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

Abstract

Granulicella tundricola hydroxynitrile lyase (GtHNL) catalyses the synthesis of chiral (R)-cyanohydrins and (R)-β-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 hours 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 hours on stream. A similar yield was observed during 15 hours 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/mgenzyme) of 0.10 g L−1 h−1 mgenzyme−1 whereas the flow system displayed 0.02 and 0.003 g L−1 h−1 mgenzyme−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.

Hydroxynitrile Lyase Discovery, Engineering, and Promiscuity towards Asymmetric Synthesis: Recent Progress

“…improved enzymatic activity and broad substrate selectivity, synthesized (R)-2-chloro MN, (R)-2-hydroxy-4-phenylbutyronitrile, (R)-2hydroxy-4-phenylbut-3-ene nitrile, and (S)-2-(furan-2-yl)-2-hydroxyacetonitrile [47] GtHNL SDM GtHN-A40H/V42T/Q110H immobilized on Celite R-633 improved rate, % ee and conv. in (R)-MN synthesis in a continuous flow reactor, while higher STY of (R)-NPE synthesis in a batch system [49,50] GtHNL& AcHNL SDM A40H, A40R, A40H-V42T-Q110H of GtHNL& AcHNL (except GtHNLA40H, rest of the above mutants) increased % conv and % ee in the synthesis of (R)-NPE. broad substrate selectivity, synthesized (R)-1-(2-chloro phenyl)-2-nitro ethanol, (R)-1-cyclohexyl-2-nitroethanol and (R)-1-nitroheptan-2-ol, also increased % conv, % ee and % de in diastereoselective synthesis of (R)anti product i. e. (1R, 2S)-2-NPP [48] AtHNL SSM Y14F, Y14M, F179N, F179W and Y14M-F179W…”

“…[49] The Celite R-633 immobilized GtHNL-3 V was used in monophasic MTBE saturated with 100 mM KPi buffer pH 7 for the synthesis of (R)-NPE in batch and continuous flow systems. [50] The batch system has produced 82 % of (R)-NPE with > 99 % ee in 24 h of reaction time and was successfully recycled for five times. This method exhibited a STY/mgenzyme of 0.10 g L À 1 h À 1 mgenzyme À 1 whereas the flow system displayed only 0.02 and 0.003 g L À 1 h À 1 mgenzyme À 1 at 0.1 and 0.01 mLmin À 1 , respectively.…”

“…in 4 minutes at 0.1 mL min −1 ) and enantioselectivity (>99 %) were achieved within minutes along with high operational stability [49] . The Celite R‐633 immobilized Gt HNL‐3 V was used in monophasic MTBE saturated with 100 mM KPi buffer pH 7 for the synthesis of ( R )‐NPE in batch and continuous flow systems [50] . The batch system has produced 82 % of ( R )‐NPE with >99 % ee in 24 h of reaction time and was successfully recycled for five times.…”

Can a Hydroxynitrile Lyase Catalyze an Oxidative Cleavage?

“…Two manganese containing cupins have attracted special attention as interesting biocatalysts. Granulicella tundricola hydroxynitrile lyase ( Gt HNL) catalyzes both the synthesis of cyanohydrins (Scheme A) and, as a promiscuous activity of all known HNLs, the nitro-aldol reaction (Henry reaction). − Both reactions are catalyzed by the Lewis acidic Mn­(II) that activates the carbonyl compound, whereas the His106 residue acts as base to deprotonate the nucleophiles (Figure ). The cupin Tm 1459 from Thermotoga maritima (Figure ) has been reported to oxidatively cleave alkenes with tert -butyl hydroperoxide (TBHP) in the presence of oxygen from air (Scheme B). , Hydrogen peroxide was not a suitable oxidation reagent, making it a rather unusual reaction in biocatalysis.…”

Hydroxynitrile lyase engineering for promiscuous asymmetric Henry reaction with enhanced conversion, enantioselectivity and catalytic efficiency

“…They are Hevea brasiliensis HNL (HbHNL), AtHNL, Granulicella tundricola (GtHNL), Acidobacterium capsulatum (AcHNL) and Baliospermum montanum HNL (BmHNL). [13][14][15][16][17][18][19] Among them, only AtHNL, and two metal dependent cupin fold HNLs, GtHNL, and AcHNL exhibit (R)-selectivity. Enantioselective Henry reaction of GtHNL, AcHNL, and their variants is restricted to only four substrates.…”