The Isopentenylation Of Aromatic Compounds Such As 10-hydroxycamptothecin By Recombinant Isopentenyl Transferase AtPT And The Sulfation Of Naringenin By Mucor

Part One:Prenylation of magnolol,honokiol,9-amino camptothecin and 10-hydroxycamptothecin with an aromatic prenyltransferase AtPTAromatic prenyltransferases are key enzymes involved in biosynthesis of the active prenylated aromatics.In our previous research,a new aromatic prenyltransferase gene AtPT has been cloned from the genome of Aspergillus sp.(A8-4),and it has been found that AtPT possessed amazing substrate promiscuity,suggesting that it may be used as a general tool enzyme for the synthesis of structurally diverse prenylated aromatics.With the aim of obtaining of new derivatives with better activity,higher bioavailability and even lower toxicity,which would be of considerable leads or candidates for drug discovery,in this dissertation,9-amino camptothecin,10-hydroxycamptothecin,magnolol,honokiol were used as the prenyl acceptors,and three different C5 unit diphosphates-DMAPP,GPP and FPP were used as prenyl donors for the enzymatic prenylation.The main results have been achieved as follows.(1)Twenty aromatics were screened as the acceptors and DMAPP,GPP and FPP were used in the prenylation of AtPT.The HPLC-MS analyses results showed that several enzymatic products with considerable yield in the enzymatic reaction,such as magnolol incubated with DMAPP and GPP,honokiol with GPP,10-hydroxycamptothecin with GPP and FPP,and 9-amino camptothecin with GPP.(2)The enzymatic products were obtained by semi-preparative HPLC and their structures were elucidated on the basis of HR-ESI-MS,1D and 2D NMR spectroscopic data.Totally,ten prenylated derivatives of magnolol or honokiol and six prenylated derivatives of camptothecin were identified as 3-(3-dimethylallyl)-magnolol(?),3-dimethylallyl-magnolol(?)2-O-dimethylallyl-magnolol(?),3-dimethylallyl-3'-(3-dimethylallyl)-magnolol(?),3,3'-di-(3-dimethylallyl)-magnolol(?),2-O-dimethylallyl-3'-(3-dimethylallyl)-magnolol(?),3-(3-geranyl)-magnolol(?),3-geranyl-magnolol(?),2-O-(3-geranyl)-honokiol(?),2-O-geranyl-honokiol(X),9-geranyl-10-hydroxycamptothecin(?),10-(O-geranyl-camptothecin(?),9-famesyl-10-hydroxycamptothecin(??),9-amino-10-geranyl-camptothecin(??),9-amino-12-geranyl-camptothecin(??),and 9-amino-10,12-digeranyl-camptothecin(??).(3)For the kinetic study,time curve in the enzymatic reaction of magnolol and GPP with AtPT was investigated and the result showed that the product formation increased linearly in the first forty minutes.The Km and Vmax values for acceptor and donor in each incubation were determined respectively.In the case of magnolol and honokiol as the prenyl acceptors,AtPT had stronger affinity to DMAPP as prenyl donor than to GPP.In the case of DMAPP as prenyl donor,AtPT had stronger affinity to magnolol,while with GPP as prenyl donor,honokiol had stronger affinity.10-Hydroxycamptothecin showed stronger affinity to enzyme than 9-Aminocamptothecin.Part Two:Sulfation of naringenin by Mucor sp.In this part,Naringenin(1)was transformed to three metabolites(2-4)by Mucor sp.Based on LCMSn-IT-TOF and NMR spectroscopic data,2,3 and 4 were identified as naringenin-7-O-sulfate,naringenin-4'-O-sulfate,and naringenin-5-O-sulfate,respectively.These results might provide hints to the possibility of Mucor sp.as the mammalian/human metabolism microbial model of naringenin and other compounds.