Neuropeptide Y acylation chemistry in aqueous solution: significance to synthesis of a peptide-based photoaffinity label

Abstract

Treatment of neuropeptide Y (NPY, 1) for 20 hr with a 20 equivalent excess of N-propionyl succinimide (2) in 10 mM phosphate buffer, pH 6.0, yields NPY and N alpha-propionyl-NPY (3) as major products, and at pH 7.5 the major product is N alpha, N epsilon-dipropionyl-NPY. However, acylation of NPY with one equivalent of N-(5-azido-2-nitrobenzolyloxy)-succinimide (5) is more rapid, yielding N alpha-(5-azido-2-nitrobenzoyl)-NPY (6) in 70% conversion yield after only 5 min. Thus, in spite of its increased reactivity, the N-hydroxysuccinimide active ester shows enhanced alpha- vs. epsilon-NH2 selectivity relative to 2. The activities of 3, 4, and 6 as reversible, competitive ligands at rat brain NPY binding sites and of 6 as an irreversible photoaffinity label are reported.