文稿教案生化上spps shanym

1、1Solid phase peptide synthesisYaming Shan College of Life ScienceJilin University Changchun, China2Outline Why peptide synthesis is necessarySolid phase peptide synthesis (idea, comparison with the synthesis in solution);3Applications of synthetic peptidesImmune peptides:synthetic antigens;vaccinesd

2、iagnostic toolsimmunostimulator peptides;muramyl dipeptidetuftsin derivativesHormones:oxytocinvasopressininsulin somatostatinGnRHetc.Neuropeptides:substance PcholecystokininneurotensinAntibiotics:tachykiningramicidine SToxins:conotoxinsspider toxinssnake toxinsionchanel blockersEnzymes and enzyme in

3、hibitors:Ribonuclease ACarriers:templatesminiproteinsPeptides for structural studies:turn mimicking cyclic peptidesTransporter peptides:penetratin oligoarginineHIV-Tat protein4Why chemists are needed?Gene expression is very popular, relatively easy and cheap method: it is good for long linear peptid

4、es or proteins containing L-amino acids.However: no D-amino acids no unnatural amino acids no post translation (Hyp, Pyr, glyco- and phosphopeptides) no branches no cyclic peptides no fluorescent or isotop labelingPeptides as drugs: there are not too many, because of the price and their fast biodegr

5、adation.“Peptides have and will continue to be important sources of lead compounds in manydrug discovery programs. However, due to their generally poor pharmacokinetic properties and hydrolytic instability, natural peptide structures are usually substituted with mimics of the actual peptide constuct

6、ion.”5PEPTIDE SYNTHESISCoupling of amino acids:NH2-CH(R)-COOH + NH2-CH(R)-COOH - H2ONH2-CH(R)-CO-NH-CH(R)-COOH; NH2-CH(R)-CO-NH-CH(R)-COOH;NH2-CH(R)-CO-NH-CH(R)-COOH; NH2-CH(R)-CO-NH-CH(R)-COOH;+ oligomers and polymers with different composition Protecting groups: amino-; carboxyl-; side chain prote

7、cting groupsX-NH-CH(R)-COOH + NH2-CH(R)-COOY - H2OX-NH-CH(R)-CO-NH-CH(R)-COOY;Removal of the protecting groups together or selectively6Synthesis in solution Synthesis on resin (SPPS) time consuming; manual; 1.1-1.2 equiv amino acid derivatives and coupling reagent for acylation; side chain protectin

8、g groups for Lys, Asp, Glu, (Cys); coupling: less than 90% conversion; purification after each steps; large scale; cheap. fast; synthesizer (or manual); 3-10 equiv amino acid derivatives and coupling reagents for acylation; side chain protecting groups for all functional groups coupling: over 99.5%

9、conversion; purification at the end; rather small scale; expensive.Synthesis of ah-ACTH (1-39) in solution took months for several chemists;A 39-mer peptide by SPPS 2 days, 1 day cleavage, 1-2 days purification,1 week altogether for 1 chemist.7SOLID PHASE PEPTIDE SYNTHESISBruce Merrifield published

10、in 1963 Nobel Prize in Chemistry in 1984The idea:TPAA1RXanchoringTPAA1RdeprotectionPAA1RTPAA2coupling (-H2O)TPAA2PAA1R8TPAA2PAA1Rdeprotectioncouplingrepetitive cycleTPAAnPAA2PAA1RAAnAA2AA1cleavage +final deprotection9Applications of Boc/Bzl strategy Resins; Protecting groups; Synthetic protocol; Mon

11、itoring; Cleavage procedures;10STRATEGIESBoc/Bzl:CH2NHCHCH2COOCONHCH2CONHCHCOOCH2OCH2ClClCH2RCH3COCH3CH3COTFAHFHFBoc-Asp(OBzl)-Gly-Tyr(2,6-Cl2Bzl)-Merrifield resinBocBzl2,6-Cl2Bzl11Fmoc/tBu:NHCCH2OCNHCHCH2COOCOHCCH3CH3CH3NHCHCOOCH2CH2OCH2OOCCH3CH3CH3CRFmoctert-butyl.piperidineTFAWang-resinFmoc-Asp(O

12、tBu)-Tyr(tBu)-Wang resin12RESINS Can be functionalized; Chemical stability (it must be inert to all applied chemicals); Mechanical stability (it shouldnt brake under stirring); It must swell extensively in the solvents used for the synthesis; Peptide-resin bond should be stable during the synthesis;

13、 Peptide-resin bond can be cleaved effectively at the end of the synthesis;The basic of the most common used resins: polystyrene-1,4-divinylbenzene (1-2%) copolymer +polymerisation13Type of resins for Boc-chemistryCH2ClPMerrifield (chloromethyl) resinNH3CH2NH2PAminomethyl resinStarting resin for the

14、 synthesis of many other resinsCH2OHCH2CONHCH2PPAM resin (phenyl-acetamidomethyl)p-hydroxymethyl-phenyl-acetic acid (handle)+ DIC14CH2ClPBoc-Aaa-O-Cs+DMF, 50oC, 48hCH2OHCH2CONHCH2PCH2OBoc-Aaa-CH2CONHCH2PBoc-Aaa-OHDIC + 10%DMAP RT DCM-DMF (1:3) 1hPeptide-PAM resin bond is more TFA stable than Peptide

15、-Merrifield resin bond.The final cleavage results in peptides with carboxyl (COOH) group at the C-terminus.Attachment of the first amino acid to Merrifield and PAM resinsCH2OBoc-Aaa-CH2COOHCH2NH2PDIC +CH2Boc-Aaa-OP15The final cleavage results in peptides with carboxamide (CONH2) group at the C-termi

16、nus.CHNH2PBoc-Aaa-OH DCC/HOBtCHNHPCCH(R)NHOBocBenzhydrylamine resin (BHA):CHNH2PBoc-Aaa-OH DCC/HOBtCHNHPCCH(R)NHOBoc4-Methyl-benzhydrylamine resin (MBHA):CH3CH3too stable under acid cleavage conditions(only; HF!)16Applied side chain protecting groups in Boc-chemistry benzyl (Bzl)CH2-OH (Ser, Thr, Ty

17、r)Side chain functional group protecting group name (abbreviation)OOCH2ClC2-chlorobenzyl- oxycarbonyl(2-Cl-Z)OOCH2Cbenzyloxycarbonyl(Z)eNH2 (Lys)Z is not stable enoughin TFA; branches in the peptide !wCOOH (Asp, Glu)OCH2Obenzyl(ester)(OBzl)cyclohexyl(ester)(OcHex)OBzl is not stable enoughin TFA; lea

18、d to ringclosure side reaction !17-C-C-C-C-AcmAcmSS-C-C-C-C-SSSSSide chain functional group protecting group name (abbreviation) 4-methylbenzyl (Meb)-SH (Cys)CH2CH3CH2CH3O 4-methoxylbenzyl (Mob)Stability vs TFA is not good enough;not for longer peptides!CH2NHCOCH3acetamidomethyl(Acm)For selective de

19、protection-C-C-C-C-AcmAcmMebMebHF-C-C-C-C-AcmAcmSHSHairoxidationI2 or Tl(tfa)3Hg(II)- or Ag(I)-salt !Eg.18Synthetic protocol of Boc-strategyWash the resin 3x with DCM; 0.5-1.0 min eachCleavage of Boc protection with 33%TFA/DCM; 2+20minWash the resin 5x with DCM; 0.5-1.0 min each(Shrinking the resin

20、with 25%dioxan/DCM)Neutralisation 3-4x with 5-10%DIEA/DCM; 1 min eachWash the resin 4x with DCM; 0.5-1.0 min eachCoupling: Boc-amino acid derivative-DCC-HOBt in DCM-DMF * (3 equiv each calculated to the resin capacity); 60 minWash the resin 2x with DMF; 0.5-1.0 min eachWash the resin 2x with DCM; 0.

21、5-1.0 min eachNinhydrin monitoring *(-) yellow (+) blue* The ratio of DCM and DMF depends on the solubility of the amino acid derivatives; DCM-DMF = 4:1 or 2:1 (V/V) in most cases. However, in case of Arg, Gln, Asn DCM:DMF 1:4 or 1:2 (V/V) is prefered.*When coupling is carried out to Pro, the ninhyd

22、rin assay cant be used. Application of isatin test or bromophenol blue test is necessary.19Ninhydrin monitoringOOHOHO2+ NH2-ROOHNOOblue l(570nm)O-OHNOO+In case of Pro:There is no difference between the colour of ninhydrine and the productyellow20Boc cleavage flow chartDoes the peptide contain His(Dn

23、p)?yesnoRemove DnpDoes the peptide contain N-terminal Boc group?yesnoRemove BocIs the peptide (protecting groups)compatible with HF, TMSOTf, TFMSA?HFDoes the peptide contain Trp(For)?yesnoDeformylate Trp(For) or”Low-high” HF cleavageHF cleavageTMSOTfTFMSADoes the peptide contain Trp(For) or Met(O)?T

24、MSOTf cleavagenoyes”Low-high” TFMSA cleavageStandard TFMSA cleavage21Why is it necessary to remove Boc-group before cleavage with strong acids? tert-butyl cation is a very effective alkylating agent; long cleavage time, high cation concentration; the best scavanger to trap the tert-butyl cation is w

25、ater; however water cant be used with strong acids because of splitting of peptide bonds; there are some special side reactions, eg. in case of peptides containing Met at the C-terminal (homoserine lactone formation);CH3SNHOORHFCH3SNHOHO+ONHOM = Mcalc- 47.022Problems with the cleavage proceduresHF :

26、 needs a special teflon instrument. However all the applied protecting groups can be cleaved. Cleavage time is 45-60 min at 0oC, but in case of Arg(Tos), Cys(Meb), Asp(OcHex), Glu(OcHex) 90 min is mended. Anisole, p-cresol and DTT as scavangers are used.TMSOTf : 1 M TMSOTf-thioanisole/TFA solution i

27、n the presence of m-cresol and EDT at 0oC for 120 min. Arg(Tos), Cys(Meb), Asp(OcHex), Glu(OcHex) and BHA resin are not cleavable under these conditions.TFMSA : 10% TFMSA- 10% thioanisole in TFA at RT for 1.5-2hrs. EDT and m-cresol are mended as scavangers. Arg(Tos), Cys(Meb), Asp(OcHex), Glu(OcHex)

28、 and BHA resin are not compatible with this method. More side reactions than in case of TMSOTf. Desalting is necessary at the end.23Application of Fmoc/tBu strategy Resins; Protecting groups; Synthetic protocol; Monitoring; Cleavage technics; Side reactions;24Fmoc/tBu:NHCCH2OCNHCHCH2COOCOHCCH3CH3CH3

29、NHCHCOOCH2CH2OCH2OOCCH3CH3CH3CRFmoctert-butyl.piperidineTFAWang-resinFmoc-Asp(OtBu)-Tyr(tBu)-Wang resin25Type of resins for Fmoc-chemistryThere are many different resins and most of them are used for special cases and in individual laboratories. Here only the most widely applied resins will be prese

30、nted. 4-Alkoxybenzyl alcohol (Wang) resin:CH2OPCH2HOAttachment of the first amino acid: The final cleavage results in peptides with COOH group at the C-terminus26Rink Amide Resin: synthesis of peptides with CONH2 C-terminusCHNFmoc-HOCH2-POCH3COH327Applied side chain protecting groups in Fmoc-chemist

31、ry-OH (Ser, Thr, Tyr)Side chain functional group protecting group name (abbreviation)CH3CCH3CH3tert-butyl (tBu)-SH (Cys)trityl (Trt)28Side chain functional group protecting group name (abbreviation)tert-butyloxycarbonyl(Boc)eNH2 (Lys)OOCCCH3CH3CH3Selectively removable protecting groups for preparati

32、on of modified peptides (labeled, functionalised, branched or cyclic peptides): eNH2 (Lys)CH34-methytrityl(Mtt)Mtt can be removed selectively with 1%TFA/DCM solution in the presence of 3-5% TES (triethylsilane) at RT in 15-30 min.Trt groups may be not stable enough under this condition.29wCOOH (Asp,

33、 Glu)Side chain functional group protecting group name (abbreviation)OCCH3CH3CH3tert-butyl ester (OtBu)wCONH2 (Asn, Gln)trityl (Trt)30Wash the resin 3x with DMF; 0.5-1.0 min eachCleavage of Fmoc protection with 10% piperidine DMF; *Wash the resin 8x with DMF; 0.5-1.0 min each*Coupling: Fmoc-amino ac

34、id derivative-DIC-HOBt in DMF* (3 equiv each calculated to the resin capacity); 60 minWash the resin 2x with DMF; 0.5-1.0 min eachWash the resin 2x with DCM; 0.5-1.0 min eachNinhydrin monitoring Synthetic protocol of Fmoc-strategy(-) yellow (+) blue* Piperidine is for the capture of dibenzofulvene 2

35、0% or 50% piperidine in DMF, 50% morpholine or DEA in DMF and 20mM TBAF in DMF are also used as cleavage mixture.* An unefficient removal of base from the resin may cause Fmoc cleavage in the next coupling step.* DIC is used instead of DCC in this method, because of the limited solubility of DCU in

36、the applied solvents. 31N,N-diisopropylcarbodiimide (DIC, DIPCDI)Coupling agentsNNCN,N-dicyclohexylcarbodiimide (DCC)NNCCHHCCH3CH3H3CH3CNNHCOCOCHX-NHRX: Boc, FmocO-acyl-isourea derivativesNNHCOCOCHX-NHRN-acyl-urea derivativesO-N acyl shiftNHNHCOurea derivatives: DCU, DIUCOCHX-NHROBtHOBtin situ active ester+32Fmoc cleavage flow chartDoes the peptide contain N-terminal Fmoc group?yesnoRemove Fmoc Does the peptide contain Arg, Met, Trp or Trt?yesnoDoes the peptide contain Arg, Met?Use cleavage mixtu