多肽合成基础知识汇编1.pdf

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1、多 肽 合 成基础知识汇编编制:合成部、多 肽 合 成 概 论1.多肽化学合成概述：1963年,R.B.Merrifield 1创立了将氨基酸的C末端固定在不溶性树脂上,然后在此树脂上依次缩合氨基酸，延长肽链、合成蛋白质的固相合成法，在固相法中，每步反应后只需简单地洗涤树脂，便可达到纯化目的.克服了经典液相合成法中的每一步产物都需纯化的困难，为自动化合成肽奠定了基础.为此，M errifield获 得1984年诺贝尔化学奖.今天，固相法得到了很大发展,除了 M errifield所建立的Boc法（Boc:叔丁氧谈基）之外，又发展了 Fmoc固相法（Fmoc:9-笏甲氧谈基）.以这两种方法为基础

2、的各种肽自动合成仪也相继出现和发展，并仍在不断得到改造和完善.M errifield所建立的Boc合 成 法 2是采用TFA（三氟乙酸）可脱除的Boc为a-氨基保护基，侧链保护采用节醇类.合成时将个Boc氨基酸衍生物共价交联到树脂上，用TFA脱除B oc,用三乙胺中和游离的氨基末端，然后通过Dec活化、耦联下个氨基酸，最终脱保护多采用HF法或TFMSA（三氟甲磺酸）法.用Boc法已成功地合成了许多生物大分子，如活性酶、生长因子、人工蛋白等.多肽是涉及生物体内各种细胞功能的生物活性物质。它是分子结构介于氨基酸和蛋白质之间的一类化合物，由多种氨基酸按照一定的排列顺序通过肽键结合而成。到现在，人们已

3、发现和分离出一百多种存在于人体的肽，对于多肽的研究和利用，出现了 个空前的繁荣景象。多肽的全合成不仅具有很重要的理论意义，而且具有重要的应用价值。通过多肽全合成可以验证一个新的多肽的结构；设计新的多肽，用于研究结构与功能的关系：为多肽生物合成反应机制提供重要信息：建立模型酶以及合成新的多肽药物等。多肽的化学合成技术无论是液相法还是固相法都已成熟。近几十年来，固相法合成多肽更以其省时、省力、省料、便于计算机控制、便于普及推广的突出优势而成为肽合成的常规方法并扩展到核甘酸合成等其它有机物领域。本文概述了固相合成的基本原理、实验过程，对其现状进行分析并展望了今后的发展趋势。从1963年M errif

4、ield发展成功了固相多肽合成方法以来，经过不断的改进和完善，到今天固相法已成为多肽和蛋白质合成中的一个常用技术，衣现出了经典液相合成法无法比拟的优点。其基本原理是：先将所要合成肽链的羟末端氨基酸的羟基以共价键的结构同一个不溶性的高分子树脂相连，然后以此结合在固相载体上的氨基酸作为氨基组份经过脱去氨基保护基并同过量的活化段基组分反应，接长肽链。重复（缩合一洗涤一去保护一中和及洗涤一 卜.一轮缩合）操作，达到所要合成的肽链长度，最后将肽链从树脂上裂解下来，经过纯化等处理，即得所要的多肽。其中a-氨基用BOC（叔丁氧段基）保护的称为B0C固相合成法，-氨基用FM O C （9-笏甲氧谈基）保护的称

5、为FMOC固相合成法，2.固相合成的基本原理多肽合成是一个重复添加氨基酸的过程，固相合成顺序一般从C端（段基端）向N端（氨基端）合成。过去的多肽合成是在溶液中进行的称为液相合成法。现在多采用固相合成法，从而大大的减轻了每步产品提纯的难度。为了防止副反应的发生，参加反应的氨基酸的侧链都是保护的。段基端是游离的，并且在反应之前必须活化。化学合成方法有两种，即F m o c和t B o c。由于F m o c比t B o c存在很多优势，现在大多采用F m o c法合成，如图：RiPJTOC-XII-CH-COOH i OCH IRiFmoc-今RiH s N-a i-C O O -R*RlFnoe

6、-XH-CH-CON1I-CH-CJOO CH;Q,1HAMCC10H8NO2174.27-Amido-4-trifluoromethyl-coumarylrlH&AFCC10H5F3NO2228.25-(2-Aminoethyl)amino-naphthalene-1-sulfonic acid(sodH1EDANSC12H13N2O3S265.3BenzoylroBzC7H5O105.1Benzylc0BzlC7H791.1BenzyloxycarbonylCrAZ(Cbz)C8H7O2135.1BenzyloxymethylBomC8H9O121.2(+)-BiotinylVBiotin

7、C10H15N2O2S227.3o2-Bromobenzyloxycarbonyler o2-Br-ZC8H6BrO2214.0tert-Butyl小tBuC4H957.1tert-Butyloxycarbonyl6人CHBocC5H9O2101.1tert-ButylthioGTCM,StBuC4H9S89.22-Chlorobenzyloxycarbonyl1Cl2-CI-ZC8H6CIO2169.6CyclohexylcHexC6H1183.12,6-DichlorobenzylCl2,6-di-CI-BzlC7H5CI2160.04-(4-Dimethylaminophenyl-azo

8、)benzoyl10一x ArDABCYLC15H14N3O252.32,4-DinitrophenylDnpC6H3N2O4167.19-FluorenylmethylFmC14H11179.1u9-Fluorenylmethyloxy-carbonylTosC7H7O2S155.2Tritylo)TrtC17H15243.3c5Xanthylo wpv XanC13H9O181.2五、多肽常识Reconstitution and Storage of PeptidesPeptides are usually supplied as a fluffy,freeze-dried materia

9、l in serum vials.Store peptides in a freezerafter they have been received.In order to reconstitute the peptide,distilled water or a buffer solutionshould be utilized.Some peptides have low solubility in water and must be dissolved in other solventssuch as 10%acetic acid for a positively charged pept

10、ide or 10%ammonium bicarbonate solution for anegatively charged peptide.Other solvents that can be used for dissolving peptides are acetonitrile,DMSO,DMF,or isopropanol.Use the minimal amount of these non-aqueous solvents and add water orbuffer to make up the desired volume.After peptides are recons

11、tituted,they should be used as soon aspossible to avoid degradation in solution.Unused peptide should be aliquoted into single-use portions,relyophilized if possible,and stored at-20.Repeated thawing and refreezing should be avoided.Methods to Dissolve PeptidesThe best way to dissolve a peptide is t

12、o use water.For peptides that are not soluble in water,use thefollowing procedure:1.For acidic peptides,use a small amount of base such as 10%ammonium bicarbonate todissolve the peptide,dilute with water to the desired concentration.Do not use base forcysteine-containing peptides.2.For basic peptide

13、s,use a small amount of 30%acetic acid,dilute with water to the desiredconcentration.3.For a very hydrophobic peptide,try dissolving the peptide in a very small amount of DMSO,dilute with water to the desired concentration.4.For peptides that tend to aggregate(usually peptides containing cysteines),

14、add 6 M urea,6 Murea with 20%acetic acid,or 6 M guanidine*HCI to the peptide,then proceed with thenecessary dilutions.Preparation of HBTU/HOBt Solution for the Peptide Synthesizer1.Preparation of 0.5 M HOBt in DMF:o Weigh 13.5 g anhydrous HOBt(0.1 mol,MW 135.1)100 g,AnaSpec Catalog#21003;500 g,AnaSp

15、ec Catalog#21004 into a 250 mL graduated cylinder.o Add DMF until the 200 mL level is reached.2.Preparation of 0.45 M HBTU/HOBt solution:o Add the solution prepared in step 1 to 37.9 g HBTLI(0.1 mol,MW 379.3)100 g,AnaSpec Catalog#21001;500 g,AnaSpec Catalog#21002 contained in a beaker oran Erlenmaye

16、r flask.3.Stir for about 15 min with a magnetic stirring bar until HBTU is dissolved.4.Filter the solution through a fine pore size sintered glass funnel.5.Pour the filtered solution into an appropriate bottle for attachment to a peptide synthesizer.*This solution is stable at room temperature for a

17、t least six weeks.Biotinylation of Amino Group1.Wash 0.1 mmol resin with DMF.2.Dissolve 0.244 g(+)-biotin(1 mmol,MW 244.3)1 g,AnaSpec Catalog#21100;5 g,AnaSpecCatalog#21101 in 5 mL DMF-DMSO(1:1)solution.A little warming is necessary.3.Add 2.1 mL0.45 M HBTU/HOBt solution and 0.3 mL DIEA to the soluti

18、on prepared in step 2.4.Add the activated biotin solution to the resin and let stir overnight.5.Check resin to make sure coupling is complete as evidenced by negative ninhydrin test(colorless).6.Wash resin with DMF-DMSO(1:1)(2x)to remove excess(+)-biotin.7.Wash resin with DMF(2x)and DCM(2x).8.Let th

19、e resin dry before proceeding to cleavage.Procedure for Loading Fmoc-Amino Acid to 2-Chlorotrityl Chloride Resin1.Weigh 10 g 2-chlorotrityl chloride resin(15 mmol)1 g,AnaSpec Catalog#22229;5 g,AnaSpecCatalog#22230 in a reaction vessel,wash with DMF(2x),swell the resin in 50 mL DMF for 10min,drain ve

20、ssel.2.Weigh 10 mmol Fmoc-amino acid in a test tube,dissolve Fmoc-amino acid in 40 mL DMF,transfer the solution into the reaction vessel above,add 8.7 mL DIEA(50 mmol),swirl mixturefor 30 min at room temperature.3.Add 5 mL methanol into the reaction vessel and swirl for 5 min.4.Drain and wash with D

21、MF(5x).5.Check substitution.6.Add 50 mL 20%piperidine to remove the Fmoc group.Swirl mixture for 30 min.7.Wash with DMF(5x),DCM(2x),put resin on tissue paper over a foam pad and let dry at roomtemperature overnight under the hood.Cover the resin with another piece of tissue paper,presslightly to bre

22、ak aggregates.8.Weigh loaded resin.9.Pack in appropriate container.Procedure for Checking Substitution of Fmoc-Amino Acid Loaded Resins1.Weigh duplicate samples of 5 to 10 mg loaded resin in an eppendorf tube,add 1.00 mL 20%piperidine/DMF,shake for 20 min,centrifuge down the resin.2.Transfer 100 pL

23、of the above solution into a tube containing 10 mL DMF,mix well.3.Pipette 2 mL DMF into each of the two cells(reference cell and sample cell),setspectrophotometer to zero.Empty the sample cell,transfer 2 mL of the solution from step 2 intothe sample cell,check absorbance.4.Subs=101(A)/7.8(w)A=absorb

24、ancew=mg of resin5.Check absorbance three times at 301 nm,calculate average substitution.Manual Fmoc Synthesis(0.25 mmol)1.Wash resin with DMF(4x)and then drain completely.2.Add approximately 10 mL20%piperidine/DMF to resin.Shake for one min and drain.3.Add another 10 mL 20%piperidine/DMF.Shake for

25、30 min.4.Drain reaction vessel and wash resin with DMF(4x).Make sure there is no piperidine remaining.Check beads using ninhydrin test,beads should be blue.5.Coupling Step-Prepare the following solution:1 mmol Fmoc-amino acid2.1 mL0.45 M HBTU/HOBT(1mmol)348 pL DIEA(2 mmol)Add above solution to the r

26、esin and shake for a minimum of 30 min.This coupling step can belonger if desired.6.Drain reaction vessel and wash resin with DMF(4x).7.Perform Ninhydrin test:o If negative(colorless),proceed to step 2 and continue synthesis.o If positive(blue),return to step 5 and re-couple the same Fmoc-amino acid

27、.Increasethe coupling time if necessary.Synthesis of Phosphotyrosine-Containing Peptides Using Fmoc-PhosphotyrosineReagent:N-a-Fmoc-O-phosphotyrosine 1 g,AnaSpec Catalog#20254;5 g,AnaSpec Catalog#202551.For 0.1 mmol or 0.25 mmol synthesis,use 0.483 g Fmoc-Tyr(PO3H2)-OH(1 mmol,MW 483.4).For ABI synth

28、esizers,pack Fmoc-Tyr(PO3H2)-OH in a cartridge.2.The cycle program for coupling Fmoc-Tyr(PO3H2)-OH is the same as for other Fmoc-aminoacids except for the coupling time(see step 3).(Note:ABI synthesizers use HBTU/HOBT as theactivating reagent.)3.The coupling time for Fmoc-Tyr(PO3H2)-OH needs to be i

29、ncreased.For ABI model 430Apeptide synthesizer,insert several steps(i.e.,vortex on,wait 990 sec,vortex off,to increase thecoupling time).For ABI model 431A peptide synthesizer,add additional Ts.Overnight couplingmay be necessary for some sequences.4.After the coupling step for Fmoc-Tyr(PO3H2)-OH,per

30、form ninhydrin test to ensure completecoupling.Negative(colorless)ninhydrin test indicates complete coupling,while a positive(blue)ninhydrin test indicates incomplete coupling.5.Increase the coupling time of the amino acid residues after the phosphotyrosine or performdouble coupling.(Note:The coupli

31、ng of amino acids after the phosphotyrosine can be difficult.)6.There is a limit on the number of amino acid residues that can be coupled after thehosphotyrosine.Since the phospho group is unprotected,side reactions are likely to ccur.(Note:Peptides have been successfully coupled with sequences cont

32、aining up o ten additional aminoacids following the phosphotyrosine residue.)Simultaneous Synthesis of Peptides Which Differ in the C-Termini Using 2-Chlorotrityl Resin andWang Resin*Peptides which differ in the C-termini can be simultaneously synthesized in one reaction vessel byemploying resins th

33、at possess different cleavage properties.The resins used were the weak acid labile2-chlorotrityl resins and the TFA labile Wang resins.The success of this approach was shown by theco-synthesis of ACTH(4-10)with ACTH(4-11)and Neuropeptide Y,a C-terminal amide peptide with itscorresponding C-terminal

34、free acid analog.*Hong A.,Le T.,and Phan T.Techniques in Protein Chemistry VI,531-562(1995).Cleavage Protocol to Produce Fully Protected PeptideStarting Resin:Chlorotrityl resinsReagents for 1 g Peptide-Resin:1 mL acetic acid(AcOH)2 mL trifluoroethanol(TFE)7 mL dichloromethane(DCM)1.Prepare above mi

35、xture.2.Add peptide-resin to the mixture and let it stir at room temperature for 1 h.3.Filter and wash resin with 10 mL TFE:DCM(2:8)(2x)to ensure that all of the product isrecovered.4.Evaporate the solvent until there is less than 5 mL of liquid.5.Add ether to a test tube containing about 100 pL of

36、the above solution.Check solubility of thefully protected peptide in ether.If the product precipitates,proceed to step 6.If no precipitate isobserved,proceed to step 7.6.Add cold ether to the residual liquid in step 4 to precipitate the fully protected peptide.Filterthrough a fine sintered funnel to

37、 obtain the product.7.Some fully protected peptides are soluble in ether.In this case,add water to precipitate themout.Filter through a fine sintered funnel to obtain the product.Procedure for FITC Labeling of PeptidesReagents:FITC 1 g,AnaSpec Catalog#20151Fmoc-e-Ahx-OH 1 g,AnaSpec Catalog#20957;5 g

38、,AnaSpec Catalog#209581.Couple Fmoc-e-Ahx-OH to the amino terminal of the peptide-resin using standard couplingconditions.2.De-Fmoc with piperidine using the standard 20%piperidine procedure.3.Wash resin with DMF(3-4x).4.Swell resin with DCM and drain.5.Prepare solution of 1.1 equivalent of FITC in

39、pyridine/DMF/DCM(12:7:5).Use just enoughsolution to form a slurry with the resin.Do not use too much solution since the rate of thereaction is proportionate to the concentration of the solution.6.Add the solution prepared in step 2 to the resin.7.Let mix overnight.8.Check the completion of the react

40、ion using ninhydrin test.9.If the coupling of FITC to the amino group is not complete,ninhydrin test will give a blue color.Repeat the coupling with FITC(steps 5-7)if necessary.10.Wash resin with DMF(2x),isopropanol(2x),and DCM(2x).Procedure for Removing Mtt group from Fmoc-Lys(Mtt)on Solid PhaseRea

41、gent:Fmoc-Lys(Mtt)-OH 1 g,AnaSpec Catalog#20093;5 g,AnaSpec Catalog#200941.Swell resin in DCM.2.Wash resin with 3%TFA/DCM(2x)(since the resin is swollen in DCM,this step of washing theresin quickly with 3%TFA/DCM ensures that the actual concentration of TFA is 3%).3.Shake the resin in 3%TFA for 10 m

42、in.4.Repeat step 3.5.Wash resin with DCM(3x),DMF(3x),isopropanol(3x),and DCM(3x).6.Let the resin dry in air.Procedure for Fluorescein Labeling of PeptidesReagent:5-carboxyfluorescein(5-FAM)0.1 g,AnaSpec Catalog#24623;0.5 g,AnaSpec Catalog#24624)Use standard coupling method to couple 5-carboxyfluores

43、cein to the amino group of the peptide.For costsaving purposes,use 2x excess compared to the mmol of resin,instead of the standard 4x excess usedfor Fmoc-amino acids.For 0.1 mmol synthesis,use 75 mg 5-carboxyfluorescein,76 mg HBTU,and 70mL DIEA.六、常用试剂及非天然氨基酸(一)缩合剂Name:EDC.HCICategory:PeptideCoupling

44、 ReagentCH3:N-CH2cH2cH2N=C=NCH2cH3,HCIH3C 2 32.6-氯-1-羟基-苯并-三氮哇Name:CI-HOBtCategory:Peptide Coupling ReagentClName:N,N-Dii so propyl carbodiim ide(DIG)Category:Peptide Coupling ReagentcH-C H3c H N=C ：=NOH产 CHCH,4.二环己基碳化二亚胺Name:Dicyclohexylcarbodiimide(DCC)Category:Peptide Coupling Reagents5.Name:BOP

45、ReagentCategory:Peptide Coupling Reagent6.六氟磷酸苯并三哇-i-基-氧基三毗咯烷基磷Name:PyBOPCategory:Peptide CouplingReagent7.N,N-蝶基二咪嚏Name:N,N-Carbonyldiim id azole(CDI)Category:Peptide Coupling ReagentName:DEPBTCategory:Peptide Coupling Reagent0O C H 2cH 3J IN 加 0 cH 2cH 3Name:4,5-Dicyanoim idazoleCategory:Peptide C

46、oupling ReagentNC、rNC人io.Name:HBTUCategory:Peptide CouplingReagent11.Name:HOBt(anhydrous)Category:Peptide Coupling Reagent12.Name:HOOBtCategory:Peptide Coupling Reagent13.Name:TBTUCategory:Peptide Coupling Reagent（二）链接剂Name:Sieber LinkerCategory:Linkers for SolidPhase SynthesisName:Weinreb LinkerCat

47、egory:Linkers for SolidPhase SynthesisName:DHP LinkerCategory:Linkers for SolidPhase SynthesisName:HMP LinkerCategory:Linkers for SolidPhase Synthesiso5.Name:Rink Am ide LinkerCategory:Linkers for SolidPhase SynthesisHO（三）树 脂 ResinName:2-ChlorotrityIChloride ResinCategory:ResinsName:Am inom ethylpol

48、ystyrene ResinCategory:Resins3.（用于合成肽醇）Name:DHP HM ResinCategory:Resins4.Name:HMPA-AM ResinCategory:Resins5.（用于合成肽酰胺）Name:Knorr CH,Resin jA sCategory:Resins U JmFmoc oO-CH,6.（用于合成肽酰胺）Name:Knorr-2-ChlorotritylResinCategory:ResinsName:MBHA ResinCategory:Resins8.Name:Merrifield ResinCategory:Resins9.Na

49、me:Oxime ResinCategory:Resinsio.Name:PAM ResinCategory:ResinsOHii.Name:Rinkamide-AM ResinCategory:ResinsHJC-OXX。片 为o12.Name:Rinkam ide-MBHAResinCategory:ResinsHjC-013.Name:Sieber ResinCategory:Resins14.Name:Wang ResinCategory:Resins1 5.（用于合成肽醛）Name:Weinreb AM ResinCategory:Resins即P 0=F mJ q铲1.9-笏甲醇（

50、四）保护剂2.Boc-酸醉Name:Boc AnhydrideCategory:N-ProtectingReagents3.4,4i二甲氧基三苯基氯甲烷Name:DMT-CICategory:N-ProtectingReagentsCH,O O CH,H jC-i-O-iJ-O -U-O-A-C HJ如 CH,HjC-O4.Name:N,N-Disuccinim idylcarbonate(DSC)Category:N-ProtectingReagents005.笏甲氧液酰氯Name:Fm oc-CICategory:N-ProtectingReagents6.笏甲氧谈酰琥珀酰亚胺Name:F