MICROBIOLOGICAL PROCESS FOR THE PREPARATION OF AMIDES

Abstract

A microbiological process for the preparation of amides from the corresponding nitriles by enzymatic hydrolysis with nitrile hydratase enzyme from a bacterial strain of Rhodococcus biphenylivorans species is described.

Claims

1. A process for the preparation of amides of formula
R(R)(R)CCONH.sub.2(I) comprising reacting a nitrile of formula
R(R)(R)CCN(II) with the nitrile hydratase enzyme produced by the bacterial strain Rhodococcus biphenylivorans named Palladio 22 deposited in the Collection of Microorganisms BCCM-LMG with deposit number LMG P-29520, wherein at least one of R, R and R is not hydrogen; R, R and R, are the same or different from each other, and are independently selected from the group consisting of hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 alkoxycarbonyl, C.sub.2-C.sub.6 alkenyl, C.sub.3-C.sub.6 cycloalkyl optionally containing a O group, RCO, heterocycle with 3 to 6 atoms of which is N or 0, arylsulfonyl, C.sub.1-C.sub.6 alkylsulfonyl, aryl, arylaminocarbonyl and C.sub.1-C.sub.6 alkylaminocarbonyl; R is C.sub.1-C.sub.6 alkyl, aryl, amino, hydrazino; and each of R, R, R and R, when not from hydrogen, is optionally substituted by one or more substituents selected from the group consisting of halogen, nitrile, amino, C.sub.1-C.sub.6 alkylamino, hydroxy, C.sub.1-C.sub.6 alkoxy, and aryl groups; provided that the compound of formula I is not acrylamide.

2. The process according to claim 1, wherein at least one of R, R, R and R is or contains a functional group sensitive to hydrolysis or solvolysis under acid, basic or neutral catalysis or environment and is selected from the group consisting of an additional nitrile group, an ester group, an amide group, a haloalkyl group, and an ammonium salt.

3. The process according to claim 1, wherein the enzyme is used in the form of a biomass as a paste having a dry residue at 105 C. within a range from 20 to 45%.

4. A microbiological The process according to claim 1 wherein the enzyme is used in the form of a dried/lyophilized biomass having a dry residue at 105 C. from 90 to 100%.

5. The process according to claim 3, herein said biomass is immobilized onto a solid-type substrate.

6. The process according to claim 1, wherein said process is carried out in aqueous solution, and optionally in the presence of an organic co-solvent.

7. The process according to claim wherein the co-solvent is selected from the group consisting of methanol, ethanol, tetrahydrofuran, methyl-tetrahydrofuran, 1,4-dioxaeo, toluene, and t.BuOMe.

8. The process according to claim 6 wherein the amount of co-solvent is between 2% and 20% v/v.

9. The process according to claim 1, wherein the reaction is carried out at a pH from 6.0 to 9.0.

10. The process according to claim 1 wherein the reaction is carried out at a temperature from 10 to 45.

11. The process according to claim 1, wherein the enzyme is used in the form of a biomass as a paste having then a dry residue at 105 C. within a range from 25 to 40%.

12. The process according to claim 1, wherein the enzyme is used in the form of a dried/lyophilized biomass having a dry residue at 105 C. from 95 to 99%.

13. The process according to claim 7, wherein the co-solvent is methanol or toluene.

14. The process according to claim 8, wherein the amount of co-solvent is between 5% and 15%.

15. The process according to claim 14, wherein the amount of co-solvent is about 10%.

16. The process according to claim 9, wherein the reaction is carried out at a pH from 6.5 to 7.5.

17. The process according to claim 10, wherein the reaction is carried out at a temperature from 15 to 30 C.

Description

EXAMPLES

[0051] The reactions were carried out in phosphate buffer (10 mM, pH=7.4) using 10% of co-solvent to increase the nitrile solubility.

[0052] General procedure: the substrate (final concentration 50 mM) was added in 1 mL of MeOH to a suspension of the enzyme (NHase 4 mg) in buffer (9 mL). In order to ensure the dissolution, sonication or heating were applied, if needed. The reactions were followed in time (1 mL samples) by different analytical methods. Every sample was quenched with MeOH, filtered and analyzed.

Example 1

[0053] 2-Chloroacetamide

[0054] NHase was grinded in a mortar and added to a potassium phosphate buffer (10 mM, pH=7.4) (9 mL) in a 50 mL Erlenmeyer. The suspension was mixed by vortex and 2-chloroacetonitrile (38 mg, 0.5 mmol) was added to the suspension dissolved in MeOH (1 mL). The reaction was stirred by orbital shaking at 25 C. and 250 rpm. The reaction was followed by .sup.1H-NMR. Samples (1 mL) were analyzed after 1.5, 2.5, 4 and 7 hours.

[0055] Samples were mixed with MeOH (2 mL), filtered and evaporated under vacuum. Total conversion towards 2-chloroacetamide was observed after 1.5 hours.

[0056] .sup.1H-NMR (CD.sub.3OD, 300.13 MHz): 4.05-4.83 (s, 2H), comparable to a commercially available sample of 2-chloroacetamide.

Example 2

[0057] 2,2-Diethoxyacetamide

[0058] NHase was grinded in a mortar and added to a potassium phosphate buffer (10 mM, pH=7.4) (9 mL) in a 50 mL Erlenmeyer. The suspension was mixed by vortex and 2,2-diethoxyacetonitrile (65 mg, 0.5 mmol) was added to the suspension dissolved in MeOH (1 mL). The reaction was stirred by orbital shaking at 25 C. and 250 rpm. The reaction was followed by .sup.1H-NMR. Samples (1 mL) were analyzed after 1.5, 2.5, 4 and 7 hours. Samples were mixed with MeOH (2 mL), filtered and evaporated under vacuum. Total conversion towards 2,2-diethoxyacetamide was observed after 1.5 hours.

[0059] .sup.1H-NMR (CD.sub.3OD, 300.13 MHz): 1.44 (t, 6 H, .sup.3J.sub.HH=6.0 Hz), 3.61-3.70 (m, 4 H), 4.81 (s, 1 H), comparable to a commercially available sample of 2,2-diethoxyacetamide.

Example 3

[0060] tert-Butyl 4-carbamoylpiperidin-1-carboxylate

[0061] NHase was grinded in a mortar and added to a potassium phosphate buffer (10 mM, pH=7.4) (9 mL) in a 50 mL Erlenmeyer. The suspension was mixed by vortex and tert-butyl 4-cyanopiperidin-1-carboxylate (105 mg, 0.5 mmol) was added to the suspension dissolved in MeOH (1 mL). The reaction was stirred by orbital shaking at 25 C. and 250 rpm. The reaction was followed by HPLC-MS. Samples (1 mL) were analyzed after 1.5, 2.5, 4 and 7 hours. Samples were mixed with MeOH (2 mL), filtered and directly injected to the HPLC. 40% conversion towards tert-butyl 4-carbamoylpiperidin-1-carboxylate was observed after 7 hours.

[0062] MS (ES.sup.+): m/z: 173.0 [M-55]. MS (ES.sup.): m/z: 227.0 [M1].

[0063] The HPLC chromatogram was comparable to a synthetized sample of tert-butyl 4-carbamoylpiperidin-1-carboxylate. RRT.sub.3=1, RRT.sub.3a=0.87.

Example 4

[0064] 2-(Phenylsulfonyl)acetamide

[0065] NHase was grinded in a mortar and added to a potassium phosphate buffer (10 mM, pH=7.4) (9 mL) in a 50 mL Erlenmeyer. The suspension was mixed by vortex and 2-(phenylsulfonyl)acetonitrile (91 mg, 0.5 mmol) was added to the suspension dissolved in MeOH (1 mL). The reaction was stirred by orbital shaking at 25 C. and 250 rpm. The reaction was followed by HPLC-MS. Samples (1 mL) were analyzed after 1.5, 2.5, 4 and 7 hours. Samples were mixed with MeOH (2 mL), filtered and directly injected to the HPLC.

[0066] MS (ES.sup.+): m/z: 200.1 [M+1], 217.1 [M+18]. RRT.sub.4a=0.36.

Example 5

[0067] Methyl 3-amino-3-oxopropanoate

[0068] NHase was grinded in a mortar and added to a potassium phosphate buffer (10 mM, pH=7.4) (9 mL) in a 50 mL Erlenmeyer. The suspension was mixed by vortex and methyl 2-cyanoacetate (91 mg, 0.5 mmol) was added to the suspension dissolved in MeOH (1 mL). The reaction was stirred by orbital shaking at 25 C. and 250 rpm. The reaction was followed by HPLC-MS and .sup.1H-NMR. Samples (1 mL) were analyzed after 1.5, 2.5, 4 and 7 hours. Samples were mixed with MeOH (2 mL), filtered and directly injected to the HPLC. The rest of the sample that was not injected was evaporated under vacuum and analyzed by .sup.1H-NMR. Total conversion was observed after 1.5 hours.

[0069] .sup.1H-NMR (CD3OD, 300.13 MHz): 3.77 (s, 2 H), 3.98 (s, 3 H). MS (ES.sup.+): m/z: 118.1 [M+1].

Example 6

[0070] 3-(Phenylamino)propanamide

[0071] NHase was grinded in a mortar and added to a potassium phosphate buffer (10 mM, pH=7.4) (9 mL) in a 50 mL Erlenmeyer. The suspension was mixed by vortex and 3-(phenylamino)propanenitrile (73 mg, 0.5 mmol) was added to the suspension dissolved in MeOH (1 mL). The reaction was stirred by orbital shaking at 25 C. and 250 rpm. The reaction was followed by HPLC-MS. Samples (1 mL) were analyzed after 1.5, 2.5, 4 and 7 hours. Samples were mixed with MeOH (2 mL), filtered and directly injected to the HPLC. Total conversion was observed after 2.5 hours. MS (ES.sup.+): m/z: 165.1 [M+1]. RRT.sub.8=1, RRT.sub.8a=0.51.

Example 7

[0072] 3,3-Dimethoxypropanamide

[0073] NHase was grinded in a mortar and added to a potassium phosphate buffer (10 mM, pH=7.4) (9 mL) in a 50 mL Erlenmeyer. The suspension was mixed by vortex and 3,3-dimethoxypropanenitrile (58 mg, 0.5 mmol) was added to the suspension dissolved in MeOH (1 mL). The reaction was stirred by orbital shaking at 25 C. and 250 rpm. The reaction was followed by GC-MS. Samples (1 mL) were analyzed after 1.5, 2.5, 4 and 7 hours. Samples were mixed with MeOH (2 mL) and filtered. Water (2 mL) was added to the sample and the samples were extracted with EtOAc (4 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and directly injected to the GC-MS. Almost total conversion was observed after 7 hours.

[0074] MS (ES+): m/z: 156.1 [M+23]. RRT.sub.9=1, RRT.sub.9a=1.22.

Example 8

[0075] 2-(2-Aminophenyl)acetamide NHase was grinded in a mortar and added to a potassium phosphate buffer (10 mM, pH=7.4) (9 mL) in a 50 mL Erlenmeyer. The suspension was mixed by vortex and 2-(2-aminophenyl)acetonitrile (66 mg, 0.5 mmol) was added to the suspension dissolved in MeOH (1 mL). The reaction was stirred by orbital shaking at 25 C. and 250 rpm. The reaction was followed by HPLC-MS. Samples (1 mL) were analyzed after 1.5, 2.5, 4 and 7 hours. Samples were mixed with MeOH (2 mL), filtered and directly injected to the HPLC. Total conversion was observed after 2.5 hours.

[0076] MS (ES.sup.+): m/z: 151.1 [M+1]. RRT.sub.10=1, RRT.sub.10a=0.48.

Example 9

[0077] 2-Amino-2-oxoacetohydrazide

[0078] NHase was grinded in a mortar and added to a potassium phosphate buffer (10 mM, pH=7.4) (9 mL) in a 50 mL Erlenmeyer. The suspension was mixed by vortex and 2-cyanoacetohydrazide (50 mg, 0.5 mmol) was added to the suspension dissolved in MeOH (1 mL). The reaction was stirred by orbital shaking at 25 C. and 250 rpm. The reaction was followed by .sup.1H-NMR. Samples (1 mL) were analyzed after 1.5, 2.5, 4 and 7 hours. Samples were mixed with MeOH (2 mL), filtered, evaporated under vacuum and analyzed by .sup.1H-NMR. Total conversion was observed after 1.5 hours.

[0079] .sup.1H-NMR (CD.sub.3OD, 300.13 MHz): 3.48-3.58 (m, 2 H).

Example 10

[0080] 2-(2-Cyanophenyl)acetamide

[0081] NHase was grinded in a mortar and added to a potassium phosphate buffer (10 mM, pH=7.4) (9 mL) in a 50 mL Erlenmeyer. The suspension was mixed by vortex and 2-(cyanomethyl)benzonitrile (71 mg, 0.5 mmol) was added to the suspension dissolved in MeOH (1 mL). The reaction was stirred by orbital shaking at 25 C. and 250 rpm. The reaction was followed by HPLC-MS and .sup.1H-NMR. Samples (1 mL) were analyzed after 1.5, 2.5, 4 and 7 hours. Samples were mixed with MeOH (2 mL), filtered and directly injected to the HPLC. The rest of the sample that was not injected was evaporated under vacuum and analyzed by .sup.1H-NMR. Total conversion was observed after 1.5 hours.

[0082] .sup.1H-NMR (CD.sub.3OD, 300.13 MHz): 3.81 (s, 2 H), 7.49 (t, 2 H, .sup.3J.sub.HH=7.6 Hz), 7.70 (d, 2 H, .sup.3J.sub.HH=7.6 Hz).

[0083] MS (ES.sup.+): m/z: 161.1 [M+1]

Example 11

[0084] 2-(Isopropylsulfonyl)acetamide

[0085] NHase was grinded in a mortar and added to a potassium phosphate buffer (10 mM, pH=7.4) (9 mL) in a 50 mL Erlenmeyer. The suspension was mixed by vortex and 2-(isopropylsulfonyl)acetonitrile (74 mg, 0.5 mmol) was added to the suspension dissolved in MeOH (1 mL). The reaction was stirred by orbital shaking at 25 C. and 250 rpm. The reaction was followed by HPLC-MS and .sup.1H-NMR. Samples (1 mL) were analyzed after 1.5, 2.5, 4 and 7 hours. Samples were mixed with MeOH (2 mL), filtered and directly injected to the HPLC. The rest of the sample that was not injected was evaporated under vacuum and analyzed by .sup.1H-NMR. Total conversion was observed after 1.5 hours.

[0086] .sup.1H-NMR (CD.sub.3OD, 300.13 MHz): 1.35 (s, 3 H), 1.38 (s, 1 H), 3.52-3.61 (m, 1 H), 4.60 (s, 2 H, overlapped with the water of CD.sub.3OD).

[0087] MS (ES.sup.+): m/z: 166.1 [M+1]. RRT.sub.14=1, RRT.sub.14a=0.54.

Example 12

[0088] Ethyl (R)-5-amino-3-hydroxy-5-oxopentanoate

[0089] NHase was grinded in a mortar and added to a potassium phosphate buffer (10 mM, pH=7.4) (9 mL) in a 50 mL Erlenmeyer. The suspension was mixed by vortex and ethyl (R)-4-cyano-3-hydroxybutanoate (79 mg, 0.5 mmol) was added to the suspension dissolved in MeOH (1 mL). The reaction was stirred by orbital shaking at 25 C. and 250 rpm. The reaction was followed by .sup.1H-NMR. Samples (1 mL) were analyzed after 1.5, 2.5, 4 and 7 hours. Samples were mixed with MeOH (2 mL), filtered, evaporated under vacuum and analyzed by .sup.1H-NMR. 34% Conversion was observed after 7 hours.

[0090] .sup.1H-NMR (CD.sub.3OD, 300.13 MHz): 1.35 (t, 3 H, .sup.3J.sub.HH=2.3 Hz), 2.41-2.77 (4 H, m, overlapped with the signals of the starting compound), 3.70 (s, 2 H), 4.40-4.43 (m, 1 H).

[0091] MS (ES.sup.+): m/z: 176.1 [M+1].

Example 13

[0092] N.sup.1-Phenylmalonamide

[0093] NHase was grinded in a mortar and added to a potassium phosphate buffer (10 mM, pH=7.4) (8 mL) in a 50 mL Erlenmeyer. The suspension was mixed by vortex and 2-cyano-N-phenylacetamide (80 mg, 0.5 mmol) was added to the suspension dissolved in MeOH (1 mL). The reaction was stirred by orbital shaking at 25 C. and 250 rpm. The reaction was followed by HPLC-MS. Samples (1 mL) were analyzed after 1.5, 2.5, 4 and 7 hours. Samples were mixed with MeOH (2 mL), filtered and directly injected to the HPLC. Low conversion (13%) was observed after 4 hours.

[0094] MS (ES.sup.+): m/z: 176.9 [M1]. RRT.sub.17=1, RRT.sub.17a=0.61.

Example 14

[0095] Methyl 1 -carbamoylcyclopentan-1 -carboxylate

[0096] NHase was grinded in a mortar and added to a potassium phosphate buffer (10 mM, pH=7.4) (9 mL) in a 50 mL Erlenmeyer. The suspension was mixed by vortex and methyl 1-cyanocyclopentan-1-carboxylate (77 mg, 0.5 mmol) was added to the suspension dissolved in MeOH (1 mL). The reaction was stirred by orbital shaking at 25 C. and 250 rpm. The reaction was followed by GC-MS. Samples (1 mL) were analyzed after 1.5, 2.5, 4 and 7 hours. Samples were mixed with MeOH (2 mL) and filtered. Water (2 mL) was added to the sample and the samples were extracted with EtOAc (4 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and directly injected to the GC-MS. About 72% conversion was observed after 7 hours.

[0097] MS (ES.sup.+): m/z: 171.9 [M+1]. RRT.sub.19=1, RRT.sub.19a=1.11.

Example 15

[0098] 6-Bromo-2,2-dimethylhexanamide

[0099] NHase was grinded in a mortar and added to a potassium phosphate buffer (10 mM, pH=7.4) (9 mL) in a 50 mL Erlenmeyer. The suspension was mixed by vortex and 6-bromo-2,2-dimethylhexanenitrile (102 mg, 0.5 mmol) was added to the suspension dissolved in MeOH (1 mL). The reaction was stirred by orbital shaking at 25 C. and 250 rpm. The reaction was followed by GC-MS. Samples (1 mL) were analyzed after 1.5, 2.5, 4 and 7 hours. Samples were mixed with MeOH (2 mL) and filtered. Water (2 mL) was added to the sample and the samples were extracted with EtOAc (4 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and directly injected to the GC-MS. About 64% conversion was observed after 7 hours.

[0100] MS (ES+): m/z: 223.9 [M+1]. RRT.sub.20=1, RRT.sub.20a=1.12.

Example 16

[0101] (E)-5-Cyanopent-3-enamide

[0102] NHase was grinded in a mortar and added to a potassium phosphate buffer (10 mM, pH=7.4) (9 mL) in a 50 mL Erlenmeyer. The suspension was mixed by vortex and (E)-hex-3-enedinitrile (53 mg, 0.5 mmol) was added to the suspension dissolved in MeOH (1 mL). The reaction was stirred by orbital shaking at 25 C. and 250 rpm. The reaction was followed by .sup.1H-NMR. Samples (1 mL) were analyzed after 1.5, 2.5, 4 and 7 hours. Samples were mixed with MeOH (2 mL), filtered, evaporated under vacuum and analyzed by .sup.1H-NMR. 97% Conversion was observed after 1.5 hours. From the .sup.1H-NMR spectrum the presence of the corresponding diamide in a percentage of about 7% can be hypothesized.

[0103] .sup.1H-NMR (CD.sub.3OD, 300.13 MHz): 3.00-3.19 (m, 2 H), 3.21-3.30 (m, 2 H), 5.55-5.50 (m, 1 H), 5.90-6.00 (m, 1 H). MS (ES.sup.+): m/z: 142.1 [M+18].

Example 17

[0104] 2-((Cyanomethyl)amino)acetamide

[0105] NHase was grinded in a mortar and added to a potassium phosphate buffer (10 mM, pH=7.4) (9 mL) in a 50 mL Erlenmeyer. The suspension was mixed by vortex and 2,2-azanediyldiacetonitrile (48 mg, 0.5 mmol) was added to the suspension dissolved in MeOH (1 mL). The reaction was stirred by orbital shaking at 25 C. and 250 rpm. The reaction was followed by .sup.1H-NMR. Samples (1 mL) were analyzed after 1.5, 2.5, 4 and 7 hours. Samples were mixed with MeOH (2 mL), filtered, evaporated under vacuum and analyzed by .sup.1H-NMR. Total conversion was observed after 1.5 hours. From the .sup.1H-NMR spectrum the presence of the corresponding diamide (about 30%) cab be hypothesized.

[0106] .sup.1H-NMR (CD3OD, 300.13 MHz): 3.336 (s, 2 H), 3.68 (s, 2 H).

[0107] MS (ES.sup.+): m/z: 113.9 [M+1].

Example 19

[0108] N.sup.1-Benzylmalonamide

[0109] NHase was grinded in a mortar and added to a potassium phosphate buffer (10 mM, pH=7.4) (9 mL) in a 50 mL Erlenmeyer. The suspension was mixed by vortex and N-benzyl-2-cyanoacetamide (80 mg, 0.5 mmol) was added to the suspension dissolved in MeOH (1 mL). The reaction was stirred by orbital shaking at 25 C. and 250 rpm. The reaction was followed by HPLC-MS. Samples (1 mL) were analyzed after 1.5, 2.5, 4 and 7 hours. Samples were mixed with MeOH (2 mL), filtered and directly injected to the HPLC. 80% Conversion was observed after 4 hours.

[0110] MS (ES.sup.+): m/z: 193.1 [M+1]. RRT.sub.25=1, RRT.sub.25a=0.65.

Example 20

[0111] 2-Phenylacetamide

[0112] NHase (4 mg) was added to a solution of 2-phenylacetonitrile (59 mg, 0.5 mmol) in potassium phosphate buffer (10 mM, 1% MeOH, pH=7.4) (10 mL) in a 50 mL Erlenmeyer. The reaction was stirred by orbital shaking at 25 C. Samples (200 L) were measured by HPLC-MS and .sup.1H-NMR. The reaction was stopped after 4 hours. Reactions were extracted with DCM (15 mL2). The organic layers were combined, dried over Na.sub.2SO.sub.4, filtered and evaporated under vacuum. 2-Phenylacetamide was isolated in 37% yield as a white solid.

[0113] .sup.1H-NMR (CDCl.sub.3, 300.13 MHz): 3.70 (s, 2 H), 5.16 (bs, 1 H), 5.73 (bs, 1 H), 7.27-7.43 (m, 5 H).

Example 21

[0114] 3-Phenylpropanamide

[0115] NHase (4 mg) was added to a solution of 3-phenylpropanenitrile (66 mg, 0.5 mmol) in potassium phosphate buffer (10 mM, 1% MeOH, pH=7.4) (10 mL) in a 50 mL Erlenmeyer. The reaction was stirred by orbital shaking at 25 C. Samples (200 L) were measured by HPLC-MS and .sup.1H-NMR. The reaction was stopped after 4 hours.

[0116] Reactions were extracted with DCM (15 mL2). The organic layers were combined, dried over Na.sub.2SO.sub.4, filtered and evaporated under vacuum. 3-Phenylpropanamide was isolated in 99% yield as a white solid. .sup.1H-NMR (CDCl.sub.3, 300.13 MHz): 2.55 (t, 2 H, .sup.3J.sub.HH=8.2 Hz), 3.00 (t, 2 H, .sup.3J.sub.HH=8.2 Hz), 5.16-5.47 (bs, 1 H), 5.73 (bs, 1 H), 7.17-7.36 (m, 5 H).

Example 22

[0117] Ethyl 3-amino-3-oxo-2-phenylpropanoate

[0118] NHase (4 mg) was added to a solution of () ethyl 2-cyano-2-phenylacetate (59 mg, 0.5 mmol) in potassium phosphate buffer (10 mM, 1% toluene, pH=7.4) (10 mL) in a 50 mL Erlenmeyer. The reaction was stirred by orbital shaking at 25 C. Samples (500 L) were extracted with DCM and measured by UPC-MS. The reaction was analyzed at different times. The conversion observed in these conditions after one hour was 30% and ethyl 3-amino-3-oxo-2-phenylpropanoate was obtained with 97% ee.

[0119] The comparison with the .sup.1H-NMR spectrum of the amide obtained by synthetic route confirmed the structure.

Example 23

[0120] 2-Phenylpropanamide

[0121] NHase (4 mg) was added to a solution of () 2-phenylpropanenitrile (66 mg, 0.5 mmol) in potassium phosphate buffer (10 mM, 1% toluene, pH=7.4) (10 mL) in a 50 mL Erlenmeyer. The reaction was stirred by orbital shaking at 25 C. Samples (500 L) were extracted with DCM and measured by GC-MS. The reaction was analyzed at different times. The conversion observed in these conditions after one hour was 83% and (S)-2-phenylpropanamide was obtained with 82% ee.

[0122] The comparison with the .sup.1H-NMR spectrum of the amide obtained by synthetic route confirmed the structure.

Example 24

[0123] 5-Cyanopentanamide

[0124] NHase (16 mg) was grinded in a mortar and added to demineralized water (40 mL) in a 100 mL Erlenmeyer. The suspension was mixed by vortex and adiponitrile (224 L, 0.5 mmol) was added to this suspension dissolved in toluene (400 4). The reaction was stirred by orbital stirring at 25 C. and 250 rpm. The reaction was followed by GC-MS. Samples (1 mL) were analyzed in time. Samples were mixed with MeOH (2 mL), filtered, evaporated under vacuum and dissolved in MeOH. 5-Cyanopentanamide (92 mg) was isolated in 37% yield.

[0125] RRT.sub.amide=1.12, RRT.sub.nitrile=1.

[0126] .sup.1H-NMR (CD.sub.3OD, 300.13 MHz): 1.59-1.84 (m, 4H), 2.26 (t, 2H, .sup.3J.sub.HH=7.0 Hz), 2.49 (t, 2H, .sup.3J.sub.HH=7.0 Hz).

[0127] MS (El): m/z: 126.1 [M]