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) 13 C and 15 N NMR, in combination with 1 H− 13 C dipolar modulation and 15 N− 15 N dipolar restoration at the magic angle, were used to examine hornworm pupal exuviae labeled with β-[ 15 N]alanine and either [β- 13 C]dopamine or [α- 13 C, 15 N]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.