ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 329, No. 1, May 1, pp. 56–64, 1996 Article No. 0191 Characterization of Products from the Reactions of N-Acetyldopamine Quinone with N-Acetylhistidine Rongda Xu,* Xin Huang,* Thomas D. Morgan,† Om Prakash,‡ Karl J. Kramer,† , ‡ ,1 and M. Dale Hawley* Departments of *Chemistry and ‡Biochemistry, Kansas State University, Manhattan, Kansas 66506; and †U.S. Grain Marketing Research Laboratory, Agricultural Research Service, United States Department of Agriculture, 2 Manhattan, Kansas 66502 Received December 18, 1995, and in revised form February 12, 1996 alanyldopamine are oxidized to quinones that undergo When insects harden or sclerotize their exoskele- nucleophilic addition by amino acid side chains in cutic- tons, quinones of N-acetylated catecholamines such as ular proteins (1 – 3). Covalent bonds that are formed N-acetyldopamine (NADA) undergo nucleophilic addi- between the aromatic ring of NADA and cuticular pro- tion reactions with amino acids such as histidine in tein amino acids, such as histidine and lysine, have cuticular proteins. To determine the products that been detected in insect cuticle using solid-state double might form when this type of reaction occurs during cross polarization 13 C and 15 N NMR (4). Cross-links cuticle sclerotization, the reactions between electro- between the b-carbon (C7) in the side chain of dopa- chemically prepared NADA quinone and N-acetylhisti- mine derivatives and histidine residues have been dine (NAcH), a protein model nucleophile, have been found in cuticle by rotational echo double resonance investigated at pH 7. Two major products, 6-[N-(N-ace- NMR (5). Catecholamine-containing proteins have tylhistidyl)]-N-acetyldopamine and 2-[N-(N-acetylhis- been isolated from insect cuticle undergoing sclerotiza- tidyl)]-N-acetyldopamine, were purified by semiprep- tion (6). Cuticle-catalyzed coupling between NADA and arative reversed-phase liquid chromatography and N-acetylhistidine (NAcH) has been studied in vitro, and identified by mass spectrometry and nuclear magnetic both b- and ring C6-addition adducts have been de- resonance spectroscopy. The relative molar ratio of tected (2). the C(6) mono-addition adduct to the C(2) mono-addi- The goal of this study was to determine the initial tion adduct is 87:13. UV/vis spectroscopic analysis steps of the oxidation reaction pathway of NADA when shows that both products have an absorption maxi- a nucleophile is present in excess. To mimic the cuticle- mum at 284 nm. Cyclic voltammetry shows that these catalyzed reactions, we investigated a model system adducts are oxidized less readily than NADA.  1996 for cuticle sclerotization in which electrochemically Academic Press, Inc. prepared NADA quinone was reacted with a typical Key Words: quinone; catecholamine; cuticle; histi- protein nucleophile, NAcH. Reactions were studied us- dine; protein; sclerotization; adduct; oxidation; addi- tion. ing reversed-phase liquid chromatography (LC), cyclic voltammetry (CV), and UV/vis spectroscopy. The two major products, 6-[N-(N-acetylhistidyl)]-N-acetyldo- N-Acylcatecholamines are important catecholamine pamine and 2-[N-(N-acetylhistidyl)]-N-acetyldopam- metabolites used by insects to sclerotize or harden their ine, were characterized by fast atom bombardment exoskeletons. N-Acetyldopamine (NADA) 3 and N-b- mass spectroscopy (FAB-MS), NMR spectroscopy, CV, and UV/vis spectroscopy. 1 To whom correspondence should be addressed. Fax: (913) 537- 5584. E-mail: kramer@crunch.usgmrl.ksu.edu. 2 The Agricultural Research Service, USDA is an equal opportu- teine; LC, liquid chromatography; FAB-MS, fast atom bombardment mass spectrometry; NMR, nuclear magnetic resonance spectroscopy; nity/affirmative action employer and all agency services are available without discrimination. CV, cyclic voltammetry; EC, electrochemical; DEPT, distortionless enhancement by polarization transfer; COSY-LR, long range corre- 3 Abbreviations used: DA, dopamine; NADA, N-acetyldopamine; NAcH, N-acetylhistidine; NAcH-NADA-I, 6-NAcH-NADA, 6-[N-(N- lated spectroscopy; TOCSY, total correlation spectroscopy; HMQC, heteronuclear multiple quantum coherence; HMBC, heteronuclear acetylhistidyl)]-N-acetyldopamine; NAcH-NADA-II, 2-NAcH-NADA, 2-[N-(N-acetylhistidyl)]-N-acetyldopamine; NACySH, N-acetylcys- multiple-bond correlation. 56 0003-9861/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.