6 Conclusion
Water, as an abundant, cost-efficient, environmentally compatible, nontoxic, and nonflammable substance, is widely recognized as an important solvent for organic synthesis. In this study, a variety of saturated α-substituted β ketoesters were efficiently prepared though the alkylation of EAA and aldehydes via a one-pot tandem reaction catalyzed by an only NerA in H2O. In the first sub-step, the amino acid residues on the surface of NerA promoted the Knoevenagel condensation, and then, the enzyme reduced the received intermediates immediately and shifted the equilibrium toward the final products, so that the substrates could be converted continuously. This method was conducted under mild conditions, and eliminated the use of organic solvents and toxic metals, consistent with the concept of green chemistry. The results also offer an efficient, practical, and environmentally friendly way to prepare value-added saturated α-substituted β ketoesters from readily available compounds. Moreover, the transformation was suitable for the high yield and preparative-scale synthesis of pharmaceutical precursor. All in all, our approach is facile and easy to scale up, has great potential for “greener” syntheses of pharmaceutical precursor, compared to the established methods.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (21572212, 51821006, 51961135104), the National Key R&D Program of China (2018YFB1501604), the Major Science and Technology Projects of Anhui Province (18030701157), the Strategic Priority Research Program of the CAS (XDA21060101), and the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01N092), the Natural Science Foundation of the Anhui Higher Education Institutions of China (KJ2019A0273).
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Scheme 1. Design for the one-pot synthesis of saturated α-substituted β ketoesters catalyzed by single NerA in water.
Figure 1. The reduction activity of ERs toward 2a-2k .[a]Conditions: 2a-k (10 mM), NADH (0.1 mM) for NerA or NADPH (0.1 mM) for OYE 2.6 and IPR, enzyme (0.2-2.5 mg mL-1), sodium phosphate buffer (100 mM, pH 7), 25 °C,[b]The reducing activity was determined by measuring the decrease of NAD(P)H at 340 nm.[c]One unit of activity is as the amount of enzyme that convert 1 μmol NAD(P)H into NAD(P)+ per minute at 25 °C.
Figure 2. The effect of different substrate concentration on the yield of intermediate. [a]Condition:1b (2.5 mM, 5 mM, 10 mM, 20 mM, 50 mM), EAA (5 mM, 10 mM, 20 mM, 40 mM, 100 mM), NerA (2.4 mg mL-1), GDH (1.5 mg mL-1), H2O (1 mL) at 25 ºC.[b]The yield was determined by external standard method by GC.
Figure 3 . [a]Conditions: All reactions were carried out with 1b (50 mM), EAA (100 mM), glucose (100 mM) and solvent (2 mL) at 25 ºC. [b]For 3A, NADH (5 mM), 30% DMSO (V/V), NerA (2.4 mg mL-1) or GDH (1.5 mg mL-1) [c]For 3B and 3C, NADH (1 mM), NerA (2.4 mg mL-1), and GDH (1.5 mg mL-1); [d]For 3D, NADH (1 mM), NerA (2.4 mg mL-1) and GDH (1.5 mg mL-1) in 30% DMSO, NerA (3 mg mL-1) and GDH (1.5 mg mL-1) in water. All the yields were calculated by external standard method, and the external substance was 2-amyl acetoacetate.
Figure 4. Ethyl-2-acetylhept-2-enoates in the binding site. A stereo view of the region of the NerA molecule close to the ethyl (E )-2-acetylhept-2-enoate (A) and ethyl (Z )-2-acetylhept-2-enoate (B) molecule. Residues in close proximity to the substrate are represented as stick models. Oxygen and nitrogen atoms are colored in red and blue respectively. Ethyl-2-acetylhept-2-enoates are represented as cyan, and the distance (Å) between 2b and amino acids and N5 of FMN were measured and shown as yellow dashed lines.
Figure 5. Preparative-scale synthesis of Ethyl 2-[(4-chlorophenyl)methyl]-3-oxobutanoate.[a]Conditions: 1g (50 mM), EAA (100 mM), glucose (100 mM), NAD+ (1 mM), NerA (3 mg mL-1), GDH (1.5 mg mL-1) and water (10 mL), 25 ºC, 28h.
Table 1. Optimize the dosage of NerA and the molar ratio of substrate. [a]Conditions: 1b (50 mM), GDH (1.5 mg mL-1), glucose (100 mM), water (1 mL), NADH (1 mM) at 25 ºC.[b]The yield was determined by external standard method using GC.
Table 2. Substrate scope of the domino Knoevenagel condensation. [a]Condition: 1a-k (50 mM), β-keto esters (100 mM), glucose (100 mM), NAD+ (1 mM), NerA (3 mg mL-1), GDH (1.5 mg mL-1) for coenzyme recycle, water (10 mL) 25 ºC.