Pii: S0040-4020(00)00949-2

ÐCuticle sclerotization or tanning is a vital process that occurs during each stage of insect development to harden and stabilize the newly secreted exoskeleton. The structural polymers protein and chitin make up the bulk of the cuticle, and chemical interactions between these biopolymers with quinonoid tanning agents are largely responsible for the physical properties of the mature exoskeleton. The oxidative conjugation of catechols with cuticular proteins plays an important role in this metabolism. The main hypothesis for cuticle sclerotization involves the formation of adducts and cross-links between nucleophilic imidazole nitrogens of histidyl residues in the proteins and electrophilic ring or side-chain carbons of ortho-quinones and para-quinone methides derived from the catechols, N-acetyldopamine, N-betaalanyldopamine, and 3,4-dihydroxyphenylethanol. C±N and C±O linkages between these quinone tanning agents and proteins in cuticles from a variety of insects from several orders have been elucidated. cDNAs for both the tyrosinase and laccase types of phenoloxidases that catalyze the cross-linking reactions have been isolated and sequenced. The sequences of laccase cDNAs from two insect species were more similar to fungal laccases than to those from plants. These results provide insights into how insects use structural proteins, catechols, and oxidative enzymes to form catechol±amino acid adducts during sclerotization. Published by Elsevier Science Ltd. The o-quinone and p-quinone methide metabolites derived from their catechol precursors by the action of phenoloxidases represent a class of reactive intermediates that can lead to a variety of effects in vivo, including exoskeletal hardening and stabilization, cytotoxicity, immunotoxicity, and carcinogenesis depending on the type of quinonoid and its modi®ers as well as the physiological system of interest. For hardening and stabilization, insects utilize N-acyldopamines and other catecholic compounds and the activation of their aromatic rings and side chains by tyrosinases and laccases for nucleophilic addition reactions with proteins. This chemistry optimizes the mechanical and chemical properties of exoskeletons, so that species can survive and adapt to their environments. The newly secreted cuticle must be selectively stiffened and hardened by sclerotization processes to provide the necessary mechanical properties for locomotion and protection of each developmental stage. Sclerotization involves in part the formation of covalent bonds between the quinone derivatives of three catechols, N-acetyldopamine (NADA), N-b-alanyldopamine (NBAD), and 3, 4-dihydroxylphenylethanol (DOPET), and side-chain functional groups of histidine and possibly other amino acid residues of cuticular proteins. During sclerotization as catechols and/or quinone metabolites replace water and interact with the structural proteins by forming several types of covalent conjugates, the cuticle becomes increasingly dehydrated, dense, hydrophobic, and insoluble. Although insects as a group produce a wide variety of structurally diverse cuticles that differ in chemical and physical properties according to their functional adaptations, little is known about the molecular organization of any of these because of the complexity of their structures and their resistance to degradation for structural analysis. Acid hydrolyses of sclerotized cuticles have yielded products that indicate the types of catechol±protein interactions involved in stabilization. Arterenone (3, 4-dihydroxyphenylketoethylamine) has been shown to represent NADA dimers and oligomers involving benzodioxin-type linkages in cuticle by acting as longer cross-links or ®ller materials. Norepinephrine derivatives represent NADA and NBAD bonded to proteins by their side-chain b-carbons (C-7) through oxygen Tetrahedron 57 (2001) 385±392 Pergamon TETRAHEDRON 0040±4020/01/$ see front matter. Published by Elsevier Science Ltd. PII: S0040-4020(00)00949-2