COMMUNICATIONS TO THE EDITOR
April 5, 1956
1734, 1688, 1664, 1600 ern.-', (Anal. Calcd. for C18H2203:C, 75.49; H, 7.75. Found: C, 75.22; H, 7.74) was obtained. Alkaline rearrangementI2 of I11 afforded a 176hydroxyestrane-3,6-dione (IX), m.p. 145-146', [ a ] D -114' (methanol), ~2:' 3290, 1715, 1704 ern.-', (Anal. Calcd. for C18H2603: C, 74.44; H, 9.03. Found: C, 75.12; H, 9.23). From the third fraction (20% acetone) was obtained 1.1 g. of 1la-hydroxy-19-nortestosterone (IV), m.p. 167-168°,13 [ a ] -46' ~ (chloroform), XalcohoI mar. 242 mp (15,475), 3345, 1650, 1610 cm.-', (Anal. Calcd. for C18H2603: C, 74.44; H, 9.03. Found: C, 74.37; H, 8.95). On acetylation it gave a diacetate (X), m.p. 190.5-191.5', [a]=-39.6' (chloroform). Oxidation afforded 19noradrenosterone (XI), m.p. 213.5-215', [ a ] +145' (methanol), X:z:O' 240 mp (14,600), 1732, 1698, 1665, 1612 cm.-l, (Anal. Calcd. for C18H2203: C, 75.49; H , 7.75. Found: C, 75.09; H, 7.81). The structure assignment for 11a-hydroxy-19nortestosterone is based upon (1) molecular rotation data, (2) infrared, ultraviolet and chemical properties of I V and its derivatives, and (3) a consideration of the microbiological data obtained from fermentation of a large number of steroids with Rhizopus nigricans. l4 v=,U$'
~2::'
1513
Dowex-1 chloride, the activity of the enzyme is dependent on the addition of a boiled extract of the organism; this can be replaced by a number of folic acid derivatives (Table I). Sagers, et al.,' using a similar preparation, have also reported the activation of this reaction by tetrahydrofolic acid. TABLE I STIMULATION OF FORMIMINOGLYCINE DEGRADATION BY PTERIDINEDERIVATIVES Compound
No addition Boiled extract'
Ptetidine,a pmoles/ml.
Activityb
0 0.024 .24
1.6 3.8 8.4 Folic acid .4 3.0 Tetrahydrofolic acidd .9 5.5 .3 6.2 ~Teropt erin" N-10-Formylfolic acid' .I4 2.9 N-5-Formyltetrahydrofolic acido .06 7.1 Diglutamyl-N-IO-formylfolic acidh .04 6.8 a Calculated from t h e molar extinction coefficient a t t h e absorption maximum in 0.1 N KOH. This was assumed t o be 26,000 a t 260 mp for the boiled extract, which on this basis contained 2.4 pmoles per ml. * @Moles of F I G utilized in 20 min. a t 37" in a system containing 10 pmoles of FIG, 50 pmoles of potassiun phosphate, PH 7.0, 0.5 pmole of NalS, 2 pmoles of ferrous sulfate, 0.4 ml. of enzyme, and the additions shown, in 1 ml. F I G was determined colorimetrically as described elsewhere.* The enzyme was an alumina-ground extract of C. acidi-urici treated with Dowex-1 chloride a t 0' for 15 min. This preparation con(12) E. Ellis and V. A. Petrow, J. Chem. Soc., 1078 (1939). tained 21 mg. of protein per ml. e Prepared by heating 2.5 (13) A higher melting polymorph, m.p. 185-187', was subsequently g. of lyophilized cells of C. acidiurici in 50 ml. of 0.01 M obtained. KPOd, PH 7, 0.02 M cysteine in a boiling water-bath for 5 (1.1) Since the preparation of this M.S., F . B. Colton (U.S. Patent min. Prepared by Dr. T. Miles from purified folic acid 2,729,654) has described the preparation of epimeric lO-hydroxy-19Gift of Dr. H. P. Broquist, nortestosterone v i a the epoxide derived from 17@-hydroxy-5(10)- by catalytic reduction.' Lederle Laboratories, purified by Dr. B. E. Wright. f A estren-3-one Although distinguishing constants for the 10-17 diols sample obtained from t h e Lederle Laboratories and purified are not given, one of the 10-hydroxy-10,17-ketoneshe describes correas previously described.6 This was provided by Dr. M . sponds reasonably well with that derived from the 10.17-diol produced Silverman. 0 A gift of Leucovorin from Dr. H. P. Broquist. microbiologically. A sample isolated from C. cylindrosporum by Dr. B. E. R . L. PEDERSON Wright.E RESEARCH LABS. J. A . CAMPBELL THEUPJOHNCOMPANY J. C. BABCOCK Dowex treated extracts prepared from lyoKALAMAZOO, MICHIGAN S. H. EPPSTEIN H. C. MURRAY philized cells of C. cylindrosporum which had been A. WEINTRAUB stored for over 2 years a t - 10' show an additional R. C. MEEKS requirement for ADP and orthophosphate (Table P. D. MEISTER 11). UDP, CDP, I D P , and G D P (tested a t 2 L. M . REINEKE D. H. PETERSON TABLE I1 RECEIVEDFEBRUARY 23, 1956 REQUIREMENTS OF FORMIMINOGLYCINE UTILIZATION
vztp
ATP' FORMATION ACCOMPANYING FORMIMINOGLYCINE UTILIZATION Sir :
FIG utilized, irmoles/ml.
Formiminoglycine (FIG) is formed from 4aminoimidazole or xanthine by extracts of Clostridium cylindrosporum. Washed cell suspensions of Clostridum acidi-urici convert FIG to acetic acid and carbon dioxide.3 Extracts of this organism or of C. cylindrosporum have now been obtained which carry out the partial reaction in which FIG is converted to glycine, formic acid, and ammonia. When the extracts of C. acidi-urici are treated with
Complete systema 6.1 Omit N-5-formyltetrahydrofolic acid 1.9 Omit A D P 1.7 1.6 Omit pi" ,. The complete system contained, per ml., 50 pmoles of potassium phosphate, p H 7.0, 10 pmoles of FIG, 1 pmole of ferrous sulfate, 0.5 pmole of 2-mercaptoethanol. 0.2 pmole of N-5formyltetrahydrofolic acid, 5 pmoles of ADP, an extract of lyophilized cells of C. cylindrosporum equivalent t o 6 mg. of protein, prepared in maleate buffer and treated with Dowex-1 chloride. Tubes were incubated a t 37" for 30 min. * T h e phosphate was replaced by 25 pmoles of maleate buffer, p H 6.8, which showed no inhibition.
(1) The following abbreviations have been used: A T P , adenosine triphosphate; A D P , adenosine diphosphate; CDP. cytidine diphosphate; IDP, inosine diphosphate; GDP. guanosine diphosphate; UDP, uridine diphosphate; G-6-P, glucose 6-phosphate. (2) J. C. Rabinowitz and W. R . Pricer, Jr., in preparation. (3) J. C. Rabinowitz and W E Pricer, Jr., Federation Proc., 16, in press (1956).
(4) H. P. Broquist, M. J. Fahrenbach. J. A. Brockman, Jr.. E . I.. R. Stokstad and T. H. Jukes, THISJ O U R N A L , 73, 3535 (1951). (5) M. Silverman, J. C. Keresztesy and G. J. Koval, J . B i d . Chcm., a i l , 53 (1954). (6) B. E. Wright, i b i d . , in press. (7) R . D. Sagers, J. V. Beck, W. Gruber and I. C. Gunsalus, TEIS JOURNAL, 78, 694 (1956).
COMMUNICATIONS TO THE EDITOR
1514
pmoles per nil.) did not replace ADP. A T P ( a t 5 pmoles per ml.) was completely inactive, unless added together with hexokinase, glucose, and LfgCln, which generated A D P from the ATP. These observations suggested that ADP was acting as a phosphate acceptor for a phosphorylation accompanying the con\-ersion of F I G to glycine. partially purified fraction of C. cylindrosporum was prepared by acetone fractionation. Using this preparation, the conversion of F I G to glycine and formic acid is accompanied by the formation of an equivalent amount of -4TP (Table 111). The reaction may be summarized b!- the equation : FIG ADP P, + glycine HCOOH SHs ATP. This reaction with the purified fraction, which had not been treated with Dowex, is not stimulated by the addition of N-5-formyltetrahydrofolic acid (Leucovorinj ; however, the amount of A T P formed was doubled by the addition of a boiled extract of C. ncidi-urici, which may contain the true coenzyme forin of folic acid." The activity of the purified fraction is completely dependent on the addition of .IDP.
+ +
+
+
+
Vol. 78
reaction is not known, formylglycine is not an intermediate since it is not converted to glycine by these extracts. Subsequent steps in the conversion of glycine to acetic acid may also provide energy for growth, and the mechanism of these reactions is under investigation.14 (11) I s o t o p e experiments' i n d i c a t e q u a l i t a t i v e di5erences with the conversion of glycine t o a c e t a t e r e p o r t e d by T. C. S t a d t m a n in C l o s t r i dium s t i c k l o n d i (personal c o m m u n i c a t i o n ) ,
XATIONAL ISSTITUTEOF ARTHRITIS METABOLIC DISEASES XATIONAI. INSTITUTESOF HEALTH
AND
JESSE
C. RABINOIVITZ
\V. E. PRICER, JR.
U S I T E D STATES P U B L I C H E A L T H S E R V I C E BETIIESDA. LIARYLAND
RECEIVED MARCH 5 , 1956 T H E REACTION OF CAMPHENE WITH HYDROGEN CYANIDE
Sir:
On treating racemic camphene with hydrogen cyanide under strongly acidic conditions differing somewhat from those reported by Iiittcr, wc were surprised t o obtain 3-formarnidoisocamphanc (1, 1n.p. 173-176') instead of the expected K-formylisoTABLE I11 bornyl'amine. Use of excess cyanide and :L remSTOICHIOMETRY I S FORMIMISOGLYCINE DEGRADATION tion temperature below j r -resulted in a high yicld of the new compound while alkaline hydrolysis of thr The incubation mixture contained 250 pinoles of potassium phosphate, pH 7.0, 25 pmoles of .\DP (Sigma, sotliuin salt), mother liquors followed by careful isolation yieltletl 2.5 mg. of hexokinase (Pahst), 123 prnolcs of glucose, 51, only traces of isoborny1:iniiiie. pmoles of MgCI?, 10 pmoles of ferrous sulfate, 30 prtioles of Compound I is isomeric with the expccted forin2-mereaptoethanol, 1 . 0 inl. of a boiled extract oi C. n c i d i ylisobornylamine, ,-I m i . Calcri. for CI1H1 &,SO: urici (see Table I), 50 pmoles of FIG, 0.5 mi. of an :icetone precipitate of an extract of C. cylindrospo~ziii!containing C, 7 2 . S 6 ; 10.56: S , 7.73. Found: C, T3.0ti; ,XO mg. of protein in a total volume of 5.0 ml. T h c mixture H,10.29; S , i . 4 2 . I t was fouiitl hoiiiogenetius was incubated a t 37" for the time indicatrd. (purity over 9.Sp;j by solubility analysis,' arid w:is FIGn Glycineb IICOOHC A'TPd Xilie saponified i n high yield to an aiiiine (IT),m . p 175.Time utilized formed, formed, formed. filrmed, IC min. pmoles/ml. pmoles / m l . pmoles,'ml. iimoles /nil. pmole/ml. I I 0 , which was analyzed as the hyc:rochloridr; 15 3.9 4.0 4.3 40 .. .4na/. Cnlcil. for ClaH2,XCl: C, G3.30: H, 1O.M; 30 5,6 5.0 C .7 5 .8 .. S , 7.38. Found: C,OR.T,!); H , IO.T!1; N ,7.01;. 60 8.1 7.8 9 6 8.2 7 9 Comparison of the infrared spectra :mtl other pliysical constants of both the amine hydrocliloritie :inti a Determined by a colorimetric procedure described elsewhere.? * Determined by a modification? of the method of formyl derivative with the corresponding tlcriraAlexander, et a1.8 The values h a r e beeti corrected for the tives of authentic bornyl :ind isol,orii!-laiiiiIic.s rcDeblank which contained 3.1pmoles per ml. of glycine. termined manometrically with formic l~ydrogeiilyase.~ vcaled Iiiarketf differences. Oxidation of the amine with potassium perinanThe values have been corrected for t h e blank which contained 3.0 pmoles per ml. of formic acid. .\TP v ; t s de- ganate rtsulted in :I small ainount of nitro C O I ~ I termined 3 5 the G - 6 - P formed through the action of liexo- pound, ti1.p. 19s"; . I t i d . C:ilcd. for C l ~ , H ~ : K ( b : kinase (:-6-P w3s measured using Z : ~ i s c h e ~ I ~ ~ i / prei c n r C, G . . j l ; H , !). 11; N , 7.6-l. Fouii(1: C ,(i--i.,j+i; pared by t h e method of Kornberg.IG These values h a v e been corrected for the G-6-P forrned i n a control tubc iron1 H , 9 . l ~ i : S , 7.43, which could lie hytlrcigenaletl omitted 'The :i:nount i i ~ m i c dhi thik con- back to starting ~riaterial. 1Tith this iiit!ic:itiiiIl oi mately 2 to :3 pmolci per ml. mid was du\- to tertiarl; carhinarnitie structure,3 11 \vas teritati\.cl>deny1:itc kinase e Dctermiried b y n c 4 c r i i.c., tho l i i i i zation after adsorption on S E 64 K'nnd elatiriii \\it11 0 2 .Y idcntifictl ;is 3-:~iiiinoiscic:!iripli:~iic, m n g r d structure rcsuitiiig iroiii :icltlitioii o f liytlroSaOII. Ken cy:initlc t o carriphcnc. The steps leading to the formation of FIG from SHR xanthine which hxre been previously tleinoii. ' ' ; + Lc 11~, I , I1 = C I I O stratetl?~'"'"appear to be hydrolytic i i i nature, 11, I< = 11 and thus are not likcly to provide energy for the 111, I< ClTr growth of thc org:mis:ii. The ])resent rc:ictioI:, which produces -ITP, provides such :I source of c n e r p . \\'bile the (Ivt:iile(l nicchanisiii of this \f-hilc racemic :S-alilinoisoc3IiipI-lnnc is iiiircportctl, F1iici;c.l nntl Sertlel p r e p r c d a11 optically ( 8 ) 73 -\lexnndcr, C, r.Lnr!ni.i7r 3mi .I 11 Selix.rn,in J 160, 31 (1!4&:), 3ctiL-t. isoiiicr froiii ti-carnphcrie hydrcdiloritic l i ? reaction with silver nitrite folloncc! s, ~ 1 i u 1 1an(! i 1 -
2
Prrss, l3aliimorr. 19.iL'. >I
,522
(3) 5.. K ~ j r n l , l t > m a n d I< J C l i i t t e r , THISJ O U R N A L , 76, 4.1'41 -11 \I' I I u c L t I ,tmI I , A t r ~ l ~. ?~) I l; ,> , 5 2 8 , 37 ( l X 3 7 : .
(l!l:4,.
