Study On Asymmetric Reduction Of3-chloropropiophenone With Immobilized Acetobacter Sp. CCTCC M209061Cells

Enantiopure chiral alcohols are of tremendous application potential. For example,(S)-3-chlor-1-phenylpropanol is an essential intermediate for the preparations ofantidepressants (S)-fuoxetine, nisoxetine and (R)-tomoxetine. At present, asymmetricreduction of3-chloropropiophenone to (S)-3-chlor-1-phenyl-propanol can be achieved byeither chemical catalysis or biocatalysis. However, the latter offers obvious advantages overthe former such as milder reaction condition, higher enantioselectivity and higher efficiency.And use of whole-cell as the biocatalysts instead of isolated enzymes needs no enzymepurification or costly co-enzymes addition. In addition, immobilized cells are more suitablefor the reaction than non-immobilized cells considering that they can simplify the process andlower the cost of production because of their recyclability and reusability. To date, thebiocatalytic asymmetric reduction of3-chloropropiophenone to (S)-3-chlor-1-phenyl-propanol has remained unexplored largely, with only few accounts where the results obtainedwere not satisfactory. In this dissertation, a comparative study was made of the biocatalyticasymmetric reduction of3-chloropropiophenone to (S)-3-chlor-1-phenyl-propanol withimmobilized Acetobacter sp. CCTCC M209061cells in various reaction systems, especiallyin deep eutectic solvents (DES)-containing systems; then the effects of relevant factors onasymmetric reduction of3-chloropropiophenone were investigated systematically; meanwhile,the operational stabilitys of this biocatalyst in various reaction media were also examined, andthe biocatalytic process of3-chloropropiophenone to (S)-3-chlor-1-phenylpropanol in deepeutectic solvents (DES)-containing systems was evaluated on a preparative scale. Finally, thenovel biocatalytic reaction system for highly efficient and enantioselective synthesis of(S)-3-chlor-1-phenylpropanol was established.Among the14micoorganisms tested, the Acetobacter sp. CCTCC M209061isolated andpurified from China kefir grains bacteria by our group showed great potential for thebiocatalytic asymmetric reduction of3-chloropropiophenone to (S)-3-chlor-1-phenylpropanolwith the highest product yield and product e.e.. Meanwhile, Acetobacter sp. CCTCCM209061cells immobilized on PVA-sodium sulfate exhibited markedly enhanced stabilityincluding thermal stability, pH stability and recyclability, and the product yield obtained with immobilized Acetobacter sp. CCTCC M209061cells was equal to that observed with freecells (82.9%vs83.9%), with the product e.e. remaining above99.0%. So Acetobacter sp.CCTCC M209061cells immobilized on PVA-sodium sulfate were the best choice for theformation of (S)-3-chloro-1-phenyl-propanol.In aqueous monophasic system, the optimal co-substrate and its concentration, buffer pH,reaction temperature and substrate concentration for this bioreduction of3-chloro-propiophenone to (S)-3-chlor-1-phenyl-propanol were glucose (50mmol/L),5.5,30°C and3.0mmol/L, respectively. Under these conditions, the initial reaction rate, the maximum yieldand product e.e. were1.77mM/h,88.9%and above99.0%, respectively. But the substrateconcentration was poor due to pronounced inhibitory effects from the product.In order to solve these problems, a new type of green reaction medium with betterbiocompatibility, deep eutectic solvents, similar to ionic liquids, were added to the aqueoussystem. Research demonstrated that [ChCl][U] showed the best biocompatibility withAcetobacter sp. CCTCC M209061cells, and the results obtained in [ChCl][U]-containingsystem were the best among seven DESs tested. It was found that the volume content of[ChCl][U] had a significant effect on this reaction and the initial reaction rate and themaximum yield reached the highest for this reaction when it was5%. The optimalco-substrate concentration, substrate concentration, buffer pH and reaction temperature in[ChCl][U]-containing systems were found to be glucose (60mmol/L),10.0mmol/L,5.5,30°C, respectively. And under these optimal conditions, the initial reaction rate, themaximum yield and product e.e. were3.08mM/h,82.3%and above99.0%, respectively. The[ChCl][U]-containing systems could improve the reaction efficiency compared with aqueousmonophasic system (3.08mM/h vs1.77mM/h). The initial reaction rate, the maximun yieldand the product e.e. were3.05mM/h,81.8%and above99.0%when the reaction scale wasenlarged on500-mL preparative scale under the optimized conditions.Although the addition of [ChCl][U] into aqueous system could improve the reactionefficiency, the optimal substrate concentration was unsatisfactory because of pronouncedinhibitions of product. Therefore, various organic solvents and water-immiscible ILs wereadded to the aqueous system, which help extract reactants and products and improve reactionefficiency. It turned out that various second phases exhibited varied influences on asymmetric reduction of3-chloropropiophenone to (S)-3-chloro-1-phenyl-propanol with immobilizedAcetobacter sp. CCTCC M209061cells, and C4MIM·PF6showed the best biocompatibility.The reaction efficiency was the best when the volume percentage of C4MIM·PF6was20%.The optimal co-substrate and its concentration, substrate concentration and buffer pH for thisbioreduction in C4MIM·PF6/buffer biphasic system were glucose (70mmol/L),16.0mmol/Land4.5, respectively. And under these optimal conditions, the initial reaction rate, themaximum yield and product e.e. were1.50mM/h,89.9%and above99.0%, respectively.Accordingly, C4MIM·PF6/buffer biphasic system was more suitable for this bioreduction thanaqueous monophasic system. Meanwhile, effects of the addition of [ChCl][U] intoC4MIM·PF6/buffer biphasic system on this bioreduction were investgated, with the resultsthat this could considerably improve the initial reaction rate (3.02mM/h vs1.50mM/h), andthereby enhance the reaction efficiency in spite of the fact that the substrate concentration forthis bioreduction was not significantly increased (18.0mmol/L vs16.0mmol/L).Among the three reaction systems investigated above, the operational stability ofimmobilized Acetobacter sp. CCTCC M209061cells in5%v/v [ChCl][U]-containingsystems and in C4MIM·PF6/buffer biphasic system, where the immobilized cells stillremained around80.0%of relative activity after5batches, was much better than that (only50.0%) in aqueous monophasic system.This study not only contributes to enriching the theoretical foundation of biocatalysis indeep eutectic solvents (DESs)-containing systems, but also provides a new approach toenantiopure (S)-3-chlor-1-phenylpropanol with high efficiency.