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Enzymatic synthesis of sitagliptin intermediate using a novel ω-transaminase
Kim, Geon-Hee,Jeon, Hyunwoo,Khobragade, Taresh P.,Patil, Mahesh D.,Sung, Sihyong,Yoon, Sanghan,Won, Yumi,Choi, In Suk,Yun, Hyungdon Elsevier 2019 Enzyme and microbial technology Vol.120 No.-
<P><B>Abstract</B></P> <P>Enantiopure β-amino acids are essential precursors of various pharmaceuticals, agrochemicals and other industrially important chemicals. In this study, we selected sixteen potential ω-Transaminases (ω-TAs) by BLAST and phylogenetic tree analysis. These ω-TAs were cloned, purified and tested for their reactivity for the synthesis of model β-amino acid (<I>R</I>)-3-amino-4-(2,4,5-triflurophenyl) butanoic acid [3-ATfBA], a key precursor for sitagliptin. In an enzymatic cascade, lipase converted β-ketoester substrate to β-keto acid, which was subsequently aminated by the selected ω-TA to its corresponding β-amino acid. A potent enzyme from <I>Ilumatobacter coccineus</I> (ω-TAIC) was identified for the production of 3-ATfBA. The pH dependency of the product inhibition suggested that lowering the reaction pH to 7.0 can circumvent the inhibition of ω-TAIC by 3-ATfBA and about 92.3% conversion of 100 mM β-keto ester substrate could be achieved. The applicability of this enzymatic system was further evaluated at the scale of 140 mM, wherein 3-ATfBA was generated with excellent conversion (81.9%) and enantioselectivity (99% <I>ee</I>). Furthermore, ω-TAIC was successfully used for the synthesis of various β-amino acids from their corresponding β-keto ester substrates.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A potent ω-TA was identified using bioinformatic tools from 16 potential enzymes. </LI> <LI> 3-ATfBA was produced with good conversion (81.9%) and 99% <I>ee.</I> </LI> <LI> ω -TAIC was successfully used for the synthesis of various β-amino acids. </LI> <LI> Lowering the reaction pH to 7.0 can circumvent the inhibition of ω -TAIC by 3-ATfBA. </LI> </UL> </P>
Biocatalytic Cascade for Synthesis of Sitagliptin Intermediate Employing Coupled Transaminase
Taresh P. Khobragade,Pagar Amol D.,GIRI PRITAM DEVIDAS,SARAKSHARADCHANGDEO,전현우,주상우,고영환,박부수,윤형돈 한국생물공학회 2023 Biotechnology and Bioprocess Engineering Vol.28 No.2
Transaminases (TAs) are employed in synthesizing various enantiopure β‐amino acids, key precursors for various pharmaceuticals. Sitagliptin, an oral hyperglycemic drug, is a well-known example. Herein, we developed the coupled enzyme cascade to synthesize the sitagliptin intermediate by fusing two different TAs to regenerate the amino donor. In a cascade system, ethyl 3-oxo-4-(2,4,5- trifluorophenyl) butanoate (1) was converted by esterase from Pseudomonas stutzeri (EstPS) to respective β-keto acid (2) which was subsequently converted by first TA to sitagliptin intermediate (3) using (S)-α-MBA as an amino donor and the acetophenone formed in the reaction was recycled by the second TA to (S)-α-MBA. A single wholecell system was established by the co-expression of esterase and TA fusion protein. The whole-cell biotransformation reaction was performed with varying substrate concentrations from 50-200 mM. The excellent conversion of the product was achieved, ranging from 62-to 100% at the expense of only 25 mM (S)-α-MBA. Notably, our designed system with fusion protein can produce ~5-fold higher product at the expense of 0.5 equivalent (S)-α-MBA. Finally, a preparative scale reaction was performed with 98% conversion.