Detection of Intercatechol Cross-Links in Insect Cuticle by Solid-State Carbon-13 and Nitrogen-15 NMR

Solid-state NMR has been used to detect an intercatechol covalent bond in sclerotized cuticle of the tobacco hornworm (Manduca sexta). Rotational-echo, double-resonance (REDOR) 13C and 15N NMR, in combination with 1H−13C dipolar modulation and 15N−15N dipolar restoration at the magic angle, were used to examine hornworm pupal exuviae labeled with β-[15N]alanine and either [β-13C]dopamine or [α-13C,15N]dopamine. The REDOR spectra showed the incorporation of N-β-alanyldopamine into insect cuticle by the formation of a variety of covalent bonds. One of these bonds links the terminal nitrogen of one N-β-alanyldopamine molecule to the β carbon of another to form an intercatechol covalent bond. This result is interpreted in terms of a novel structure for stabilized cuticle that postulates protein−catechol−catechol−protein cross-links. REDOR spectra also showed oxygen substitution at both α and β carbons of N-β-alanyldopamine, consistent with the possibility of intercatechol oxygen bridges in dimeric or oligomeric forms of catecholamines in the cuticle.