The Preparation Of Catalytic Antibodies And Its Application In Catalyzing Selective Hydrolysis And Asymmetric Reduction Reaction

Catalytic antibody, abzyme, is an immunoglobulin that provided with catalytic activities ofcatalyzing a chemical reaction similarly to enzymes and highly selectivity similar to antibody. It is anewly developed research area derived from chemistry and biology. The development of catalyticantibody indicates that the preparation of man-made enzyme with highly effective multiple functionscatalyssis by rational design is possible. These work gave rise to a new area of investigation ofmimic enzyme and new domain of catalyst. It is great value both in theoretical and practical sciencesuch as medical science and chemistry and biology and pharmaceutics and so on. How to design andprepare catalytic antibodies and the enzymological properties and catalysis properties of the catalyticantibodies applied in non-aqueous medium are one of important things for catalytic antibodiesapplication. In this research, the special catalytic antibodies which were application in resolution ofchirle drug, ibuprofen, and selective reduction to prepare chirle pharmacuetical intermediate,(S)-3-chloro-1-phenyl- propanol, were generated from synthetic haptens that designed for thesereactions. Some reaction systems and media were investigated in detail, and emphasis was given tomicroemulsion, cosolvent systems. This study mainly includes the following contents:Firstly, based on the hydrolysis reaction mechanism, the two sulphate haptens and twophosphate haptens were designed and synthetized. These haptens was similar as TSA in structureand electric properties. In the space, they all has the tetrahedron structure. A catalytic antibody whichaccelerates the rate of enantioselective hydrolysis of ibuprofen methyl ester was successfully elicitedagainst an immunogen which was these haptens attached to bovine serum albumin (BSA). The rateconstant enhancement factor k_cat/k_uncat is about 1.6×10⁴. As a result, the catalytic antibody canacceleratory catalysis S-ibuprofen methyl ester but R-ibuprofen methyl ester. The most active is atabout pH 7.0～8.0 and temperature 30～40℃. In the result of thermostable test, the catalyticantibody is very sensitive in hot. The catalytic activity almost lost at 55℃. The reaction kineticsequation was established according to the selective hydrolysis of ibuprofen methyl ester catalysed bythe catalytic antibody. The results shows that the kinetics conform to the Michaelis-Menten kineticequation. The values of k_cat and K_m obtained from the empirical data by the least squares method fitting analysis were 1.01 s^-1 and 28.31μmol·l^-1, respectively.Secondly, catalytic antibody catalyzed enantioselective hydrolysis of ibuprofen ester in W/Omicroemulsion was studied. The experiments reveal that the catalytic antibodies retains theircatalytic function after their entrapment in AOT/isooctane microemulsion. The enantioselctivity wastotal for S-enantioer of ibuprofen in this work. Optimal antibody activity was observed at a w_o valueof 21. Temperature effect, pH profile was determined. The optima temperature and pH are 30～40℃and 8.0 respectively. Kinetic analysis of the catalytic antibody catalyzed reaction was found to bepossible in this system. Kinetic studies showed that the hydrolysis in microemulsion systemfollowed Michaelis-Menten kinetics. Base on the analysis of partition of insoluble substrate indifferent phases in microemulsion, the results deduced theoretically reveal that parameters V_max andk_cat^app aqueous but K_m was increased. The experimental results were confirmed thisconclusion well. The catalytic active influence of surfactant in this system is the competitiveinhibition, the K_i is 1.5×10^-3 mol·l^-1.Thirdly, the catalytic activity of the catalytic antibody in the water-miscible organic-solventsystem composed of a buffer solution and N, N-dimethylformamide (DMF) was studied. With 6%DMF in the buffer solution (containing catalytic antibody 0.25μmol, 0.2 M phosphate buffer, pH 8)at 37℃for 10 h, a good conversion (41.7%) and high enantiomeric excess (>99%) could bereached. The kinetic analysis of the catalytic antibody-catalyzed reaction showed that the hydrolysisin the water-miscible organic-solvent system with DMF in buffer solution followed theMichaelis-Menten kinetics.Fourthly, the catalytic activity of enantioselective hydrolysis of ibuprofen methyl ester bycatalytic antibodies and the Candida cylindrucea lipase coated with didodecylN-D-glucono-L-glutamate in the water-miscible organic-solvent system composed of buffersolution and DMF was studied. The main variables affecting the resolution reaction by thelipid-coated catalytic antibody and lipid-coated lipase were optimized, including organic solvent,temperature, pH and so on. The optimal pH and temperature for lipid-coated catalytic antibodieswere 7.5～8.5 and 35～40℃. Under this condition, the total conversion rate and the enantiomericratio could reach 44.5% and >500, respectively. The optimal pH and temperature for lipase-coated lipid were 7.5 and 40℃. Under this conditions, the total conversion rate and the enantiomeric ratiocould reach 46.3% and 241 respectively. The kinetic results showed that the hydrolysis in thewater-miscible organic-solvent system followed Michaelis-Menten kinetics. The catalytic rateconstant (k_cat) and V_max of the lipid-coated catalytic antibodies were 5.7 min^-1 and 0.56mmol·min^-1·g^-1. The Micheaelis contant (K_m) of the lipid-coated lipase was only half that of thenative lipase while the maximum velocity (V_max) was 1.4 times higher. The deactivationkinetics of lipid-coated catalytic antibodies in the water-miscible organic-solvent system and inaqueous phase were studied. Two deactivation kinetics deduced and fitted by experimental datawere consistent with first kinetic model. The deactivation kinetic equation were(?) ( in the water-miscible organic-solvent system) and(?) (in aqueous phase).Fifthly, based on analysis of the asymmetric reduction mechanism of3-chloropropiophenone to (S)-3-Chloro-1-phenylpropanol, the N-oxide hapten as TSA wasdesigned and synthesized. A catalytic antibody was successfully elicited against an immunogenwhich was this hapten attached to bovine serum albumin (BSA). A high enantiomeric excess(96.2%) and conversion (83.2%) of (S)-3-chloro-1-phenylpropanol can be achieved byasymmetric reduction of 3-chloropropiophenone using the catalytic antibodies.The catalytickinetics was investigated. The result indicated that the reaction is consistent withreaction-in-sequence mechanism. The kinetic model was:The model simulating curves were in good agreement with the experimental data.In a word, the preparation, characterization and application of catalytic antibodies werecarried out in this thesis. Some important information was obtained and the differentmechanism in microemulsion and the water-miscible organic-solvent system etc. was discussed,which would certainly be useful for the application of catalytic antibodies.