Formula omitted]: Biosynthesis in insect cuticle and possible role in sclerotization

Abstract

N-β- alanylnorepinephrine (NBANE), a metabolite of N-β- alanyldopamine (NBAD) in pupal cuticle of the tobacco hornworm, Manduca sexta, was identified by analysis of hydrolytic products, by comparison of HPLC retention times with a synthetic standard and by fast atom bombardment mass spectrometry. NBANE and NBAD were abundant in the brown cuticle of M. sexta pupae, Periplaneta americana adults, Tribolium castaneum adults and Musca domestica puparia. Side chain modification of NBAD was rapid during the first few hours of sclerotization as evidenced by a dramatic increase in NBANE extracted from M. sexta pupal cuticle with cold 1.2 M HCl. In later stages of sclerotization less NBANE was recovered indicating further metabolism and possible involvement in the formation of crosslinks to cuticular components. Although similar amounts of the two catecholamines were extracted by 80% methanol from pupal cuticle which was just starting to tan, over 15-fold more NBANE than NBAD was recovered with cold dilute HCl. Low pH may be necessary to remove physically entrapped NBANE from the insoluble polymeric matrix of the cuticle, or NBANE released by the acid treatment may be a hydrolytic product from NBAD covalently linked by its β-carbon to cuticular components. NBANE was synthesized from NBAD by an insoluble enzyme in tanning pupal cuticle that also catalyzed to a lesser extent the β-hydroxylation of N- acetyldopamine (NADA). However, NADA was a better precursor than NBAD for compounds that yield 3,4-dihydroxphenylketoethanol after heating in 1 M HCl. Therefore, two pathways were observed in tanning cuticle for side chain metabolism of the N- acylated dopamines, and the type of acyl group influenced the relative amounts of different sclerotizing agents produced.