Aug. 5, 1959
COMMUNICATIONS TO THE EDITOR
4115
but approximately 1% non-pentose sugar was noted during the sulfuric acid-cysteine reaction. The purified helenine was found t o be unstable to lyophilization and to repeated freezing and thawing. Removal of Mg++ by dialysis caused a loss of activity. Helenine was more stable when stored in 0.25 ilrl' sucrose solution but, even under these conditions, inactivation occurred. Our best preparations were heterogeneous when examined by electrophoresis and ultracentrifuga(14) P. Gautschi and K . Bloch, J B i d . Chenz., 233, 1343 (1958) tion.6 Three major components with sedinientaROSCOEB. JACKSON MEMORIAL LAB. A . A . KANDUTSCH BARHARBOR, MAIXE .\, E. RUSSELL tion constants ranging from 43s to IOOS usually were observed during ultracentrifugation. ElecRECEIVED J U X E 8, 1959 trophoresis showed one major peak (70%) and several minor ones. The mobility of the major PURIFICATION AND CHARACTERIZATION OF T H E component was -5.2 X 10-5 cm.2/volt/sec. a t ANTIVIRAL AGENT HELENINE p H 7 . It seems likely that the inhomogeneity Sir: is caused by alteration of the native helenine and %'e wish to report a purification procedure for the not by extraneous impurities in the usual sense of antiviral agent helenine, and data which suggest the word, since dissociation of ribonucleoproteins that it is a ribonucleoprotein. It is of interest that is well known.' Attempts a t further purification this uniquely active agent is apparently of the same were made using diethylaminoethyl-cellulose colclass of chemical compounds as are the viruses umns, but singly eluted peaks readily dissociated against which i t acts. into smaller components. Because of this heteroHelenine is a product of the mold Penicillium geneity, final characterization of helenine cannot be funiculosum. I t has been reported by Shopel made now. All the physical and chemical observato protect mice against Columbia SK encephalo- tions, however, including stability data, are conmyelitis and Semliki Forest viruses and to prevent sistent with the hypothesis that helenine is a ribodevelopment of poliomyelitis in monkeys. The nucleo-protein. assay used to follow the fractionation was an in vivo (6) We wish t o thank Dr. D. E. Williams for these studies. test in mice, a modification by McClelland of that ( 7 ) A. Tissieres and J. D. Watson, Nntuve, 162, 778 (1958) described by Sh0pe.l To obtain more definitive U. J . LEWIS MERCK SHARPt3 DOHME assay values, a 24-hour pre-treatment dose was RESEARCH LABORATORIES EDVARDL. RICKES substituted for the 24-hour post-treatment dose MERCK& Co., INC. LAURELLA MCCLELLAND described, the observation period was extended to RAHWAY, XEW JERSEY N O R M AG. N BRINK 15 days, and survivors were evaluated statistically. RECEIVED JULY 1, 1959 Helenine was extracted from the mycelium by homogenization in 0.005 M tris-(hydroxymethy1)ON T H E EFFECT O F DOSE RATE ON T H E aminomethane or phosphate buffer, pH 7 , containRADIOLYSIS O F LIQUID HYDROCARBONS' ing 0.005 M Mg++, precipitated by addition of one volume of acetone, and then taken up in more of the Sir : same buffer. About 20 g. of such material was Recent studies of the radiolysis of cyclopentaneobtained from an 80-gal. fermentation. All frac- cyclohexane mixtures with cobalt-60 y-rays have '. tionation processes were performed near 0 indicated that secondary reactions between primary The acetone-precipitated material was further radicals and solvent play an important role in purified by repeated ultracentrifugation for two determining the path of this radiolysis.? Cyclohours a t 110,000 X g. Helenine was sedimented hexyl radicals apparently preferentially abstract completely. From every gram of acetone-precipi- hydrogen atoms from cyclopentane and increase the tated material, 40 t o 50 mg. of pellet was obtained, relative yield of cyclopentyl radicals. From this with a 25-fold increase in potency. The material one expects an effect of dose rate on the over-all was active a t 50 to 100 pg. in mice. A well-defined radiolysis since the lifetime of the radicals and 260-mp absorption peak was noted with this frac- therefore the probability of abstraction decreases tion. It contained about 40% protein3 and gave a with increasing dose rate. Preliminary experipentose test with orcinol4 and with sulfuric acid- ments were therefore undertaken to examine the cysteine.j No deoxyribose was detected. The dose rate dependence of the secondary reactions in perchloric acid hydrolysate5 contained guanine, this system and are briefly reported here.3 No hexose conadenine, cytosine and ~ r a c i l . ~ Degassed mixtures of equal volumes of Phillips tamination was detected during the orcinol test, research grade cyclopentane and cyclohexane were irradiated to a total dose of 5 X lo6 rads. (3 X 10" (1) R . E . Shope, J . E x p . M e d . , 97! 601 (1953). (2) K . W . Cochran and T. Frances, Jr., J . Phavmacol. E x p . Therap.. ev./g.) with cobalt-60 y-rays a t absorbed dose rated into I more rapidly than into cholesterol by preputial gland tumor slices incubated in vitro (unpublished). I is the probable precursor for the A7-isomer (1111,which has been found in rat feces]' and in cactus,8 but has not been identified in the tumor. The precursor of I is less obvious, since the pathway from lanosterol may lead through either dihydrolsnosterol or through 14-norlanosterol.14
116. 13 (1956). (3) E. W. Sutherland C. F . Cori, R . Haynes and N. S. Olsen, J . B i d . Chem., 180, 826 (1949) (4) 2. Dische in "The Piucleic Acids" edited by E. Chargaff and J. N. Davidson, Vol. I. Academic Press, Iac., New York, X. Y . , 1955, p. 285. ( 5 ) A . Bendich in "Methods in Enzymology" edited by S. P. Colowick and N. 0. Kaplan, Vol 111, Academic Press, l a c . , New York, Pi. Y . , 1957, p. 715.
(1) Supported in part by t h e U. S. Atomic Energy Commission. (2) G. A. Muccini and R. H Schuler, t o be published. (3) H. A. Dewhurst and E. H. Winslow, J . Chem. P h y s . , 2 6 , 969 (1957), previously have compared the radiolysis of a simple hydrocarbon (%-hexane) b y y-rays and by fast electrons and have reported a difference in product ratios presumably due t o t h e widely different dose rates involved in t h e comparison. C/. also H A. Dewhurst and R . H. Schuler, THISJOURNAL, in press.
rates of lo4, 7 X IO4 and lo6 rads./h. A similar irradiation was carried out with 2 Mev. Van de Graaff electrons a t a current of 1 microampere (a dose rate in the irradiation zone of 7 X lo8 rads./ h.). After irradiation the samples were examined gas chromatographical!y with attention focussed on the products in the Clo to C12 region (cf. Table I).
the formation of C7Hs+ ion from 1-phenylbutane, now has been found to show a high degree of specificity. 1-Phenylpropane, to a slight extent, and higher 1-phenylalkanes, to a great extent, give rise not only to the expected C7H7+ ion of mass 91, but also to C7Hs+ of mass 92.* This ion must contain a hydrogen atom from the side chain in addition to TABLE I the elements of the benzyl group. We suspected EFFECTOF DOSE RATEON THE RADIOLYSIS OF CYCLOPEN- t h a t the hydrogen comes from the gamma carbon, TANE-CYCLOHEXANE MIXTURES' because this migration would lead directly to the Dose Relative yieldsc formation of a stable 1-olefin as the neutral product Source rateb CioHiee C u H d CnHzzQ Rd of the dissociation. Co60 y-rays 0.01 1.65 1.75 0.60 1.71 T o determine the origin of the migrating hydroCow y-rays 0.07 1.51 1.83 0.66 1.54 gen atom, we made use of two independent apCobs y-rays 1.0 1.39 1 . 9 5 0.76 1.30 proaches. The first consisted of labeling the 3 mev. electrons 700 0 . 7 0 2.04 1.26 0.75 molecule with deuterium in known positions, and hlixtures 0.46 mole fraction in cyclohexane. hlegarads examining the fragment ions for deuterium retenper hour in irradiation zone. Normalized to a t,)tal ~ second was blocking with methyl R, the ratiu of twice the Cloconiponent t i ~ n . The relative yield of 4. -+ the CI1 coinponent to the Cll component -+ twice the CI? groups the positions from which the migrating ~ l J I I l p l l l l ~ l l t e~~~~ . Respectively, cyclupentyl cyclr~peiitatie, hydrogen atom was thought to come. r y I [ ~ p e ~ i t c> y l cliillexane aiid c y c l ~ ~ h e xcycli~liexane. yl Table I shows partial mass spectra of unlabeled I t is seen t h a t a very pronounced increase in the 1-phenylbutane and 1-phenylbutane-3-d and -44. relative yield of cyclopentylcyclopentane and a The 3-d species gives rise to C7Hs+ ions t h a t are corresponding decrease in cyclohexylcyclohexane 03.2/190 or 48.6y0 labeled, close to the 50c70 accompanies the decrease in intensity from the fast expected on the assumption t h a t the migrating atom electron to the y-ray experiments. \Ye have comes solely from the gamma carbon and t h a t no taken as a quantitative measure of this change the isotope effect is involved. The discrepancy, though ratio (R)of the total Cg component to the Ca small, is shown to be real by the 4-d spectrum, in which 4.5/196 or 2.3% of the C7Hs+ions are labeled. component represented in the Cla to C ~ region. Z Irradiation of a similar (z.e., 50/50) cyclopentane- Thus, about 9E17~of the migrating hydrogen atoms cyclohexane mixture containing 2 mM. iodine with originates on the gamma carbon. y-rays (at 70,000 rads./h.) indicated t h a t the ratio TABLEI of cyclopentyl to cyclohexyl radicals initially P A R T I A L M A S S SPECTRA O F DEUTERATED 1-PHESYLBUTANES produced was 0.70 in good agreement with the ratio Corrected for naturally occurring CI3 I? observed in the fast electron experiments. It is -3-d -44 nr/e Colaheled concluded therefore t h a t a t the high intensities 100.0 100.0 135 represented by the Van de Graaff irradiations ( l o x 0.2 0.6 134 100.0 rads./h. and above) secondary reactions are not 95.3 4.5 93 product determining. The observed products 119.4 200 93 196 therefore represent reactions of radicals initially 420 432 91 438 produced by the radiation. At lower intensities Table I1 shows partial mass spectra of 1-phenylsecondary reactions occur here and may be expected to be of general importance in the radiolysis of pure butane, 3-methyl-1-phenylbutane and 3,3-dimethyl1-phenylbutane. The first two show large yields liquid hydrocarbons. J
RADIATIOS RESEARCH LABORATORIES ROBERTH. SCHULER TABLE I1 MELLOSINSTITUTE, PITTSBURGH, PA.,AND MASSSPECTRA OF METHYLATED ~-PHENYLBUTANES DEPARTMEST OF CHEMISTRP G. A , MCCCISI PARTIAL BROOKHAVES NATIOYAL LABORATORY Corrected for naturally occurring C i 3 U P T O N , L O N G I S L A S D , XSW Y O R K m/c Unsubstituted 3-Methyl3.3-DimethylRECEIVED M A R C H 16, 1959 Parent 22.8 24.0 28.4 0.3 30.0 106 0.3 13.3 23.6 105 8.2 HYDROGEN MIGRATION I N GASEOUS ORGANIC 132.4 5.0 92 44.8 CATIONS 100.0 100.0 91 100.0 Sir:
Many ionic dissociations induced by electron impact involve migration of a hydrogen atom from one part of a molecule to another. In compounds containing oxygen, a favorably oriented hydrogen usually is pictured as migrating to the oxygen atom, which is considered trivalent by virtue of localization of the charge in a non-bonding orbital.' In hydrocarbon ions, on the other hand, hydrogenmigration processes are generally thought to be rather non-specific. However, one such process, ( 1 ) See f o r exilmplr, F. \V, lfrT.atTerty, A x u i . Ckcm , 31, 82 (1959).
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of C7Hs+. Moreover, the mass-92 intensities shown define only lower limits for these yields; a pronounced metastable peak 90.0 (92+) (91+) 1 in both spectra indicates that the C7H7+ ion intensities a t mass 91 are due, a t least in part, to further dissociation of C7Hs+ions, I n sharp contrast, the spectrum of the 3,3-dimethyl compound shows but little C7Hst and no more than a suggestion of ;L metastable peak a t 90.0. The process leading to
+
(2) S. Meyerson. A p p l . Spectroscopy, 9 , I 2 0 (19:;). ( 3 ) S . hieyerson and P. N . Rylaodcr, J . P h y i i ' h e m , 62, 2 (1958)
