Efficient Enzymatic Synthesis Of(S)-N-Boc-3-Hydroxypiperidine In The Aqueous/Organic Biphasic System

Nitrogen heterocycles are ubiquitous structure of small molecule active drugs.?S?-N-Boc-3-hydroxypiperidine,one of six-membered nitrogen heterocycles,is the building block for the synthesis of various drug intermediates.Oxidoreductases can act as potential catalyst in the asymmetric synthesis of?S?-N-Boc-3-hydroxypiperidine,but still warrant improvements on their catalytic performance,e.g.,incompatibility of activity and selectivity,low efficiency of cofactor regeneration,and low substrate load.The study aims to achieve highly efficient asymmetric synthesis of high optical purity of?S?-N-Boc-3-hydroxypiperidine in high substrate load,and the detailed study has been highlighted as followed:1.The medium-chain carbonyl reductase from Rhodococcus erythropolis?ReCR?exhibited outstanding catalytic properties.ReCR showed the highest specific activity of 85.5 U/mg and k_cat/K_m value of35.96 s^-1mM^-1in the reduction of N-Boc-3-piperidone,but displayed no activity in the oxidation of?S?-N-Boc-3-hydroxypiperidine.The specific activity and k_cat/K_m value for 2-octanone reduction were 173.95 U/mg and43.23 s^-1mM^-1,and those for 2-octanol oxidation were 88 U/mg and 13.05s^-1mM^-1.Furthermore,ReCR had good thermostability.The enzyme kept54.26%of the initial activity at 55?after 6 h,and 90%of the initial activity at 35?after 12 h.It also retained 68%of the initial activity in the presence of 40%?v/v?2-octanol.In addition,ReCR exhibited high stereoselectivity since it only afforded?S?-N-Boc-3-hydroxypiperidine in the asymmetric reduction of N-Boc-3-piperidone.2.ReCR-catalyzedasymmetricsynthesisof?S?-N-Boc-3-hydroxypiperidine in high substrate load was driven by a novel cofactor regeneration system,in which 2-octanol was used as co-substrate,co-solvent and organic phase.The optimized conditions for whole-cell catalyzed reduction of for 10%?w/v?substrate were listed as followed:pH 8.0,35?,300 rpm,0.4 mM of external cofactor NAD⁺,1:4for the mole ratio of substrate and co-substrate,and 6:4 for the volume ratio of aqueous phase and organic phase.The whole-cell catalyzed reductionofN-Boc-3-piperidonedisplayedexcellent stereoselectivity?>99%?.When the reduction of high concentration of substrate?10%and 20%?w/v??was carried out at pH 8.0,35?and 200rpm,the product yields after 10 h reached up to 99%and 94%,respectively.When the substrate load was increased to 30%and40%?w/v?,the yields were 76%and 56%,respectively.When the rotation speed was 300 rpm,the 6 h reduction of 30%?w/v?substrate led to the yield of 82%.3.The engineering of recombinant ReCR was conducted through the strategy of rational design.Among nine constructed mutants,two mutants F43Y and Y54F showed higher activity.Particularly,the mutant Y54F displayed significant improvements on the specific activities of N-Boc-3-piperidone reduction?112.68 U/mg?and 2-octanol oxidization?121.7 U/mg?.The k_cat/K_m values of N-Boc-3-piperidone and 2-octanol were 49.25 s^-1mM^-1and 56.54 s^-1mM^-1.When the whole cells of Y54F catalyzed the reduction of 30%?w/v?N-Boc-3-piperidone in the presence of 60%?v/v?2-octanol for coenzyme regeneration for 6 h,the yield,the total turnover number and the space-time yield were 91.46%,1142 and1096.8 g L^-1d^-1,respectively.The final yield after 12 h reaction reached up to 93.92%,which was 11.8%higher than that of the native one.The docking analysis of the mutant Y54F and the substrate?N-Boc-3-piperidone or 2-octanol?illustrated that both the carbonyl oxygen of N-Boc-3-piperidone and hydroxy oxygen of 2-octanol were closer to the Zn²⁺ion,which might facilitate transferring hydrogen to coenzyme.4.In the ReCR-catalyzed asymmetric synthesis of?S?-N-Boc-3-hydroxypiperidine,alcohol dehydrogenase ADH-A-driven coenzyme regeneration was investigated using 2-octanol as co-substrate.ADH-A from Rhodococcus ruber DSM 44541 were over-expressed in Echerichia coli BL21?DE3?and had high specific activity of 113.15U/mg for 2-octanol oxidation at pH 8.5 and 75?.The purified ADH-A showed a good adaptation for broad range of pH?7.0¹0.0?and temperature?30?⁸5??.Two plasmids carrying the encoding genes of ADH-A and ReCR were co-expressed in the same host strain.Either enzymatic catalysis or whole-cell catalysis was investigated for the reduction of 20%?w/v?N-Boc-3-piperidone using both ADH-A and ReCR,and the results were unsatisfactory.In the enzymatic catalysis,8 U/mL ReCRcoupledwith4U/mLADH-Areduced20%?w/v?N-Boc-3-piperidone for 5h and the yield was 49.78%,meanwhile 8 U/mL ReCR alone caused the yield of 51.42%of yield.In the whole-cell catalysis,0.15 g/mL ADH-A and ReCR co-expressed whole cells led to the yield of 44.3%after 5 h reudction of 20%?w/v?N-Boc-3-piperidone,but the only ReCR-expressed whole cells afforded the yield of 66.47%.