COMMUNICATIONS TO THE EDITOR
Sept. 5 , 1057
4809
with metallic lithium4 thereby causing reduction of Such a square link is assumed in Eq. (1: to conboth the triple bond and the aromatic A ring. Hy- stitute one cross link as well as one square link. drolysis with mineral acid followed by chromatog- By "ring" links we mean intramolecular bonds raphy yielded 3-(3-oxo-l7~-hydroxy-19-nor-4-anformed by reactions such as drosten-17a-y1)propionic acid y-lactone (V), m.p. -CH~CH~CIX~CH~&H ---f ~126.5-127" (137-138'), [ a ] D +22.7' (CHC13) ; -CHCHzCHzCHzCH+ H P (3) Anal. Calcd. for C2,H=03: C, 76.79; H, 8.59. Found: C, 76.49; H, 5.34. (E represents the electron). Equation ( 3 ) is -,=O similar to a reaction proposed earlier.3 Other ring forming reactions could be proposed. Cross links and vinylene groups formed by irradiation have been recognized since 19,50,3but ring or square links have never before been observed in the irradiation of any substance :as far as we know. It is the purpose of this nota to present evidence for cyclization during irradiation. 1,2-Disubstituted cyclopentane and cyclohexane Bioassay5-reversal of the effect of desoxycorticosterone (DOC) on the urinary Na-K ratio of derivatives absorb4 in the infrared approximately adrenalectomized rats was used as a measure of a t wave lengths of 10.2 p with extinction coeffipotency. If progesterone, recently described as cients varying between 6 and 25 1. cm-I. mole-'. capable of reversing the effect of DOC,6is assigned In Marlex-50 the initial absorption a t 10.0 and an activity of 1, then I11 and V were found to have 10.98 p due to the vinyl group rapidly falls with iran activity of approximately S and 27,respectively. radiation a t room temperature and in ZJUCUO(initial In similar tests 111 and V also reversed the elec- G(-vinyl) equal to 7.7), revealing the gmwth of another absorption a t 10.1p which is approximately trolyte effects of aldosterone. the wave length a t which trans 1,2-disubstituted (4) A. L. Wilds and N. A. Nelson, THISJOURNAL, '75, 5360 (1953). cyclo pentanes and hexanes absorb. We have at( 5 ) T h e details of bioassay will be reported elsewhere. tributed the growth of this peak to the formation (6) R . L. Landau, D . M Bergenstal, K. Lugibihl, and M. E. Kaschl, J. Clin. Endocrind. and Metabolism, 15, 1194 (1955). of ring links of this size (other ring or squa.re links which might absorb a t this wave length are not exDIVISIOSSOF CHEMICAL AXD BIOLOGICAL RESEARCH G. D. SEARLE & Co. J. A. CELLA cluded). A similar growth in the peak at: 10.1 CHIC.4G0, ILLISOIS C. M. KAGAWA was found also in the case of a Ziegler-type polyRECEIVED J U L Y 24, 1957 ethylene. An order of magnitude estimate of G (trans ring links) is 1.0 which is of sufficient magnitude to show the necessity of considering cyclization in atCYCLIZATION IN GAMMA RAY IRRADIATED H I G H tempting to achieve a material balance. DENSITY POLYETHYLENE I n the case of a low density polyethylene, the Sir: vinylene peak a t 10.3 p is broader than in the case Material balance in the irradiation of poly- of Marlex-50 and possibly obscures the peak: due to ethylene has never been convincingly attained, es- the ring systems. The 10.3 1peak is unsyrnmetripecially a t liquid nitrogen temperatures where cal, however, which suggests that ring links are G(H2) and G(-CH=CH-) are essentially equal produced in low as well as high density polyto their values a t room temperature while cross link ethylenes. This research was supported by a grant fsom the formation is sharply reduced.' As adapted to the high density polyethylene, U S . Atomic Energy Commission. Marlex-50, and considering no chain scission, the (3) hi. Dole, Report of Symposium IV, "Chemistry a n 3 Physics of Radiation Dosimetry," Army Chemical Center. Md., Saptemher, material balance equation can be written 1950. [Hz] [Vi0 - Vi] = I/z[RI [Vl] (4) "Catalog of Infrared Spectral Data" of the American Petroleum
+
+
[C.L.]
+ + + [E.L.] + [S.L.] + [R.L.]
(1)
where brackets signify moles per gram of substances produced during the irradiation except for Via - Vi which represents moles/g. of vinyl groups destroyed. R., V1, C.L., E.L., S.L. and R.L. signify free radicals, vinylene groups, cross, end, square and ring links, respectively. By "square" links we mean double cross links formed by a reaction such as ?-CH=C€I-
+-CH-CHI
-CH-CH-
Institute Research Project, Carnegie Institute of Technolosy. Pittsburgh, Pennsylvania. ( 5 ) Fulbright Fellow, 1956. (6) On leave from A.E.R.E., Harwell, England,
DEPARTMEST OF CHEMISTRY MALCOLM DOLE NORTHWESTERN UNIVERSITY D. C. M I L N E R ~ EVANSTON, ILLISOIS T. F. W1:LI.IAMSe RECEIVED JUSE 17, 1957
(2)
1
(1) A. Charlesby and W. H. T . Davison, Chem. and I n d . , 232 (1957). ( 2 ) A. Charlesby has proposed the term "end link," Proc. R o y . Soc., A2S1, 521 (1955).
ENZYMATIC OXYGEN FIXATION INTO ACETATE CONCOMITANT W I T H T H E ENZYMATIC DECARBOXYLATION OF L-LACTATE
Sir: The enzymatic incorporation of molecular oxygen into various organic substrates recently has been
reported from several laboratories. These “oxygenases”2 are generally concerned with the transformation (hydroxylation, ring cleavage, cyclization) of various ring compounds such as aromatic amino acids, phenols and steroids. I n this communication we wish to report a novel type of decarboxylation reaction in which atmospheric oxygen is incorporated into a simple aliphatic compound. L-Lactate was incubated with a crystalline lactic oxidative decarboxylase prepared from JIycobacterium phlei3 in an atmosphere uf 0 2 l s and with H2016 as a medium. Potassium acetate isolated from the incubation mixture was found to have incorporated approximately one atom of atmospheric oxygen (Table I). The OI8 enrichment of the incorporated oxygen atom corresponded to approximately 82y0of that of the atmospheric oxygen used in this experiment, assuming only one atom was inserted into the product. On the other hand, when the reaction was carried out in the medium of H201S and with O2I6 as a gas phase, potassium acetate isolated did not contain appreciable Ols. Carbon dioxide did not contain 0 l s in the first TABLE I ESZIWATIC IXCORPORATIOS ore 0 ’ 8 m r o ACETATE Expcriment I : Crystalline lactic oxidative decarboxylase3 (3!350 units), 2 millimoles of DL-lithium lactate and 2 inillimoles of potassium phosphate buffer ( p H 6.0) were incubated in a total volume of 50 ml. of v a t e r in a special flask designed for this type of experiment.a An 0218-helium gas mixture ( 2 : 3 ) was used as a gas phase.* Experiment 11: T h e same reaction components as in Experiment I were cmployed except t h a t HzOi8 was used as a solvent and 0 2 1 6 helium gas mixture ( 2 : 3 ) was used as a gas phase. The incubation was carried out a t 37” for 30 minutes with vigorous shaking. After the oxygen and carbon dioxide xere removed for mass spectrometric analyses, the incubation mixture was immediately chilled a t 4’ and pH was adjusted to 3.5 with several drops of 2?; H z S O ~ . T h e solution was then frozen and water and acetic acid were distilled from the frozen state. The distillate was neutralized with 1iY KOH t o p H 6.5 aud water n-asremoved by lyophilization. The residue dried over P2Oj was pyrolyzed“ and the 0 ’ 6 content determined with a mass spectrometer.d Bxpt.
Type
Atom Yo excess
Cpd. ;inalyzed
case, but was highly enriched in the second experiment. This incorporation of 0ls from HzOI8into carbon dioxide probably was caused by a nonenzymatic exchange reaction as reported by Cohn and Urey?and more recently by Rothberg and Stein-berg in their studies of various microbial decarboxyla3es.j The enzyme which catalyzes “oxidative” decarboxylation of L-lactate has been described froin various microorganisms.6 The over-all reaction involves oxidation and decarboxylation of L-lactate to form stoichiometric quantities of acetate and carbon dioxide, but the mechanism of this unique enzymatic reaction has not yet been completely understood. The available evidence suggests that a t least one atom of atmospheric oxygen was incorporated into acetate when the C1-C2 bond of lactate was cleaved and that the other atom of the oxygen molecule probably was reduced to water CH-CHOH-COOH
+
09’8
--+ CHjC00”H
+ CO? -t I-IrO“
The mechanism of this reaction may be analogous to that of the so-called “mixed function oxygenase”lFJ except that instead of reduced pyridine nucleotides, the substrate itself is providing elcctrons to reduce one atom of oxygen. (4) R I . Cohn and H. C . Urey, T a m J o c ~ i i a ~60, . , 679 (1W8). ( 5 ) S. Rothberg and D. Steinberg, ibid., 1 9 , 3274 (10.571. ( 6 ) (a) S . L. Edson, Biochem. J , 4 1 , 143 (19-17); (b) S. I,. 1311sull and F. B. Cousins, S a t w e , 171, 702 (10Z3): ( c ) Y.Yamamura, XI. Kusunose a n d E. Kusunose, z 6 i d . , 170, 207 (1992); J . Biochein , J Q p Q l Z ,39, 227 (1932); (d) W B . Sutttlvr, F r d i ’ i n r , 1 6 , c t niopliys. A r l i i , 2 0 , 2fi9 (I%;(\); 302 (l!15(!); ( c ) l i , 1;. l k c r i , J I , ,A‘,!otr(, 111, 71, ( 2 ) I , A fIvl,pc!, 1’ 1 O r t i / : ~ 1 1 , 1 S ~ I ~ ~ I i\u i. ,
