A method of enantiomerically enriching chiral cyanohydrins is disclosed that involves selective cleavage of the unwanted enantiomer into its cleavage products HCN and the corresponding aldehyde or ketone by use of an enantioselective dehydrocyanation catalyst, coupled with simultaneous removal of at least one of the dehydrocyanation products.
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1. An enzymatic process for the enantiomeric enrichment of a chiral cyanohydrin of the structure ##STR16## comprising the steps: (a) bringing said mixture of chiral cyanohydrins into contact at a ph of 3.5 to 5.5 with an enantioselective dehydrocyanation catalyst selected from the group consisting of (R)- and (S)- oxynitrilase and cyclic peptides until one of said chiral cyanohydrins is converted in a dehydrocyanation reaction to dehydrocyanation reaction products comprising hydrogen cyanide and an aldehyde or ketone of the structure ##STR17## (b) simultaneously reducing the concentration of at least one of said dehydrocyanation reaction products by a method selected from a liquid-liquid extraction, a liquid-gas extraction, a membrane-based separation, a chemical conversion, and combinations thereof where
(i) R1 and R2, taken together, form a diyl hydrocarbon chain containing 3 to 5 carbons, or (ii) R1 and R2 are different and R1 is selected from straight and branched chain alkyl, cycloalkyl, heterocyclic and aryl groups, said cycloalkyl, said heterocyclic and said aryl groups being unsubstituted or substituted with a substituent selected from the groups consisting of halo, hydroxy, lower alkyl, lower alkoxy, lower alkylthio, cycloalkyl, carbamoyl, trifluoromethyl, phenyl, phenoxy, nitro, alkylsulfonyl, arylsulfonyl, alkylcarboxamide, and . .acrylcarboxamido,.!. arylcarboxamido, and R2 is selected from hydrogen and R1.
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The government has a nonexclusive, nontransferable, royalty-free license to practice this invention under Contract No. ISI-8960705 awarded by the National Science Foundation.
This application is a Reissue of 07/848,023 filed Mar. 9, 1992, now U.S. Pat. No. 5,241,087. to achieve trapping involves including reagents and/or catalysts that effect the reaction of the aldehyde or ketone dehydrocyanation products. Exemplary aldehyde or ketone removal reactions include reaction with bisulfite to form bisulfite adducts; reaction with amines or diamines to form Schiff bases; reaction with hydroxylamine to form hydroxamates; reaction with hydrazine or substituted hydrazines to form hydrazones; and reaction of aldehydes with oxidants such as molecular oxygen, peroxide, perchlorate, perbromate, periodate, chlorate, bromate, iodate, chlorine, bromine, and iodide to form carboxylic acids. These reagents can be used dissolved in solution or in an immobilized form.
Catalyst activity may be expressed as units/mg for solid catalysts and units/ml for dissolved catalysts. A unit of catalyst activity is conveniently defined as the dehydrocyanation of one micromole of cyanohydrin per minute. For purposes of uniformity, benzaldehyde cyanohydrin is used as the standard cyanohydrin. For example, one unit of (R)-oxynitrilase activity corresponds to the amount of enzyme that converts benzaldehyde cyanohydrin to benzaldehyde and hydrogen cyanide at a rate of one micromole per minute.
The following standardized assay was utilized to measure the activity of oxynitrilase set forth in the Examples which follow. A solution of enzyme was prepared in 0.1M (pH 5.5) citrate buffer. A 3-ml sample of enzyme solution was combined with 30 microliters of 0.21M benzaldehyde cyanohydrin and the rate of increase in absorbance at 250 nm recorded. Then, the same procedure was followed with a 3-ml sample of 0.1M (pH 5.5) citrate buffer to obtain the background, non-enzymatic, reaction. The activity in units/ml was calculated by subtracting the slope of the non-enzymatic reaction from the slope of the enzymatic reaction and converting the absorbance change with time to concentration change with time using a molar extinction coefficient of 13,200 M-1 cm-1.
The progress of the dehydrocyanation reaction was monitored by one of two methods. In the first method, reaction aliquots were removed, acidified with sulfuric acid to stabilize the cyanohydrin, and extracted with chloroform. The organic extract was analyzed by liquid chromatography on a C-18 reverse phase column (25 cm×2 mm) employing isocratic elution using a mixture of methanol and 0.05M aqueous trifluoroacetic acid (60/40; vol/vol) with detection at 250 nm. In the second method, the cyanohydrin was extracted with chloroform as described above, then treated with bis(trimethylsilyl)acetamide to convert the cyanohydrin to the trimethylsilyl derivative and analyzed by capillary gas chromatography on an HP-1 dimethylpolysiloxane column (25 m×0.2 mm) with flame ionization detection.
The enantiomeric excess of cyanohydrins was measured by one of two methods. In the first method, the specific rotation was compared to the specific rotation of samples of known enantiomeric excess. In the second method, samples of cyanohydrin were allowed to react with (-)-alpha-(trifluoromethylphenyl)methoxyacetyl chloride according to the method reported by Mosher et al. in 34 J. Org. Chem. 2543 (1969) and the resulting diastereomers were separated by capillary gas chromatography on an HP-1 dimethylpolysiloxane column (25 m×0.2 mm) with flame ionization detection.
Partition coefficient studies of cyanohydrins and their corresponding aldehydes in organic solvents and aqueous buffer solutions were made and confirmed that the aldehydes partition much more strongly into the organic phase than do the corresponding cyanohydrins.
Relative rates of dehydrocyanation of different cyanohydrins subjected to the action of the soluble catalyst (R)-oxynitrilase were determined from initial rate studies. A solution of (R)-oxynitrilase was combined with a 0.21 mM solution of cyanohydrin and the rate of increase of absorbance at the wavelength corresponding to the absorption maxima was monitored as described above in connection with the measurement of catalyst activity. The measured activity was calculated using the appropriate extinction coefficient for the corresponding aldehyde. The results, summarized in Table 2, demonstrate that (R)-oxynitrilase catalyzes dehydrocyanation of a range of cyanohydrin structures and that it is unexpectedly more effective with several unnatural substrates than with its natural substrate, benzeldehyde cyanohydrin.
| TABLE 2 |
| ______________________________________ |
| Cyanohydrin Substrate Relative Rate |
| ______________________________________ |
| 5-Methylfurfural cyanohydrin |
| 2.8 |
| Furfural cyanohydrin 2.5 |
| Piperonal cyanohydrin 2.1 |
| Benzaldehyde cyanohydrin |
| 1.0 |
| Crotonaldehyde cyanohydrin |
| 0.90 |
| P-Tolualdehyde cyanohydrin |
| 0.45 |
| m-Anisaldehyde cyanohydrin |
| 0.19 |
| p-Anisaldehyde cyanohydrin |
| 0.17 |
| p-Ethylbenzaldehyde cyanohydrin |
| 0.13 |
| Cinnamaldehyde cyanohydrin |
| 0.05 |
| ______________________________________ |
Two 60 mM solutions of racemic benzaldehyde cyanohydrin in pH 5.5 citrate buffer were incubated at 25°C with the enzyme (R)-oxynitrilase present in a concentration of 2.1 units/ml, and the optical rotation of the solutions were measured over approximately 45 minutes. One of the solutions, designated as solution A, was treated with the supported-gas membrane system of substantially the same type shown and discussed above in connection with FIG. 1, for the removal and trapping of the dehydrocyanation products HCN and benzaldehyde. The other solution, designated as solution B, had no dehydrocyanation product removed, and was simply allowed to approach thermodynamic equilibrium. The optical rotation of the solutions in both cases was negative due to the enantiomerically selective depletion of (R)-benzaldehyde cyanohydrin. In the case of solution B, the optical rotation leveled off and began to decrease over time as a result of racemization due to the back-reaction (or hydrocyanation reaction) of the dehydrocyanation products HCN and benzaldehyde. The results are shown in the plot comprising FIG. 3. As is apparent, the optical rotation of solution A leveled off at a much higher value than that of solution B, suggesting more extensive cleavage of (R)-benzaldehyde cyanohydrin. As is also apparent from FIG. 3, the optical rotation of solution A did not decrease over time, strongly suggesting that back-reaction of the dehydrocyanation products was insignificant because the concentrations of dehydrocyanation products was very low due to removal and trapping of the same.
Example 1 was repeated with removal and trapping of the dehydrocyanation products in the same manner as was done for Solution A in that Example, and the solution was monitored for enantiomeric enrichment by optical rotation and for the total amount of benzaldehyde cyanohydrin and benzaldehyde by High Pressure Liquid Chromatography (HPLC). Based on measured concentrations of these moieties and the optical rotation, the enantiomeric excess of (S)-enantiomer was calculated over the course of the reaction (approximately 90 minutes in duration). The results are shown in the plot comprising FIG. 4. As is apparent, the total concentration of benzaldehyde cyanohydrin BCH! leveled off at approximately half its starting value, consistent with selective dehydrocyanation of (R)-benzaldehyde cyanohydrin. Also consistent with such enantioselective dehydrocyanation was the observation that the enantiomeric excess of the desired enantiomer (S)-benzaldehyde cyanohydrin approached 100%. The results also showed that the concentration of benzaldehyde B! rose during the early part of the reaction, but fell in the latter stages as it was removed and trapped via the supported-gas membrane.
Example 1 was substantially repeated, with the exceptions noted. The aqueous feed reservoir of the supported-gas membrane loop was charged with 500 ml of pH 5.5 citrate buffer, 5.32 grams of racemic benzaldehyde cyanohydrin (40 mmol), and sufficient (R)-oxynitrilase to obtain an activity of 7.4 units/mi. The strip reservoir was charged with 2 L of 0.5M aqueous sodium hydroxide. The feed reservoir and strip reservoir solutions were recirculated by peristaltic pumps at a flow rate of approximately 350 ml/min. Reaction progress was monitored by measuring optical rotation of the feed reservoir, which became progressively negative with time and approached a fixed value of -0.19°. After 80 minutes, contents of the feed reservoir were collected, the reaction quenched by the addition of concentrated sulfuric acid, and the product was isolated by extracting the mixture with two successive portions of chloroform (100 ml and 50 ml). The organic extracts were combined and dried over sodium sulfate. The organic extract had an optical rotation of -0.399°. HPLC analysis using a C-18 reverse phase column employing isocratic elution using methanol (0.05M aqueous trifluoroacetic acid) (60/40 vol/vol) with detection at 250 nm showed that the concentration of (S)-benzaldehyde cyanohydrin was 7.6±0.4 g/ml. On the basis of the rotation at that concentration, the measured specific rotation of (S)-benzaldehyde cyanohydrin was -52.5°±-2.8°. Given the literature value for (R)-enantiomer of +49°, this was estimated to correspond to an enantiomeric excess (S)-enantiomer of approximately 100%.
Example 3 was substantially repeated, varying the cyanohydrin starting material, with the results noted in Table 3.
| TABLE 3 |
| __________________________________________________________________________ |
| Specific Rotation |
| Reaction of Cyanohydrin |
| Example Time Conversion |
| (degrees) ee |
| No. Cyanohydrin |
| (hr) (%) Measured |
| Literature* |
| % |
| __________________________________________________________________________ |
| 4 p-Anisaidehyde |
| 2 50 -47.6 +48 99 |
| cyanohydrin |
| 5 Piperonal |
| 2 50 -46.3 +47 99 |
| cyanohydrin |
| 6 5-Methylfurfural |
| 106.5 |
| 61 -38 +79 48 |
| cyanohydrin |
| 7 m-Anisaldehyde |
| 3 66 -30.9 +36.6 84 |
| cyanohydrin |
| 8 Furfural 2 63 -44.4 +51 87 |
| cyanohydrin |
| __________________________________________________________________________ |
| *Highest specific rotation cited in the literature for the (R)enantiomer. |
Trapping by liquid-liquid extraction during dehydrocyanation was demonstrated. Ten ml of racemic m-phenoxy benzaldehyde cyanohydrin was combined with 100 ml of aqueous 20 mM citrate buffer (pH 5.5) containing 4000 units of (R)-oxynitrilase. Because m-phenoxybenzaldehyde cyanohydrin and its dehydrocyanation product, m-phenoxybenzaldehyde, are immiscible with water, the mixture forms a two-phase reaction system comprising an enzyme-containing aqueous buffer phase and an unreacted-cyanohydrin-containing organic phase. In this case, the dehydrocyanation reaction took place in the aqueous phase and the unreacted (R)- and (S)-cyanohydrin itself acted as an organic extractant to remove and trap the dehydrocyanation product (R)-m-phenoxybenzaldehyde across the organic/aqueous interface. The two-phase mixture was maintained at ambient temperature and stirred for 48 hours, and the reaction progress was monitored by HPLC to 50% conversion. The desired enantiomeric product (S)-m-phenoxybenzaldehyde cyanohydrin was identified by NMR, isolated by extraction with diethyl ether, dried, freed of solvent and its optical rotation measured. The specific rotation in toluene was found to be -23.4°, corresponding to an enantiomeric excess of 96% of the (S)-enantiomer.
Example 9 was substantially repeated, varying the starting cyanohydrin and organic extractant, with the results noted in Table 4.
| TABLE 4 |
| __________________________________________________________________________ |
| Specific Rotation |
| Reaction of Cyanohydrin |
| Exantiomeric |
| Example Time Conversion |
| (degrees) Excess |
| No. Cyanohydrin pH Extractant |
| (hr) (%) Measured |
| Literature* |
| (%) |
| __________________________________________________________________________ |
| 11 Furfural 5.5 |
| cyanohydrin |
| 48 39 -29 +51 57 |
| cyanohydrin |
| 12 m-Anisaldehyde |
| 5.5 |
| cyanohydrin |
| 72 41 -24 +36.6 65 |
| cyanohydrin |
| 13 p-Anisaldehyde |
| 5.5 |
| cyanohydrin |
| 48 48 -27.4 +48 57 |
| cyanohydrin |
| 14 Piperonal 5.5 |
| cyanohydrin |
| 48 57 -26.6 +47 57 |
| cyanohydrin |
| 15 m-Phenoxybenzaldehyde |
| 5.5 |
| cyanohydrin |
| 48 54 -16.2 -24 67 |
| cyanohydrin |
| 16 m-Phenoxybenzaldehyde |
| 5.25 |
| cyanohydrin |
| 48 53 -17.2 -24 72 |
| cyanohydrin |
| 17 m-Phenoxybenzaldehyde |
| 5.0 |
| cyanohydrin |
| 48 51 -19.2 -24 81 |
| cyanohydrin |
| 18 p-Anisaldehyde |
| 4.5 |
| cyanohydrin |
| 48 51 -36.2 +48 75 |
| cyanohydrin |
| 19 Piperonal 4.5 |
| cyanohydrin |
| 48 58 -48 +47 100 |
| cyanohydrin |
| __________________________________________________________________________ |
| *Positive rotations correspond to highest specific rotation cited in the |
| literature for the (R)enantiomer. |
The following examples demonstrate the need for pH control during dehydrocyanation. Example 9 was substantially repeated, except for a lower enzyme concentration and varying the pH of the aqueous citrate buffer. The results, summarized in Table 4, shows that although a decrease in pH decreases the rate of dehydrocyanation, evidenced by a decrease in the extent of conversion, there is a measurable improvement in the apparent selectivity, evidenced from the enantiomeric excess of the cyanohydrin remaining at the end of the reaction.
Examples 12 and 13 were substantially repeated, with exception that the pH of the aqueous citrate buffer was lowered from 5.5 to 4.5. The results, summarized in Table 4, show the expected improvement in enantiomeric excess.
The following procedures exemplify trapping by chemical reaction during dehydrocyanation with a soluble enantioselective catalyst to effect enantiomeric enrichment. Example 9 was substantially repeated, varying the cyanohydrin starting material and either including or keeping out one molar equivalent of cyclohexanone, which chemically trapped the hydrogen cyanide produced in the dehydrocyanation reaction. The results, summarized in Table 5, show that including cyclohexanone increases the extent of conversion and improves the enantiomeric excess of the cyanohydrin remaining at the end of the reaction.
| TABLE 5 |
| __________________________________________________________________________ |
| Specific Rotation |
| Chemical |
| Reaction of Cyanohydrin |
| Exantiomeric |
| Example Trapping |
| Time Conversion |
| (degrees) Excess |
| No. Cyanohydrin |
| pH |
| Reagent (hr) (%) Measured |
| Literature* |
| (%) |
| __________________________________________________________________________ |
| 20 Benzaldehyde |
| 5.5 |
| none 48 25 -0.40 -49.0 1 |
| cyanohydrin |
| 21 Benzaldehyde |
| 5.5 |
| cyclohexanone |
| 48 36 -8.26 -49.0 24 |
| cyanohydrin |
| 22 5-Methylfurfural |
| 5.5 |
| none 48 44 -21.7 -79 27 |
| cyanohydrin |
| 23 5-Methylfurfural |
| 5.5 |
| cyclohexanone |
| 48 61 -38 -79 48 |
| cyanohydrin |
| 24 Piperonal |
| 5.5 |
| none 48 57 -26.6 -47 57 |
| cyanohydrin |
| 25 Piperonal |
| 5.5 |
| cyclohexanone |
| 48 76 -41.6 -47 89 |
| cyanohydrin |
| __________________________________________________________________________ |
| *Positive rotations correspond to highest specific rotation cited in the |
| literature for the (R)enantiomer. |
The following procedure exemplifies use of a combination of trapping methods (gas-liquid extraction and liquid-liquid extraction) during dehydrocyanation to effect enantiomeric enrichment.
A 100 ml three-necked flask was charged with 5 ml of racemic m-phenoxybenzaldehyde cyanohydrin and 50 ml of aqueous 20 mM citrate buffer (pH 5.5) containing 2000 units of (R)-oxynitrilase. The contents of the flask were stirred with a magnetic stirrer and sparged continuously with a flow of nitrogen to remove hydrogen cyanide produced in the dehydrocyanation. To prevent the loss of water in the reaction mixture through evaporation, nitrogen gas was presaturated with water by bubbling through a water trap. The two-phase mixture was maintained at ambient temperature and the reaction progress was monitored by gas chromatography. After 24 hours, the reaction had progressed to 43% conversion and the enantiomeric excess of m-phenoxybenzaldehyde cyanohydrin remaining was 81% of the (S)-enantiomer.
The following procedure exemplifies the use of another combination of trapping methods (supported-gas membrane and membrane-based liquid-liquid extraction) during dehydrocyanation to effect enantiomeric enrichment. A combination membrane contactor/supported-gas membrane of the type shown in and discussed above in connection with FIG. 2 was used to remove and trap the dehydrocyanation cleavage reaction products. The organic feed reservoir was charged with a solution of 20 grams of racemic benzaldehyde cyanohydrin in 140 ml of toluene/heptane (80/20; vol/vol); the aqueous enzyme reservoir was charged with 500 ml of aqueous 0.1M sodium citrate buffer (pH 5.5) containing 6 units/ml of (R)-oxynitrilase; and the aqueous strip reservoir was charged with 3 L of 0.17M aqueous sodium hydroxide. The temperature of the reservoirs was maintained at about 4°C and peristaltic pumps were used to circulate the solutions at approximately 150 ml/min.
At various times, aliquots were removed from the organic feed and aqueous enzyme reservoirs, and the optical rotations of the samples were measured. The results from the aqueous enzyme are shown in the plot comprising FIG. 5 where the dashed horizontal line comprises the calculated optical rotation of the solution that corresponds to 100% enantiomeric excess of (S)-cyanohydrin. As is apparent, after approximately three hours the optical rotation of (S)-benzaldehyde cyanohydrin leveled off at a value approaching 100% enantiomeric excess (actual measured was 92%). (S)-benzaldehyde cyanohydrin in the organic reservoir was isolated and its optical rotation measured, which was found to corresponded to an 82% enantiomeric excess. Overall yield of the (S)-cyanohydrin enantiomer was 82%.
The terms and expressions which have been employed in the foregoing specification are used therein as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding equivalents of the features shown and described or portions thereof, it being recognized that the scope of the invention is defined and limited only by the claims which follow.
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