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support (A. E.), and Mr. B. O’Leary for technical assistance with the irradiation.
standard procedure was dissolved in dilute aqueous acetic acid, the solvent removed in vacuo, the residue taken up in a minimum quantity of pure water, and DEPARTMENT OF PHYSICAL CHEMISTRY A . EKSTROM J . L. GARNETT the solution allowed to stand for three weeks, completely THEUN~VERSITY OF KEW SOUTHWALES transparent, beautifully formed, individual paralleloKENSINGTON, N.S.W., AUSTRALIA pipeds separated. This material was shown to be RECEIVED J U L Y 13, 1964 tetrodotoxin through the identity of its infrared spectrum (KBr) and Debye-Scherrer diagram with those of an authentic sample. Although the crystals were The Structure of Tetrodotoxin still very small (maximum extension 2 0.06m m . ) , it was possible to mount several of them for single Sir: crystal X-ray diffraction studies, using Cu Kcr radiation. Recently we presented the evidence which led us to The crystals were found to be monoclinic through deduce the structure I for the Spheroides poison tetrodoobservation of C B h diffraction symmetry in oscillation toxin.1s2 The very interesting alternative suggestion3 and precession photographs, and to belong to space that the toxin possesses the structure 11, in which two group C22-P21, since tetrodotoxin is optically active, units of the structure I are joined together by an and systematic extinctions were found only for OkO, k odd ( k 13),in Weissenberg photographs. Precession -. photographs taken of the h0l and hll reciprocal lattice planes established the axial lengths a = 6.72 + 0.04 A,,c = 6.49 f 0.02 K . , and the interaxial angle p = 113’56’ 2 2 ‘ . The length of the unique axis, b = 14.66 0.02 A,, was determined through observation of the Ok0 reciprocal lattice line, after suitable reorientation of the same crystal which provided the above information. The standard deviations were ascertained through superimposing lead nitrate powder diagrams upon the precession photographs. Satisfactory confirmatory values were obtained for all three axial lengths from oscillation photographs taken on different crystals mounted along each of the crystallographic axes. The density of the crystals was found ethereal oxygen atom, deserves serious consideration. to be 1.792 0.006 g . / ~ m by . ~ the flotation method; The problem of differentiating between the two this is almost certainly a minimum value, since obstructures is not accessible to study ’ by the usual servations were made on aggregates of the parallelomethods of solution chemistry, since tetrodotoxin is pipeds, rather than on the exceedingly small and very all b u t completely insoluble in all solvents except acids, difficultly observable single crystals. and the latter might well effect cleavage of the ether The volume of the unit cell of the tetrodotoxin link in 11, whose lability would have to be accepted in crystal is therefore 585 =t 4 A.3,and the weight is 632 view of the fact that all transformation products of f 6 on the atomic scale. Further, for a nonpolymeric established constitution are derived from the Cll substance, the crystal s y m m e t r y requires the presence structure I rather than a C22molecule. We now wish w i t h i n the u n i t cell of an even number of complete, disto present evidence which permits an unequivocal crete, unconnected molecules. These conditions are decision in favor of the structure I for tetrodotoxin. fully met by a cell containing two molecules of the Tetrodotoxin is ordinarily crystallized by addition structure I (2 X mol. wt. = 63S), and cannot in any of ether and ethanol or methanol to solutions of the way be reconciled with the alternative ether structure toxin in dilute aqueous acetic acid.* The aggregates 11, which would require a minimum cell weight of of very small crystals prepared in this way are not 1240. suitable for fundamental crystallographic studies. However, when material thrice recrystallized by the Acknowledgment.-We are pleased to acknowledge generous support by the National Institutes of Health (1) Lecture presented o n April 1 4 , 1961. in K y o t o a t t h e 3 r d I n t e r n a t i o n a l and the Army Research Office/Durham, and the kindS y m p o s i u m o n t h e C h e m i s t r y of I i a t u r a l P r o d u c t s ; K . B. W o o d w a r d , Puve A p p l . C h e m . , 9, 1 9 (1964). T h e s t r u c t u r e t h e r e presented is a c t u a l l y t h e ness of Professor William N . Lipscomb, who placed his mirror image of I. Professor I i i t t a a n d his colleagues reported a t t h e s a m e counsel and equipment freely a t our disposal. s y m p o s i u m t h a t extension of their X - r a y crystallographic studies o n h r o m o -
