SPPS-固相合成肽,蛋白

1、Solid phase peptide synthesisIntroduction Definition SPPS is quick and easy approach to synthesizing peptides and small proteins. The C-terminal amino acid is attached to a cross-linked polystyrene resin via an acid labile bond with a linker molecule. This resin is insoluble in the solvents used for

2、 synthesis, making it relatively simple and fast to wash away excess reagents and by-products. The N-terminus is protected with the Fmoc group, which is stable in acid, but removable by base. Any side chain functional groups are protected with base stable, acid labile groups. .IntroductionThe Nobel

3、prize for chemistry in 1986- for his development of methodology for chemical synthesis on a solid matrixRobert Bruce MerrifieldRockefeller UniversityFlowing diagram of Fmoc-based SPPSX: FmocDeprotect: 20% piperidine in DMFY: Activating group Choice of functionalized resinsRink amide MBHA resin Resin

4、 for peptide amides:95% TFAPeptide amidesp-alkoxybenzyl alcohol polystyrene resin(Wang resin) Choice of functionalized resinsResin for peptide acids:95% TFAPeptide acids2-Chlorotrityl chloride resin 1% TFAPeptide acidsChoose of N-Fmoc-amino acidActivation of amino acidsHBTUGenerally use: Carbodiimid

5、es: DIC Phosphonium: PyBOP Uronium salts: HBTU,HATUChoose of cleavage cocktail Depend on the protecting group present Three commonly used cleavage mixtureA: 95% TFA + 5% H2OB: 95% TFA + 2.5% H2O + 2.5% TIPS (Triisopropylsilane)C: 90.5% TFA + 5% Phenol + 2.5% H2O + 2% TIPS A for peptides not containi

6、ng problematic amino acids B for peptides containing tBu, OtBu, Trt, Boc side chain protection group or MetC for peptides containing Arg or TrpReaction times : peptides containing Arg, 4-12 h Monitoring the synthesis Fmoc deprotection monitoring Coupling reaction monitoringCoupling efficiency and final yieldKaiser ninhydrin testReagent: Ninhydrin, Phenol, KCN + peptide-resin110 , 5 minutes Positive results : a