title: In situ neutralization in Boc-chemistry solid phase peptide synthesis. Rapid, high yield assembly of difficult sequences.
abstract: Simple, effective protocols have been developed for manual and machine-assisted Boc-chemistry solid phase peptide synthesis on polystyrene resins. These use in situ neutralization [i.e. neutralization simultaneous with coupling], high concentrations (> 0.2 M) of Boc-amino acid-OBt esters plus base for rapid coupling, 100% TFA for rapid Boc group removal, and a single short (30 s) DMF flow wash between deprotection/coupling and between coupling/deprotection. Single 10 min coupling times were used throughout. Overall cycle times were 15 min for manual and 19 min for machine-assisted synthesis (75 residues per day). No racemization was detected in the base-catalyzed coupling step. Several side reactions were studied, and eliminated. These included: pyrrolidonecarboxylic acid formation from Gln in hot TFA-DMF; chain-termination by reaction with excess HBTU; and, chain termination by acetylation (from HOAc in commercial Boc-amino acids). The in situ neutralization protocols gave a significant increase in the efficiency of chain assembly, especially for "difficult" sequences arising from sequence-dependent peptide chain aggregation in standard (neutralization prior to coupling) Boc-chemistry SPPS protocols or in Fmoc-chemistry SPPS. Reported syntheses include HIV-1 protease(1-50,Cys.amide), HIV-1 protease(53-99), and the full length HIV-1 protease(1-99).
authors: Scripps Research Institute, La Jolla, CA.
authors: M
authors: M
authors: Schnölzer
authors: P
authors: P
authors: Alewood
authors: A
authors: A
authors: Jones
authors: D
authors: D
authors: Alewood
authors: S B
authors: SB
authors: Kent
mesh_terms: D000107:Acetylation; D000213:Acyl Carrier Protein / Q000138:chemical synthesis; D002851:Chromatography, High Pressure Liquid; D005563:Formic Acid Esters / Q000737:chemistry*; D005973:Glutamine / Q000737:chemistry; D016333:HIV Protease / Q000138:chemical synthesis; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000138:chemical synthesis; D010455:Peptides / Q000138:chemical synthesis*; D011137:Polystyrenes; D012117:Resins, Synthetic; D013237:Stereoisomerism; D013997:Time Factors
chemical_list: D000213:Acyl Carrier Protein; D005563:Formic Acid Esters; C078983:HIV-1 protease (44-48); C078984:HIV-1 protease (89-99); D010446:Peptide Fragments; D010455:Peptides; D011137:Polystyrenes; D012117:Resins, Synthetic; C000345:t-butyloxycarbonyl group; D005973:Glutamine; C078985:acyl carrier protein (65-74); D016333:HIV Protease