Lub hauv paus ntsiab lus thiab kev ua haujlwm ntawm cov khoom khov -Phase Peptide Synthesis (SPPS)
Khoom -phase peptide synthesis (SPPS)yog ib txoj hauv kev los tsim cov peptides ntawm kev txhawb nqa khoom. Cov tswv yim tseem ceeb ntawm SPPS suav nrogTxoj kev Bocthiab covFmoc txoj kev. Peptide synthesis yog cov txheej txheem rov ua dua ntawm cov amino acid txuas ntxiv thiab feem ntau ua los ntawmC-terminus (carboxyl kawg)mus rauN-terminus (amino kawg). Ua ntej, pawg carboxyl ntawm thawj cov amino acid ntawm lub hom phiaj peptide yog covalently txuas nrog cov khoom txhawb nqa (resin). Siv cov amino pawg ntawm cov amino acid no raws li qhov pib, N- pab pawg tiv thaiv lub davhlau ya nyob twg raug tshem tawm, thiab cov saw peptide loj hlob yog elongated los ntawm cov tshuaj tiv thaiv nrog ntau tshaj ntawm cov khoom siv thib ob amino acid. Lub voj voog no -coupling → ntxuav → deprotection → neutralization thiab ntxuav → tom ntej no coupling- rov ua dua kom txog thaum qhov ntev peptide xav tau tiav. Thaum kawg, cov saw peptide yog cleaved los ntawm cov cob thiab purified kom tau lub hom phiaj peptide. Thaum cov -amino pawg tiv thaiv nrogBoc (tert{0}}butyloxycarbonyl), txoj kev yog hu uaBoc-raws li solid-phase peptide synthesis. Thaum cov -amino pawg tiv thaiv nrogFmoc (9-fluorenylmethyloxycarbonyl), nws yog lub npe hu uaFmoc-raws li solid-phase peptide synthesis.
Boc-raws li Khoom-Phase Peptide Synthesis (Boc Method)
HauvTxoj kev Boc, Boc pawg tshem tau los ntawm trifluoroacetic acid (TFA)yog siv los tiv thaiv -amino pawg, thaumbenzyl- hom tiv thaiv pab pawgyog ua hauj lwm rau sab chains. Thaum lub sij hawm synthesis, ib tug Boc- tiv thaiv -amino acid yog thawj covalently txuas rau cov resin. Cov pab pawg Boc raug tshem tawm nrog TFA, thiab cov txiaj ntsig pub dawb amino terminus yog nruab nrab nrog triethylamine. Tom ntej no amino acid yog qhib sivDCC (dicyclohexylcarbodiimide)thiab txuas ntxiv mus txuas ntxiv cov saw peptide. Tom qab ua tiav ntawm kev sib dhos, lub hom phiaj peptide yog cleaved los ntawm cov resin siv lub zog acid, feem ntauAnhydrous hydrogen fluoride (HF)los yogTrifluoromethanesulfonic acid (TFMSA).Nyob rau hauv txoj kev Boc synthesis, rov qab kho cov kua qaub yuav tsum tshem tawm cov pab pawg tiv thaiv ua ntej txhua kauj ruam txuas. Qhov no tuaj yeem ua rau ntau qhov kev tawm tsam, xws li kev tawg ntxov ntxov ntawm peptide los ntawm cov resin thiab instability ntawm amino acid sab chains nyob rau hauv acidic tej yam kev mob, uas ua rau undesirable sab kev xav.
Fmoc-raws li Khoom-Phase Peptide Synthesis (Fmoc Method)
Hauv1978, Meienhofer thiab Athertontsim lub tswv yim peptide synthesis sivFmoc (9-fluorenylmethyloxycarbonyl)raws li -amino tiv thaiv pab pawg, hu uaFmoc txoj kev. Hauv txoj hauv kev no, - pawg amino yog tiv thaiv nrogbase-labile Fmoc, thaum sab chains tiv thaiv nrogacid-labile Boc- hom tiv thaiv pab pawg.Qhov txiaj ntsig tseem ceeb ntawm Fmoc ua ib qho amino- pab pawg tiv thaiv yog nwsstability nyob rau hauv acidic tej yam kev mob; nws tsis cuam tshuam los ntawm kev kho mob nrog reagents xws litrifluoroacetic acid (TFA)thiab tuaj yeem xaiv tshem tawm sivme ntsis puag. Nyob rau tib lub sijhawm, sab- cov saw tiv thaiv cov pab pawg xws li Boc tau ruaj khov nyob rau hauv cov xwm txheej yooj yim thiab raug tshem tawm raws li kev kho acidic. Thaum kawg, peptide yog cleaved ntau ntawm cov resin siv.TFA / dichloromethane (DCM), zam kev siv cov kua qaub uas muaj zog thiab yog li txhim kho kev nyab xeeb thiab kev sib raug zoo nrog cov peptide synthesis niaj hnub.
Kev Txhim Kho Keeb Kwm ntawm Khoom -Phase Peptide Synthesis (SPPS)
1.Foundational Stage (Early 20th Century to 1963)
Hauv1902, Emil Fischeryog thawj zaug tshawb nrhiav peptide synthesis, tab sis kev nce qib tau qeeb vim muaj kev txwv. Thaum ntxov syntheses ua haujlwmbenzoyl thiab acetyl tiv thaiv pawg, uas nyuaj rau tshem tawm thiab feem ntau coj mus raupeptide chain cleavage.
Hauv1932, Max Bergmannthiab co-cov neeg ua haujlwm tau qhia txogbenzyloxycarbonyl (Z) pawgraws li -amino tiv thaiv pab pawg. Pab pawg no tuaj yeem raug tshem tawm hauv qabcatalytic hydrogenation los yog acidic tej yam kev mob, muab cov cuab yeej txhim khu kev qha ntau dua rau peptide synthesis thiab tso lub hauv paus rau kev txhim kho cov khoom sib txuas -theem peptide synthesis hauv xyoo 1960.
Thaum lub sij hawm1950s, cov kws tshawb fawb tau ua tiav ntau yam ntawmbiologically nquag peptides, zoo lioxytocin thiab insulin. Cov kev ua tiav no tau ua tiav qib siab peptide chemistry thiab tsim lub hauv paus rau kev tshwm sim ntawm cov khoom sib txuas -theem peptide synthesis.
2. Pioneering Theem (1963)
Hauv1963, Robert B. Merrifieldnpaj lubsolid-phase peptide synthesis (SPPS) txoj kev, hauv covC-terminal amino acidyog anched rau kev txhawb nqa resin, thiab cov saw peptide yog elongated los ntawm kev rov ua dua ntawmdeprotection thiab coupling. Txoj hauv kev no ua kom yooj yim rau cov txheej txheem peptide synthesis thiab ua tustxoj kev nyiamrau peptide sib dhos.
Merrifield sivBoc (tert{0}}butyloxycarbonyl)los tiv thaiv -amino pawg. Los ntawm lig 1960s, nws kuj tsim lubthawj tag nrho automated peptide synthesizerthiab ntse synthesizedbiological proteinsxws liribonuclease (124 amino acids). Rau cov kev ua tau zoo tshaj plaws no, Merrifield tau txais txiaj ntsigNobel nqi zog hauv Chemistry hauv 1984.
3.Technological Innovation Theem (1972)
Hauv1972, Lou Carpinotsim lub9-fluorenylmethyloxycarbonyl (Fmoc) tiv thaiv pawg, uas taumaj mam muab tshem tawm nyob rau hauv tej yam kev mob, txo cov kev mob tshwm sim. Qhov no ua rau nws tshwj xeeb tshaj yog haum raucomplex peptidesthiabautomated synthesis.
CovFmoc txoj kevmaj mam hloov txoj kev Boc raws li lub tswv yim tseem ceeb. Automated peptide synthesizers raws li ob qho tib siFmoc thiab Boc chemistrytau tom qab tsim, tsav lubautomation ntawm peptide synthesisthiab zoo heev txhim kho efficiency thiab reproducibility.
4.Maturity and Expansion Stage (1990s to Tam sim no)
Resin thiab Reagent Optimization:Kev loj hlob ntawmsiab -loading, hydrophilic PEG- hloov kho resinsthiab muaj txiaj ntsig coupling reagents xws liHBTUthiabHATUtau txhim kho qhov ua tau zoo thiab cov txiaj ntsig ntawm cov khoom siv -theem peptide synthesis (SPPS).
Kev koom ua ke thev naus laus zis:Txoj kev tshiab zoo limicrowave- pab synthesisthiabflow chemistrymuaj shortened cov tshuaj tiv thaiv lub sij hawm thiab ntxiv nce yields.
Basic Principles thiab Workflow of SPPS:
Lub hauv paus ntsiab lus tseem ceeb ntawm cov khoom -theem peptide synthesis yog qhovstepwise kev tsim kho ntawm peptide saw ntawm kev txhawb nqa khoom, sivtiv thaiv pab pawg tswv yimthiabcondensation (coupling) cov tshuaj tiv thaivkom ua tiavua tau zoo thiab tswj tau peptide sib dhos.
1.Lub luag haujlwm ntawm Kev Txhawb Khoom (Resin) hauv Khoom -Phase Peptide Synthesis (SPPS)
A insoluble polymeric cob-xws lipolystyrene-divinylbenzene cross-linked resin, Wang resin, lossis Rink amide resin- raug xaiv raws li kev txhawb nqa hauv SPPS. Cov resin nto muajpab pawg ua haujlwm(piv txwv li, hydroxyl lossis amino pawg) uas tuaj yeem tsimcovalent bonds nrog ib kawg ntawm peptide saw, feem ntau covC-terminus, anchoring peptide mus rau cov khoom txhawb nqa.Qhov kev sib txuas lus no ua kom cov peptide loj hlob tuaj.tseem khi rau cov resin thoob plaws hauv lub synthesis, uas zoo heevpab txhawb cov kauj ruam tom ntej, ntxuav, thiab ntxuav
2.Kev Tiv Thaiv Pab Pawg Strategy hauv Khoom -Phase Peptide Synthesis (SPPS)
Thaum lub sij hawm peptide synthesis, lub- pawg amino, pawg carboxyl, thiab sab reactive-chain functional pawgntawm cov amino acids (xws li thiol, carboxyl, thiab amino pawg) yog nquagsab kev xav. Txhawm rau tiv thaiv cov kev xav tsis zoo no,tiv thaiv pab pawgsiv. Cov pab pawg tiv thaiv muaj xws li:-Amino Protecting Groups:
Boc (tert -butyloxycarbonyl):Acid-labile, yooj yim tshem tawm hauv qabacidic tej yam kev mob.
Fmoc (9-fluorenylmethyloxycarbonyl):Ruaj nyob rau hauv acidic tej yam kev mob, yuav raug tshem tawm nyob rau hauvyooj yim tej yam kev mob(piv txwv li, piperidine / DMF tov).Sab -Chain Tiv Thaiv Pawg:Xaiv raws li cov khoom siv tshuaj lom neeg ntawm cov amino acid sab chain.ommon pawg suav nrogbenzyl (Bn), tert-butyl (tBu), trityl (Trt), etc.Cov pab pawg no yogtshem tawm thaum kawg ntawm synthesis, feem ntau thaum peptide cleavage los ntawm cov cob, sivacid lossis lwm yam mob tshwj xeeb.
3.C-Terminus rau N-Terminus Synthesis hauv SPPS
Hauvsolid-phase peptide synthesis (SPPS), peptide sib dhos feem ntau pib ntawm lubC-terminus (carboxyl kawg)thiab mus stepwise mus rau lubN-terminus (amino kawg). Qhov no yog vim qhovC-terminal amino acid yog thawj zaug txuas rau cov khoom txhawb nqa, thiab cov amino acids tom ntej yog ua ke rau covdawb amino pawg ntawm peptide saw twb khi rau cov cob, maj mam elongating peptide saw.
4.Coupling Reaction in Solid-Phase Peptide Synthesis (SPPS)
Kev tiv thaiv amino acids yuav tsum muajqhibyog li ntawd lawvcarboxyl pawg ua reactive, tso cai rau lawv tsim ibpeptide daim ntawv cog lusnrog cov amino pawg ntawm cov resin - khi peptide. Feem ntauactivating reagentssuav nrogcarbodiimides(xws li DCC, DIC),HOBt, HATU, thiabPIB. Thaum ua haujlwm, pawg carboxyl reacts nrog cov amino pawg dawb hauv acondensation tshuaj tiv thaiv, forming ibamide daim ntawv cog lusthiab yog li txuas cov amino acid tshiab rau cov saw peptide loj hlob.
5.Repetitive (Cyclic) Kev Ua Haujlwm hauv Khoom -Phase Peptide Synthesis (SPPS)
Cov txheej txheem koom nrogrov ua lub voj voog ntawm kev tiv thaiv → coupling → ntxuav, ntxivib qho amino acid rau ib lub voj voogkom maj mam elongate cov saw peptide. Tom qab txhuakev sib txuas lus, resin yogntxuav(piv txwv li, nrog cov kuab tshuaj xws li DMF lossis DCM) kom tshem tawm cov tshuaj tsis ua haujlwm, los ntawm - cov khoom, thiab cov amino acids ntau dhau. Cov-Amino pawg ntawm cov residue uas tau ntxiv tshiab yuav raug tiv thaiv, nthuav tawm ib qho dawb amine los npaj rau kev sib txuas lus tom ntej. Lub voj voog no rov ua dua kom txog rau thaum qhov xav tau peptide ib ntus tag nrho.
6.Cleavage hauv Khoom-Phase Peptide Synthesis (SPPS)
Thaum cov saw peptide tau mus txog qhov xav tau ntev, nws yogcleaved los ntawm cov khoom txhawb nqasiv cov reagent tsim nyog, thaum tib lub sijhawmtshem tawm txhua pab pawg tiv thaiv. Feem ntau siv cleavage reagent yogtrifluoroacetic acid (TFA), uas tsis tsuas yogrhuav tshem kev sib txuas ntawm peptide thiab cov cobtab sis kujtshem tawm cov pab pawg tiv thaivxws li Fmoc thiab Boc, kho cov peptide rau nwshaiv neeg lub xeev. Thaum cov saw peptide tau mus txog qhov xav tau ntev, nws yogcleaved los ntawm cov khoom txhawb nqasiv cov reagent tsim nyog, thaum tib lub sijhawmtshem tawm txhua pab pawg tiv thaiv. Feem ntau siv cleavage reagent yogtrifluoroacetic acid (TFA), uas tsis tsuas yogrhuav tshem kev sib txuas ntawm peptide thiab cov cobtab sis kujtshem tawm cov pab pawg tiv thaivxws li Fmoc thiab Boc, kho cov peptide rau nwshaiv neeg lub xeev.
Los ntawm cov kauj ruam no,solid-phase peptide synthesis (SPPS)enables covmuaj txiaj ntsig thiab tswj kev sib dhos ntawm peptide chains ntawm kev txhawb nqa khoom, zam qhov nyuaj nruab nrab purification cov kauj ruam uas yuav tsum tau nyob rau hauv cov tshuaj ib txwm siv -theem synthesis thiab lojtxhim kho synthesis efficiency thiab tawm los.
Los ntawm cov kauj ruam no,solid-phase peptide synthesis (SPPS)enables covmuaj txiaj ntsig thiab tswj kev sib dhos ntawm peptide chains ntawm kev txhawb nqa khoom, zam qhov nyuaj nruab nrab purification cov kauj ruam uas yuav tsum tau nyob rau hauv cov tshuaj ib txwm siv -theem synthesis thiab lojtxhim kho synthesis efficiency thiab tawm los.
