can be added subsequently to 
the culture. The enzymatic reaction can also be carried out with the 
so-called resting cells. In this case the bacterial cells are recovered 
from the culture medium, washed and suspended in an appropriate buffered 
medium to which the racemic hydantoin is added. 
In addition, it is possible to use preparations which contain the 
hydrolases, such as extracts or concentrates of them, raw or purified 
hydrolase preparations which have been obtained from cells of the above 
enumerated microorganisms. Lastly, raw or purified hydrolases can be used, 
which have been immobilized through combinations with macromolecular 
compounds. 
Other procedures will become apparent from the ensuing examples which are 
given only for the purpose of illustrating the present invention.

EXAMPLE 1 
To 100 mls of a broth culture in the above reported medium of the strain 
Pseudomonas sp. 942 in a 500-ml flask there were added at the 24th hour of 
incubation (orbital stirring) at 30.degree. C., 100 mls of a phosphate 
buffer (0.14 M) pH = 8.5, which contained 30 micromol/ml or 
5-(D,L)-phenyl-hydantoin. 
After 5 additional hours of incubation, under the same conditions, the 
as-formed N-carbamylphenylglycine was determined. 
From 530 milligrams of 5-(D,L)-phenylhydantoin, there were formed 525 
milligrams of N-carbamylphenylglycine, which correspond to a yield of 
about 90%. 
EXAMPLE 2 
A culture broth was prepared, having the above reported composition and 
which contained 1 gram per liter of 5-(D,L)-methylhydantoin. 
The pH was adjusted to 7.2 with soda and the medium was distributed into 
50-ml portions in 250-ml flasks. Upon sterilization at 110.degree. C. 
during 30 mins., the flasks were inoculated with a culture of Pseudomonas 
sp. 942 from a slant which contained the same medium with the 2% of agar 
(DIFCO) and incubated at 30.degree. C. during 20 hours with orbital 
stirring (220 rpm). 
With this pre-culture (Optical Density, at 550 nm: 0.400; dilution 1:10) 
there were inoculated 5 mls in 500-ml flasks which contained 100 mls of 
the same medium and the culture was incubated at 30.degree. C. with 
orbital stirring (220 rpm) during 18 hours (last phase of the exponential 
growth). The cells were then collected by centrifugation (5,000 g, 20 
mins) and washed three times in a buffered physiological salt solution and 
finally suspended in a phosphate salt buffer pH = 8.5, 0.07 M: resting 
cells. 
For the enzymatic hydrolysis there were incubated at 30.degree. C. with 
orbital stirring (220 rpm) in 250-ml flasks, 64 mls of the reaction 
mixture composed of: 260 milligrams of bacteria (dry weight) and 20 
micromols/ml of D,L-phenylhydantoin (= 3.52 milligrams/ml). At various 
time intervals, the product of the hydrolysis, i.e. 
D-carbamylphenylglycine, was determined with a colorimetric method (J. 
Biol. Chem. 238, 3325 (1963) at 438 nm. FIG. 1 shows the results, in terms 
of percent of the theoretical yield of the as-formed carbamylderivative: 
on the abscissae there are reported the times (hours) and on the ordinates 
the percentages of D(-)-N-carbamylphenylglycine which had been formed. 
EXAMPLE 3 
There was prepared a semisynthetic medium having the following composition: 
______________________________________ 
Na.sub.2 HPO.sub.4 
7.05 grams per liter 
KH.sub.2 PO.sub.4 2.72 grams per liter 
(NH.sub.4).sub.2 SO.sub.4 
5.0 grams per liter 
MgSO.sub.4 0.2 grams per liter 
MnSO.sub.4 0.45 milligrams per liter 
FeSO.sub.4 . 7H.sub.2 O 
5.5 milligrams per liter 
Glucose 10 grams per liter 
Yeast Extract 0.2 grams per liter 
5-(D,L)-methylhydantoin 
1.0 grams per liter 
______________________________________ 
The medium was distributed in 50-ml portions in 250-ml flasks, and in 
100-ml portions in 500-ml flasks and sterilized during 30 mins. at 
110.degree. C. 
For the pre-culture, the 250-ml flasks were inoculated from slant with a 
culture of the Pseudomonas sp. 942 strain and incubated as in Example 2 
during 24 hours at 30.degree. C. 
From this culture (Optical Density at 550 nm : 0.245; dilution 1 : 10) 
there were inoculated 5 mls in 500-ml flasks. After a 19-hour incubation 
at 30.degree. C., as above, the resting cells were prepared as in Example 
2. 
Enzymatic hydrolysis was carried out at 30.degree. C. in a reaction mixture 
which contained in 64 mls of buffer, 280 milligrams of bacteria (on dry 
weight basis) and 20 micromols/ml of 5-(D,L)-phenylhydantoin. After 5, 10 
and 15 minutes, the quantity of the as-formed carbamyl derivative was 
determined. 
TABLE 2 
______________________________________ 
Time minutes 5 10 15 
______________________________________ 
micromol/ml of the formed 
carbamyl derivative 
1.15 2.05 3.00 
______________________________________ 
EXAMPLE 4 
Bacterial cells were prepared of the strain Pseudomonas sp. 942 as 
described in Example 2. A suspension of them (42 milligrams per milliter 
of dry cells) in a phosphate salt buffer, 0.1 M, pH = 8, was subjected to 
mechanical break by using a "Manto-Gaulin" homogenizer working at the 
pressure of 850 kg/sq. cm at a temperature below 24.degree. C. 
The cellular debris was separated from the extract by centrifugation 
(25,000 g, 30 mins.). 
To 950 mls of phosphate salt buffer, pH = 7.7, containing 2.12 grams of 
5-(D,L)-phenylhydantoin there were added 50 mls of the extract which 
contained 8,500 U of the enzyme. (1U is the amount of enzyme which 
converts in a phosphate buffer, pH = 7.7 at 30.degree. C., containing 12 
micromol/ml of 5-(D,L)-phenylhydantoin, 1 micromol/ml of the substrate, in 
60 minutes). 
After 1 hour at 30.degree. C. there had been formed 1.7 grams of 
D-carbamylderivative corresponding to about 80% of the total hydrolysis. 
EXAMPLE 5 
As in Example 4, there were added to 993 mls of substrate, 7 mls of a 
preparation of the extract which had been purified about 7 times. After 1 
hour at 30.degree. C. there had been formed 1.7 grams of D-carbamyl 
derivative corresponding to about 80% of the total hydrolysis. 
EXAMPLE 6 
Under the same conditions as in Example 2, there were obtained, after 30 
minutes at 30.degree. C., with the strain Pseudomonas sp. 945, from 352 
milligrams of 5-(D,L)-phenylhydantoin, 220 milligrams of 
N-carbamylphenylglycine, which correspond to about 57% of the theoretical 
yield.