Aug., 1951
4051
C O M M U N I C A T I O N S T O T H E EDITOR PERCHLORIC ACID SALT OF VITAMIN Biz
Sir :
its formation reveals the presence of a t least six weakly basic groups in the vitamin molecule. SQTJIBB INSTITUTE FOR MEDICAL RESEARCHJ. F.ALICINO
During the course of an investigation of the tiNEWJERSEY tration of vitamin B1z in glacial acetic acid with per- XEWBRUNSWICR, RECEIVED JUNE 28, 1951 chloric acid, i t was observed that the addition of an excess of the reagent resulted in the formation of an amorphous orange-colored precipitate. Vitamin THE PREPARATION OF RADIOACTIVE VITAMIN BrJ B12a responded in a like manner, except that it BY DIRECT NEUTRON IRRADIATION' forms a red-colored product. Sir : A well-defined product of constant composition It has been found possible to activate the cobalt could be obtained only when more than 8 equiva- atom in crystalline vitamin B 1 2 by the Co6$(n, lents of perchloric acid were added. I n a typical y)Cos0reaction with high Cos0retention and small experiment, 20 mg. of Blz was dissolved in 5 ml. of over-all loss of biological activity. The one other glacial acetic acid and 20 ml. of 0.01 N perchloric preparation of vitamin B12-Co60 has been accomacid in glacial acetic acid added. The orange pre- plished by biosynthesis.2 That the cobalt atom is cipitate was centrifuged after standing for ten min- firmly bound in the vitamin is evidenced by its utes, and washed with 5 d. of glacial acetic acid complete lack of exchange with inorganic cobalt and then successively with four 10-ml. portions of compounds, in agreement with the diamagnetic beanhydrous ether. After preliminary drying in a havior of vitamin B1za3 It seemed reasonable, vacuum desiccator the material was dried to con- therefore, to expect that some radio- and bioactive stant weight a t 56' over phosphorus pentoxide a t Blz molecules would result from the neutron activa2 mm. tion of this crystalline solid.4 As will be shown, Anal. Calcd. for C~aH~$\T140~~PCo~6HC104 (mol. purification of irradiated B I Z yields a fraction with wt. 1953): C, 38.7; H, 4.6; N, 10.0; P, 1.5; Co, -80% of the original specific radioactivity and 3.0; C1, 10.9; CN, 1.4. Found: C, 38.4; H, 4.5; 100 f 15% of the specific biological activity based N, 10.1; P, 1.6; Co,3.2; C1,10.6; CN,l1.4. on L.L.D. assay. A sample of crystalline vitamin B12,6weighing 8.8 That this product represents a simple salt of the unchanged vitamin with six moles of perchloric acid mg., sealed in a quartz ampoule in vacuo, was subis furthermore evidenced by the following facts : jected to a thermal neutron flux of 1 X l O l 3 neuAfter ir(1) back titration with 0.01 N potassium acid trons/sq. cm. for seven days a t phthalate2 of the perchloric acid remaining in the radiation, the sample was let stand for two months supernatant showed that an amount corresponding to allow short-lived activities to decay. The crysto 5.93 and 6.02 equivalents had entered the precip- tals were not visibly altered and were completely itate; (2) in an experiment in which anhydrous soluble in water (Solution I). To determine the ether was added for quantitative precipitation, 5.94 purity of this aqueous solution and the subsequent mg. of vitamin yielded 8.60 mg. of the perchloric fractions mentioned below, small aliquots were apacid salt (calcd. 8.59 mg.) ; (3) after decomposition plied to sheets of Whatman No. 1 filter paper. of the salt with water (reappearance of pink color) These prepared sheets were then developed with the aqueous solution consumed alkali corresponding ethyl acetate-acetic acid-water' in a descending to 6 equivalents of the amount of vitamin used; (4) chromatographic system. The developed and dried while qualitatively the ultraviolet absorption spec- paper sheets were placed in contact with X-ray film trum of the aqueous solution of the salt was identi- for several days to locate the areas having radioaccal with that of the vitamin, a t 360 my was tivity and then the identical sheets were placed on solid agar plates prepared for the L.L.D. assays to 68% of the value given by the latter (calcd. 69.2). locate areas having bioactivity. Solution I gave a Similarly, in the microbiological assay the salt chromatogram exhibiting both radio- and bioactivshowed 66-690/, of the potency of the crystalline ity a t the origin and in an elongated area extending B I Z standard; (5) the infrared spectrum of the salt (1) Work carried out under the auspices of the Atomic Energy (Nujol) exhibited a band a t 4.70 y , characteristic Commission. (2) L. Chaiet, C. Rosenblum and D T. Woodbury, Science, 111, 601 for the cyano group. The perchloric acid salt described above repre- (1950). (3) (a) J. C. Wallmann, B. B. Cunningham and M. Calvin, ibid. sents to our knowledge the first well-defined deriva- 113, 55 (1951); (b) for a discussion of exchange in cobalt complexes as tive of vitamin Blz having an intact cobalti-cyano related to their structure, see B. West, Nafurc, 165,122 (1950). (4) For data concerning retention in liquids, see J. M. Miller, J. W. linkage,a*4i5 and is of structural interest insofar as Gryder and R. W. Dodson, J . Chem. Phys., 18,579 (1950). (1) E. A. Schulek, Anal. Chcm., 63, 337 (1923). (2) P. C. Markunas and J. A. Riddick, i b i d . , 89, 337 (1951). (3) N. G. Brink, F. A. Kuehl, Jr.. and K. Folkers, Science, 112, 354 (1950). (4) E. A. Kaczka, D. E. Wolf, F. A. Ruehl, Jr., and K. Folkers, %bid., 118, 354 (1950). (5) R. P. Buhs, E. G. Newstead and N. R. Trenner, ibid., 118, 825 (1851).
(5) We wish to thank the following members of the Chas. Pfizer and Co., Inc. staff, J. H. Kane and J. Snell, for providing several samples of Bin; and T. Lees, for data on the L.L.D. plate assay. (6) We wish to thank the personnel at the National Research Council for the irradiation performed a t Chalk River, Ontario, Canada. (7) M. A. Jermyn and F. A. Isherwood, Biochcm. J . , 44,402 (1949). ( 8 ) See note 5. Similar to the method described by W. F. J. Cuthbertson, H. F. Pegler and J. T. Lloyd, The Analyrf, 76, 133 (19 51).
COVTMUNIC~TIONS TO THE EDITOR
4052
to the limit of the solvent migration. Consequently, an elution chromatogram of a portion of Solution I was made by diluting with acetone and running on a silica gel column (0.9 X 30 cm.) using acetone-water mixtures. The radioactivity of the fractions was measured by counting evaporated drops of the eluate on aluminum discs with a C.M. counter. One fraction (Solution IX) behaving in the column in a manner similar to authentic B12, was then subjected to further study. This solution exhibited absorption maxima a t 2780 and 3610 A. No radioactivity could be detected in the organic phase when IX was extracted with dithizone in chloroform. X paper chromatogram of IX revealed a concentrated spot with little tailing, coincident for both the radio- and bioactivity. Tlie Rf of this spot (0.79-0.SO) was in agreement with :I BIZ standard run simultaneously. The radioactivity of the original and purified samples was shown to be Co60 (5.3 y) by measurement of the gamma ray spectrum in a scintillation spectrometer. On a G . X . counter (geometry -9%), Solution I had 6813 =t 16 cpm./y, solution IX 567 i 30 cpm./y, giving a retention value of -80%. By employing serial dilutions of I and IX against standard B12, i t was determined that the bio-activity in Solution I was 80 i 20% and in 15’3 of the standard by Solution I X was 100 weight.
1701. 73
TABLE I Wheat Germ DNA; Intact Preparation and Enzymatically Produced Cores (as moles per 100 moles P ) Constituent
Adenine Guanine Cytosine 5-Methylcytosine Thymine
Total Purine to pyrimidine ratio
Intact DNA
19%; Core
8% Core
26.3 21.8 16.2 5.8 26.1 96.2
33.2 20.0 11.8 4.3 26.2 95.5
35.4 19.8 10.3 3.6 23.4 92.5
1 ,00
1.26
1,4X
TABLE I1 Liberation of Adenine (as moles per 100 moles P) Agerit
1 iV HeSol, 100”, 1 hr. 98% HCOOH, 175”, 2 lir. 7.5 AT HClOd, loo”, 1 hr.
Intact DNA
19% Core
8% Core
26.5 26.3
31.2 33.3 33.2
31.6 35.4
Several points appear of interest. The ratio of cytosine to 5-methylcytosine remained constant in all stages, viz.,2.8. I n the intact DNA, the sum of these two pyrimidines equalled the molar concentration of guanine, a relationship observed with respect to the ratio of guanine to cytosine in almost all DNA specimens ~ t u d i e d . ~As judged from the extent of its liberation by various hydrolyzing agents, adenine seems to occur in two types of linkage, one of which is enriched in the cores (Table CHEMISTRY DEPARTMENT BROOKHAVEN XATIONAL R . CHRISTIAN ANDERSON 11). The procedures employed for the isolation LABORATORY YVETTEDELABARREand purification of the DNA will soon be discussed UPTON,L. I., N. Y. in detail. The enzyme used was supplied by the RECEIVED MAY14, 1951 Worthington Biochemical Laboratory, Freehold, N. J. The analytical methods have been described before. ENZYMATIC DISINTEGRATION OF WHEAT GERM This work was supported by research grants DESOXYPENTOSE NUCLEIC ACID from the U. S. Public Health Service and the RockeSir : feller Foundation. Previous work in this Laboratory1 has shown that IS. Chargaff, Enpericntia, 6, 201 (1050); J . Cell. Coirzp P h v i o l . , the degradation of calf thymus desoxypentose nuc- i n (3) press; Fedel alioii Pioc., 10, in press. leic acid (DNA) by crystalline pancreatic desoxy(4) E . Vischer and E. Chargaff, J . B i d . Chem., 176, 703, 715 (1948); ribonuclease proceeded according to a complex E. Chargaff, E. Vischer, R. Doniger, C. Green and F. Misuni, ibid., pattern, resulting in the formation of dialyzable 177,405 (1949); E. Chargaff, R. Lipshitz, C. Green and M. E. Hodes, fragments and of a non-diffusible core which was i b i d . , 192,in press. OF BIOCHEMISTRY characterized by greatly increased ratios of adenine DEPARTMENT OP PHYSICIANS AND SURGEONS to guanine, thymine to cytosine, and purines to COLLEGE COLUMBIA UN~VERSITY GEORGEBRAWERMAN pyrimidines. ERWINCHARCAFF XEIVYORK32, N. Y. A study of the generality of this phenomenon RECEIVED JUNE 18, 1951 appeared important, since it offers an additional tool for the differentiation between DNA preparations of different origin and for the understanding of ON THE SYNTHESIS OF CORTISONE ACETATE the relationship between nucleotide sequence and Sir : enzyme action. ’CVheat germ DNA4,highly polyWe wish to report the synthesis of cortisone merized and entirely free of pentose nucleic acid, was employed. This DNA contains an appreciable acetate from allopregnane-3P-ol-l1,20-dioneacequantity of a third pyrimidine, 3-methylcytosineI2 tate, I, made available recently from As,6 steroids, and provides thereby one more marker, useful for devoid of functional groups in ring C.’j2 Hydroxylation of the allopregnane I a t the the study of the enzymatic attack. The results 17-position by conversion into its enol acetate and summarized in Table I indicate the trend of degradation; “19Y0 core” and “Sy0core” refer to the treatment with perbenzoic acid followed by caustic dialvsis residues recovered after 81 and 927* of saponification3 yielded allopregnane-3/3,17cr-diol(1) Chamberlain, Ruyle, A. E. Erickson, Chemerda, Aliminosa, the DNA, respectively, had been convertei to R. L. Erickson, Sita and Tishler, THISJOURNAL,73,2396 (19.51). dialyzable products. (2) Stork, Romo, Rosenkranz and Djerassi, i b i d . , 73, 3546 (1951).
*
_I
(1) S. Zamenhof a n d E . Charraff, J . A i d . Chem., 178, 531 (1949);
187,1 (19.50). ( 2 ) G . R. Wyatt. F % x h ~ m. I , , 48, 6Xi ~1Cl;l).
(3) Kritchevsky and Gallagher, J . Biol. Chrm., 179, 507 (1949); Marshall, Kritchevsky, Lieherman and Gallagher, THISJOURNAL, 70, 1837 (1948).
