Complete Proton and Carbon-13 NMR Assignment of the Alkaloid Gephyrotoxin through the Use of Homonuclear Hartmann-Hahn and Two-Dimensional NMR Spectroscopy

Three different types of modem NMR techniques have been used to obtain a complete proton and carbon13 assignment of the alkaloid gephyrotoxin. In addition to the two-dimensional phase-sensitive homoand heteronuclear shift correlation methods, use of the recently proposed one-dimensional homonuclear Hartmann-Hahn difference experiment was crucial for obtaining the required long-range connectivity. Guidelines are presented for optimal use of these techniques. Experiments were performed at 500-MHz 'H frequency, using 8 mg of sample. The introduction of two-dimensional (2D) N M R techniques has greatly simplified the NMR analysis of many natural products, permitting the study of problems hitherto considered intractable. As an example, we present here a study of the alkaloid gephyrotoxin (GyTx), extracted from the skin of the frog Dendrobates histrionicus. Routine application of standard 2D NMR techniques is not sufficient in this case because of the complexity of the 'H spectrum in the methylene region. A second limitation is imposed by the small amount of sample available, which prohibits the application of most of the more advanced, but less sensitive, I3C N M R experiments. Frogs of the family Dendrobatid produce a wide range of unique Many of the alkaloids exhibit pharmacological activity on nerve and m ~ s c l e . ~ . ~ These alkaloids have been categorized by structural similarities into five major classes: the batrachotoxins, the histrionicotoxins, the gephyrotoxins, and the pumiliotoxin C and pumiliotoxin A classes. Gephyrotoxin (GyTx), a major alkaloid of the perhydrobenzoindolizidine-type structure, is a tricyclic compound with the empirical formula C19H29N04 (I). There has, to the best of our knowledge, been no successful attempt at establishing the complete 'H and I3C NMR assignment of GyTx. The present effort is to identify the proton and carbon resonances of this biologically active compound5j6 as a reference for isotopic labeling studies and to add to the database of alkaloids *Address correspondence to this author at the Laboratory of Chemical Physics. derived from amphibians, which may aid in the structural elucidation of new alkaloids of unknown structure. The strategy for complete assignment is to use the newly developed HOHAHA relay experiment' along with standard homoand heteronuclear two-dimensional shift correlation techniques. Special attention is paid to the experimental optimization of the various N M R experiments, both from the resolution and sensitivity points of view. Experimental Approach Double-Quantum Filtered COSY. The first step in the N M R analysis of GyTx is the recording of a 'H-IH connectivity map. Because of the high resolution provided by the double-quantum filtered6 absorption mode9J0 COSY method, this technique is (1) Daly, J. W.; Witkop, B.; Tokuyama, T.; Nishikawa, T.; Karle, I. L. (2) Daly, J. W.; Brown, G. B.; Mensa Dwumah, M.; Myers, C. W. ToxHela Chim. Acta 1977, 60, 1128-1 140. icon 1978, 16, 163-188. Exp. Neurol. 1975, 47, 558-578. B.; Albuquerque, E. X . Biochemistry 1978, 17, 5474-5484. querque, E. X . Mol. Pharmacol. 1984, 25, 395-400. Pharmacol. 1984, 25, 384-394. (3) Lapa, A. J.; Albuquerque, E. X.; Sarvey, J. M.; Daly, J.; Witkop, B. (4) Eldefrawi, M. E.; Eldefrawi, A. T.; Mansour, N. A,; Daly, J.; Witkop, ( 5 ) Souccar, C.; Varanda, W. A.; Aronstam, R. S . ; Daly, J. W.; Albu(6) Souccar, C.; Varanda, W. A.; Daly, J. W.; Albuquerque, E. X. Mol. (7) Davis, D. G.; Bax, A. J. Am. Chem. SOC. 1985, 107, 7197-7198. (8) Piantini, U.; Sorensen, 0. W.; Ernst, R. R. J . Am. Chem. SOC. 1982, 104, 6800-6801. This article not subject to U.S. Copyright. Published 1986 by the American Chemical Society I3C N M R Assignment of the Alkaloid Gephyrotoxin J . Am. Chem. SOC., Vol. 108, No. 5, 1986 919