Process for the preparation of optically active chiral amines

Abstract

The present invention relates to the production of optically pure secondary amines, which can be used as intermediate products in a synthesis of for instance pharmaceutical products.

Claims

1. A process for the preparation of a compound selected from a group consisting of 3-aminopyrrolidone derivatives, chephalosporine, derivatives of cephalosporine, heterocyclic boronic acids, L-dihydroxphenylalanine (L-Dopa), -methyldopa, D-phenylglycine, -hydroxyphenylglycine, phosphinothricine, pyrimido derivatives and pyrrolidone derivatives, the process comprising: a) providing an amino acceptor and an amino donor, b) reacting the amino acceptor and the amino donor with a transaminase, c) obtaining a desired optically active chiral amine and a ketone by-product, and d) converting the optically active chiral amine to the compound, and wherein the amino acceptor is selected from a group consisting of phenylpyruvic acid, a salt thereof, pyruvic acid, a salt thereof, 2-ketoglutarate, 3-oxobutyrate, 2-butanone, 3-oxopyrrolidine, 3-pyridylmethylketone, 3-oxobutyric acid ethyl ester, 3-oxopentanoic acid methyl ester, N-1-boc-3-oxopiperidinone, N-1-boc-3-oxopyrrolidine, 3-oxo-piperidine, alkyl-3-oxo-butonoates, methoxy-acetone and 1-oxotetralone.

2. The process according to claim 1, wherein the transaminase is a (R)- or (S)-selective transaminase.

3. The process according to claim 1, wherein the amino donor is selected from the group consisting of amines or amino acids, in particular from -alanine, alanine, -methylbenzylamine (-MBA), glutamate, phenylalanine and -aminobutyrate, glycin, 3-aminobutyrate, isopropylamine, 2-aminobutane and a salt, for instance a chloride, of any one thereof.

4. The process according to claim 1, wherein the obtained amines are amines, in particular mono- or bicyclic amines, in particular amines of 5 to 6-membered cyclic or S-, O-, or N-substituted heterocyclic hydrocarbons or aromatic amines, in particular alkyl- or alkoxy-substituted aromatic amines.

5. The process according to claim 1, wherein the obtained amines are selected from the group consisting of phenylalanine, alanine, 3-am inopiperidine, alkyl-3-amino-butanoates, 3-aminopyrrolidine (3-AP), 3-pyridyl-1-ethylamine (3-PEA), N-1-boc-3-aminopyrrolidine (B3AP), 3-aminobutyric acid ethyl ester (3-ABEE), 3-aminopentanoic acid methyl ester (3-APME), -methylbenzylamine (-MBA), 1-aminotetraline, -methyl-4-(3-pyridyl)-butanamine, glutamate, -aminobutyrate, sec-butylamine, methoxyisopropylamine, derivatives of 3-aminopyrrolidine, 1-N-boc-3-aminopiperidin, cephalosporine and derivatives of cephalosporine.

6. The process according to claim 1, wherein the transaminase is from Vibrio fluvialis, Alcaligenes denitrificans, Klebsiella pneumoniae or Bacillus thuringiensis.

7. The process according to claim 1, wherein the ketone by-product obtained in step c is pyruvate.

8. The process according to claim 1, wherein the ketone by-product obtained in step c) is in a further process step e) removed from by reaction with at least one enzyme.

9. The process according to claim 8, wherein the enzyme used in step e) is a decarboxylase.

10. The process according to claim 8, wherein the at least one enzyme used in step e) is a synthase.

11. The process according to claim 8, wherein the enzyme used in step e) is a dehydrogenase.

12. The process according to claim 8, wherein the enzyme is a pyruvate decarboxylase (PDC).

13. The process according to claim 8, wherein the enzyme is a lactate dehydrogenase (LDH).

14. The process according to claim 8, wherein the enzyme is an acetolactate synthase.

15. The process according to claim 12, wherein acetaldehyde formed by the action of the PDC is removed.

16. The process according to claim 15, where acetaldehyde is removed by reaction with at least one enzyme.

17. The process according to claim 16, wherein the enzyme is an alcohol dehydrogenase.

18. The process according to claim 15, wherein the acetaldehyde is removed by feeding gaseous nitrogen into the reaction mixture.

19. The process according to claim 15, wherein the acetaldehyde is removed by applying a reduced pressure to the reaction mixture.

20. The process according to claim 15, wherein the acetaldehyde is removed by chemical methods.

21. The process according to claim 1, further comprising removing the optically active chiral amine obtained in step c).

22. The process according to claim 1, wherein the process is carried out in a reaction mixture having a pH from 5.0 to 9.5, preferably 6.0 to 7.0, preferably 6.0 to 6.9.

23. The process according to claim 1, wherein the process is carried out for a reaction time of 40 to 70 minutes.

24. A process for the preparation of a compound selected from 3-aminopyrrolidone derivatives, the process comprising: a) providing an amino acceptor and an amino donor, b) reacting the amino acceptor and the amino donor with a transaminase, c) obtaining a desired optically active chiral amine and a ketone by-product, and d) converting the optically active chiral amine to the compound, and wherein the amino acceptor is N-1-boc-3-oxopyrrolidine.

25. The process according to claim 1, wherein the amino donor is alanine.

26. The process according to claim 12, wherein the acetaldehyde formed by the reaction of the PDC is removed by feeding gaseous nitrogen (N.sub.2) into the reaction mixture.

27. The process according to claim 12, wherein the reaction is carried out in the presence of at least one pyruvate decarboxylase and at least one alcohol dehydrogenase.

28. The process according to claim 12, wherein the reaction is carried out in the presence of at least one pyruvate decarboxylase, at least one alcohol dehydrogenase and additionally in the presence of gaseous nitrogen (N.sub.2).

Description

(1) The accompanying figures illustrate the present invention.

(2) FIG. 1 shows a thin layer chromatogramm.

(3) FIG. 2 shows the relative activity of V. fluvialis -TA in dependence from the pH-value.

(4) FIG. 3 shows the relative reduction of the B3AP concentration for incubations with various substances.

(5) FIG. 4 shows the relative reduction of the B3AP conversion in the presence of pyruvate and acetaldehyde.

(6) FIG. 5 shows the conversion of B3OP to B3AP over the time for various pressures.

(7) FIG. 6 shows the relative conversion of B3OP to B3AP in the presence of various PDC's.

(8) FIG. 7 shows the effect of an increased alanine concentration and an increased PDC-concentration on asymmetric B3AP synthesis.

(9) FIG. 8 shows the conversion of B3OP to B3AP at increased alanine concentrations.

(10) FIG. 9 shows the relative PLP-dependent conversion of B3OP to B3AP.

(11) FIG. 10 shows the relative conversion of B3OP to B3AP dependent upon N.sub.2-presence.

(12) FIG. 11 shows the relative conversion of B3OP to B3AP in the presence of an ADH.

EXAMPLE 1

Asymmetric Synthesis of B3AP

(13) The asymmetric synthesis of B3AP was carried out in 1.5 ml reaction tubes. B3OP as amino acceptor was used in a concentration of 5 mM (7.5 mol). The concentration of the used amino donor L-alanine was 5 mM. The reagents and reaction conditions used are evident from table 1 below.

(14) TABLE-US-00001 TABLE 1 Reaction conditions for the asymmetric (S)-B3AP synthesis using (S)- transaminase for transaminating the amino group from alanine to B3OP 1 2 3 4 5 6 B3OP, 20 mM [l] 375 375 375 375 375 375 D,L-Ala, 150 26 mg 150 150 150 150 100 mM [l] D,L-Ala TA7/TA8 [l] 18 111 18 111 111 18 111 18 111 18 111 LDH [l] 500 l + 60 l 250 mM NADH PDC1 [l] 200 (15 U) PDC2 [l] 34 (20 U) 34 (20 U) Buffer [l] 957 864 1218 1125 664 923 830 923 830 397 304

(15) The buffer used was 50 mM sodium phosphate, pH 7. TA7 designates the -transaminase from Vibrio fluvialis (Jlich Fine Chemicals, Germany). TA8 designates the transaminase from Alcaligenes denitrificans (Jlich Fine Chemicals, Germany). As lactate dehydrogenase an extract of Escherichia coli was used. In addition, NADH was added to a final concentration of 10 mM. The concentration of pyruvate decarboxylase was varied. 1.5 units (20 l) and 15 units (200 l) of the pyruvate decarboxylase of Saccharomyces cerevisiae (PDC1) were used. 2 units (3.4 l) and 20 units (34 l) of the pyruvate decarboxylase of Zymomonas mobilis (PDC2) were used.

(16) TABLE-US-00002 TABLE 2 Conversion and optical purity obtained using TA8 for the asymmetric synthesis of B3AP. The calculations were based on GC-analysis (+/5%) Conversion % ee.sub.s [%] Run Enzyme [%] (S)-enantiomer 1 TA7 or TA8 alone 1.3 99.4 2 Excess of Alanine (50-fold) 10.1 99.6 3 PDC Saccharomyces cerevisiae 4.6 99.5 4 PDC Zymomonas mobilis 34.0 99.6 5 PDC Zymomonas mobilis (72 hrs) 73.0 99.4 6 LDH Escherichia coli 66.5 99.9

(17) Referring now to table 2, above, it is evident that in each of the six runs using the -transaminase TA8, a very high degree of optical purity for the obtained (S)-B3AP could be achieved. It was also observed that independently from using either TA7 or TA8 the degree of conversion was only moderate, if the equilibrium of the reaction was not influenced (run 1). Using alanine in a 10-50-fold excess only slightly improved the conversion. In runs 3, 4, 5 and 6 the ketone product of the reaction, that means pyruvate, was, during the transamination reaction, removed from the equilibrium reaction. The use of TA8 together with lactate dehydrogenase from E. coli (run 6) led to an extremely improved degree of conversion while maintaining and even improving the enantioselectivity. Essentially the same holds valid for the enantioselectivity provided by the pyruvate decarboxylase from Zymomonas mobilis (runs 3 to 5). PDC1, however, only slightly increased the conversion, PDC2 moderately increased the conversion rate (run 4) if reacted for 24 hrs while in a 72 hr reaction (run 5) the conversion was drastically improved. All the reactions took place for 24 hrs except for run 5, which took place for 72 hrs.

(18) The figure shows the thin layer chromatogram of reactions carried out according to table 1. A designates the -transaminase from Alcaligenis denitrificans while V the -transaminase from Vibrio fluvialis. K designates run 1 using TA7 or TA8 alone (run 1). PDC1 designates the run with Saccharomyces cerevisiae pyruvate decarboxylase (run 3), LDH the run with lactate dehydrogenase from Escherichia coli (run 6) and PDC2 the run with Zymomonas mobilis pyruvate decarboxylase (after 24 and 72 hrs) (run 4 and 5). Thus, the results clearly show that the production of (S)-B3AP from the prochiral ketone B3OP could be carried out with a very high enantioselectivity. Using the -transaminases as the sole enzymes in the preparation process, however, leads to a moderate conversion. This moderate conversion rate could be greatly improved by removing pyruvate from the equilibrium, in particular using lactate dehydrogenase or pyruvate decarboxylase. Using pyruvate decarboxylase has inter alia the advantage that no co-factor recycling (NADH) was necessary. It further advantageously provides the enzymatic removal of pyruvate with PDC and thereby provides the additional advantage of removing or avoiding product inhibition (product ketone) and pulling the reaction equilibrium to the right achieving higher conversion (ideal case 100%).

EXAMPLE 2

pH-Dependency of -Transaminase Activity in the Conversion Reaction of (S)-MBA to Acetophenone

(19) The synthesis was carried out in a quarz cuvette using 50 l 100 mM pyruvate, 4 units/ml of -TA Vibrio fluvialis (in the following also Vfl) (12 l) and 388 l of sodium phosphate buffer, 50 mM with pH-variations from pH 6.0 to pH 7.4 in 0.2 steps. The reaction was started with 50 l 100 mM (S)-MBA as amino donor and the increase in absorption was measured at 250 to 260 nm. The increase in absorption is due to the acetophenone formed. The other substrates only insignificantly contribute to the absorption so that the velocity of the reaction can be determined by measuring the absorption of acetophenone. The value reached at pH 7.4 was set as 100% and the relative activity for the other pH-values was calculated as is evident from FIG. 2. FIG. 2 shows the relative activity of V. fluvialis -TA in dependence from the given pH-value.

(20) FIG. 2 shows that at lower pH-values such as 6.0, 6.2 or 6.4 there is still considerable activity present, for instance 11% at pH 6.0. Thus, this result demonstrates that even at a low pH, it is possible to obtain a significant transaminase activity, allowing to react the substrates at a lower pH, which in turn allows to increase the conversion by using a PDC, which is sensitive to higher pH-values.

EXAMPLE 3

Asymmetric Synthesis of B3AP at Different pH-Values

(21) In this example, the asymmetric synthesis of B3AP from alanine and B3OP is shown in the presence and absence of a pyruvate decarboxylase (PDC).

(22) For each pH-value 6.0, 6.4 and 7.0 three runs of experiments were conducted. Run 1 used the PDC of Zymomonas mobilis (wild-type cell extract), run 2 used the Zymobacter palmae (recombinant in E. coli) and run 3 was a control without PDC, employing only the transaminase. To obtain comparable results, the activities of both of the PDC's have been determined at pH 6 with an alcohol dehydrogenase assay and the same quantity of activity of the PDC's was used in the runs identified above.

(23) Table 3 gives the volumes of the used substance in l. Each reaction run was carried out three times at pH-values 6.0, 6.4 and 7.0. The pH-value was adjusted by the buffer of the B3OP substrate solution. The activity of the PDC was about 2.5 units/ml at pH 7. The substrate and enzyme concentrations are also evident from table 3 below. After 10 minutes, 30 minutes, 60 minutes and 120 minutes a sample of 100 l was taken and the reaction stopped by the addition of 100 l 1 M NaOH. The quantification of the B3AP-concentration was done using CE (capillary electrophoresis).

(24) TABLE-US-00003 TABLE 3 Run 1 Run 2 Run 3 10 mM B3OP in 100 mM 500 500 500 buffer of corresponding pH-value 400 mM D,L-alanine 25 25 25 PDC Z. mobilis 290 PDC Zb. palmae 18.32 V. fluvialis -TA at pH 7.0 21.4 21.4 21.4 V. fluvialis -TA at pH 6.4 72.3 72.3 72.3 V. fluvialis -TA at pH 6.0 110 110 110 4 mM TPP 25 25 25 4 mM PLP 25 25 25 40 mM MgCl.sub.2 25 25 25 Water ad 1000 l

(25) Table 4 below shows the conversion at different pH-values for the different PDC's. It is evident that the use of the PDC increases the conversion. It is also evident that the PDC from Z. palmae (Zpa) causes a somewhat higher conversion than the PDC from Z. mobilis (Zmo). It is also evident that at lower pH-values, such as 6.0 or 6.4, a remarkable conversion increase in the runs employing PDC's is to be observed, which is not to be seen in the PDC-free control. In all reaction runs it could be observed that after 120 minutes the conversion decreased.

(26) TABLE-US-00004 TABLE 4 Conversion Run 1 (Zmo) Run 2 (Zpa) Run 3 (control) Time pH pH pH pH pH pH pH pH pH (min) 6 6.4 7 6 6.4 7 6 6.4 7 10 7.5 5.5 2.4 12.1 9.8 4.9 2.5 2.4 2.0 30 11.4 14.2 9.6 16.0 17.8 15.2 5.1 4.8 4.6 60 8.3 n.d. 12.4 11.0 n.d. 16.3 7.0 n.d. n.d. n.d. = not determined

EXAMPLE 4

Stability of B3AP in the Presence of Various Reactants of an Asymmetric Synthesis Reaction

(27) To show the stability of B3AP in the presence of various reactants incubations of 1 mM B3AP were conducted in a reaction tube for 3 hrs in the presence of various substances as listed in table 5 below.

(28) The example was carried out at a pH of 6 and 7 (sodium phosphate buffer). Directly after reacting the substances being a first sample T.sub.0 was taken, and another sample, T.sub.1, after 3 hrs. After the extraction the amine concentration was determined with an internal standard (MBA) by CE. From the difference of the concentrations obtained, the %-decrease of the B3AP concentration was calculated (see FIG. 3).

(29) TABLE-US-00005 TABLE 5 Run 1 mM B3AP and: Reactants 1 50 mM Na-P-buffer 2 5 mM B3OP 3 10 mM D,L-alanin 4 cofactors (0.1 mM PLP, TPP, 5 mM Mg) 5 cofactors + B3OP + alanine 6 145 l Z. mobilis cell extract (50% glycerine) 7 10 l E.coli cell extract (Zpa PDC recombinant) 8 55 l/11 l Vfl-TA (pH6/7) 9 50 l Ade-TA 10 Vfl-TA + B30P + cofactors 11 1 mM acetaldehyde 12 Vfl-TA + acetaldehyde 13 Ade-TA + acetaldehyde

(30) From FIG. 3 it is evident that the different reactants do not significantly affect the B3AP-concentration (runs 1 to 9). Reaction runs 11 to 13 show the influence of acetaldehyde. From run 11 it is evident that in the absence of a transaminase there is no reduction in the B3AP concentration, while in the presence of a transaminase and acetaldehyde a strong reduction in B3AP-concentration can be observed. Acetaldehyde functions in the transaminase reaction as an amino acceptor, such as pyruvate, and obviously leads to a reduction in B3AP concentration.

EXAMPLE 5

Reaction of B3AP with the Amino Acceptors Pyruvate and Acetaldehyde

(31) In this example the transaminase activity of V. fluvialis and A. denitrificans -TA for the substrates B3AP and pyruvate and for B3AP and acetaldehyde is shown.

(32) 2 mM B3AP was reacted with 2 mM pyruvate or 2 mM acetaldehyde (36 l Alcaligenes denitrificans (Ade) or 6 l Vibrio fluvialis (Vfl)-transaminase per 0.5 l reaction volume, corresponding to 2 units/l transaminase was reacted for 30 minutes). FIG. 4 shows the results. Accordingly, B3AP was converted to B3OP by both enzymes, both with pyruvate and acetaldehyde, without any significant differences.

EXAMPLE 6

Asymmetric Synthesis of B3AP Under Reduced Pressure at pH 6

(33) In this example, a reduced pressure was applied to the reaction mixture for an asymmetric synthesis reaction to form B3AP. As a control, the same reaction was carried out under normal pressure and without PDC.

(34) Reaction Conditions:

(35) Final volume: 1.5 ml

(36) 50 mM sodium phosphate buffer, pH 6

(37) 300 l Vibrio fluvialis-transaminase

(38) 60 l Zpa-PDC (corresponds to 8 units/ml at pH 7)

(39) 5 mM B3OP

(40) 10 mM D,L-alanine

(41) 0.1 mM TPP, PLP

(42) 5 mM MgCl.sub.2

(43) The reduced pressure was applied using a rotary evaporator (150 mBar). The measurement was done using CE.

(44) FIG. 5 shows the conversion of B3OP to B3AP over the time for various pressures. It is evident that the conversion is almost independent from the pressure applied. The conversion at a pressure of 150 mbar is, with regard to the maximal conversion reached, similar to the conversion at a pressure of 1000 mbar.

EXAMPLE 7

Comparison of Various Pyruvate Decarboxylases

(45) In this example, three different pyruvate decarboxylases were used for the asymmetric synthesis of B3AP. The reaction conditions correspond to those of example 6, except that a pH of 7 was used. PDC's from Z. mobilis, Z. palmae and a PDC from Biocatalytics (catalogue no. PDC-101) were used. The activities of the PDC's in the ADH-assay were identical (1.6 units/ml).

(46) FIG. 6 shows that all three PDC's essentially result in comparable conversions.

EXAMPLE 8

Influence of Enzyme and Co-Substrate Concentrations on the Conversion of B3OP to B3AP

(47) In this example the influence of the concentration of PDC on the conversion of B3OP to B3AP using alanine as amino donor is shown. Furthermore, the influence of the alanine concentration on the conversion of B3OP to B3AP is shown.

(48) The reaction conditions are given in example 6, except that a pH of 7 was used and except if otherwise stated. Zymobacter palmae TA was used.

(49) As is evident from FIG. 7, in a reaction without PDC a 5-fold increase of the alanine concentration from 5 mM to 25 mM results in a duplication of the conversion (12% in contrast to 5.3% conversion after 2 hrs). In the presence of PDC, the conversion increases at a 5-fold alanine excess to the duplicate (30% in contrast to 17% after 90 minutes). In case the amount of PDC is increased at the usual alanine concentration from 1.6 units/ml to 50 units/ml, the conversion is also increased, however, only by a factor <2 (29% in contrast to 17% after 40 minutes).

(50) In a further run of experiments, the influence of a combined alanine excess and a PDC excess was shown, both at a pH of 6 and 7. The reaction is faster at a pH of 6, whereas after reaching a conversion of 49%, the B3AP concentration also decreases faster. At a pH of 7 the conversion increased up to 56%.

EXAMPLE 9

Influence of the Alanine Concentration on the Asymmetric Synthesis of B3AP

(51) In four reactions, alanine concentrations of 5 mM, 25 mM, 110 mM, 300 mM and 500 mM were used. For each run, one control without PDC and one reaction with PDC was carried out. The pH-value was adjusted to pH 7.0. Samples were taken every half hour for a reaction time of 3 hours. The reaction times for the conversions given in FIG. 8 were for 5 mM 40 minutes, for 25 mM 60 minutes and for 110 to 500 mM 90 minutes.

(52) FIG. 8 shows the conversion of the asymmetric B3AP-synthesis at increased alanine concentrations.

(53) FIG. 8 clearly shows that the conversion can reach 60 to 70%. It is evident that increasing the alanine concentration from 25 to 110 mM has a significant effect on the conversion from B3OP to B3AP and that a further increase up to 500 mM only slightly influences the conversion. The influence of PDC on the conversion decreases with increasing alanine concentration.

(54) From the data of the control reaction, the equilibrium constant of the B3AP synthesis was calculated as follows:
[B3AP]=[Pyr]
[B3OP]=c.sub.0,B3OP[B3AP] and
[Ala]=c.sub.0,Ala[B3AP]

(55) Thus, using the measured B3AP concentration, the equilibrium constant was calculated as:

(56) $K=\frac{\left[B3\mathrm{AP}\right].Math.\left[\mathrm{Pyr}\right]}{\left[B3\mathrm{OP}\right].Math.\left[\mathrm{Ala}\right]}=\frac{{\left[B3\mathrm{AP}\right]}^2}{\left(c_{0,B3\mathrm{OP}}-\left[B3\mathrm{AP}\right]\right).Math.[c_{0,\mathrm{Ala}}-\left[B3\mathrm{AP}\right]}$

(57) TABLE-US-00006 TABLE 6 c.sub.0, [Ala] [B3AP] K .Math. 10.sup.3 5 5 3.2 25 12 3.1 110 24 3.5 300 35 3.2 500 40 2.8 Mean of K: 3.1

(58) Table 6 shows the calculated values. Thus the equilibrium constant for the reaction with B3AP is 3.110.sup.3. Thus, the substrate B3OP is a suitable substrate for the asymmetric synthesis in contrast to other ketones.

EXAMPLE 10

Influence of PLP on the Asymmetric Synthesis of B3AP

(59) In this example, the influence of PLP (pyridoxal-5-phosphate) on the conversion of B3OP to B3AP is shown. The following three reaction runs have been examined. a) Run 1 using 0.1 mM PLP without addition of further PLP. b) In run 2 PLP was added during the reaction as soon as the yellow colour, which is due to the presence of PLP in the reaction medium, has faded. For this purpose, 1 to 2 l of a saturated PLP solution is added, thereby regaining a strong yellow colour. The influence on the amine concentration through this slight increase in volume is considered to be below 1% and can therefore be neglected. c) No PLP present and no PLP added.

(60) Reaction conditions: 1 ml final volume, 37 l Vfl-transaminase, 5 mM B3OP, phosphate buffer pH 7.0. The L-alanine concentration was 110 mM. The measurements were taken by CE and -MBA was used as internal standard.

(61) FIG. 9 shows the conversion over the time. There appears to be no significant influence on the maximal conversion due to the PLP addition in run b) compared to run a). The reaction without PLP appears to be slower, although it reached the same conversion as the other reactions. The addition of PLP in run b) causes a slightly greater reduction in the amine concentration as compared to the control run.

EXAMPLE 11

Asymmetric Synthesis of B3AP with Removal of Acetaldehyde by the Addition of Nitrogen

(62) The following example details one way to improve the conversion of the asymmetric synthesis of B3AP by the removal of acetaldehyde.

(63) Reaction Conditions:

(64) Substrates:

(65) 5 mM B3OP

(66) 500 mM L-alanine

(67) 32 U/ml Zpa-PDC

(68) 37 l/ml Vfl-transaminase

(69) Sodium phosphate buffer pH 7.0

(70) 0.1 mM PLP and TPP (thiamine diphosphate)

(71) 5 mM MgCl.sub.2

(72) Since the reaction solution contained a significant amount of protein, there was a strong tendency to the formation of foam. To suppress said foaming, 0.6 l of an antifoam A concentrate (Sigma, silicone-polymer) was added to the reaction run. The concentrate suppressed the foam generation to a large extent but could not inhibit it completely. To exclude that said antifoam concentrate inhibits the enzymes, a control run without the addition of nitrogen was supplemented with antifoam A.

(73) Since the addition of dry nitrogen led to an evaporation of water from the reaction solution, the nitrogen was wetted.

(74) FIG. 10 shows the calculated relative B3AP conversions. The control with antifoam A (N.sub.2-control: Vfl-TA, Zpa-PDC, antifoam, no nitrogen N.sub.2) without nitrogen addition corresponds exactly to the reaction run without antifoam A (Vfl-TA, ZpaPDC). Thus, the enzymes are not influenced by the addition of the antifoam concentrate. The run (run N.sub.2: Vfl-TA, Zpa-PDC, antifoam, N.sub.2) treated with nitrogen showed an increased conversion after 60, 90 and 180 minutes.

EXAMPLE 12

Asymmetric B3AP Synthesis Under Various Conditions

(75) Reaction Conditions:

(76) Final volume: 1 ml

(77) 37 l Vfl-transaminase

(78) 32 units/ml Zpa-PDC or Biocatalytics PDC or 3.2 units/ml Zmo PDC

(79) 5 mM B3OP

(80) 500 mM L-alanine

(81) Cofactors 0.1 mM PLP and TPP, 5 mM MgCl.sub.2

(82) pH 7, sodium phosphate

(83) FIG. 10 shows the conversion of B3OP to B3AP for the above-identified reaction runs combining the Vfl-transaminase with each PDC.

(84) In FIG. 10 the run designated N2 is the run containing Vfl-transaminase and Zpa-PDC treated with nitrogen and antifoam A. The N2 control is a sample containing Vfl-transaminase, Zpa-PDC and antifoam A without nitrogen treatment. It is evident that there is a significant increase in conversion from the N2 control to the N2-sample due to the presence of nitrogen, which has been fed into the reaction solution. Thus, feeding nitrogen in gaseous form into the reaction medium significantly increases the conversion from B3OP to B3AP.

EXAMPLE 13

Asymmetric Synthesis of B3AP in the Presence of Alcohol Dehydrogenase (ADH)

(85) To remove the acetaldehyde produced by the PDC reaction from the reaction mixture, ADH can be used to convert the acetaldehyde to ethanol.

(86) Reaction Conditions:

(87) 110 mM L-alanine

(88) 5 mM B3OP

(89) 37 l/ml Vfl-transaminase

(90) 32 units/ml Zpa PDC

(91) 0.1 mM PLP and TPP

(92) 5 mM MgCl.sub.2

(93) Sodium phosphate buffer, pH 7

(94) The following reaction runs are shown:

(95) Reaction run 1: reaction with PDC and transaminase

(96) Reaction run 2: reaction with PDC and transaminase with 5 mM ethanol (final concentration) and NADH-addition

(97) Reaction run 3: reaction with PDC, ADH and NADH

(98) The ADH used was the ADH from Saccharomyces cerevisae with an activity between 50 and 100 units/ml. The PDC-activity was 32 units/ml.

(99) At the beginning of the reaction absolute ethanol has been added to reaction run 2 in a final concentration of 5 mM. At the beginning of the reaction 5 mol NADH were added to the reaction runs 2 and 3 corresponding to a final concentration of 5 mM NADH. After 10 minutes each, a further addition of 2.4 mol NADH (4 l of a 0.6 M NADH-solution in 50 mM phosphate buffer, pH 8.5) was added. The NADH solution was stored in ice and prepared immediately before use.

(100) The results are given in FIG. 11 and table 5. The effect of the ADH is clearly evident. The conversion increases up to approximately 90%. Control run 2 without ADH and 5 mM ethanol only slightly deviates from control run 1. Thus, the addition of ADH greatly increases the conversion in the asymmetric synthesis of B3AP from B3OP.