天然药物化学第二讲课件

Introduction to the Studies of Biosynthesis of Natural Products Yuemao SCollege of Pharmaceutical Sciences,Shandong UniversitySeptember 13,20103.Three examplesThe Biosynthesis of TaxolTechniques we applied:A.Stable isotope labeling,feedingB.Radioactive isotope tracing in Enzymatic acetylation Conclusion:10-deacetylbaccatin-III is a precursor of taxol02000400060008000CPM05101520Retention time,min Figure B-210g Of 10-deacetylbaccatin-III incubated with 1Ci of 3H-acetyl-CoA plus100nmole unlabeled acetyl-CoA and 300l of crude cell free extract fromTaxus media cell cultures collected on the 5th day and 7th day,respectively.300ul of boiled 7th days crude cell free extract300ul of 7th days crude cell-free extract300ul of boiled 5th days crude cell-free extract300ul of 5th dayscrude cell-free extractbaccatin-IIIHPLC conditions:Solvents:A.H2O;B.AcetonitrileFlow rate:1ml/minGradient:(T min,B%),(0,40),(5,40),(15,70)UV detection at 230nmHypothetic multiple pathways towards taxolAcOOHOHAcOOOONHOHOOOBzAcOOHOHAcOOOHOOBzTaxolBaccatin-IIIHOOHOHAcOOOONHOHOOOBzHOOHOHAcOOOHO10-DeacetyltaxolOBz10-Deacetylbaccatin-IIIABABThe biosynthesis of rifamycin BTwo possible pathways,A and B,towards rifamycin B.A demanded that protorifamycin I is the key intermediate and the oxygen at the C-8 position is introduced later.B required that the oxygen at C-8 is of the same origin as the carbon of C-8,i.e.,from the carboxyl group of AHBA.OHH3COOCH2CO2HNHOHOHOHCH3CH3OOCH3CH3CH3CH3CH3COOCH3OOOHNH2OOOH OH OH OHEnzSOOHNHOHOHOHOHOOOOHOOHNHOHOHOHOHOOOOHOOHABrifamycin B 8x propionate 2x acetateprotorifamycin Iproansamycin Xrif PKSs888C.6HOONH2OHAHBA+Methods and Techiques:DNA mutagensis;stable isotope labeling,feeding;13C NMR assay;LC-ESI-MSConclusions:A.The C-8 oxygen is the same origin as the C-8 carbonB.The formation of naphthoquinone precedes the lactamizationC.The biosynthesis of type I polyketides occurs progressivelyThe sequence of the formation of the naphthoquinone and lactamEnz-SOHOHOHOHNH2OHOHOONH2OHOHOOtype I PKSsThe mutant HGF010,in which the genes from rifF to rifN and part of rifE were deleted.Upon feeding AHBA,this mutant should produce C.15 or related compounds.rifArifBrifCrifDrifEC.15PEP+E4PrifG,rifH,rifI,rifK,rifL,rifM,rifNAHBA(3-amino-5-hydroxybenzoic acid)rifA,rifB,rifC,rifD,rifErifFOHOHOHOHNH2OHOHOONH2OHOHOOEnz-SOONH2HOOOHOHOHOHHOOtype I PKSsThe mutant HGF018,in which the rifF gene was deleted,thus it might accumulate C.10 or C.10a or related compounds.rifArifBrifCrifDrifEC.10PEP+E4PrifG,rifH,rifI,rifK,rifL,rifM,rifNAHBA(3-amino-5-hydroxybenzoic acid)rifA,rifB,rifC,rifD,rifEC.10arifFOONH2MeOMeOOOOHNH2OHOOHOOONH2HOHOOOSY5OHNHOHOOHOCHOOHNHOHOOHOCCH3OOONH2HOOOOHOOONH2HOOOHHOOOONH2HOOOOHOHOHOOONH2HOOOHOHOHHOOOONH2HOHOOOOHOHOHOHSY4aSY4b16111517SY5SY41816111517123456789124356123456789124356123456789124356SY7SY618201111171919212421SY9SY822241111212323253027SY101324567891011131517192123252627282930313218The structures of new polyketides from the HGF018 mutant(Scheme C-10).C.16C.17C.18C.19C.19C.20C.22C.21C.23C.25type I PKSsrifArifBrifCrifDrifEPEP+E4PrifG,rifH,rifI,rifK,rifL,rifM,rifNNH2OHOHOAHBA(3-amino-5-hydroxybenzoic acid)rifA,rifB,rifC,rifD,rifEOONH2HOOOHOHOHOHHOOC.27rifFOONH2HOOOHOHOHOHHOOC.26OHOONH2HOOOONH2HOOOHH+SY5-10The suspected conversion of the original compounds produced by the HGF018 mutan to SY5-10 by dehydration during workup.C.28C.29Table 5 Ions selected in LC-ESI-MS analysis for the methanol extract of the HGF018 mutant.280b SY4 (Scheme C-12)298 P8/1-OG(Scheme C-11)320a SY4a (Scheme C-12)334a SY4b (Scheme C-12)344a SY5 (Scheme C-12)372 SY5a(Scheme C-16)362 SY5b (Scheme C-17)390 SY5c (Scheme C-18)402a SY6 (Scheme C-12)430 SY6a (Scheme C-16)420 SY6b (Scheme C-17)448 SY6c (Scheme C-18)442a SY7 (Scheme C-12)470 SY7a (Scheme C-16)460c SY7b (Scheme C-17)SY7 (Scheme C-17)488 SY7c (Scheme C-18)518a SY8 (Scheme C-12)500b SY8 (Scheme C-17)546 SY8a (Scheme C-16)536 SY8b (Scheme C-17)564 SY8c (Scheme C-18)558a SY9 (Scheme C-12)586 SY9a (Scheme C-16)576c SY9b (Scheme C-17)SY9 (Scheme C-17)604 SY9c (Scheme C-18)602a SY10 (Scheme C-12)630 SY10a (Scheme C-16)620 SY10b(Scheme C-17)648 SY10c (Scheme C-18)642 C.26 (Scheme C-13)670 C.26a (Scheme C-14)684 C.26b (Scheme C-14)660 C.27 (Scheme C-13)688b SY11a(Scheme C-14)702 C.27b (Scheme C-14)662 C.28 (Scheme C-13)666 C.29 (Scheme C-13)650 C.30 (Scheme C-13)a.Detected by LC-ESI-MS in the HGF010 and HGF018 mutant,and isolated from the HGF018mutant.b.Only detected by LC-ESI-MS in the HGF018 mutant.c.Each has two candidate structures,SY7b and SY7,and SY9b and SY9,respctively.Accordingto the retention time of respective peak in HPLC,the two compounds detected in this experimentwere SY7 and SY9 rather than SY7b and SY9b.OHH3COOCH2CO2HNHOHOHOHCH3CH3OOCH3CH3CH3CH3CH3COOCH3OOOHNH2OOOHOHOHOHEnzSOOHNHOHOHOHOHOOOOHOOHNHOHOHOHOHOOOOHOOHrifamycin B AHBA 8x propionate 2x acetateprotorifamycin Iproansamycin Xrif PKSs888C.6ABPathway B is correctOHH3COOCH2CO2HNHOHOHOHCH3CH3OOCH3CH3CH3CH3CH3COOCH3OORifamycin BThe rifamycin B biosynthetic pathway deduced from the chemical data obtained from the studies on the components accumulated by the HGF018 mutant and the feeding experiment in the HGF003 mutant.PEP+E4PNH2OHOHOAHBA(3-amino-5-hydroxybenzoic acid)amide synthaseProansamycin XNHOHOHOHOHOOOOHOOHOONH2HOOOHOHOHOHACP-SOOHPKSsA speculative structure only its formamide was likely detected by LC-ESI-MS in the HGF018 mutantA speculative structureSNH2HOOHOOSNH2HOOHOSNH2HOOOOHOSOOOHOHOHOHOOHOSOOHOHOHOHOOOHOSOOHOHOHOHOOOHOSOOHOHOHOOOHOSOOHOHOOOHOSOOHOOOHOSOOSNH2HOOHOOO Scheme C-20Scheme of rifPKS showing functional domains in the five-component polyketides,and scheme of acyl chain growth showing structures as ACP thioester at the end of each condensation/reduction cycle.Modules are shown to indicate component functions.Scheme of processive syntheses of rif polyketides.showing the formation of naphthoquinonebetween tetraketide and pentaketide(see Scheme C-15 for detailed information about the proposed mechanism of this transformation).Ten polyketides should be produced by rifPKS,among them,SY4a-10 have been isolated by the author.Their structures were elucidatedon the bases of 1D and 2D NMR spectra.Starter unit,AHBA is coded with pink.Polyketide extension units are coded with deep blue.Abbreviations:ACP,acyl carrier protein;AT,acyltransferase;DH,dehydratase;KS,-ketoacyl ACP synthase;KR,-ketoreductase;DH,KR,nonfunctional DH and KR domain.After selecting AHBA as stater unit,the procedures of the biosynthesis of rif polyketide can be classified to three phases:Stage A,B and C.NH2NH2OHOHOHNH2NH2OHOHNH2OHNH2OHNH2OOHOOOHOOOHNH2ONH2NH2HOOH KS AT DH KR ACP KS AT ACP KS AT DH KR ACPKS AT DH KR ACP KS AT KR ACP KS AT DH KR ACPKS AT KR ACPKS AT DH KR ACPKS AT DH KR ACP KS AT DH KR ACPAT ACPSY5SY6SY7SY8SY9SY10SY11aSY4aSY4bDehydrationSY4Acylation orLactonizationAcylationOHStage BThe releasement of intermediates under the action of the editingfunction of thioesteraseStage CModificationsproductsintermediatesStage AThe assembly ofpolyketide chainThe biosynthesis of Streptomyces spore pigmentPlasmid constructions and resulting polyketide productsplasmid genes productspIJ4293 whiE-ORFIII,-ORFIV,-ORFV TW93a,TW93b,TW93c,TW93d,TW93e,TW93f,TW93g,TW93hpIJ4294 whiE-ORFIII,-ORFIV,-ORFV,actIII TW94a,TW94b,TW94c,TW94dpIJ4295 whiE-ORFIII,-ORFIV,-ORFV,-ORFVI TW95a,TW95bpIJ4296 whiE-ORFIII,-ORFIV,-ORFV,-ORFVI,-ORFVII TW95a,TW95bpIJ4297 whiE-ORFII,-ORFIII,-ORFIV,-ORFV TW93a,TW93b,TW93c,TW93d,TW93e,TW93f,TW93g,TW93hpIJ4298 whiE-ORFII,-ORFIII,-ORFIV,-ORFV,-ORFVI TW95a,TW95bpIJ4300 whiE-ORFII,-ORFIII,-ORFIV,-ORFV,-ORFVI,-ORFVII Blue pigment(not identified)pIJ4301 sch1,sch2,sch3 TW93a,TW93b,TW93c,TW93d,TW93e,TW93f,TW93g,TW93hpIJ4302 sch1,sch2,sch3,actIII TW94a,TW94b,TW94c,TW94dpIJ4303 curA,curB,curC TW93a,TW93b,TW93c,TW93d,TW93e,TW93f,TW93g,TW93hpIJ4304 curA,curB,curC,actIII TW94a,TW94b,TW94c,TW94dpIJ4305 curA,curB,curC,curF,curG TW95a,TW95bOOHOHOHOOOHOHOHO222497O22OOHOO2272OOOHOHTW94cTW94bTW94dTW94aOrigins of Building BlocksCommon Building BlocksC1C2C5Common Building BlocksC6C3C6C2C6C1C6C2NCommon Building BlocksC4NC5NIndole-C2NCommon FAsAcyl ThioesterGeneral ChemistryAldol and Claisen CondensationAldol and Claisen CondensationAldol and Claisen CondensationMalonyl CoA:activated acetyl CoAMalonyl CoA:activated acetyl CoAOverall Biosynthetic SchemeHOOCSCoAOHOOCS-ACPORS-CoAORS-KSORS-ACPOORROOOOOHnROHOnATKSKRDHERCO2CondensationABCDKetoReductionDehydrationEnoylReductionFatty acidReduced polyketidePolyketideAcyl transferFatty acid synthase(FAS)and Polyketide synthase(PKS)KS=Beta-ketoacyl synthase KR=Beta-ketoacyl reductase ACP=Acyl carrier proteinAT=Acyl transferase DH=Dehydratase ER=Enoyl reductase TE=ThioesteraseTEROROACPStarter unitExtension unit+ATRS-ACPOHORS-ACPORS-ACPOTEKSBacterial Aromatic PolyketidesBacterial Aromatic PolyketidesBacterial Aromatic PolyketidesBacterial Reduced PolyketidesA,Acetate;P,Propionate;B,Butyrate;G,Glycerol;S,Starter from branched chain or cyclic unitsBacterial Reduced PolyketidesBacterial Reduced PolyketidesFungal Aromatic Polyketides(Non-reduced/Partially Reduced Polyketides)Orsellinic acidTetrahydroxynaphthalene(THN)6-Methylsalicylic acid(MSA)Norsolorinic acidFungal Aromatic Polyketideswith unusual features-mixed reduced/non-reducedUnusual acetate labelUnusual starterUnusual mixFungal Highly Reduced PolyketidesT-toxinMevacorStatins hydroxy-methylglutaryl-CoA(HMG-CoA).Fungal Aromatic Polyketides(Non-reduced/Partially Reduced Polyketides)Orsellinic acidTetrahydroxynaphthalene(THN)6-Methylsalicylic acid(MSA)Norsolorinic acidPlant Polyketides(Bacterial polyketides made by the same type of PKS)chalcone in higher plants 1,3,6,8-tetrahydroxynaphthalene(THN)in Streptomyces griseus and S.coelicolor 3,5-dihydroxyphenylacetic acid(DHPA)in Amycolatopsis mediterranei and A.orientalis Plant Polyketides(Starter:Phenylpropanhoid Pathway)chalconePlant Polyketides(Bacterial polyketides made by the same type of PKS)chalcone in higher plants 1,3,6,8-tetrahydroxynaphthalene(THN)in Streptomyces griseus and S.coelicolor 3,5-dihydroxyphenylacetic acid(DHPA)in Amycolatopsis mediterranei and A.orientalis Nonribosomal PeptidesAntibioticsNonribosomal PeptidesAntibioticsNonribosomal PeptidesImmunosuppresantsNonribosomal PeptidesToxinsNonribosomal PeptidesNonribosomal PeptidesNonribosomal PeptidesNonribosomal PeptidesNonribosomal PeptidesNonribosomal PeptidesAntibioticsNonribosomal PeptidesAntibioticsNonribosomal PeptidesLast resort for against drug-resistant pathogens Nonribosomal PeptidesHybrid NRP-PK Nonribosomal PeptidesHybrid NRP-PK-C-aminated Nonribosomal PeptidesHybrid NRP-PK-N-acylatedNonribosomal Peptides