Gerald P. Schwartz

[47] The synthesis of peptides by homogeneous solution procedures

Publisher Summary This chapter presents a procedure for the synthesis of peptides by homogeneous solution. The synthesis of peptides by homogeneous solution procedures involves four stages: (1) the synthesis of the carboxyl component by blocking the amino group of an amino acid or peptide with a group Y; (2) the synthesis of the amino component by blocking the carboxyl group of another amino acid or peptide with a group Z; (3) the activation of the carboxyl group of the carboxyl component (or, very rarely, of the amino group of the amino component) with a group X and the coupling of the so-formed active intermediate with the amino component (or carboxyl component) to yield a protected peptide; and (4) the removal of the blocking groups, Y and Z, from the protected peptide to form the free peptide. The synthesis of peptides containing polyfunctional amino acid residues, such as cysteine, arginine, lysine, histidine, tyrosine, serine, threonine, glutamic acid, or aspartic acid, requires additional steps. The secondary functional group is often protected during the various synthetic steps and is deprotected in the final step. The chapter also presents the methods for the elongation of a peptide chain. In general, three approaches are available for this purpose: (1) stepwise synthesis from the amino terminal of the growing peptide chain; (2) stepwise synthesis from the carboxyl terminal of the growing peptide chain; and (3) fragment condensation—that is, ioining together of small peptide subunits to form a larger peptide.

5-Hydroxytryptophan: An absorption and fluorescence probe which is a conservative replacement for [A14 tyrosine] in insulin

Use of insulins intrinsic tyrosine absorption and fluorescence to monitor its interaction with the insulin receptor is limited because the spectral properties of the receptor tryptophan residues mask the spectral properties of the hormone tyrosine residues. We describe the synthesis of an insulin analog where A14 tyrosine is replaced by a tryptophan analog, 5-hydroxytryptophan. This insulin is spectrally enhanced since 5-hydroxytryptophan has an absorption band above 300 nm which is at lower energies than the absorption of tryptophan. Steady-state and time-resolved fluorescence parameters indicate that 5-hydroxytryptophan reports the same information about the environment of the A14 side chain as does the corresponding tryptophan-containing insulin. The synthetic hormone is a full agonist compared to porcine insulin, but has slightly reduced specific activity. Consequently, this spectrally enhanced insulin analog will be useful for hormone-receptor interaction studies since it can be observed by both absorption and fluorescence even in the presence of the tryptophan-containing receptor.

Synthesis of a biologically active truncated insulin. Des(pentapeptide B26–30) human (porcine) insulin

An analogue of human (porcine) insulin which differs from the parent molecule in that the C-terminal pentapeptide sequence of the B chain has been eliminated has been synthesized. For this purpose, des(pentapeptide B26–30) B chain S-sulphonate was synthesized by the fragment condensation approach and isolated in highly purified form. Interaction of this compound with the thiol form of the A chain of human (porcine) insulin yielded des(p entapeptide B26–30) human (porcine) insulin, which was purified by chromatography on a carboxymethylcellulose column with an exponential sodium chloride gradient. The des(pentapeptide B26–30) insulin shows potencies of 8·5–9 I.U. mg–1 when assayed by the mouse convulsion method and of 11 I.U. mg–1 by the radioimmunoassy method (cf. 23–25 I.U. mg–1 for the natural hormone). This indicates that the C-terminal pentapetide sequence of the B chain does not participate functionally in the mechanism of the action of insulin.

Insulin peptides. Part XXIV. A novel synthesis of the human insulin B chain S-sulphonate

The fragment condensation method was employed to construct the protected triacontapeptide which contains the amino-acid sequence of the human B chain. The four peptide fragments used in this synthesis were prepared stepwise. The protected triacontapeptide was deblocked by treatment with liquid hydrogen fluoride and the resulting reduced peptide chain was converted into the S-sulphonated derivative by oxidative sulphitolysis. The S-sulphonated chain, purified by chromatography on a CM-cellulose column with a urea–acetate buffer at pH 4·0. was obtained in 33% overall yield, based on the amount of the protected B chain used; a yield of 6–7% was obtained in our original synthesis.

Glutamine B16 insulin: Reduced insulin-like metabolic activity with moderately preserved mitogenic activity

An analogue of insulin in which the naturally occurring tyrosine residue in position B16 is replaced by a glutamine residue has been synthesized. Glutamine appears in the corresponding position in the B-domain of the insulin-like growth factors. This analogue displays 9% of the potency of insulin in binding to the insulin receptor from rat liver plasma membranes, 17% in stimulating the conversion of [3-3H] glucose into lipids in rat adipocytes, and 23% in insulin radioimmunoassay, but 40% of the potency of insulin in stimulating DNA synthesis in cultured chick fibroblasts. The analogue is a more potent mitogen than is a hybrid molecule which contains the A-chain of insulin and the entire B-domain sequence of IGF-I.

The importance of the B10 amino acid residue to the biological activity of insulin. [Lys10-B] human insulin

An analog of human insulin, which differs from the parent molecule in that the histidine residue at position 10 of the B chain (B10) is replaced by lysine, has been synthesized and isolated in purified form. This analog, [10-lysine-B] insulin ([Lys10-B] insulin), in stimulating lipogenesis and in radioimmunoassays, exhibited potencies of 14.2% and 14.7%, respectively, as compared to the natural hormone. In insulin receptor binding in rat liver membranes, [Lys10-B] insulin was found to possess a potency of ∼17% compared to insulin. We have shown previously that substitution of the B10 polar residue histidine with the nonpolar leucine results in an analog exhibiting inin vivo assays ∼50% of the activity of the parent molecule. It is speculated that in insulin the relative size of the amino acid residue at B10, rather than its polarity, is the most important factor in maintaining a structure commensurate with high biological activity.

Insulin peptides. Part XXIII. The synthesis of a hexadecapeptide derivative related to the B chain of human insulin

A protected hexadecapeptide with the amino-acid sequence found at the C-terminal region of the human insulin B chain has been prepared by condensation of N-terminal hexapeptide and C-terminal decapeptide fragments, which were prepared stepwise. All the protecting groups employed are labile to hydrogen fluoride.

A synthetic human insulin analogue modified at position B22. [Lys22-B] human insulin

A human insulin analogue which differs from the parent molecule in that the amino-acid residue arginine at position 22 in the B chain is replaced by lysine has been synthesized. For this purpose a human B chain analogue, in which the arginine residue at position 22 was replaced by a lysine residue, was prepared by the fragment condensation approach and isolated as the S-sulphonate. Combination of the latter compound with the thiol form of the human (porcine) insulin A chain yielded the [Lys-22B] human insulin. This analogue was isolated in highly purified form by chromatography on a carboxymethylcellulose column with an exponential sodium chloride gradient. [Lys-22B] insulin shows potencies of 13–14 I.U. mg–1 when assayed by the mouse convulsion method and of 8 I.U. mg–1 by the radioimmunoassay method (cf. 23–25 I.U. mg–1 for the natural hormone).