P-24 Concise Synthetic Method of v-Coelenterazine Analogues Toward Near-Infrared Bioluminescence Imaging(Poster Presentation)

In vivo molecular imaging enables non-invasive observation of molecule of interest in organisms has been attracting much attention and has been widely used in life science. In particular, imaging technique utilizing near-IR bioluminescence has been developed recently. Coelenterazine (CTZ, 1) is known as a luciferin of several marine-derived luciferases such as Renilla, Oplophorus, and Gaussia, as well as a light emitting substrate for same Ca^<2+>-binding photoproteins. So far, more than fifty CTZ analogs have been synthesized and their luminescent properties were examined. Among them, v-coelenterazine (v-CTZ, 2), having a fused consecutive tetracyclic core structure shows red-shifted light emitting character favorable for in vivo molecular imaging. In fact, in vivo molecular imaging of mice was achieved by using v-CTZ in combination with a mutant Renilla luciferase. Although Kishi and Shimomura et al. reported the first and only synthesis of v-CTZ in 1988, the experimental details including procedures, reagents, yields and intermediates were not mentioned. Because more, efficient substrate with further red-shifted emitting character and increased stability was desired from practical viewpoint, we embarked on the synthetic study of v-CrZ and its analogs. Herein, we disclose a concise synthetic method of v-CTZ, which can be readily applied to the synthesis of a diverse array of analogs. We developed an efficient, synthetic route of v-coelenteramine (v-CTM), the precursor of v-CTZ. The route is based on the use of three continuous regioselective cross幼oupling reactions, namely, Negishi, Suzuki柚iyaura, and Stille coupling, followed by ring closing metathesis. This strategy allows facile preparation of broad range of analogs by replacing the coupling partners. The 1H NMR spectrum of synthesized v-CTZ was fully consistent with that of the reported one. The synthetic v-CTZ showed similar emission characters with previous reports when it was used as a substrate of Renilla luciferase and its mutant. In the previous study, we found that trifluoromethyl group placed instead of hydroxy group on the C2-phenyl group of CTZ contributes to improving the stability of CTZ without (detriment of light emitting properties. Thus, we also synthesized the trifluoromethyl-congener of v-CTZ, cf3-v-CTZ (22), which was also found to work as a substrate of Renilla luciferases.