Batch and Flow Nitroaldol Synthesis Catalysed by Granulicella tundricola Hydroxynitrile Lyase Immobilised on Celite R-633

Granulicella tundricola hydroxynitrile lyase (GtHNL) catalyses the synthesis of chiral (R)-cyanohydrins and (R)-beta-nitro alcohols. The triple variant GtHNL-A40H/V42T/Q110H (GtHNL-3V) was immobilised on Celite R-633 and used in monophasic MTBE saturated with 100 mM KPi buffer pH 7 for the synthesis of (R)-2-nitro-1-phenylethanol (NPE) in batch and continuous flow systems. Nitromethane was used as a nucleophile. A total of 82% of (R)-NPE and excellent enantioselectivity (>99%) were achieved in the batch system after 24 h of reaction time. GtHNL-3V on Celite R-633 was successfully recycled five times. During more recycling steps a significant decrease in yield was observed while the enantioselectivity remained excellent over eight cycles. The use of a flow system enabled the continuous synthesis of (R)-NPE. A total of 15% formation of (R)-NPE was reached using a flow rate of 0.1 mL min(-1); unfortunately, the enzyme was not stable, and the yield decreased to 4% after 4 h on stream. A similar yield was observed during 15 h at a rate of 0.01 mL min(-1). Surprisingly the use of a continuous flow system did not facilitate the process intensification. In fact, the batch system displayed a space-time-yield (STY/mg(enzyme)) of 0.10 g L-1 h(-1) mg(enzyme)(-1) whereas the flow system displayed 0.02 and 0.003 g L-1 h(-1) mg(enzyme)(-1) at 0.1 and 0.01 mL min(-1), respectively. In general, the addition of 1 M nitromethane potentially changed the polarity of the reaction mixture affecting the stability of Celite-GtHNL-3V. The nature of the batch system maintained the reaction conditions better than the flow system. The higher yield and productivity observed for the batch system show that it is a superior system for the synthesis of (R)-NPE compared with the flow approach.

Automated summary

The Granulicella tundricola hydroxynitrile lyase (GtHNL) can catalyze the synthesis of chiral (R)-cyanohydrins and (R)-beta-nitro alcohols. In this study, the GtHNL-3V variant was immobilized on Celite R-633 and used in batch and continuous flow systems for the synthesis of (R)-2-nitro-1-phenylethanol. The batch system showed higher yield and productivity compared to the flow system, indicating that the batch system is superior for the synthesis of (R)-NPE.