Vol. 78
COMMUNICATIONS TO THE EDITOR
1310
T h e Bz03 and B were mixed in various ratios, ball milled and pressed into pellets. The pellets were thoroughly baked out in the system b y preheating at 900' until a pressure of less than I O 4 mm. was obtained. When the temperature was increased to above 1050' and the pressure had dropped to lop4 mm. or less, an amber glassy deposit began to form on the wall of the glass heating chamber and on the metal cold finger above the heated crucible. The most favorable ratio of boron to oxygen in the mixture was found t o lie between 3 : 1 and 4 : 1. In this range a yield of 65y0 (based on available oxygen) was obtained in two hours a t 1350' with an average yield of 0.5 g. per run. The purity of the product, expressed as the ratio B : 0, fell within the limits of 1 : 1.00 and 1:1.014. T h e material which condensed on the cold finger was found to be pyrophoric in a few instances in more than 100 production runs and ignited spontaneously in the course of its removal. Boron monoxide obtained by this method is a polymorphic glass which gave an X-ray diffraction patte-n showing only amorphous rings. Attempts to deb elop a crystalline pattern by heating the polymer a t temperatures below 400" were unsuccessful. A t temperatures above 450' its color changed as it disproportionated to boric oside and a black amorphous insoluble non-reducing solid which was probably boron. T h e rate of disproportionation increased with temperature. The density of the polymer, determined by the flotation method, is 1.765 =t 0.001 g . / ~ r n . ~ .X o solvent has been found for the polymer. It reacts vigorously with water and alcohols with the evolution of hydrogen which contains traces of boranes. T h e rate of reaction with alcohols decreases with increasing molecular weight of the alcohol. The resulting solutions in water and alcohol have reducing character which decreases with aging. The rate of decrease i n reducing ability is a function of pH and is affected by the presence of dissolved oxygen in the solutions.
The enzymes catalyzing these reactions have been purified 30 and 50 fold, respectively. T h e crude extracts are also capable of converting IMP to an adenine derivative. Extracts of A . aerogenes strain P-14, a guanine requiring mutant which excretes ~ a n t h o s i n e ,are ~ unable t o aminate XMP (reaction 2). Consequently, i t appears t h a t reactions 1 and 2 are essential steps in the biosynthesis of nucleic acid guanine. They are also essential in the conversion of exogenous adenine to nucleic acid guanine, since mutant P-I4 incorporates adenine into xanthosine, b u t not into nucleic acid guar~ine.~ The following observations indicate t h a t the conversion of exogenous guanine to nucleic acid adenine does not proceed through a reversal of these steps: (a) Reactions (1) and (2) are not reversible; (b) xanthine, which is converted to both nucleic acid adenine and guanine in mutant PD-1 blocked a t an early stage of purine biosynthesis6 (Table I, Exp. 2), cannot be converted to either of the nucleic acid purines in P-14, b u t is excreted exclusively as xanthosine (Table I, Exp. 8); (c) exogenous guanine is a precursor of nucleic acid adenine in P-145(Table I, Exp. 7 ) . IKCORPORATION
OF
ACID
Rxp.
TABLE I VARIOUS P R E C U R S O R S I N T O NUCLEIC PURISES A N D HISTIDINE
Supplement added
Strain
-Relative Guanine
100 103 100
specific activityAdenine Histi(line
1" P D - l 5 3 PD-1 3 PD-1 4 PD-1 5 PD-1 6 PD-1
Guanine-8-C" XY:mthine-8-C1@ Guanine-2-C'4c
7" P-14' 8 P-14 9 P-14
Guanine-8-CI4 Xanthine-S-C14' G~anine-2-C'4~
97 0 96
38 0 24
Xanthine-8-Ci4' Gunnine-2-C14" Hirtidine-2-C1':d
107 111)
94 106
10 11 12
HP-1' HP-1 IIP-1
~
1
4
0
~
~
HC1400Hd Glycine-2-Ci4d
0 3
0.1 0 7
100 103 30 0.4 0.1 7.4
0 38 2 5 1.7 rl 4
19 0
BB' 65a
0 2 F. -1.KANDA A . J. KING a See reference 5. b A . aerogenes mutant requiring V.A. RUSSELL guanine, adenine, hypoxanthine, xanthine or 4-amin0-~5~VALTER KATZ imidazok carboxamide. Kindly supplied by Drs. G . I3 Brown and M. E. Balis, Sloan-Kettering Institute, S e \ v York. d Unlabeled guanine added. e 8 . aerogenes mutant requiring guanine or 2,6-diaminopurine. E . coli mutant INTERCONVERSION OF PURINES IN T H E BIOSYNrequiring guanine or xanthine; spared by histidine. u 1,:ihel T H E S I S OF NUCLEIC ACID ADENINE AND GUANINE located in position 2 of t h e imidazole ring. AND OF HISTIDINE'
DEPARTMENT OF CHEMISTRY SYRACUSE UNIVERSITY SYRACUSE, NEW YORK
'
Sir: Cell free extracts of Aerobacter nerogenes and of Escherichia coli were found to convert I h l P ? to G M P by the following reactions:
+
+ +
I M P c DPS+ +);LIP DPSH IX+ (113 AMP P P (2) SLIP ATP S I X 4 --f C,IfP
+
+
f I ) T h i s work x a s supported in p a r t liy on institutional g r a n t t o H a r v a r d University from t h e American Cancer Suciety, b y a research g r a n t (SSF-GISS5) from t h e h-atiiinnl Science 1:uundation. a n d b y f u n d s received f r o m t h e E u g e n e IIififiins T r u s t . ( 2 ) Abbreviations. I L l P , inosinr-j'-phosph3te: D P S + a n d DPX I f , oxidized a n d reduced diphosphopyridine nucleotide; X?v\.IP, ranthosine~;l'-phosph3te; A T P , adenosine t r i p h o s p h a t e ; GhZP, guanosine-5'-phosphate; A l f I ' , adenosine-5'-phosphate; PP, pyrophosphate. ( 3 ) I. B. Geliring a n d I3 llnfixs:anik, Trrrs J U I ~ H N ~ I 7 . ,7 . 4655 (1955).
The pathway of the conversion of guanine to adenine was explored by experiments with C14 labeled compounds (Table I ) . In strain PD-1 carbon X of exogenous guanine or xanthine is transferred to nucleic acid adenine without dilution (Expt. l , 2), while carbon 2 of guanine contributes only 4070 (Exp. 3 ) . The methylene carbon of glycine, but not formate or COz, is incorporated into nucleic acid adenine when guanine is the purine source (Exp. 4,6). Similar results are obtained in strain P-14, except that here some of the nucleic acid adcnine is formed by synthesis de. novo5 (Exp. 7-!2). (1) B . M a g a s a n i k a n d M. S. Brooke, J . Bioi. Chem , 206, 8 3 (19541 ( 5 ) h l . E Ralis, M. S Brooke. G. B. Brown a n d B. Magasanik J Btol Chrm , in press. ( 6 ) PIZ. S Brook? B \T:u~is:inik, J . B a c l . , 6 8 , 727 (19541.
April 5, 1956
COMMUNICATIONS TO TIIE EDITOR
1511
Carbon 2 of guanine and the methylene carbon of glycine are incorporated equally into nucleic acid adenine and into histidine (Exp. 3, 6, 9), suggesting t h a t they enter a pool from which 1-carbon units are drawn for the biosynthesis of the precursors of purines and of h i ~ t i d i n e . ~ This assumption gains support from the results obtained in mutant HP-1, a strain with an unusually high guanine requirement which is spared by histidine. Here carbon 2 of guanine is incorporated into adenine and into the amidine carbon of histidine without dilution (Exp. 10-12), indicating t h a t it is the only available source of the 1-carbon units required for the formation of these compounds; i t would seem t h a t in this mutant the guanine requirement reflects a genetic block which prevents the production of these 1-carbon units from other sources. The experiments presented here suggest that the conversion of guanine to adenine occurs through the replacement of the amino-substituted carbon 2 of guanine b y a single carbon unit. An alternative possibility, the reduction of carbon 2 of guanine by a pathway not involving reactions (1) and (2) followed by interchange with single carbon units, is not rigorously excluded b u t appears less likely because i t would not account for the equal incorporation of carbon 2 of guanine into adenine and histidine.
3a,4,5,6,7,7a,8-decachloro-3a,4,7,7a-tetrahydro-4,~methanoindene (V), m.p. 21j0, both of which give good yields of their respective monomers on therinal degradation. I n contrast, the chlorocarbon ( I ) undergoes pyrolysis only a t very high temperatures (500” or above) giving largely carbonaceous material and chlorine with only a small amount of 11. Compound I V can be isomerized to I by aluminum chloride. Neither I nor 111 contain a carbon-carbon double bond. N o , addition of chlorine, bromine, or other reactivk species has been noted. T h e infrared spectra of I and 111, in contrast to the spectra of IV and V, showed no absorption in the 6-7 region where the carboncarbon double bond is known to absorb unless a selection rule is in operation because of the presence of certain elements of symmetry in the m o l e c ~ l e . ~ Ultraviolet absorption spectra are not subject to such selection rules. T h e ultraviolet spectrum of I contrasts sharply with the spectra of IV, 1,2,3,4,5,6,7,7-octachlorobicyclo [2.2,1Ihept-2-ene and octachlorocyclopentene which absorb in the region above 210 mp, a characteristic of highly chlorinated cyclic monoenes and dienes.6 T h e ketone I11 shows a maximum of 307 mp which is ascribed to an unconjugated carbonyl group,7 but no maxima in the “double bond” region. This is in contrast to the spectrum of octachloro-3a,4,7,7~i-tetr3hytlro7,7-methanoindene- 1$-&me, the structure of which has been estahlishc~l.~ (7) T h e t r a n s f e r of carbon 2 of guanine to t h e amidine carbon of histidine h a s recently been observed in Laclobacillus casei; C. M i t o m a Compound I is unaffected by the following: a n d E. E. Snell, Proc. N a t . A c a d Sci., 41, 801 (1955). zinc dust antl hydrochloric acid, acetic acid, or DEPARTMENT OF BACTERIOLOGY methanol; silver nitrate and ethanol for long A N D IMMUNOLOGY BORISMAGASANIKperiods; alkaline reagents such as potassium HARVARD MEDICALSCHOOL H. S. MOYED DORISKARIBIAN hydroxide in methanol or lithium aluminum BOSTON15, MASSACHUSETTS hydride in ether; oxidizing agents such as ozone, RECEIVED J A N U A R27, Y 1956 potassium permanganate, chromic acid, sulfur trioxide, sulfuric acid, nitric acid or nitric acid with AN INVESTIGATION OF THE CHLOROCARBON, selenium. Compound 111 does not undergo the CloClz, M.P. 485’ AND THE KETONE, CioClloO. M.P.349’ haloform reaction which would be expected for a dichloromethylene group adjacent to a carbonyl Sir: A chlorocarbon, CloCllp (I), m.p. 485’, has been group. The presence of a bridged carbonyl is exobtained from the reaction of 1,1,2,3,3,4,5,5-octa- cluded by the fact t h a t 111 does not lose carbon chloropentene and aluminum chloride and directly monoxide on heating to 200’. Compound IT1 fails from hexachlorocyclopentadiene (11) by reaction to exhibit olefinic properties. The presence of a with aluminum chloride in boiling methylene chlo- reactive carbonyl group is demonstrated by the ride or carbon t e t r a c h l ~ r i d e . ~When ~ ~ ~ ~ 11 was preparation of several hemi-ketals, thioherniketals, treated with sulfur trioxide, and the product and amine adducts. However, 111 fails to react hydrolyzed, a ketonic material, CIL!lloO (111), with Caro’s acid or hydrazoic acid. T o mcet the m.p. resulted. Compound 111 was originally requirements of the formula ClnCl,2a n d , excluding thought to be perchloro-3a,4,7,7~~-tetrahydro-4,T-the carbonyl dOUhlc bond, ClnClloO,the structures methanoindene. When compound IIJ was treated must contain six rings and/or double bond. Prewith phosphorus pentachloride, compound I was liminary X-ray diffraction stutlies on I inclicate a ~ b t a i n e d . ~I t was initially thought t h a t compound highly symmetrical inolccule crystdlizing in the cubic systeni or i n an arrangement closely approxiI was perchloro-3a,4,7,7a-tetrahydro-4,7-inethanoindene (IV), the “Diels-=llcler” dimer of cornpound mating the cubic system. From the prcceding el-itlence, aromatic systems, 11. Recently however, the Diels-Alder dimer (11’) of I1 has been prepared4; thus the compound aliphatic olefins and acctylencs and alicylic olefins described as compound I , n1.p. 4S3’, is not the seem to be excluded. The only remaining possiDiels-Alder dimer. The high 1n.p. of I contrasts bility is a “caged” structure possessing a total of sharply with t h a t of IT.’, n1.p. 221’ and with 1,2,3,- six saturated rings. 0:1 the basis of the informa-
( 1 ) H. J. Prins, Rec. l r a v . chim.,66, 455 (19.16). (2) J. S. h-ewcomer a n d E. T . McBee, THIS J O U R N A L , 71, 952 (1949). (3) E. E. G i l b e r t and S . L G i < ~ l i t oI!., S P a t e n t 2.GI17.928(19321. (1) E. T. hlcBec. J. D. Idol, J r . , and C .IV. Rolierts, T i n s J O I I I ( N A I . , TT, 4375 (1955).
( 5 ) F. A . Miller in Gilrnan, ’ Organic Chcrnistry, An Advanced Treatise,” Vol. 111, John Wiley a n d Sons, New York, N.Y., P. 1 2 2 fl. ( 6 ) J . D . Idol, J r . , C. W. R o b e r t s a n d E. T. hfcllree, J. O ~ KC. h r i n . . 2 0 , 1743 (19.55). (7) E. T. SIcBee. D . K . S m i t h antl 11, I ? , 1inKn:i‘le. ’l‘lrls J u U K N A L , 77,5,59 (1955).
