Synthesis and biological activity of locust AKH-I and its analogues with modifications at the threonine residues.

Abstract

A convenient method of synthesis, using a combination of solid and liquid phase methodology, for locust Adipokinetic Hormone-I (AKH-I) and its analogues with modifications at the threonine residues are reported. The N-terminal nonapeptide acid of AKH-I is synthesized in the solid phase using the 2-chlorotrityl chloride resin and the Fmoc/t-Bu strategy. Quantitative cleavage of the nonapeptide acid from the resin, with the tert-butyl type side-chain protection intact, is achieved with a mixture of acetic acid/trifluoroethanol/dichloromethane. The nonapeptide acid is then coupled in solution to the threonine derivatives, H-Thr-NH2 or H-Thr(Bzl)-NH2, with the DCC/HOBt method. The efficiency of this approach in the synthesis of AKH-I is demonstrated by the high yields and purity of the synthesized peptides. All the synthesized peptides were tested in two ways: first, in a lipid mobilization assay in locusts in vivo; and second, in a novel assay in vitro concerned with the uptake of radiolabelled acetate into locust tissue. Replacement of the hydroxyl hydrogen in Thr5 of locust AKH-I by the bulky and highly lipophilic tert-butyl group reduced the potency markedly, whereas efficacy is unaffected, but when the hydroxyl hydrogen of Thr10 in AKH-I is replaced by a benzyl group, the activity of the resulting analogue is identical to that of the natural peptide. Structure-activity relationships are discussed.