We developed a multienzyme biocatalytic cascade with high atom efficiency and a self-sufficient redox network for the synthesis of nylon monomers without adding auxiliary enzymes to recycle cofactors.
Here, we report a bienzymatic cascade to produce β-amino acids as an intermediate for the synthesis of the leading oral antidiabetic drug, sitagliptin. A whole-cell biotransformation using recombinant Escherichia coli coexpressing a esterase and transaminase were developed, wherein the desired expression level of each enzyme was achieved by promotor engineering. The small-scale reactions (30 ml) performed under optimized conditions at varying amounts of substrate (100-300 mM) resulted in excellent conversions of 82%-95% for the desired product. Finally, a kilogramscale enzymatic reaction (250 mM substrate, 220 L) was carried out to produce β-amino acid (229 mM). Sitagliptin phosphate was chemically synthesized from β-amino acids with 82% yield and > 99% purity.
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.
Two polynuclear end‐to‐end dicyanamide (dca) bridged copper(II) Schiff base complexes [Cu(L1)(μ1,5‐dca)]n (1) and [Cu(L2)(μ1,5‐dca)]n (2) {[HL1=(1‐(2‐(diethylamino)ethylimino)ethyl) naphthalene‐2‐ol] and [HL2=(1‐(2‐(dimethylamino)ethylimino)methyl) naphthalene‐2‐ol]} were synthesized and X‐ray characterized. Complex 1 crystallizes in chiral space group C2 and complex 2 crystallizes in achiral space group P21/c. Interactions of both complexes with calf thymus DNA (CT DNA) were studied by UV‐vis and circular dichroism spectroscopy. Molecular docking studies were also carried out for both complexes to find their binding affinity using Alpha PMI as the placement methodology. Although docking studies indicated that the binding constants of both complexes with CT DNA were more or less same, UV‐Vis spectral data indicated that the binding constant of complex 1 with CT DNA is considerably large compared to that of complex 2. As complex 1 is chiral and complex 2 is achiral, it may be concluded that the chirality of complex 1 played very significant role to binding of chiral DNA molecule. The antimicrobial activity were estimated by the determination of the minimal inhibitory concentration (MIC) using the broth microdilution method. The complexes showed high in vitro cytotoxicity against MDA‐MB 468 cells.
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