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

Taresh P Khobragade,Hyunwoo Jeon,Sharad Sarak,Hyungdon Yun,Pritam Giri,Amol D Pagar

doi:10.3389/fbioe.2021.757062

Taresh P Khobragade, Hyunwoo Jeon + Show 4 more

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https://doi.org/10.3389/fbioe.2021.757062

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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.

Highlights

Biocatalysts continue to be explored for consideration as green alternatives to alleviate the environmental issues encountered with the use of methods pertaining to traditional organic chemistry (Patil et al, 2018; Pagar et al, 2021)
Few TAs showed comparable activity, the considerably lower conversion could be attributable to enzyme inhibition by benzaldehyde, which was generated as a deaminated co-product of benzylamine during the reaction
Literature reports have evidenced that aldehyde reductase (AHR) demonstrates good activity towards benzaldehyde and can efficiently catalyze its conversion into benzyl alcohol at the expense of NAD(P)H; we selected AHR and formate dehydrogenase (FDH) for recycling of the depleted NAD(P)H cofactor

Summary

Introduction

Biocatalysts continue to be explored for consideration as green alternatives to alleviate the environmental issues encountered with the use of methods pertaining to traditional organic chemistry (Patil et al, 2018; Pagar et al, 2021). Sitagliptin is a well-reported example of a drug containing a β-amino acid moiety as a key component. Merck has described the industrial manufacturing method of sitagliptin phosphate. The hydroxyl group was transformed into protected amino acid through several steps, followed by direct coupling to triazolopiperazine to afford sitagliptin. The complete process involves eight steps to enable the production of sitagliptin phosphate with an overall yield of 52% (Hansen et al, 2005; Hansen et al, 2009). The second-generation process comprised a three-step one-pot synthesis of dehydro-sitagliptin. The synthesis was initiated with trifluoro phenylacetic acid, followed by serial and controlled addition of other reactants and reagents, resulting in the formation of sitagliptin phosphate. The product was crystallized and separated via simple filtration and resulted in the obtainment of 82% overall yield (Hsiao et al, 2004; Xu et al, 2004; Clausen et al, 2006)

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