Synthesis of Sitagliptin Intermediate by a Multi-Enzymatic Cascade System Using Lipase and Transaminase With Benzylamine as an Amino Donor

Abstract

Herein, we report the development of a multi-enzyme cascade using transaminase (TA), esterase, aldehyde reductase (AHR), and formate dehydrogenase (FDH), using benzylamine as an amino donor to synthesize the industrially important compound sitagliptin intermediate. A panel of 16 TAs was screened using ethyl 3-oxo-4-(2,4,5-trifluorophenyl) butanoate as a substrate (1). Amongst these enzymes, TA from Roseomonas deserti (TARO) was found to be the most suitable, showing the highest activity towards benzylamine (∼70%). The inhibitory effect of benzaldehyde was resolved by using AHR from Synechocystis sp. and FDH from Pseudomonas sp., which catalyzed the conversion of benzaldehyde to benzyl alcohol at the expense of NAD(P)H. Reaction parameters, such as pH, buffer system, and concentration of amino donor, were optimized. A single whole-cell system was developed for co-expressing TARO and esterase, and the promoter engineering strategy was adopted to control the expression level of each biocatalyst. The whole-cell reactions were performed with varying substrate concentrations (10–100 mM), resulting in excellent conversions (ranging from 72 to 91%) into the desired product. Finally, the applicability of this cascade was highlighted on Gram scale, indicating production of 70% of the sitagliptin intermediate with 61% isolated yield. The protocol reported herein may be considered an alternative to existing methods with respect to the use of cheaper amine donors as well as improved synthesis of (R) and (S) enantiomers with the use of non-chiral amino donors.

A Plurizyme with Transaminase and Hydrolase Activity Catalyzes Cascade Reactions

“…Furthermore, we tested 3‐OTfBE as a substrate. This ester is used to synthesize precursors of gliptins, namely, β‐amino acids obtained through bienzymatic cascade reactions catalyzed by esterases and transaminases [22, 23] . TR 2 E 2 was shown to efficiently hydrolyze 3‐OTfBE ( k cat : 32.47±1.13 min −1 ).…”

“…We further revealed that this bioinspired design facilitates the one‐step synthesis of a sitagliptin intermediate, specifically the conversion of 3‐OTfBE into 3‐ATfBA in a single one‐pot reaction (Scheme 2). This β‐amino acid is commonly synthesized through a bienzymatic cascade reaction that involves an esterase and a transaminase or a recombinant Escherichia coli strain coexpressing both enzymes [22, 23, 38] . A series of chiral 3‐substituted cyclo‐hexylamine derivatives, capsaicin analogs and nylon‐6 monomer 6‐aminohexanoic acid, to cite some significant examples, have also been synthesized by combining hydrolases with ωTAs as separate catalytic entities [39–41] .…”

“…This β-amino acid is commonly synthesized through a bienzymatic cascade reaction that involves an esterase and a transaminase or a recombinant Escherichia coli strain coexpressing both enzymes. [22,23,38] A series of chiral 3-substituted cyclo-hexylamine derivatives, capsaicin analogs and nylon-6 monomer 6-aminohexanoic acid, to cite some significant examples, have also been synthesized by combining hydrolases with ωTAs as separate catalytic entities. [39][40][41] It is worth mentioning that to date, only three native hydrolases, namely, those from Candida rugosa, Pseudomonas avellanae and P. stutzeri, [22,23,37] have been reported as capable of hydrolyzing 3-OTfBE and used in the cascade synthesis of 3-ATfBA.…”

“…We designed a PluriZyme , hereafter referred to as TR 2 E 2 , able to catalyze the one‐pot cascade synthesis of the β‐amino acid ( R )‐3‐amino‐4‐(2,4,5‐trifluorophenyl)butanoic acid (3‐ATfBA), a key precursor in the synthesis of gliptins (Scheme 1). [22, 23] Our results show that the design of this catalytically efficient PluriZyme is feasible and facilitates the generation of 3‐ATfBA from the methyl 3‐oxo‐4‐(2,4,5‐trifluorophenyl)butanoate (3‐OTfBE) β‐keto ester with an excellent conversion rate (>99 %; conc. : 14 mM) and enantioselectivity (e.e.…”

“…Therefore, as a model reaction, we chose to synthesize 3-ATfBA from 3-OTfBE; this molecule is an intermediate of gliptins. [22,23] Simulation of the reaction by a local PELE exploration showed efficient 3-ATfBE and 3-OTfBE catalytic (hydrolytic) binding poses: 23 catalytic events for 3-ATfBE and 42 for 3-OTfBE when the serine-substrate distance threshold was set to 4 Å (Figure S12). Glyceryl tripropionate, an example of an ester commonly hydrolyzed by most esterases, [36] had 121 catalytic events with an associated experimental k cat of 388.1 min À 1 (Table S5).…”