Aug. 5 , 1953
4170
COMMUNICA'I'IONS TRANSFORMATION OF INOSINIC ACID TO ADENYLIC AND GUANYLIC ACIDS IN A SOLUBLE ENZYME SYSTEM'
+Yir:
We have shown recently that a soluble enzyme extract from rabbit bone marrow was capable of synthesizing compounds of adenine and guanine from inosinic acid (IMP) in the presence of DPN.? Activity was lost upon dialysis and restored by addition of boiled extract. We have now found that the boiled extract may be replaced by appropriate amino donors. Three reaction sequences have been observed in the biosynthesis of adenine and guanine nucleotides from I M P in the dialyzed extract: (1) An enzyme, inosinic acid dehydrogenase, catalyzes the oxidation of I M P to xanthosine 5'-phosphate (XMP) by DPN. (2) X M P is aminated to guanosine 5'-phosphate (GMP) in the presence of I-glutamic acid or I-glutamine, 4 T P and AIg++. (3) I h l P is aminated to adenosine 5'-phosphate (AMP) in the presence of Zaspartic acid and a high energy phosphate source. When I M P was incubated with the dialyzed extract and DPN, a xanthine derivative accumulated. This was isolated by chromatography on Dowex-1-formate using the elution gradient technique of Hurlbert, et u Z . , ~ and purified by rechromatography on Dowex-1-acetate. Its specific activity (168,000 counts C1*/min./pmole) was equal to the mean I M P value. I t was identified as X M P by (1) absorption spectrum (maxima a t 263 mp and 233 mp a t pH 2 ) ; (2) analytical values of 1.02 moles P and 0.94 mole ribose per mole of xanthine; (3) ready hydrolysis with 5'-iiucleotidase of Crotalus adamanteus venom; and (4) a positive Schiff periodate test4 indicating the 2'and 3'-positions were unsubstituted. Amino donors were identified by incubating dialyzed extract (0.8 ml.) with 2 pmoles of DPN, 10 pmoles of glucose, 0.5 pmole of IMP-8-C14, and, in place of boiled extract, 5 pmoles of one of the following : 1-aspartic acid, 1-asparagine, 1-glutamic acid, 1-glutamine, 1-alanine, 1-arginine, ammonium chloride, and glycine. Of these, it was found that aspartic acid replaced the boiled extract for adenine synthesis, and glutamic acid or glutamine for guanine formation. Optimal activity in the amination of I M P required the addition of aspartic acid, 3( -)phosphoglycerate (PGA), K+, Mg++, and inorganic PO4. Phosphopyruvate can be substituted for PGA but, as indicated in Table I, ATP is not an adequate substitute for PGA, and addition of ADP as a phosphate acceptor has an apparent inhibiting (1) Supported by a grant-in-aid from t h e American Cancer Society upon recommendation of t h e Committee on Growth of t h e National Research Council, and by t h e U. S. Atomic Energy Commission under Contract A T (30-1)-1818. (2) R . Abrams a n d M. Bentley, Arch. Biochcm. and Biophys., in press. (3) R. B. Hurlbert. H. Schmitz, A. F. Brumm and V. R . Potter, J . B i d . Chem., 909, 23 (1954). (4) J. G. Buchanan, C. A. Dekker and A. G. Long, J . Chcm. SOC., 3162 (1950).
T O T H E EDITOR rather than a stimulating effect. The other nucleoside triphosphates tested (UTP and GTP) were also inferior to PGA. In previous experiments,' we have used glucose plus DPN to satisfy the energy requirement for amination. TABLE I REQUIREMENT FOR HIGH ENERGYPHOSPHATE IN THE AMINATION O F I M P Each vessel had a total volume of 1.0 ml. containing 0.75 ml. dialyzed extract, 0.5 pmole IMP-8-CI4, 10 pmoles I-aspartate, 5 pmoles MgC12, 5 pmoles potassium phosphate, 50 pmoles tris buffer, pH 7.4, and the additions shown below. Incubated at 37" for 30 min. pmoles adenine formed
Additions
None PGA, 10 pmoles ATP, 3.5 pmoles UTP, 2 pmoles GTP, 2 pmoles PGA ADP, 3 prnoles PGA UDP, 2 pmoles
0.000 IF ,022 .029 ,018
+ +
,078 .19
The requirements for the amination of XLIP are glutamic acid or glutamine, ATP and Mg++. As shown in Table 11, in contrast to I M P amination, PGA alone is ineffective unless ADP is also added, ADP. and ATP completely replaces PGA
+
TABLEI1 FOR THE AMINATIONOF X M P CONDITIOXS Extract and tris buffer as in Table I with 0.2 pmole XMP-8-Cl4, 5 pmoles /-glutamate, 5 prnoles I-glutamine, 5 pmoles MgC12, and additions shown below. Additions
pmoles guanine formed
0.001 ,007 ,035 .14 .14 .011
None PGA, 10 pmoles ADP, 1.5 pmoles PGA ADP ATP, 3.5 pmoles UTP, 1.6 pmoles
+
In the foregoing experiments free purines were isolated after acid hydrolysis. In order t o demonstrate that the hydroxypurines were indeed aminated in the nucleotide form, chromatography was carried out on Dowex-1-acetate without hydrolysis. Table I11 demonstrates that the I M P to AMP and TABLEI11 GUANINE XUCLEOTIDESFORMEDFROM I M P AND Xh.IP 10 ml. reaction volumes with reactants as indicated in Tables I and 11; incubated a t 37' for 60 min.
ADENINE
AND
pmoles
counts/min./pmole
I M P added I M P recovered AMP ADP ATP
8.1 0.8 1.4 2.4 2.7
34,100 30,300 25,500 25,000
X M P added X M P recovered GMP
3.9 1.3 1.2
40,400 39,100 25,700
24,400
4180
COMMUNICATIONS TO THE EDITOR
X M P to GMP reactions occurred with no extensive dilution of specific activity. The mechanisms of the amination reactions are being investigated. Carter and Cohen5 have reported the formation of adenylosuccinic acid from AMP and fumaric acid. It seems likely that this compound is an intermediate in AMP formation from IMP, and that the corresponding guanyloglutaric acid is an intermediate in G M P formation from XMP. ( 5 ) C. E. Carter and L. H. Cohen, THISJ O U R N A L , 77, 499 (1955).
THEINSTITUTE OF RESEARCH ABRAMS MONTEFIORE HOSPITAL RICHARD PITTSBURGH 13, PA. MARIANBENTLEY RECEIVED JUNE 21, 1955 RAUWOLFIA ALKALOIDS. XXII. FURTHER OBSERVATIONS OF THE STEREOCHEMISTRY OF RESERPINE
Sir:
Four lines of evidence have been offered recently in support of I a as representing the stereochemistry of reserpine1v2: (1) the C-3 epimerization of reserpine, (2) molecular rotational differences indicating ,&orientationof the C-3 hydrogen, 16-carbomethoxyl and 18-acyloxy groups, (3) the inability of 3-isoreserpic acid to lactonize and (4) data strengthening the presently accepted structures of allo- and 3-epialloyohimbane. We now wish to describe new findings demonstrating conclusively the validity of l a . When 3-iso-reserpinol (11)3was treated with ptoluenesulfonyl chloride in pyridine a t room temperature overnight, a substance crystallized directly from the reaction mixture in high yield. ,iln inspection of its properties made i t evident that the substance must be a quaternary salt. It has a high melting point (320-330' (dec.)) and is virtually insoluble in chloroform. Analysis indicated it to be a mixed tosylate-chloride salt. Anal. Calcd. for C22H29N202+.0.6 SOaC7Hi-.0.4C1-: C, 66.86; H, 7.09; Y , 5.96; S, 4.09; C1, 3.02. Found: C, 66.64; H, 7.10; N, 6.02; SI 3.89; C1, 2.87. Addition of an excess of sodium iodide to a hot aqueous solution gave an immediate precipitate of the crystalline iodide salt (m.p. 360-365' (dec.)). Anal. Calcd. for C22H~9Nz02+~1-:C, 54.98; H, 6.10. Found: C, 53.33; H, 6.17. A free base extractable in chloroform could not be liberated from the salt with dilute ammonia. The infrared spectrum of the mixed tosylate-chloride salt showed the bands characteristic of the p-tosylate ion4 and the presence of chloride ion was revealed by an instantaneous precipitate with silver nitrate. Formulation of this quaternary salt as 111 requires a cis relationship of the hydrogens a t C-1.5 and (2-16 restricting the stereochemistry of reserpine to Ia. Similarly, reserpinol (Ibj5 gave a quaternary tosylate best characterized as its iodide salt (m.p. 315-316" (dec.)). (1) C. F. Huebner, H. B. MacPhillarny, E. Schlittler and A. F. St. Andre, Experientia, in press. (2) E. Wenkert and L. H . Liu, ibid., in press. (3) H. B. MacPhillamy, C. F. Huebner, E. Schlittler, A. F. St. Andre and P. R. Ulshafer, THISJOURNAL, in press. (4) P. A. Diassi, F. H . Weisenborn, C . M , Dylion and 0. Wintersteiner, i b i d . , 77, 2028 (1955) (5) C. F. Huebner, H . B. MacPhillamy, A . F. St. Andre and E. Schlittler, i b i d . , 77, 472 (1955).
Vol. 77
1 118 R/'Vi\R
T
16
2
1~ I
HOCH?/~'J'
OCHI I1 R ' = -OCOCJT?(OCHa)3 3: R = -COOCHa, 1 ) : R = --CH~OH,R' = Er.
I
OCHo
111 RESEARCH DEPARTMENT PRODUCTS, INC. CIBA .PHARMACEUTICAL C. F. HUEBNER SUMMIT, NEWJERSEY DEPARTMENT OF CHEMISTRY E. WEXKERT IOWASTATECOLLEGE AMES,IOWA RECEIVED J U N E 27, 19%
STUDIES IN THE SYNTHESIS OF THE ANTIRACHITIC VITAMINS. 111. THE SYNTHESIS OF I-CYCLOHEXYLIDENE-2- [5'-METHOXY-2'-METHYLENECYCLOHEXYLIDENE- 1'1-ETHANE
Sir: Several years ago we reported' the synthesis of a simple model (I) of vitamin D. However, subsequent work in this Laboratory2 showed that the method used was impractical for the synthesis of vitamin D:! or DI. In view of work undertaken in other laboratories3 we wish to report a t this time the synthesis of 1-cyclohexylidene-2-[5'-methoxy2'-methylenecyclohexylidene-l']-ethane (111) using a method which can easily be adapted for the synthesis of all vitamin Ds. Cyclohexylidene acetaldehyde (6.8 g.) 4 ~ 6 prepared by the chromic acid oxidation6 of l-ethenylcyclohexanol-1 was allowed to condense with 14 g. of 4-methoxycyclohexanone with stirring under nitrogen in 900 cc. of methanol containing 4 g. of sodium hydroxide and 10 cc. of water. After twelve hours the mixture was acidified and from i t was obtained 6.1 g. (58.670) of the ketone (II),recrystallized from ligroin, m.p. 79.5-50.5'. Anal. Calcd. for (11): C, 76.92; H, 9.46; mol. wt., 234. Found: C, 76.77; H, 9.45; mol. w t . (in exaltone), 224; E (309 mp), 29,100. The infrared spectrum shows strong bands for the dienone. 2,3-Dinitrophenylhydrazone, m.p. 178-179'. Anal. Calcd. for C21H2BX406:C, 60.86; H, 6.33; N, 13.52. Found: C, 61.05; H, 6.68; N, 13.66. The ketone (11) was allowed to react with freshly (1) N. A . Milas and W. L. Aldercon, Jr., THISJ O U R N A L , 61, 2534 (1939). (2) Unpublished results. (3) I. T. Harrison, B. Lythgoe and S. Trippett, Chem. and I d . , April, 507 (1955). (4) J. B. Aldersley, G. Pur. Burkhardt, A. E. Gillam and h7. C. Hindley, J. C h e m . Soc., 10 (1940). (5) V. K. Paranjpe, N. L. Phalnikar and B. V. Bhide, J , L ' i i i v . B o m b a y , 28,38(1949-50). ( R ) R . Kuhn and C. Grundmann, BPI., TO, 1897 (1937).
