Enzymatic Synthesis Of Anti-tumor Drug Rhamnoside, In Vitro Activity Evaluation And Catalytic Activity Of Mutant Enzymes

Cancer is likely to remain a very formidable clinical problem.On basis of the data of National Center for Health Statistics(NCHS),more than 8 million people died from cancer every year around the world.Development of anticancer drugs has become the hotspot of the present research.Nowadays,chemotherapy drugs such as nucleoside analogues and hydroxycarbamide have become the general therapy method in cancer.Nevertheless,due to their toxicity towards normal cells,low oral bioavailability and poor targeting property,clinical applications of chemotherapy drugs have been limited.Pro-drugs,also known as prodrugs,which are inactive or less active chemical modified derivatives of normal drugs,can be selectively actived by enzymatic or chemical transformation,which provide alternative for cancer treatment.Traditionally,prodrugs are mainly synthesized by chemical methods which produce many by-products and include complicated purification steps.Enzymatic synthesis methods possess many advantages including mild reaction conditions,simple operations,and encironment friendly.However,few studies using enzymatic methods for prodrug synthesis have been reported,and those studies mainly focus on using glucose or galatose for parent drug modification.Rhamnose containing chemicals(RCCs)are important glycoside compounds and widely distributed in natural.Previous studies have reported that there are rhamnose-specific binding lectins showing high specificity to rhamnose located on the surface of some cancer cells.It suggests that the rhamnosylation of anticancer drugs may increase the targeting function of the drugs,reduce the drug dose of administration and their damage to normal cells.a-L-Rhamnosidases,belonging to glycoside hydrolases,can catalyze the hydrolysis of terminal,non-reducing L-rhamnose residues and reverse hydrolysis reaction under the thermodynamic control utilizing the L-rhamnose as a donor.In this report,for the first time we designed and synthesized two kinds of anti-cancer drug derivatives by enzymatic glycosylation and assessed anti-tumor activities of three derivatives.Alternaria sp.L1 ?-L-rhamnosidase(RhaLl)isolated and characterized in our previous work,was emploied in this thesis to catalyze glycoside synthesis via reverse hydrolysis reaction.In the reaction system,L-rhamnose was used as the glycosyl donor,and hydroxycarbamide,5-fluoro-2'-deoxyuridine and cytarabine were used as the acceptors.Syntheszed products were purified by gel filtration,TLC recycle and high performance liquid chromatography.The molecular weight of these products were determinded by MS.The chemical structures of hydroxycarbamide and cytarabine were identified by NMR analysis as 1-hydroxyureal-?-L-rhamnopyranoside and 5'-O-?-L-rhamnopyranosyl-cytarabinesylThe antitumor activities of these products were tested in vitro using human breast cancer cell lines MDA-MB-231 and MCF-7 through the MTT method.The cytotoxicities of these products incubated with or without ?-L-rhamnosidase were compaired.The results revealed that all these products were much less toxic than their parent drugs.However,when incubated with ?-L-rhamnosidase they restored the cytotoxity against the cancel cell lines through the release of parent drugs.Although we optimized the reaction conditions of synthesis of these products,the yields of target compounds were still low.So we try to improve the catalytic efficiency of RhaLl by site-directed mutagenesis.The 3D structural modeling was performed based on the crystal structure of ?-L-rhamnosidase(SaRha78A)from Streptoryces avermitilis.Through rationally design,we acquired 12 mutants(D252N,D257N,D264N,W261Y,W261A,Y302F,Y316F,W369Y,E530Q,R548A,H553A,and W555A)from the 11 selected amino acid residues.The analysis of synthesis abilities of these mutants was carried out,using D-mannitol as the acceptor and rhamnose as the glycosyl donor.Three positive mutants(Y302F,W261Y and Y316F)catalyzed higher product yield stood out.From the three positive variants,mutant W261Y accelerated the reverse hydrolysis with a prominent increase(44%)inrelative yield compared to the wild-type enzyme.The Km value of W261Y for pNPR was 1.31 mM which is almost the same as that of wild-type enzyme(1.47 mM),but kcat value was about 100-fold lower than that of wild-type enzyme.It suggested that the replacement of Trp with Tyr at position 261 evidently affected the hydrolysis activity,but enhanced the reverse hydrolysis efficiency.Based on the analysis of enzymatic properties,the optimal temperatures and thermostability of W261Y were similar with those of wild-type.Interestingly,the optimal pH was dropped from 6.5 to 3.5 and the enzyme was stable in the acidic pH buffers.We supposed that exchange of a polar residue in the active site cavity is likely to affect the pKa of the residues containing carboxyl group,leading to the change of pH dependency.