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COMMUNICATIONS TO THE EDITOR
Yol. 82
an order of magnitude faster than the ultrafast reaction of NHd+:NH3 hydrogen exchange (II).7 We infer that the chemical mobility of the electrons in these solutions matches their physical mobility (e.g., electrical conductivity), and that this may be correlated with the physical models being proposed8 to describe metal-ammonia solutions. In these solutions, the electron attack on an electronattracting entity appears to be aia a mechanism quite different from that of ordinary ionic transport.
resin and the crude dry salts extracted with npropyl alcohol to remove cyclohexylammonium j+ toluoate. Inorganic phosphate then was removed as magnesium ammonium phosphate and the resulting material crystallized as the di-(cyclohexylammonium) salt from methanol-ether or from aqueous acetone. The product, which was obtained in a yield of ca. 300j0, based on the chloride, showed [ a l Z 0of~ about +22" in water. Anal. Calcd. for C17H37N20iP(412.47): C, 49.49; H, 9.04; N, 6.79; P, 7.51. Found: C, 49.33; H, (7) Because t h e r e a c t a n t ratio of L i : N H a + is high, t h e r e is t h e possibility t h a t only t h e ammonia associated with one equivalent of 9.32; N, 6.63; P, 7.53. lithium should be considered a s exchanging with t h e ammonia associIn n-propyl alcohol-ammonia-water and in isoa t e d with ammonium ion. This seems improbable, since i t would propyl alcohol-ammonia-water the material is involve t h e mixing of all t h e NH4Br solution with only a p a r t of t h e Li chromatographically homogeneous, and possesses solution. It is more straight-forward t o assume t h a t t h e mixing involves approximately equal a m o u n t s of both solutions a t all times, t h a t the same Rfas authentic, enzymatically prepared both solutions mix fairly uniformly i n order for all t h e NHa' t o react, 2-deoxy-D-ribofuranose 1-phosphate. Fractional t h a t both reactions I a n d I 1 are diffusion controlled, a n d t h a t t h e r a t e of crystallization of the synthetic salt has not been ions b y electrons competes effectively with t h e r a t e of a t t a c k of " I + entirely successful ; however, using methanol-ether , a t t a c k (exchange) of N H a + i o n s b y NHa molecules. (8) G. Lepoutre a n d J. F. Dewald, THISJOURNAL, 78, 2953 (1956); fractions were obtained with [ a ]?OD as high as +30° E. Becker, R. H . Lindquist a n d B. J. Alder, J . Chem. Phrs., 2 5 , 971 (C, 49.44; H, 8.76; N, 6.87; P, 7.48). One of (1956); M. C . R. Symons, ibid.,3 0 , 1628 (19.59). DEPARTMENT OF CHEMISTRY C. W. KEENAN these fractions, when assayed enzymatically using THEUNIVERSITY OF TENNESSEE HENRYV.SECOR fish nucleoside phosphorylase,j had about 80% KNOXVILLE, TENNESSEE EUGENE J. KELLY of the activity of natural 2-deoxy-~-ribofiiranose JEROME F. EASTHAM 1-phosphate. RECEIVED FEBRUARY 26, 1960 Since crystalline acylated 2-deoxy-D-ribofuranosyl halides have been shown6 to give rise to THE SYNTHESIS OF 2-DEOXY-D-RIBOFURANOSE anomeric mixtures of 2-deoxy-~-ribofuranosides, I-PHOSPHATE it seems probable that the phosphate obtained here Sir : is a mixture of anomers. -4comparison of our Cleavage of a ribonucleoside with a nucleoside product and the natural material on the basis of phosphorylase was reported by Kalckarl to give a optical rotation and biological activity supports the ribose 1-phosphate. Subsequently, this product was assumption5 that the natural substance of [DID shown to be a-D-ribofuranose 1-phosphate on the +38.8" is the a-anomer and, furthermore, one may basis of certain chemical tests, as well as by chemi- calculate that the 6-anomer would have an [ a ] ~ cal synthesis of both anomers.2 In a presumably of approximately - 10". similar fashion, the action of the appropriate The authors are grateful to Dr. H. L. A. Tarr for nucleoside phosphorylases on deoxyribonucleosides carrying out the enzymatic assays and for providing leads to the formation of an extremely acid-labile a sample of enzymatically prepared 2-deoxy-D2-deoxy-~-ribofuranose1-phosphate,3 - 6 which has ribofuranose 1-phosphate. Xlso, we wish to thank been isolated as a crystalline di- (cyclohexylammo- Dr. IV.E. Scott of Hoffmann-La Roche, Inc., for nium) ~ a l t . ~The , ~ optical rotation of this material, certain as yet unpublished experimental details. [ a ] +38.8",j ~ and the manner in which i t is enzy- XATIOSAL INSTITCTE O F ARTHRITIS matically synthesized, suggest5 that, like its D-ribose UOS.4LD I,. l\IACI)OSALI) 4 S D >1ETABOLIC DISEASES IXSTITUTES OF HEALTH analog, i t possesses the cr-configuration. This XATIONAL HEWITTC;. FLETCHEK, JR conclusion is supported by the present work, in BETHESDA14, hl.4RYL.QJD RECEIVED FEBRUARY 15, 1960 which we describe the chemical synthesis of a 2deoxy-D-ribofuranose 1-phosphate of high biological THE PHOTOREDUCTION OF PORPHYRINS AND THE activity. OXIDATION OF AMINES BY PHOTO-EXCITED DYES Crystalline 3,5-di-0-p-toluoyl-2-deoxy-~-ribofuranosyl chloride6 was condensed in benzene solu- Sir: Two distinct series of hydro-porphyrins are tion with disilver phosphate7 (previous workers who have used this material for the synthesis of phos- known. The hydrogens may be located on the phates* have termed i t "monosilver phosphate"). peripheral "pyrrole" ring carbons (chlorophyll and The product of the condensation was saponified in bacteriochlorophyll), or they may be on the methine aqueous alcohol with lithium hydroxide, the lithium carbons (porphomethenes,' I and 11, and porphyreplaced by cyclohexylamine using an ion exchange rinogens) . The photoreduction of uroporphyrin produces, in addition t o a transitory compound (1) H . h l . Kalckar, J . Biol. C h e w , 167, 477 (1947). absorbing a t 140 and i 3 5 rnp, a more stable sub(2) R. S. Wright a n d H . G. Khorana, THIS J O U R N A L , 78, 811 (1956); G. M . Tener, R . S. Wright and H G. Khorana, ibid., 79, 441 stance absorbing a t 500 nip which is related to the (1957). second series of hydroporphyrins, the porpho(3) M. Friedkin and H. 51.Kalckar, J . Biol. Chem., 184, 437 (19.50). methenes. (4) M . Friedkin, ibid., 184, 449 (1950); M , Friedkin and D. Roberts, Porphyrins may be photoreduced by a wide ibid., 207, 257 (1954). (5) H. L. A. T a r r , C a n . J . Biochem. a x d Physiol., 36,517 (1958). variety of compounds, e.g., ascorbic acid, gluta(6) M. Hoffer, R. Duschinsky, J . J . Fox and N. Yung. T H r s J O U R N A L , thione and tertiary amines The reduction of 81, 4112 (1959). uroporphyrin with ethylenediaminetetraacetic acid (7) R. F l a t t a n d G. Brunisholz, Helo. Chim. Acta, 34, 692 (1951). (8) F. Lipmann and I 10 seconds) is a transitory compound having lo5) and intense absorption bands a t 440 ( E m 735 mp ( E m 5 X lo4). In the absence of light and oxygen, i t is stable for a t least thirty hours a t neutral fiH and room temperature. In the presence of air i t has a half life of about five minutes. This autoxidation is sensitive to light. The substance also reacts rapidly with iodine. Krasnovsky and co-workers2have noted an absorption band a t 740 mp on illuminating hematoporphyrin in pyridine containing ascorbic acid. They favor the possibility of a radical-ion-pair, but a variety of dihydroporphyrins exclusive of structure I are also possible. On continued illumination, an intense band a t 500 mp ( E m = 5-10 X lo4) arises as the 440-735 rnp and the uroporphyrin absorptions fall. Over 95% of the porphyrin can be reduced, and further r e d i d o n beyond the 500 mp stage appears to be very slow. Titration of this product with iodine 5% yield. About reforms the porphyrin in 95 5 moles of iodine was required per mole of reduced porphyrin. This high value may be due to side reactions of the iodine. The reaction of iodine with tertiary amines is highly sensitive to light. The recovered porphyrin was identified by the position and by the relative ratios (6 independent values) of the intensities of its absorption bands in neutral (4 bands) and acid (3 bands) solution. Molar absorption coefficients of the reduced species were calculated from changes in the porphyrin absorption assuming only two reduced products to be present. No evidence for the presence of any colorless reduction product (e.g., uroporphyrinogen) was obtained. The photoreduced porphyrin absorbing a t 500 mp has spectral reactions characteristic of the dipyrrylmethene structure’ of porphomethenes : (1) The absorption of porphomethenes is also a t 500 mp (with Em E l o b ) ; ( 2 ) the absorption band decreases in intensity and shifts to shorter wave lengths in alkaline solution with a PK of about 9.5; and (3) the absorption vanishes in the presence of sulfite ion a t pH 7. Seely and Calvin claim that the photoreduction of the zinc chelate of CY$, y,&tetraphenylporphyrin by benzoin3forms reduced porphyrins of the chlorin and bacteriochlorin series. Under our conditions described above, the zinc chelate of uroporphyrin shows little if any reaction, and the copper chelate is not visibly affected. Many secondary and tertiary amines will act as photoreducing agents toward a variety of dyes, e . g . , thiazines and flavines. Ethylenediarninetetraacetic acidj is especially reactive. The claim is
*
( 2 ) A. A. Krasnovsky, J . C h t m Pkys., 56, 968 (1958). (3) G. R . Seely and M . Calvin, J . Chem. Pkys., 23, 1068 (1Y55). (4) G. Oster and N. Witherspoon, THISJOURNAL, 79, 4836 (1957); F. Millich and G. Oster, ibid., 81, 1357 (1959).
dihydro por phyrin porphodimethene 1
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tetrahydmpor ph yrjn porphomethene I1
often made that the water molecule is split during the reactionjm6presumably with concomitant formation of the amine oxide. However, oxidation of the amine a t the CY carbon is more reasonable. In general the reactivity of the amine is related to its ease of oxidation a t the a-carbon: e.g., sparteine is very reactive, primary amines are far less reactive, and ammonium ions are unreactive. The direct isolation of the oxidized amine product is complicated by the facile autoxidation of the reduced dye and by the possible decomposition of the amine oxide if formed. In an attempt to circumvent these difficulties, the fact that small ring bridgehead amines are very resistant to oxidation a t the CYcarbon, yet readily form N-oxides, was used. No reasonable intermediate or transition state can be stabilized due to ring strain (Bredt’s rule). In fact, the rate of photoreduction of thionine by 1,4diazabicyclo [2,2,2]octane was found to be less than 1/5000ththat with N,N’-dimethylpiperazine. This bicyclodiamine is also vastly less susceptible to photooxidation by iodine. Among the more interesting non-nitrogeneous photo-reducing agents we have found for these dyes are ethyl acetoacetate and Z-carbethoxycyclopentanone. Chlorophyll has a somewhat similar active “methylene” group a t position 10 and possibly this molecule can act both’ as a photooxidant and photo-reductant. Acknowledgment.-I am grateful to Dr. S . Granick for continual encouragement, to Dr. G. Oster for valuable discussions and wish to thank the Houdry Process Corporation for a generous gift of the bicyclodiamine (DABCO). ( 5 ) J. R. Merkel and W. J. Sickerson, Biockem. Biophys. Acta, 14, 303 (1954). (6) M. Koizumi and H. Obata, Bull. C h e m SOL. Japn)z, 30, 136 (1957).
(7) E. I . Rabinowitch, “Photosynthesis,” Vol. 11, P t . 2, Interscience Publishers, Inc., h-ew York, N. Y., 1956, p. 1487.
THEROCKEFELLER INSTITUTE D. MAUZERALL NEWYORK21, S.Y . RECEIVED JANUARY 13, 1960
MEMBRANE DIFFUSION STUDIES WITH PROTEINS AND NUCLEIC ACIDS
Sir: Previous dealing with separation by differential diffusion through cellophane mem(1) L. C. Craig, T. P. King and A. Stracher, THISJ O U R N A L , 79, 3729 (1957). ( 2 ) L. C. Craig, Wm. Konigsberg, A. Stracher and T. P. King, “Symposium on Protein Structure,” Methuen & Co. Ltd., London, 1958, p. 104.
