Synthesis of insulin-like growth factor I using N-methyl pyrrolidinone as the coupling solvent and trifluoromethane sulphonic acid cleavage from the resin.
Insulin-like growth factor I (IGF-I), a protein of 70 amino acid residues and 3 cystine bridges, has been synthesized by two solid phase Boc methods. The first method used N-methylpyrrolidinone as the solvent with single coupling cycles while the second synthesis used dimethylformamide and dichloromethane as the solvents with a double-coupling protocol. In both cases, trifluoroacetic acid/trifluoromethanesulphonic acid cleavage of the peptide from the resin was employed. Purification of the cleavage products followed by removal of the S-acetamidomethyl protecting groups gave reduced peptides which were then oxidized under conditions favouring the formation of the correct disulphide bonds. The purified synthetic IGF-I peptides were full agonists of natural IGF-I in a radioimmunoassay, in an IGF-I radioreceptor assay, in a bioassay which measures the stimulation of protein synthesis in rat L6 myoblasts and in an IGF-binding protein competitive binding assay. Moreover, in each of these assays, the synthetic IGF peptides were found to be at least 70% as potent as natural IGF-I.
Bagley
C J
CJ
Biochemistry Department, University of Adelaide, SA.
Otteson
K M
KM
May
B L
BL
McCurdy
S N
SN
Pierce
L
L
Ballard
F J
FJ
Wallace
J C
JC
D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007202:Indicators and Reagents; D007334:Insulin-Like Growth Factor I / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D008698:Mesylates; D011760:Pyrrolidinones; D011956:Receptors, Cell Surface / Q000378:metabolism; D017451:Receptors, Somatomedin; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D012116:Resins, Plant; D012997:Solvents
D007202:Indicators and Reagents; D008698:Mesylates; D011760:Pyrrolidinones; D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D011994:Recombinant Proteins; D012116:Resins, Plant; D012997:Solvents; D007334:Insulin-Like Growth Factor I; C012077:trifluoromethanesulfonic acid; C038678:N-methylpyrrolidone