Date of Award

8-1-2012

Document Type

Campus Access Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Jason J. Evans

Second Advisor

Manickam Sugumaran

Third Advisor

Timothy J. Dransfield

Abstract

1,2-Dehydro-N-acetyldopamine (dehydro NADA) and its derivatives have been identified as important cuticular sclerotizing precursors responsible for the hardening of the exoskeleton of soft-bodied insects. In addition, these compounds may serve as precursors in the biosynthesis of the large number of bioactive compounds present in the exoskeleton of marine invertebrates. In this work the products of non-enzymatic oxidation of 1,2-dehydro N-acyldopamine were examined using reversed-phase high performance liquid chromatography/electrospray/tandem mass spectrometry (RP-HPLC/ESI/MS-MS). In addition, the role of tyrosinase-catalyzed oxidation of dehydro-N-acetyldopamine in the cross-linking of cuticle proteins was studied by examining two thiol-containing nucleophiles and a model peptide using RP- HPLC/ESI/MS-MS. Finally, the tyrosinase-catalyzed oxidative cyclization and polymerization of N-acetyl-1,2-dehydro Dopa was examined as a possible biosynthetic pathway for forming bioactive compounds called lamellarins present in marine invertebrates. It was found that 1,2-dehydro N-acyldopamine undergoes polymeric oxidation under non-enzymatic conditions at slightly alkaline pH to form not only dimeric, but the trimeric, tetrameric and pentameric products, similar to the previous findings observed under tyrosinase-catalyzed oxidation at physiological pH. This result has led to a revised mechanism for non-enzymatic polymeric oxidation of N-acyldopamines. In the cross-linking studies, RP- HPLC/ESI/MS-MS data provided strong evidence for the addition of N-acetylcysteine to the side chain of the dehydro NADA dimeric species. However, no conclusive evidence for side chain addition of dithiothreitol was obtained. An examination of tyrosinase-catalyzed cross-linking reaction between Angiotensin I and dehydro NADA did not yield the anticipated cross-linked product. The major product, which formed only in the presence of both dehydro NADA and tyrosinase, resulted from cleavage at the histidine to form Angiotensin II. The study investigating the tyrosinase-catalyzed oxidation of N-acetyl-1,2-dehydro Dopa verified the initial formation of the cyclization product, 3-aminoacetylesculetin, followed by its rapid oxidative polymerization to form dimeric through hexameric species, suggesting a likely pathway by which marine invertebrates synthesize lamellarins and other bioactive compounds that contain a 3,4-dihydroxy coumarin unit.

Comments

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