Human insulin-like growth factor I (somatomedin C) with 70 amino acid residues and three disulfide bridges has been synthesized by the solid-phase method. The synthetic product behaves as a pure polypeptide in paper electrophoresis, isoelectric focusing, and high-performance liquid chromatography. Its elution behavior in Sephadex G-50, isoelectric point, amino acid composition, and growth-stimulating activity in bovine adrenal cortical cells or granulosa cells are comparable to those reported for the natural product. In radioimmunoassay, the synthetic product is indistinguishable from the natural hormone when either synthetic or natural product is used as the labeled ligand.Production of somatomedins (SMs) and insulin-like growth factors (IGFs) is known to be stimulated by growth hormone (somatotropin) in animals (1, 2) and human subjects (3, 4). SM-C (5) and IGFs (6) have been purified to homogeneity from human plasma. Complete amino acid sequences for IGF-I (7) and IGF-II (8) have been proposed, but only limited sequence data for SM-C are known (5). Various bioassay and radioimmunoassay (RIA) data indicate that IGF-I and SM-C are identical (5, 9, 10). IGF-I consists of 70 amino acid residues with three disulfide bridges, and its structure is homologous to that of proinsulin (7, 11). Fig. 1 presents the primary structure of IGF-I as proposed by Rinderknecht and Humbel (7). Because chemical synthesis of any known growth factor has not been reported, this communication describes the total synthesis of IGF-I (SM-C). MATERIALS AND METHODSProtected IGF-I Resin. 4-(Boc-alanyloxymethyl)-phenylacetamidomethyl-resin (1.60 g, 0.60 mmol) prepared as described (12) was placed in a Beckman model 990 peptide synthesizer and carried through procedures of solid-phase synthesis (13) consisting of (i) 15-min deprotection with 50% (vol/vol) trifluoroacetic acid/CH2Cl2, (ii) neutralization with 5% (vol/vol) diisopropylethylamine (DIEA)/CH2Cl2 (two times). Coupling was effected with preformed symmetrical anhydrides (1.8 mmol, 3 eq) of for 20 min in CH2Cl2 except for Boc-Met(O)-OH, Boc-Arg(Tos)-OH, and Boc-Gln-OH, for which 40-min coupling was allowed and sufficient N,N-dimethylformamide was employed to give concentrations of 20, 15, and 20%, respectively. Boc-Asn-OH was coupled with dicyclohexylcarbodiimide and 1-hydroxybenzotriazole (15). In all cases a second coupling stage was performed (20 min): (i) for residues 69 through 59 and all subsequent couplings in which dimethylformamide was used 1 eq of DIEA was added (16); (ii) for all other residues 1 eq of N-methylmorpholine and sufficient trifluoroethanol to give a concentration of 20% were added (16). The following side-chain blocking groups were used: Asp, cyclopentyl (17); Thr, Ser, and Glu, benzyl; Cys, 3,4-dimethylbenzyl (18); Met, sulfoxide (19); Tyr and Lys, 2-bromobenzyloxycarbonyl; Arg, tosyl. The yield of protected peptide resin was 7.92 g (weight gain was 87% of theory).IGF-I. Protected IGF-I resin (1.336 g, 0.101 mmol) was treated with 50% trifluoroacetic ac...

The biological and immunological properties of a chemically synthesized preparation of somatomedin C (Sm-C) were compared with those of the natural product isolated from human plasma. The two preparations produced identical curves in the radioimmunoassay and radioreceptor assay for Sm-C and in the radioreceptor assay for insulin. They were identical in their ability to stimulate DNA synthesis in confluent BALB/c 3T3 cells previously exposed to plateletderived growth factor, and the biological activities of both preparations were completely neutralized by a monoclonal antibody raised against native Sm-C. These studies demonstrate that the chemically synthesized product is equivalent to the native molecule in all important respects and that it can be used interchangeably with the natural product for any studies that are contemplated.