Oxidative transformation of a tunichrome model compound provides new insight into the crosslinking and defense reaction of tunichromes Adal Abebe a , Qun F. Kuang b , Jason Evans b , William E. Robinson c , Manickam Sugumaran a,⇑ a Department of Biology, University of Massachusetts Boston, 100 Morrissey Boulevard, Boston, MA 02125-3393, United States b Department of Chemistry, University of Massachusetts Boston, 100 Morrissey Boulevard, Boston, MA 02125-3393, United States c School for the Environment, University of Massachusetts Boston, 100 Morrissey Boulevard, Boston, MA 02125-3393, United States article info Article history: Received 1 February 2017 Revised 10 February 2017 Accepted 13 February 2017 Available online 16 February 2017 Keywords: Tunichrome Tunic formation Dehydro dopa Dehydro topa Quinoinoid reactivity Crosslinking Antimicrobial activity Defense reaction abstract 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 instabil- ity and high reactivity has severely hampered the assessment of their biological role. Experiments con- ducted 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 alka- line condition such as pH 7.5 causes rapid nonenzymatic oxidation. High performance liquid chromatog- raphy 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 forma- tion 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 forma- tion of oligomeric tunichrome products. Attempts to monitor the polymerization reaction by mass spec- trometry 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. Ó 2017 Elsevier Inc. All rights reserved. 1. Introduction Sea squirts, more formally known as ascidians, are prominent marine filter feeders, many of which are important members of the marine biofouling community. They are ancient members of the Chordata (Subphylum Urochordata; Class Ascidiacea), sharing three characteristic morphological structures with other members of that phylum – gill slits, notocord and dorsal nerve cord [1]. Sev- eral species of ascidians contain a group of low molecular weight dehydrodopamine containing oligopeptides named tunichrome [2–7], found primarily in the morula cells of their blood [8]. The first members of this group, tunichromes An-1, An-2 and An-3, were obtained from whole blood cell lysates of the phlebobranch, Ascidia nigra [2,9,10]. All three were identified as tripeptides of modified tyrosines. Tunichrome An-1 is made up of Topa (3,4,5-tri hydroxyphenylalanine), dehydrotopa and dehydrotopamine. Tunichrome An-2 contains dopa, dehydrotopa and dehydro- topamine, while tunichrome An-3 possesses dopa dehydrotopa and dehydrodopamine (Fig. 1). Subsequently, tunichromes Mm-1 and Mm-2 were isolated from the stolidobranch Molgula manhat- tensis [8,10,11]. Tunichromes Mm-1 and Mm-2 are very similar to tunichrome An-3 with glycine (Mm-1) and leucine (Mm-2), respectively, substituted for the N-terminal dopa unit (Table 1). Another group of tunichromes was isolated and characterized from the phlebobranch, Phallusia mammilata [6], which differ in their structure from that of the An tunichromes in that the central dehy- dro topa units are replaced with saturated topa units (Table 1). The morula cells of Phallusia mammilata also possess an oligopeptide containing 6-bromotryptophan and dehydrodopamine called mor- ulin [12]. A modified pentapeptide tunichrome Sp-1 was identified from the hemocytes of the stolidobranch ascidian Styela plicata, whose structure is elucidated to be dopa-dopa-Gly-Pro- dehydrodopamine [13]. From this same organism an octapeptide called plicatamide was isolated and its structure was established as Phe-Phe-His-Leu-His-Phe-His-dehydrodopamine [14,15]. So far, tunichromes have been isolated or characterized from thirteen species of ascidians (2 stolidobranchs and 11 phlebobranchs) http://dx.doi.org/10.1016/j.bioorg.2017.02.008 0045-2068/Ó 2017 Elsevier Inc. All rights reserved. Abbreviations: Dehydro NADA, 1,2-dehydro-N-acetyldopamine; Topa, 3,4,5-trihydroxyphenylalanine. ⇑ Corresponding author. E-mail address: manickam.sugumaran@umb.edu (M. Sugumaran). Bioorganic Chemistry 71 (2017) 219–229 Contents lists available at ScienceDirect Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg