HIGHLY EFFICIENT ENZYMATIC PROCESS TO PRODUCE (R)-3-QUINUCLIDINOL

Abstract

The present invention relates to enzymatic reduction of 3-quinuclidinone to (R)-3-quinuclidinol (Scheme I), by reacting 3-quinuclidinone with a variant of ketoreductase enzyme derived from Rhodotorula rubra. The invention also relates to enzymatically produced (R)-3-quinuclidinol wherein the substrate loading capacity of the enzyme is not less than 100 g/L.

##STR00001##

Claims

1-20. (canceled)

21. A process to produce (R)-3-quinuclidinol comprising enzymatic reduction of 3-quinuclidinone or salt thereof using a variant of ketoreductase enzyme derived from Rhodotorula rubra, in the presence of a co-factor regenerating system variant of glucose dehydrogenase derived from Bacillus megaterium; wherein both the variant are in cell lysate; and wherein substrate loading capacity of the variant of ketoreductase enzyme is not less than about 100 g/L.

22. The process as claimed in claim 21, wherein the co-factor is selected from NAD and NADP.

23. The process as claimed in claim 21, wherein the process comprises steps of: a. reacting 3-quinuclidinone with the cell lysate containing 3-quinuclidinone reductase, in the presence of cell lysate containing glucose dehydrogenase, to give (R)-3-quinuclidinol; b. extracting and purifying (R)-3-quinuclidinol obtained in step a.

24. The process as claimed in claim 23, step b, wherein extracting and purifying (R)-3-quinuclidinol comprising the steps of: a. basifying/acidifying the reaction mixture, to obtain basified/acidified reaction mixture; b. adding acetone to the basified/acidified reaction mixture in step a, filtering the solvent mixture and removing acetone by evaporation to obtain aqueous solution of product; c. alternatively, adding celite to the basified/acidified reaction mixture in step a, stirring at 20 C.-30 C. for 20 min to 2 h and filtering to obtain aqueous solution of product; d. extracting the product from the aqueous solution obtained in step b or c using n-butanol and concentrating the extract to dryness to obtain the extracted product; e. solubilizing the extracted product obtained in step d in hot toluene at 80 C.-105 C. to obtain solubilized solution; and f. filtering the solubilized solution obtained in step e, gradually cooling the filtrate under constant stirring to room temperature to obtain pure crystals of (R)-3-quinuclidinol and recovering the crystals by filtration.

25. The process as claimed in claim 21, wherein substrate loading capacity of the enzyme is not less than 125 g/L.

26. The process as claimed in claim 21, wherein per mL of cell lysate comprises not less than 4 units of ketoreductase enzyme.

27. The process as claimed in claim 21, wherein per mL of cell lysate comprises not less than 250 units of glucose dehydrogenase.

28. A process to enzymatically produce (R)-3-quinuclidinol wherein (R)-3-quinuclidinol produced is not less than 99% pure and has greater than 99.5% enantiomeric excess, wherein the enzyme is in cell lysate; and wherein substrate loading capacity of the enzyme is not less than about 100 g/L.

29. (R)-3-quinuclidinol, produced by the process comprising enzymatic reduction of 3-quinuclidinone or salt thereof using a variant of ketoreductase enzyme derived from Rhodotorula rubra, in the presence of a co-factor regenerating system variant of glucose dehydrogenase derived from Bacillus megaterium wherein both the variant are in cell lysate; wherein substrate loading capacity of the variant of ketoreductase enzyme is not less than about 100 g/L; and the (R)-3-quinuclidinol is not less than 99% pure and having greater than 99.5% enantiomeric excess.

30. The process as claimed in claim 21, wherein substrate loading capacity of the enzyme is not less than 150 g/L.

31. The process as claimed in claim 21, wherein substrate loading capacity of the enzyme is not less than 175 g/L.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0017] In the present invention the process is said to be highly efficient due to increase in substrate loading capacity of the enzyme which leads to higher yield and reduced time of bioconversion.

[0018] The terms ketoreductase enzyme 3-quinuclidinone reductase, variant, are all used interchangeably and all refer to an enzyme derived from the amino acid sequence from Rhodotorula rubra and is used in the enzymatic reduction of 3-quinuclidinone to (R)-3-quinuclidinol.

[0019] The substrate used in the present invention is 3-quinuclidinone or salt thereof.

[0020] v/v or V/V mean volume/volume; w/v or W/V mean weight/volume and why or W/W mean weight/weight.

[0021] Time in hour or hours in indicated as h or hr or hrs. The phrase e.e. is short form of enantiomeric excess and indicates the percentage of one enantiomer compared to the other.

[0022] The terms glucose dehydrogenase and GDH are interchangeably used and refers to enzyme derived from the amino acid sequence from Bacillus megaterium (Bin), wherein GDH has the ability to regenerate co-factors like NAD or NADP using glucose as the substrate.

[0023] The main embodiment of the present invention relates to the enzymatic reduction of 3-quinuclidinone to (R)-3-quinuclidinol, by reacting 3-quinuclidinone with a variant of ketoreductase enzyme derived from Rhodotorula rubra in the presence of a suitable co-factor regenerating system consisting of a variant of glucose dehydrogenase derived from Bacillus megaterium and co-factors NAD or NADP, wherein both the variants are in the cell lysate.

[0024] According to the embodiment the co-factor regenerating system comprises a variant of glucose dehydrogenase and a co-factor NAD or NADP.

[0025] According to the aspect of this embodiment the enzymatic reduction of 3-quinuclidinone to (R)-3-quinuclidinol was carried out using the process comprising steps of: [0026] a. reacting 3-quinuclidinone with the cell lysate containing 3-quinuclidinone reductase, in the presence of cell lysate containing glucose dehydrogenase, to give (R)-3-quinuclidinol; [0027] b. extracting and purifying (R)-3-quinuclidinol obtained in step a.

[0028] According to this embodiment of invention 3-quinuclidinone is reacted with cell lysate containing 3-quinuclidinone reductase in the presence of cell lysate containing glucose dehydrogenase to obtain (R)-3-quinuclidinol, wherein the reaction mixture comprises: [0029] a) 3-quinuclidinone as substrate [0030] b) cofactor NAD or NADP; [0031] c) glucose, 1 to 2 times of the substrate concentration; [0032] d) cell lysate containing the enzyme 3-quinuclidinone reductase: [0033] e) cell lysate containing the enzyme glucose dehydrogenase; [0034] f) potassium phosphate buffer medium

[0035] Another aspect of this embodiment is a process for extracting and purifying (R)-3-quinuclidinol. The process comprises the steps of: [0036] a) basifying/acidifying the reaction mixture, to obtain basified/acidified reaction mixture; [0037] b) adding acetone to the basified/acidified reaction mixture in step a, filtering the solvent mixture and removing acetone by evaporation to obtain aqueous solution of product; [0038] c) alternatively, adding celite to the basified/acidified reaction mixture in step a, stirring at about 20 C.-30 C. for about 20 min to 2 h and filtering to obtain aqueous solution of product; [0039] d) extracting the product from the aqueous solution obtained in step 13 or c using n-butanol and concentrating the extract to dryness to obtain the product; [0040] e) solubilizing the product obtained in step in hot toluene. [0041] f) filtering the solution obtained in step c, gradually cooling the filtrate under constant stirring to room temperature to obtain pure crystals of (R)-3-quinuclidinol and recovering the crystals by filtration.

[0042] Yet another embodiment of the invention is to enzymatically produce (R)-3-quinuclidinol of not less than about 99% purity and more than about 99.5% e.e., wherein the substrate loading capacity of the enzyme is not less than about 100 g/L.

[0043] According to this embodiment the substrate loading capacity of the enzyme is at least 125 g/L, preferably at least 150 g/L and most preferably at least 175 g/L.

[0044] Accordingly, (R)-3-quinuclidinol was produced by reduction of 3-quinuclidinone using a variant of kctoreductase enzyme derived from Rhodotorula rubra in the presence of a suitable co-factor regenerating system.

[0045] The co-factor regenerating system comprises variant of glucose dehydrogenase and a co-factor NAD or NADP.

[0046] The enzymatic reduction of 3-quinuclidinone to (R)-3-quinuclidinol was carried out using the process comprising steps of: [0047] a) reacting 3-quinuclidinone with at least 4 units of 3-quinuclidinone reductase enzyme per ml of cell lysate, in the presence of cell lysate containing not less than 250 units of glucose dehydrogenase enzyme per milliliter of lysate, wherein 3-quinuclidinone loaded in the reaction wasnot less than 100 g/L; [0048] b) extracting and purifying (R)-3-quinuclidinol obtained in step a;

[0049] According to this embodiment 3-quinuclidinone is reducedusing at least 4 units of 3-quinuclidinone reductase per milliliter of cell lysate in the presence of cell lysate containing not less than 250 units of glucose dehydrogenase to obtain the product (R)-3-quinuclidinol, wherein the reaction mixture comprises: [0050] a) 3-quinuclidinone as substrate, not less than about 100 g/L of reaction mixture; [0051] b) co-factor NAD or NADP; [0052] c) glucose, 1 to 2 times of the substrate concentration; [0053] d) cell lysate containing the enzyme 3-quinuclidinone reductase; [0054] e) cell lysate containing the enzyme glucose dehydrogenase; [0055] f) potassium phosphate buffer.

[0056] Another aspect this embodiment is a process for extracting and purifying (R)-3-quinuclidinol. The process comprises the steps of: [0057] a. basifying/acidifying the reaction mixture, to obtain basified/acidified reaction mixture; [0058] b. adding acetone to the basified/acidified reaction mixture in step a, filtering the solvent mixture and removing acetone by evaporation to obtain aqueous solution of product; [0059] c. alternatively, adding celite to the basified/acidified reaction mixture in step a, stirring at about 20 C.-30 C. for about 20 min to 2 h and filtering to obtain aqueous solution of product: [0060] d. extracting the product from the aqueous solution obtained in step b or in step c using n-butanol and concentrating the extract to dryness to obtain the product; [0061] e. solubilizing the product obtained in step d in hot toluene at about 80 C.-105 C. to obtain solution; [0062] f. filtering the solution obtained in step e, gradually cooling the filtrate to room temperature to obtain pure crystals of (R)-3-quinuclidinol and recovering the crystals by filtration.

[0063] The crystals of (R)-3-quinuclidinol obtained by the present process exhibit more than about 99% purity and enantiomeric excess of greater than about 99.5%.

[0064] The process in the present invention increases the loading capacity of the enzymes involved in the conversion of 3-quinuclidinone to (R)-3-quinuclidinol, which results in increase in the yield of the product, increase in production efficiency due to higher production in shorter duration, higher yield and higher purity.

[0065] Thus the present inventors have designed a reproducible, economical, industrially feasible and efficient enzymatic process to produce (R)-3-quinuclidinol.

[0066] Also the present inventors have successfully reduced reaction time of enzymatic conversion of 3-quinuclidinone to (R)-3-quinuclidinol,

[0067] The present invention thus overcomes all the problems and achieves all the object of the invention.

EXAMPLES

[0068] Following examples of the present invention demonstrate the best mode of carrying out the present invention. These examples do not limit the scope of invention in any manner and should be considered as purely illustrative.

Example 1: Production of (R)-3-quinuclidinol Using Whole Cells

[0069] For the bioconversion of 1 g of 3-quinuclidinone, 4 g of cell mass was used in a reaction mix comprising 10 mg of NADP, 6 g of glucose, 10 mg of glucose dehydrogenase in a final volume of 40 ml. Reaction mass was mixed at 150 rpm on a rotary shaker at 25 C.1 C. for 3-4 h. pH was adjusted intermittently to 6.5-7.5 using 20% NaOH solution. The reaction was monitored for the completion by silica gel-Thin Layer Chromatography (TLC).

Example 2: Production of (R)-3-quinuclidinol Using Cell Lysate

[0070] The reaction mixture comprised of 100 ml cell lysate containing at least 4 units of ketoreductase per millilitre and 50 ml cell lysate containing not less than 250 units of glucose dehydrogenase enzyme per millilitre along with 30 mg of NADP and 1.4 g of glucose, 10 g 3-quinuclidinone. The final volume of the reaction mixture was 150 ml. Reaction mass was stirred at 150 rpm on a rotary shaker at 25 C.1 C. for 3-4 h. pH was constantly adjusted to 6.8-7.5using 20% NaOH. At 4 h, the mixture was sampled to analyze conversion of the substrate 3-quinuclidinone and determine the enantiomeric purity of the product (R)-3-quinuclidinol.

Example 3: Production of (R)-3-quinuclidinol Using Cell Lysate with Increased Substrate Loading in the Reaction

[0071] Reaction mass comprising of 60 mL of cell lysate containing at least 4 units of ketoreductase enzyme per milliliter, 30 mL cell lysate containing not less than 250 units of glucose dehydrogenase enzyme per millilitre and 30 mg of NADP was used. 3-quinuclidinone was varied from 10.0 g, 12.5 g, 15.0 g, 17.5 g and 20.0 g in a final reaction volume of 100 mL. Reaction mass was stirred at 150 rpm on a rotary shaker at 25 C.1 C. for 3-10 h. pH was constantly adjusted to 6.8-7.5 using 20% NaOH. After 10 h the mixture was sampled and analyzed the conversion of 3-quinuclidinone by TLC. Complete conversion was observed in all reactions.

Example 4: Purification of (R)-3-Quinuclidinol Using Celite

[0072] The reaction mixture (100 mL) was alkalified with 20% NaOH solution to pH 12. 5 g celite was added to the alkalified reaction mixture and stirred at 25 C. for 1 h. The reaction mixture was filtered to obtain aqueous solution of product. A 200 mL of n-butanol was added to the aqueous filtrate and mixed vigorously. Aqueous and organic layers were separated and aqueous phase was re-extracted with 200 mL of n-butanol. The n-butanol extracts were pooled, concentrated to dryness and solubilized in toluene at 90 C.-100 C. Hot toluene solution was filtered and the filtrate was allowed to gradually cool to room temperature under constant stirring. (R)-3-quinuclidinol crystals were filtered and dried at 50 C.-60 C. under vacuum. White to off-white crystals were obtained, with more than 99% purity and more than 99.9% e.e.