Organic Acid to Nitrile: A Chemoenzymatic Three‐Step Route

Abstract Various widely applied compounds contain cyano‐groups, and this functional group serves as a chemical handle for a whole range of different reactions. We report a cyanide free chemoenzymatic cascade for nitrile synthesis. The reaction pathway starts with a reduction of carboxylic acid to aldehyde by carboxylate reductase enzymes (CARs) applied as living cell biocatalysts. The second – chemical – step includes in situ oxime formation with hydroxylamine. The final direct step from oxime to nitrile is catalyzed by aldoxime dehydratases (Oxds). With compatible combinations of a CAR and an Oxd, applied in one‐pot two‐step reactions, several aliphatic and aryl‐aliphatic target nitriles were obtained in more than 80% conversion. Phenylacetonitrile, for example, was prepared in 78% isolated yield. This chemoenzymatic route does not require cyanide salts, toxic metals, or undesired oxidants in contrast to entirely chemical procedures.


1.
Additional data and figures

GC-FID analysis
For GC-FID measurements, a ZP-5 column (crosslinked 5% Ph-Me Siloxane; 30 m, 0.32 mm diameter, 0.25 μm film thickness) on a Shimadzu GC 2030 equipped with an FID was used. Sample aliquots of 1 μL were injected in split mode (split ratio 10:1) at 240°C injector temperature and 320°C detector temperature with N2 as carrier gas. The temperature gradient for 3a, 3b, E/Z-3c, 3d and 3e as well as 7a,  7b, E/Z-7c, 7d and 7e was reported previously [16] and 4a, 4b, E/Z-4c, 4d and 4e were also analyzed with this method. The temperature gradient for 1a, 1b, E/Z-1c, 1d and 1e started with a hold at 50°C for 5 min, followed by temperature gradients to 80°C at 5°C min -1 and then to 300°C at 40°C min -1 and a hold at 300°C for 2 min. The total run time was 18.5 min. The temperature gradient for 2a, 2b, E/Z-2c, 2d and 2e started with a hold at 60°C for 6 min, followed by temperature gradients to 100°C at 10°C min -1 and then to 300°C at 40°C min -1 and a hold at 300°C for 2 min. The total run time was 17.0 min. The temperature program for quantification of 8a, 8b, E/Z-8c, 8d and 8e started with a hold at 70°C for 3 min, followed by a temperature gradient to 250°C at 40°C min -1 and a hold for 5 min, continued by a temperature ramp to 300°C at 40°C min -1 and a hold for 3 min. The total runtime was 16.75 min. GC-FID results were evaluated with the GC-FID Data Analysis software LabSolution (Shimadzu). Quantification of all compounds was established through linear intrapolation from calibration curves with authentic standard.

HPLC-UV analysis
HPLC-UV analysis for compounds 5, 6 and derivatives were performed according to the methods previously reported. [1,17]

Butyraldehyde oxime (1c) synthesis and purification
Butyraldehyde oxime 1c was synthesized according to Hinzmann et al. [20] 1b was distilled prior to use. A RBF (250 mL) was charged with sodium carbonate (12.26 g, 115.7 mmol, 0.75 equiv.) which was dissolved in water (285 mL) and ethanol (15 mL). Subsequently, hydroxylamine hydrochloride (16.19 g, 233 mmol, 1.5 equiv.) and 1b (11.1 g, 154 mmol, 1 equiv.) were added. Vacuum was applied to the flask for a short time and then the flask was purged with argon. The reaction solution was stirred at rt under Ar atmosphere overnight (19 h). NMR analysis of the reaction mixture confirmed full conversion. The ethanol was removed by rotary evaporation. Subsequently the aqueous layer was extracted with diethyl ether (3 x 150 mL). The combined organic layers were washed with brine (150 mL) and then dried over Na2SO4. The solvent was removed by rotary evaporation to yield 6.57 g (50 %) 1c as colorless liquid with an E/Z-ratio of 55/45.

Pentanal oxime (2c) synthesis and purification
Pentanal oxime 2c was synthesized according to Hinzmann et al. [20] 2b was distilled prior to use. A RBF (250 mL) was charged with sodium carbonate (2.84 g, 26.7 mmol, 0.75 equiv.) which was dissolved in water (90 mL) and ethanol (4.75 mL). Subsequently, hydroxylamine hydrochloride (3.81 g, 54.8 mmol, 1.50 equiv.) and 2b (3.16 g, 35.6 mmol, 1.00 equiv.) were added. The reaction solution was stirred for 18 h at rt under argon atmosphere during which an oily layer was formed at the surface. As TLC analysis (LP:EA = 25:1, stained with KMnO4) showed incomplete conversion, a second portion of ethanol (5 mL) was added. After additional 4 h, TLC analysis confirmed full conversion. The aqueous solution was extracted with diethyl ether (3 x 100 mL). The combined organic layers were washed with brine (100 mL) and then dried over Na2SO4. The solvent was removed by rotary evaporation and a clear, oily residue was obtained. The crude product was purified by flash chromatography (DCM + 1 % MeOH, 80 g silica gel 60) to yield 1.05 g (29 %) 2c as colorless crystals with an E/Z-ratio of 54/46.

Hexanal oxime (3c) synthesis and purification
Hexanal oxime 3c was synthesized according to Hinzmann et al. [19] 3b was distilled prior to use. A RBF (250 mL) was charged with sodium carbonate (7.21 g, 68.0 mmol, 0.75 equiv.) was dissolved in water (142.5 mL) and ethanol (7.5 mL). Subsequently, hydroxylamine hydrochloride (9.54 g, 137 mmol, 1.50 equiv.) and 3b (9.02 g, 90.1 mmol, 1.00 equiv.) were added. The reaction was stirred for 19 h at rt under argon atmosphere during which an oily layer was formed at the surface. TLC analysis (LP, stained with KMnO4) confirmed full conversion. The aqueous solution was extracted with diethyl ether (3 x 100 mL). The organic layer was washed with brine (100 mL) and then dried over Na2SO4 and concentrated to yield a clear, oily residue. The crude product was purified by flash chromatography (CH2Cl2 + 1 % MeOH) and yielded 7.67 g of 3c (74 %) as colorless crystals with an E/Z-ratio of 55/45.