Oxidative transformation of tunichromes - Model studies with 1,2-dehydro-N-acetyldopamine and N-acetylcysteine.

Tunichromes are 1,2-dehydrodopa containing bioactive peptidyl derivatives found in blood cells of several tunicates. They have been implicated in metal sequestering, tunic formation, wound healing and defense reaction. Earlier studies conducted on these compounds indicate their extreme liability, high reactivity and easy oxidative polymerization. Their reactions are also complicated by the presence of multiple dehydrodopyl units. Since they have been invoked in crosslinking and covalent binding, to understand the reactivities of these novel compounds, we have taken a simple model compound that possess the tunichrome reactive group viz., 1,2-dehydro-N-acetyldopamine (Dehydro NADA) and examined its reaction with N-acetylcysteine in presence of oxygen under both enzymatic and nonenzymatic conditions. Ultraviolet and visible spectral studies of reaction mixtures containing dehydro NADA and N-acetylcysteine in different molar ratios indicated the production of side chain and ring adducts of N-acetylcysteine to dehydro NADA. Liquid chromatography and mass spectral studies supported this contention and confirmed the production of several different products. Mass spectral analysis of these products show the potentials of dehydro NADA to form side chain adducts that can lead to polymeric products. This is the first report demonstrating the ability of dehydro dopyl units to form adducts and crosslinks with amino acid side chains.

Oxidative transformation of a tunichrome model compound provides new insight into the crosslinking and defense reaction of tunichromes.

Tunichromes, small oligopeptides with dehydrodopa units isolated from the blood cells of ascidians, have been implicated in the defense reactions, metal binding, wound repair, or tunic formation. Their instability and high reactivity has severely hampered the assessment of their biological role. Experiments conducted with the model compound, 1,2-dehydro-N-acetyldopamine, indicated that the instability of tunichromes is due to this basic structure. Exposure of this catecholamine derivative to even mild alkaline condition such as pH 7.5 causes rapid nonenzymatic oxidation. High performance liquid chromatography and mass spectrometry studies confirmed the production of dimeric and other oligomeric products in the reaction mixture. The nonenzymatic reaction seemed to proceed through the intermediary formation of semiquinone free radical and superoxide anion. Ultraviolet and visible spectral studies associated with the oxidation of tunichromes isolated from Ascidia nigra by tyrosinase indicated the probable formation of oligomeric tunichrome products. Attempts to monitor the polymerization reaction by mass spectrometry ended in vain. Tunichrome also exhibited instability in mild alkaline conditions generating superoxide anions. Based on these studies, a possible role for oxidative transformation of tunichrome in defense reaction, tunic formation and wound healing is proposed.

Mass spectrometric studies shed light on unusual oxidative transformations of 1,2-dehydro-N-acetyldopa.

RATIONALE: Lamellarins are a group of over 70 plus bioactive marine natural compounds possessing a 6,7-dihydroxycoumarin moiety. Although they appear to derive from 3,4-dihydroxyphenylalanine (dopa), practically nothing is known about the metabolic fate of these compounds. Biochemical considerations indicate that they could arise from a N-acetyl-1,2-dehydrodopa precursor through oxidative cyclization reaction. METHODS: To assess the above hypothesis, we synthesized N-acetyl-1,2-dehydrodopa and conducted oxidation studies with commercially available mushroom tyrosinase and evaluated the course of the reaction with reversed-phase liquid chromatography/mass spectrometry (LC/MS). RESULTS: Mushroom tyrosinase readily oxidized N-acetyl-1,2-dehydrodopa - not to the normally expected quinone - but to an unstable quinone methide isomer, which rapidly cyclized to produce the dihydroxycoumarin product, 3-aminoacetyl esculetin. Interestingly, 3-aminoacetyl esculetin was further oxidized to a second quinone methide derivative that exhibited an addition reaction with the parent dihydroxycoumarin generating dimeric and other oligomeric products in the reaction mixture. CONCLUSIONS: LC/MS analysis of the N-acetyl-1,2-dehydrodopa oxidation reaction reveals not only a possible novel oxidative cyclization route for the biosynthesis of coumarin-type dehydrodopa compounds in marine organisms, but also unusual oxidative transformations of dehydro dopa derivatives.