The Research Of Enzyme-catalytic Asymmetric Synthesis Of The Chiral Caffeic Acid Derivatives

Caffeic acid esters and amides commonly exit in the natural plant tissue. Thesecompounds have high biological activities, such as antioxidant, antibacterial, anticanceretc. Besides, chiral caffeic acid compounds’ biological activity has also been reported. Ithas been reported three methods to synthesis Caffeic acid esters and amides: biologicalextraction, chemical synthesis and biocatalysis, but the synthesis method of chiralcaffeic acid derivatives has not been reported.In this paper, Novozym435was adopted to catalyze the synthesis of (R)-2-amylcaffate by using caffeic acid and (R, S)-2-pentanol as substrates. The reaction conditionswere also optimized. Moreover, an enzyme-catalyzed method to synthesis (R)-caffeicacid2-phenylacetamide was established. In view of the high cost of CAL-B for reaction,recombinant plasimid PA1K-pPIC9K with the target gene was successfully constructedand recombined into Pichia pastoris GS115, and expressed yeast with the target genewas obtained. After that, the expressed yeast was induced to express the target protein.The activity of the expressed protein was determined as well as the fermentationconditions were optimized.Caffeic acid and excessive (R, S)-2-pentanol were used to synthesis2-amly caffatethrough esterification catalyzed by Novozym435. Besides, the best reaction conditionswere also preliminarily optimized: reaction solvent is anhydrous isooctane; the molarratio of substrate (caffeic acid to (R,S)-2-pentanol) was1:20; the enzyme amount was70mg per ml; the reaction temperature was70℃and the reaction time was72h. Underthe optimum conditions, the final reaction conversion20%was obtained with e.e.P98%.Subsequently, the effect of the carbon chain length of the fatty alcohol on reaction wasinvestigated. With the extension of the carbon chain, the conversion was graduallyreduced, but the enantiosekectivity increased. Finally, the reutilization of theimmobilized enzyme was investigated, and the results showed that the activity and enantioselectivity of the immobilized enzyme were not found significant reduction afterfive times.On the basis of the previous studies, RSM optimization experiments were carriedout to further optimize the reaction conditions. Software of Design-Expert7.0was usedto design a5-level-4-factor experiment. By analyzing, the optimum reaction conditionswere predicted: reaction temperature was67℃; molar ratio of substrates (caffeic acid:(R,S)-2-pentanol) was1:21; reaction time was52h and enzyme amount was50.8mg.Under this condition, the predicted results were the conversion of31%and e.e.Pof99%.Then, three repeated experiments were conducted. All the e.e.Pwere99%and conversionwere34.3%,35.2%and31.9%respectively.Comparison of cinnamic acid，3,4-dinethoxy cinnamic acid and caffeic acid showedthat the3C′,4C′hydroxyl group of benzene ring could inhibit the acylation.Furthermore, MOM ester was more favorable for the acylation reaction than methyl.MOM-Cl was used to protect both the hydroxyl group and carboxyl group of caffeicacid to prepare the MOM-protected caffeic acid MOM ester. Then, Novozym435wasused to catalyze the ester and (R, S)-2-phenethylamine to synthesis R-MOM protectedcaffeic acid2-benzeneacetamide.(R)-the caffeic acid2–benzeneacetamide was obtainedby removing MOM of (R)-MOM protected caffeic acid2-benzeneacetamide.Theoptimum reaction conditions screened were that reaction solvent was anhydrousisooctane, reaction temperature was70℃, reaction time was24h, molar ratio ofsubstrates (caffeic acid:(R,S)-2-pentanol) was1:40and enzyme amount was40mg.Umder this condition, the final reaction conversion25.5%was gotten with e.e.Phigherthan99%. In addition, the recycling of the immobilized enzyme was investigated, andthe results showed that the activity and enantioselectivity of the immobilized enzymewere not found significant reduction after five times.The recombinant plasmid of PA1K-pPIC9K with target gene was constructed,linearized and transferred into Picha pastoris GS115through electric conversion method.Positive recombinants were screened out and identified via PCR.The recombinant yeastwas induced to express the CAL-B which was purified by ammonium sulfate, DEAE ionexchange chromatography and G-75gel chromatography to make it reach electrophoresis purity. And the activity of the purified protein was determined. Inaddition, the induced optimum conditions were also preliminarily investigated. Underthe optimum condition (1%of the content of methanol was1%，the desnty ofcell (OD600)was12and the pH of medium was6.0)，we got the90mg CAL-B per liter and specificactivity was376U/mg。 Throughout the purification process, the total enzyme activityrecovery was about38%。In the present study, we have successfully synthesized three chiral caffeic acidesters and found a way to synthesize chiral caffeic acid amides via enzyme catalysis. Atthe same time, the reaction conditions were also been optimized. In order to save thecost in the experiment, we used Pichia pastoris GS115to express the target protein. Wegot the electrophoretically pure protein after a series of purification steps.