Peptide Synthesis yog dab tsi

 

Peptide synthesis yog ib qho chaw ua haujlwm nyob rau hauv cov protein thiab peptide chemistry, uas feem ntau koom nrog qhov sib ntxiv ntawm cov amino acids nyob rau hauv ib qho kev txiav txim kom tsim cov peptide saw los ntawm kev siv tshuaj. Lub ntsiab synthesis txoj kev yog kua-theem thiab solid-phase synthesis. Muab piv rau LPPS, SPPS muaj qhov zoo ntawm kev ua kom nrawm dua, yooj yim dua, nrog tsawg dua kev cuam tshuam, thiab cov txiaj ntsig siab dua.

 

Cov txheej txheem SPPS, suav nrog Boc (benzyl) thiab Fmoc (tBu) cov tswv yim.

 

 

Boc (benzyl) lub tswv yim

Nyob rau hauv no synthesis mus kom ze, Chloromethylated, hydroxymethylated polystyrene thiab p-methoxybenzyl (PMA) resins yog siv los ua khoom txhawb nqa. Cov pab pawg -amino tiv thaiv nrog t-butoxycarbonyl (Boc), thaum sab saw pawg ntawm cov amino acids tiv thaiv nrog benzyl derivatives. Cov pab pawg Boc raug tshem tawm siv TFA los yog TFA zoo hauv CH2Cl2, thiab thaum kawg ntawm kev sib txuas, sab-chain tiv thaiv pawg thiab peptide-resin linkages raug tshem tawm nrog cov kua qaub uas muaj zog xws li anhydrous hydrofluoric acid (HF) lossis TFMSA.

 

Fmoc (tBu) Strategy

Nyob rau hauv no synthesis mus kom ze, Wang-resin, 2-Chlorotrityl Chloride Resin, Rink amide-AM Resin, Rink amide-MBHA resins yog siv los ua khoom txhawb nqa.Cov -amino pab pawg muaj kev tiv thaiv nrog 9-fluorenylmethoxycarbonyl (Fmoc ), thaum sab saw pawg ntawm cov amino acids tiv thaiv nrog acid-labile tiv thaiv pawg. Cov pab pawg Fmoc raug tshem tawm siv 20% piperidine hauv DMF, thiab thaum kawg ntawm kev sib txuas, sab-chain tiv thaiv pawg thiab peptide-resin linkages raug tshem tawm nrog 1%-95% TFA. Fmoc SPPS tam sim no yog txoj kev nyiam rau peptide synthesis.

 

Lub hauv paus ntsiab lus ntawm Fmoc Solid-phase Peptide Synthesis

 

Xaiv Resin

Cov khoom siv dav tshaj plaws hauv Fmoc SPPS suav nrog 4-Alkoxybenzyl cawv (Wang) resin, 2-Chlorotrityl chloride resin, Rink Amide Resin, Rink Amide-MBHA resins. Amide resins yog siv rau kev sib txuas ntawm peptides nrog requried C terminal amidation. Wang resin thiab 2-Cl Trt resin feem ntau yog siv rau synthesis peptide nrog rau C terminal dawb COOH. Tab sis thaum C-terminus ntawm peptide muaj Pro thiab Gly raws li thawj amino acid, Wang resin yuav tsum tsis txhob siv vim muaj kev pheej hmoo ntawm DKP (diketopiperazine) tsim, 2-Chlorotrityl Chloride Resin raug pom zoo siv.

 

 

Txuas cov amino acids tiv thaiv rau Resins

Thawj Fmoc amino acid txuas mus rau ib qho kev txhawb nqa insoluble ntawm cov kua qaub labile linker.

 

Deprotection ntawm -amino tiv thaiv pawg

Cov pab pawg tiv thaiv Fmoc ib ntus ntawm N-terminus ntawm peptidyl-resin raug tshem tawm los ntawm kev kho nws nrog kev daws ntawm 20% piperidine hauv DMF. Cov tshuaj tiv thaiv no feem ntau ua tiav hauv 10 mus rau 20 feeb.

 

Ntxuav cov kauj ruam

Tom qab txhua qhov tshuaj tiv thaiv, kev ntxuav cov kauj ruam yog ua kom tshem tawm cov reagents ntau dhau, cov khoom los ntawm cov khoom, thiab cov molecules unreacted los ntawm cov resin. Qhov no yuav pab tswj tau qhov purity ntawm cov saw peptide loj hlob.

 

Ua kom thiab coupling ntawm kev tiv thaiv amino acid

Kev tiv thaiv amino acid khi rau cov resin yog qhib kom txhim khu nws reactivity rau coupling nrog cov amino acid tom ntej nyob rau hauv ib theem zuj zus. Cov neeg ua haujlwm nquag muaj xws li HOAt, HOBt, PyBOP, PyAOP, HBTU thiab HATU. Kev sib txuas nrog kev tsim cov peptide daim ntawv cog lus ntawm cov activated amino acid thiab cov amino pawg ntawm cov saw peptide loj hlob. Kev kuaj Kaiser feem ntau yog siv los saib xyuas qhov ua tiav ntawm kev sib txuas lus.

 

Ntxuav cov kauj ruam

Zoo ib yam li cov kauj ruam ntxuav ua ntej, qhov no ua kom qhov kev tshem tawm ntawm cov reagents ntau dhau thiab cov khoom lag luam tom qab cov tshuaj tiv thaiv sib txuas.

 

Cleavage los ntawm resin thiab deprotection ntawm sab saw tiv thaiv pawg

Thaum qhov xav tau peptide ib ntu yog tsim los ntawm cov cob, nws yog cleaved los ntawm cov khoom txhawb nqa siv lub cleavage cocktail xws li trifluoroacetic acid (TFA) uas muaj scavengers xws li thianoanisole thiab dej. Ib txhij, sab saw tiv thaiv pawg ntawm cov amino acid residues raug tshem tawm, nthuav tawm tag nrho cov kev tiv thaiv peptide npaj rau kev ntxuav thiab tshuaj xyuas.