Enzymatic reaction medium containing surfactant

Abstract

The present invention is directed to aqueous reaction mixtures for enzymatic synthesis reactions comprising a surfactant. The surfactant in the reaction mixture increases stability and yield of the enzymatic reaction. Furthermore, method for performing an enzymatic reaction using said aqueous reaction mixtures are provided.

Claims

1. An aqueous, enzyme-catalyzed chemical synthesis reaction mixture comprising: an enzyme, a substrate of the enzyme and a surfactant, wherein the enzyme is selected from the group consisting of reductases, transferases, dehydrogenases, lipases, and esterases, further wherein the surfactant is present in the reaction mixture above its critical micellar concentration and is selected from the group consisting of: (i) surfactants having the following formula I: ##STR00027## (ii) surfactants having the following formula II: ##STR00028## and (iii) surfactants having the following formula III: ##STR00029## wherein R1 comprises a poly(C.sub.1-4-alkylene glycol) group; R2, R3, R4 and R5 are independently hydrogen or C.sub.1-4-alkyl; and R6 is C.sub.5-80-alkyl or C.sub.5-80-alkenyl.

2. The reaction mixture according to claim 1, wherein R1 is a poly(C.sub.1-4-alkylene glycol) group, which is attached to the oxygen atom of the core structure via a covalent bond or a linking group, and which optionally comprises a terminal C.sub.1-4-alkyl group.

3. The reaction mixture according to claim 2, wherein the linking group is a dicarboxylic acid which forms ester linkages to the core structure and the poly(C.sub.1-4-alkylene glycol) group.

4. The reaction mixture according to claim 3, wherein the linking group is a dicarboxylic acid having 2 to 20 carbon atoms selected from the group consisting of succinic acid and sebacic acid.

5. The reaction mixture according to claim 2, wherein the terminal C.sub.1-4-alkyl group is methyl.

6. The reaction mixture according to claim 1, wherein the poly(C.sub.1-4-alkylene glycol) group is a poly(ethylene glycol) group.

7. The reaction mixture according to claim 1, wherein the poly(C.sub.1-4-alkylene glycol) group has an average molecular weight of about 250 to 2500 g/mol.

8. The reaction mixture according to claim 1, wherein R5 is methyl.

9. The reaction mixture according to claim 1, wherein R4 is methyl.

10. The reaction mixture according to claim 1, wherein R2 and R3 are independently hydrogen or methyl.

11. The reaction mixture according to claim 1, wherein (i) the surfactant has formula (I) and R2, R3, R4 and R5 are all methyl; or (ii) the surfactant has formula (II) and R4 and R5 are methyl; or (iii) the surfactant has formula (III) and R2 is hydrogen and R3, R4 and R5 are methyl.

12. The reaction mixture according to claim 1, wherein R6 is branched or linear.

13. The reaction mixture according to claim 1, wherein (i) the surfactant has formula (I) and R6 is C.sub.8-25-alkyl or C.sub.8-25-alkenyl; or (ii) the surfactant has formula (II) and R6 is C.sub.5-20-alkyl or C.sub.5-20-alkenyl or (iii) the surfactant has formula (III) and R6 is C.sub.30-80-alkyl or C.sub.30-80-alkenyl.

14. The reaction mixture according to claim 13, wherein (i) the surfactant has formula (I) and R6 is C.sub.1-6-alkyl or C.sub.1-6-alkenyl; or (ii) the surfactant has formula (II) and R6 is C.sub.10-alkyl; or (iii) the surfactant has formula (III) and R6 is C.sub.50-alkenyl.

15. The reaction mixture according to claim 1, wherein the surfactant has formula (I) and is a vitamin E-derived compound.

16. The reaction mixture according to claim 15, wherein the surfactant is selected from the group consisting of a D-a-tocopherol poly(ethylene glycol) succinate (TPGS)-750-M (TPGS-750-M), TPGS-1000, and PEG-600/alpha-tocopherol-based diester of sebacic acid (PTS).

17. The reaction mixture according to claim 1, wherein the surfactant has formula (II) and is an ubiquinol-derived compound.

18. The reaction mixture according to claim 17, wherein the surfactant is polyethyleneglycol ubiquinol succinate (PQS).

19. The reaction mixture according to claim 1, wherein the surfactant has formula (II) and is a β-sitosterol-derived compound.

20. The reaction mixture according to claim 19, wherein the surfactant is β-sitosterol methoxyethyleneglycol succinate (Nok).

21. The reaction mixture according to claim 1, wherein the enzyme is selected from the group consisting of ketoreductases, ene reductases, transaminases, alcohol dehydrogenases, and amino acid dehydrogenases.

22. The reaction mixture according to claim 1, further comprising a co-enzyme or a co-factor.

23. The reaction mixture according to claim 22, wherein the co-enzyme or co-factor is selected from the group consisting of alcohol dehydrogenases, NAD, NADP, FAD and pyridoxal monophosphate.

24. The reaction mixture according to claim 22, wherein the enzyme is a ketoreductase, the substrate is a ketone and the co-factor is NADP.

25. The reaction mixture according to claim 22, wherein the enzyme is an ene reductase, the substrate is a compound comprising a carbon-carbon double bond and the co-factor is NAD.

26. The reaction mixture according to claim 22, wherein the enzyme is a transaminase, the substrate is a ketone and the co-factor is pyridoxal monophosphate.

27. The reaction mixture according to claim 1, wherein the concentration of the surfactant in the reaction mixture is at least 0.01% (w/w), at least 0.02% (w/w), at least 0.5% (w/w), or at least 1% (w/w).

28. The reaction mixture according to claim 1, wherein the concentration of the surfactant in the reaction mixture is in the range of 0.1% to 10% (w/w), 0.5% to 5% (w/w), 0.75% to 3% (w/w), or at about 2% (w/w).

29. The reaction mixture according to claim 1, wherein the enzyme is present in an amount in the range of 0.1% to 50%, 0.5% to 35%, or 1% to 20% of the amount of the substrate.

30. The reaction mixture according to claim 1, further comprising a buffer.

31. The reaction mixture according to claim 30, wherein the buffer is selected from the group consisting of TRIS, phosphate, citrate, acetate and ammonia.

32. The reaction mixture according to claim 1, wherein the reaction mixture has a pH value suitable for the enzymatic reaction.

33. The reaction mixture according to claim 32, wherein the pH value is in the range of from 4.0 to 10.0, from 6.0 to 8.0, from 6.5 to 7.5, or at about 7.0.

34. The reaction mixture according to claim 1, wherein the reaction mixture is of industrial scale.

35. The reaction mixture according to claim 34, wherein the volume of the reaction mixture is at least 10 L, at least 100 L, or at least 1000 L.

36. A method of performing an enzyme-catalyzed chemical synthesis reaction, comprising the steps of: (a) providing the reaction mixture as set forth in claim 1, and (b) performing the enzymatic reaction at a suitable pH and temperature.

37. The method according to claim 36, wherein the reaction is performed at a temperature of 80° C. or less.

38. The method according to claim 37, wherein the reaction is performed at a temperature in the range of 10° C. to 50° C., or 20° C. to 45° C.

39. The method according to claim 36, wherein the pH in the reaction mixture varies by at least 1.0, or at least 2.0 pH units during the reaction.

Description

FIGURE LEGEND

(1) FIG. 1 shows a comparison of the reaction kinetics in different reaction media. Reaction conditions: In each vial was added 20 mg of ketone 1, 5 mg of KRED-EW124, 5 mg GDH, 2 mg NADP, 25 mg glucose, 2 mM MgCl.sub.2 in the cosolvent systems as listed. pH=7, temperature=30° C. HPLC analyses of the reaction mixture were carried out at the specified time.

EXAMPLES

Example 1: Synthesis of Benzyl (R)-4-hydroxy-2-(4-(methoxycarbonyl)phenyl)piperidine-1-carboxylate Using Ketoreductase

(2) Surfactants have been applied in biocatalysis in modulating enzyme activities with beneficial effects, such as increasing the solubility of reactants and enhancing reaction selectivity. Although a number of surfactants including ionic liquids, SDS and Triton X have been utilized in a wide range of biotransformations, TPGS-750-M as an alternative surfactant developed in the Lipshutz lab has not been investigated in biocatalysis. During the investigations of a ketoreductase (KRED) mediated reaction as shown in scheme 1, the low solubility of both starting ketone 1 and product alcohol 2 necessitated significant efforts to search for a suitable reaction media for this heterogeneous reaction.

(3) ##STR00022##

(4) First, a number of co-solvent systems including TPGS, PEG400 and DMSO in aqueous solutions were employed under parallel screening conditions for the above reaction. As shown in FIG. 1, during the first phase of the reaction within 2.5 h, TPGS performed better than PEG400 and DMSO with a 10% conversion difference. As the reaction proceeded further, the reaction is significantly faster in TPGS and a conversion difference of around 40% was observed as compared to the reaction in PEG400 and DMSO within 6 h. After 22 h, the reaction came to essentially complete conversion in TPGS (100%) while the reaction in PEG400 and DMSO stopped at 82% and 86% conversions, respectively. This phenomenon reflected the less stability of the ketoreductase in the organic solvent additives of both PEG400 and DMSO system.

(5) With the screening results in hand, we further optimized the reaction in gram scale. Especially we compared the performance of TPGS and DMSO system. Thus the reactions were carried out in 2% TPGS and 15% DMSO aqueous solution respectively at 40° C. using a 5 w % of enzyme loading. The reaction reached 93% conversion after 18 h in TPGS system while a lower 80% conversion after 20 h in DMSO system. More importantly, the reaction continued and reached 98.8% conversion in TPGS after additional 25 h. But an additional 2 wt % enzyme has to be added to push the reaction to 98.6% conversion in DMSO (entry 1, table 1). This observation clearly demonstrated the more active and stable nature of the enzyme in TPGS as compared to DMSO. Another interesting observation was that after an accidental exposure of the enzyme under pH 4.7 for 24 h in the reaction system of TPGS and readjustment of pH to 7, the enzyme is still active enough to catalyze the reaction further with the same activity (entry 2, table 1). The superior stability could be afforded by the molecular interactions between the TPGS and protein and the catalytic active site of the enzyme is protected from the aqueous reaction media.

(6) TABLE-US-00001 TABLE 1 Comparison of ketoreductase in TPGS and DMSO Conditions 2% TPGS-750M DMSO/water Conversion @ 93% for 18 h; up to 98.8% for 80% for 20 h; up to 40° C. (with 5% additional 25 h without 98.6% for additional enzyme) additional enzymes 32 h, but 2% more enzyme necessary Enzyme activity Still active for 6 d, even pH pH should be drop to 4.7 for 24 h @ rt constant 6.9-7.1 for active catalysis

(7) Finally the reaction in TPGS is more amenable to scale up, and compound 1 can be added in solid form without preformation of a solution or milling to reduce the particle size. A typical experimental procedure is as follows: to a degassed 2% weight solution of TPGS-750-M in buffer water (34 mL, 10 v; stock solution prepared from 74 mL of 2 wt % TPGS-750-M water solution, 1.6 g of Na.sub.2HPO.sub.4.12H.sub.2O and 0.5 g of NaH.sub.2PO.sub.4.2H.sub.2O) was added glucose (3.2 g, 18 mmol, 2.0 eq) in a mechanically stirred reactor equipped with pH/ORP controller at rt. The suspension was stirred at rt for 20 minutes, and to the resulting mixture was sequentially added NADP (59 mg, 68% purity), GDH (33 mg) and ketoreductase (0.16 g, 5 wt % of substrate 1). Then substrate 1 (3.3 g, 9 mmol, 1.0 eq) was added and the pH of the reaction mixture was adjusted to 6.8-7.2 by addition of 1M aqueous NaOH at rt. The resulting reaction mixture was heated to 40° C. and stirred at 40° C. for 43 hr until completion of the reaction as determined by HPLC. As the reaction proceeded, the product 2 precipitated out from the reaction mixture and formed a suspension. The resulting suspension was filtered at 40° C., and the resulting wet cake was washed with water and dried to give product 2 as an off-white solid (2.8 g, purity 97%, yield 85%).

(8) ##STR00023##

(9) In conclusion, a beneficial effect of TPGS as an additive in a ketoreductase mediated biotransformation was shown, including superior reaction kinetics and process robustness. The underlying enzyme stability in TPGS will find similar benefits when applied to a wide range of biocatalytic transformations.

Example 2: Synthesis of (R)-5-fluoro-3-(3-fluorophenyl)-2-(1-hydroxyethyl-4H-chromen-4-one Using Ketoreductase

(10) ##STR00024##
Reaction Mixture:
20 mg substrate 3
enzyme ketoreductase KRED-EW-109 (amount: see table 2)
20 mg D-glucose
0.4 mg (2% (m/m)) glucose dehydrogenase (GDH)
0.2 mg (1% (m/m)) NADP
0.1M PBS (amount: see table 2)
2% TPGS-750-M (m/m) in 0.1M PBS (amount: see table 2)
DMSO (amount: see table 2)
Reaction Conditions: pH 7.0, 30° C.

(11) TABLE-US-00002 TABLE 2 2% TPGS DMSO Conversion No. enzyme 0.1M PBS in PBS (v/v) 17.5 h 24 h 1   1 mg 0.9 ml N.A. 0.1 ml 72.6% 67.5% 5.0% (m/m) 10% (v/v) 2 N.A.   1 ml N.A. 87.2% 87.2% 3 0.2 mg 0.9 ml N.A. 0.1 ml 3.9% 4.1% 1.0% (m/m) 10% (v/v) 4 N.A.   1 ml N.A. 11.7% 13.2% 5 0.1 mg 0.9 ml N.A. 0.1 ml 1.5% 1.5% 0.5% (m/m) 10% (v/v) 6 N.A.   1 ml N.A. 7.9% 9.3% 7   1 mg 0.9 ml N.A. 0.1 ml 63.2% 61.0% 5.0% (m/m) 10% (v/v) 8 0.5 ml 0.5 ml N.A. 89.9% 90.4% 9 0.2 mg 0.9 ml N.A. 0.1 ml 2.1% 2.5% 1.0% (m/m) 10% (v/v) 10 0.5 ml 0.5 ml N.A. 12.6% 14.4% 11 0.1 mg 0.9 ml N.A. 0.1 ml 1.2% 1.6% 0.5% (m/m) 10% (v/v) 12 0.5 ml 0.5 ml N.A. 7.6% 8.4%

Example 3: Synthesis of 1-phenylethanol Using Ketoreductase

(12) ##STR00025##
Reaction Mixture:
20 μl acetophenone
enzyme ketoreductase KRED-EW-124 (amount: see table 3)
20 mg D-glucose
0.4 mg (2% (m/m)) glucose dehydrogenase (GDH)
0.2 mg (1% (m/m)) NADP
0.1M PBS (amount: see table 3)
2% TPGS-750-M (m/m) in 0.1M PBS (amount: see table 3)
DMSO (amount: see table 3)
Reaction Conditions: pH 7.0, 30° C.

(13) TABLE-US-00003 TABLE 3 2% TPGS DMSO Conversion No. enzyme 0.1M PBS in PBS (v/v) 18 h 1 8 mg 0.9 ml N.A. 0.1 ml 53.5% 40% (m/m) 10%(v/v) 2 N.A.   1 ml N.A. 55.0% 3 4 mg 0.9 ml N.A. 0.1 ml 48.7% 20% (m/m) 10%(v/v) 4 N.A.   1 ml N.A. 52.4% 5 2 mg 0.9 ml N.A. 0.1 ml 45.2% 10% (m/m) 10%(v/v) 6 N.A.   1 ml N.A. 47.5% 7 8 mg 0.9 ml N.A. 0.1 ml 53.2% 40% (m/m) 10%(v/v) 8 0.5 ml 0.5 ml N.A. 52.5% 9 4 mg 0.9 ml N.A. 0.1 ml 47.9% 20% (m/m) 10%(v/v) 10 0.5 ml 0.5 ml N.A. 50.6% 11 2 mg 0.9 ml N.A. 0.1 ml 43.3% 10% (m/m) 10%(v/v) 12 0.5 ml 0.5 ml N.A. 45.2%

Example 4: Synthesis of (S)-3-(3-bromopyridin-4-yl)-methylcyclohexan-1-one Using Ene Reductase

(14) ##STR00026##
Reaction Mixture:
20 μl substrate 5
enzyme ene reductase ENE012 (amount: see table 4)
20 mg D-glucose
0.4 mg (2% (m/m)) glucose dehydrogenase (GDH)
0.2 mg (1% (m/m)) NAD
0.1M PBS (amount: see table 4)
2% TPGS-750-M (m/m) in 0.1M PBS (amount: see table 4)
toluene (amount: see table 4)
Reaction Conditions: pH 7.0, 30° C.

(15) TABLE-US-00004 TABLE 4 2% TPGS Toluene Conversion No. enzyme 0.1M PBS in PBS (v/v) 19.5 h 43.5 h 1 4 mg 0.8 ml N.A. 0.2 ml 45.8% 36.7% 20% (m/m) 20% (v/v) 2 N.A.   1 ml N.A. 82.7% 79.9% 3 2 mg 0.8 ml N.A. 0.2 ml 30.9% 33.8% 10% (m/m) 20% (v/v) 4 N.A.   1 ml N.A. 42.3% 69.3% 5 4 mg 0.8 ml N.A. 0.2 ml 47.8% 44.4% 20% (m/m) 20% (v/v) 6 0.5 ml 0.5 ml N.A. 62.3% 59.0% 7 2 mg 0.8 ml N.A. 0.2 ml 28.1% 28.2% 10% (m/m) 20% (v/v) 8 0.5 ml 0.5 ml N.A. 47.6% 75.2%