DETECTION OF INTERCATECHOL CROSS-LINKS IN INSECT CUTICLE BY SOLID-STATE C-13 AND N-15 NMR

Solid-state NMR has been used to detect an intercatechol covalent bond in sclerotized cuticle of the tobacco hornworm (Manduca sexta). Rotat ional-echo, double-resonance (REDOR) C-13 and N-15 NMR, in combination with H-1-C-13 dipolar modulation and N-15-N-15 dipolar restoration at the magic angle, were used to examine hornworm pupal exuviae labeled with beta-[N-15]alanine and either [beta-C-13]dopamine or [alpha-C-13, N-15]dopamine. The REDOR spectra showed the incorporation of N-beta-a lanyldopamine into insect cuticle by the formation of a variety of cov alent bonds. One of these bonds links the terminal nitrogen of one N-b eta-alanyldopamine molecule to the beta carbon of another to form an i ntercatechol covalent bond. This result is interpreted in terms of a n ovel structure for stabilized cuticle that postulates protein-catechol -catechol-protein cross-links. REDOR spectra also showed oxygen substi tution at both alpha and beta carbons of N-beta-alanyldopamine, consis tent with the possibility of intercatechol oxygen bridges in dimeric o r oligomeric forms of catecholamines in the cuticle .