3608
COMMUNIC.~TIONS TO THE EDITOR
Yul. 79
tively identified in our hydrolyzates, may be the product N F observed by Hock and Huber. It is interesting to note that the reactions leading to the formation of (I) are similar in character to two of the enzymatic reactions involved in polynucleotide synthesis.9, Recently Khorana, et al.," described a thymidine derivative analogous to (I) which they prepared by an entirely different procedure.
tatively to 5'-AMP, identified by paper chromatography and dephosphorylation by low concentrations of snake venom. The biological activity of (I) was not destroyed by incubation with pancreatic ribonuclease or spleen phosphodie~terase.~Through private communication with Dr. Leon Heppel, we learned that the structure we had tentatively proposed for (I) was identical with that proposed by Cook, Lipkin and Markham for a product isolated from the DEPARTMENT OF CHEMISTRY \TILLTAM H. COOK \\'BSHISGTOS UNIVERSITY DAVIDLIPKIX Ba(0H)t digestion of ATP.J This knowledge S T . LOUIS5 , ~ ~ ~ I S S O U R I ROYMARK HAM^^ prompted an exchange of information and samples were kindly provided for c o m p a r i ~ o n . ~ These (9) 31. Grunherg-Manago a n d S. Ochoa, THIS JOURNAL,77, 3165 samples were identical with (I) by the following (1955). criteria: (1) ultraviolet spectrum, (2) biological (IO) A. Kornberg, I. R. Lehman, M. J. Bessman a n d E. S.Simms, activity, (3) paper chromatography, (4) loss of Riochim. Biophrs. Acta, 21, 197 (1956). biological activity, when incubated with enzyme (11) H . G . R h o r a n a , et a l , THISJOWRNAL, 79, 1002 (1957). (12) On leave f r o m t h e rZgricultura1 Research Council Virus Kefractions from heart or brain, ( 5 ) quantitative search Unit. Cambridge, and wishep t o t h a n k t h e Wellcome Foundaconversion to 5'-AMP on incubation with a tion for a travel grant. partially purified enzyme from heart, and (6) conversion to adenosine on prolonged incubation with RECEIVED MAY6 , 195'7 large amounts of Crotalus adamnnteus venom (although, as reported,' moderate atnounts of Russell's viper venom did not attack (I)).fi
THE PROPERTIES OF AN ADENINE RIBONUCLEOTIDE PRODUCED WITH CELLULAR PARTICLES, ATP, Mg++, AND EPINEPHRINE OR GLUCAGON
Sir: Previous studies in this laboratory have shown that a heat-stable factor (formed by particulate fractions of liver homogenates in the presence of ATP, Mg++, and epinephrine or glucagon) stimulated the formation of phosphorylase in supernatant fractions of homogenates.' This factor was isolated by ion-exchange chromatography and proved to be an adenine ribonucleotide (I).l Similar or identical compounds have been isolated from heart, skeletal muscle and brain. Crystals formed when 2 X A1 aqueous solutions of (I) were chilled a t acid pH. (I) was not attacked by several monoesterases' and upon titration with alkali exhibited no buffering capacity between pH 5 and pH 8. Descending paper chromatography, using an ethanol-ammonium citrate (pH 4.4) solvent system, revealed that (I) moved more rapidly than 5'-XMP, but more slowly than adenosine. Heating a t 98" for more than 60 minutes in 1 Ar HC1, or more than 40 minutes in 1 N NaOH, was required to destroy completely the biological activity of (I). Biological activity of (I) was lost only after relatively prolonged heating a t 98' in 0.05 N HC1 in the presence of Dowex-50 (H+). The major products of such treatment, amounting to 85yc of the total, were (1) adenine, identified by ion-exchange chromatography and ultraviolet spectrum, and (2) a mixture of ribose-3-phosphate (60%) and ribose-2phosphate (40y0), identified by ion-exchange chromatography in the presence of borate.2 However, the biological activity of (I) was lost rapidly on incubation with crude or fractionated extracts of heart or brain. In the presence of a partially purified enzyme from heart, (I) was converted quanti(1) T \T Rall, E W Sutherland and J Berthet, J Bfol Chem 224, 403 (105;) 0)J S K h q m a n d W E L o h n , THISJ O U R N A L , 75, 1153 (1953)
I
(3) We t h a n k Dr. Leon Heppel of t h e National Institutes o f Health, Bethesda, Maryland, f o r supplying a sample of purified spleen phosphodiesterase, a n d for t h e information regarding t h e similarity of ( I ) with t h e compound reported i n t h e accompanying paper b y TT. H Cook, D. Lipkin and R . M a r k h a m . (4) W. H. Cook, D. Lipkin a n d R. M a r k h a m , THISJ O l i R N h I . , 79, 3607 (1957). ( 5 ) W e t h a n k Dr. R o y M a r k h a m for sending these samples a n d for furnishing a sample of Cvotalus adamanteus venom. (6) T h i s investigation was supported (in p a r t ) b y a research grant No. H-2745 f r o m t h e National H e a r t I n s t i t u t e of t h e Public Health Service.
DEPARTMEKT OF PHARMACOLOGY
EARLIT,SUTHERLAXD SCHOOL OF MEDICINE WESTERNRESERVE UNIVERSITY T IT.RALL CLEVELAND 6, OHIO RECEIVED MAY13, 1957
UNEQUIVOCAL SYNTHESES OF DL-cZ'S-~, IO-METHYLENEOCTADECANOIC ACID (DIHYDROSTERCULIC ~ ACID) AND D L - C ~ S -1,12-METHYLENEOCTADECANOIC ACID'
Sir: The recen t interest regarding the structure of sterculic acid2-7 prompts us to record a t this time an unequivocal synthesis of DL-C~S-9, 10-methyleneoctadecanoic acid (I) and the demonstration of its complete identity with dihydrosterculic acid. '2'
H
H
\C/ H3C-(CH2)x(I) x and y
o/---\o-(CH2),-COOH 7; (VI) x = 5 ; y = 9
=
(1) Supported b y grants f r o m t h e American Cancer Society, recommended b y t h e Committee on Growth of t h e h'ational Research Council, a n d by t h e U. S. Public Health Service. (2) J . R . N u n n , J . Ckem. Soc., 313 (1952). (3) J. P. Verma, B. K a t h a n d J . S. Aggarwal, F a t w e , 176, 84 (1955). (4) P. K. F a u r e and J. C . Smith, J . Chem. Soc., 1818 (1950). ( 5 ) D. G. Brooke a n d J. C. Smith, Chent. a n d I n d . , 49 (1097). (6) B. A. Lewis a n d R . A, Raphael, ibid., 50 (1957). (7) V . V.N a r a y a n a n a n d B. C. L. Weedon, ibid., 394 (1957). ( 8 ) K. Hofmann, 0 . Jucker, W . R . Miller, A . C vtlng, J r . and F. Taussig, Tms J O U R N A L , 76, 1789 (1931).
July 5 , 1957
COMMUNICATIONS TO THE EDITOR
3600
The addition of dibromocarbene to 1,4-cyclohex- brueckii. ~L-trans11,12-Methyleneoctadecanoic adieneg afforded 7,7-dibromonorcar-3-ene (II),m.p. acid fails to support growth of this organism under 36.8-37.0’; Anal. Calcd. for C7HsBrz: C, 33.5; identical experimental conditions. l 4 These obserH, 3.2; Br, 63.3. Found: C, 33.0; H , 3.5; Br, vations point to either D- or L-cis-11,12-methylene63.0. Oxidation converted (11) into cis-3,3-di- octadecanoic acid as the structure for lactobacillic bromocyclopropane-l,2-diacetic acid (111), m.p. acid. 179.4-181.2°; Anal. Calcd. for C7H804Br2:C, (14) K.H o f m a n n a n d C. Panos, J . B i d . Chem., 210, 687 (1951) 26.8; H, 2.5; Br, 50.5; neut. equiv., 158. KLAUSHOFMANN BIOCHEMISTRY Found: C, 26.5; H, 2.2; Br, 50.5; neut. equiv., USIVERSITYOF DEPARTMENT PITTSBURGH SALVADOR F OROCHENA 159. Hydrogenolysis of (111) gave cis-cyclopro- SCHOOL CLAYTON R‘. Y O H O OF MEDICINE PENNSYLVANIA pane-l,%diacetic acid (IV), m.p. 131-133’; Anal. PITTSBURGH, Calcd. for C7H1004: C , 53.3; H, 6.4; neut. equiv., RECEIVED MAY16, 1957 79.4. Found: C, 53.0; H, 6.2; neut. equiv., 79.6. The monomethyl ester chloride of (IV) on treatment with n-hexylcadmium followed by alkaline hydrolysis afforded ~~-cis-6-keto-3,4-methylOXIDATIONS AT MERCURY HALIDE ANODES ene-dodecanoic acid which was redwedlo to DLcis-3,4-methylenedodecanoicacid (V), b.p. 153- Sir: 154’ a t 3 mm.; Anal. Calcd. for C13H2402: It is well known in polarography that the anodic C, 73.7; H, 11.4; neut. equiv., 213. Found: potential range of a mercury electrode is severely C, 73.6; H, 11.3; neut. equiv., 208. Conversion limited by oxidation of the mercury itself. A of (V) into DL-cis-9,lO-methyleneoctadecanoicacid mercury electrode which can be used a t highly (I) was effected by procedures previously de- anodic potentials might well be considered a sigscribed,8s11 m.p. 38.6-39.6” ; Anal. Calcd. for nificant advance in polarographic practice. Such C19H3602: C, 77.0; H, 12.2; neut. equiv., 296. an electrode has been developed as a result of Found: C, 77.0; H, 12.2; neut. equiv., 298. chronopotentiometric studies a t a mercury pool in Amide, m.p. 86.4-EK7.6’; Anal. Calcd. for (219- chloride media. This electrode operates through &ON: C, 77.2; H:, 12.6; N, 4.7. Found: C, formation of an “inert” film of mercurous chloride 77.0; H, 12.3; N, 5.0. Mixed melting point de- prior to oxidation of electroactive species in the terminations and comparison of the infrared absorp- bulk of solution. This phenomena has not been tion spectra and X-ray diffraction patterns of (I) observed previously since conventional polaroand dihydrosterculic acid demonstrated the com- graphy a t the dropping mercury electrode does not plete identity of these compounds. The X-ray allow sufficient time for the proper film formation. diffraction patterns of the amide of (I) and of diThe theory of chronopotentiometry has been hydrosterculamide were identical also, and admix- thoroughly reviewed by Delahay. The equipture of the amides did not result in a depression of ment used in the present study was conventional the melting point. These findings establish the in all respects. The background electrolyte was structure of dihydrosterculic acid.I2 a solution 0.3 M in potassium chloride and 2 X In connection with studies on the structure of M in hydrochloric acid (pH 2.7). The anodic lactobacillic acid, we have converted cis-cyclopro- chronopotentiogram of this solution (Hg pool pane-1,2-diacetic acid (IV) into DL-cis-11,12-meth- area 1.47 cm.*, current 350 microamperes) shows ylene-octadecanoic acid (VI), m.p. 31.0-33.6’; four small waves a t ca. +0.07, 0.5, 0.8, and 1.2 V. Found: C, 76.5; 13, 12.0; neut. equiv., 294. vs. S.C.E. The first wave is due to formation of merAmide, m.p. 84.0-8,5.2’; Found: C, 77.0; H, curous chloride. The variation of El/, for this 12.6; N, 4.8. Although exhibiting an identical wave follows closely infrared absorption spectrum, the synthetic speciEaa = Eo - 0.059 log [C1-]/2 men differed from lactobacillic acid in melting point and X-ray diffraction pattern. The non-identity where Eo = 0.022 v., the standard potential VS. of lactobacillic acid (which has been shown to repre- S.C.E. of the reaction sent one of the stereoisomeric forms of 11,122H9 2C1- = Hg$& + 2emethyleneoctadecanoic acid13) with either cis- or trans8-DL-11,12-methyleneoctadecanoic acids dem- Further interpretation of these potential-time patonstrates clearly that: lactobacillic acid must be a terns will be reported in the near future. The chronopotentiogram of a 2 x M sohdistinct optical isomer. Synthetic DL-cis-l1,12(DPP) rnethyleneoctadecanoic acid exhibits approxi- tion of N,N’-dimethyl-fi-phenylenediamine in the same background electrolyte shows an idenmately one-half the growth promoting activity of natural lactobacillic acid for Lactobacillus del- tical first wave for mercurous chloride formation. It is followed by a clearly defined wave for the oxi(9) W. von E. Doering a n d A. K. Hoffmann, THISJOURNAL, 7 6 , dation of DPP. The E l / , of D P P (ca. +0.4 v.) 6162 (1954). wave is identical with that obtained a t a platinum (10) Huang-Minlon, ibid., 68, 2487 (1946). electrode under the same $H conditions. A plot (11) S. Stallberg-Stenhagen, Archiv Kemi Mineral. Geol., A22, No. of ri’2 vs. concentration shows excellent linearity 19, 1 (1946). (12) W e wish t o express o u r sincere appreciation t o Professor G. A. M DPP. between 4 X lop4to 3 X Jeffrey, D e p a r t m e n t of Chemistry, University of Pittsburgh, for t h e The anodic oxidations of aniline, o-phenyleneX - r a y studies. Details of this work will be presented in THISJOURdiamine and hydroquinone, which take place a t NAL.
+
(13) G. J (1956).
Marco and K . H o f m a n n . Federotion Proc., 16, 308
(1) P Delahay, “New Instrumental Methods in Electrochemistry,” Interscience Puhlishers, l n c , Piew York, N Y , 1954
