June 5 , 19SS
3167
COMMUNICATIONS TO THE EDITOR
with one mole of bromine and the resulting bromo- are irradiated with y-rays from cobalt-60 a t a dose ketone was dehydrohalogenated via semicarbazone rate of 2 x lo5 r./hr.l Ethanol and acetic acid formation. Reversal5 of the semicarbazone with are also produced later in the reaction with much pyruvic acid in aqueous acetic acid gave in addition smaller G values (Fig. 1). to (I) and (11) the ketone (111) [m.p.6 ca. 237'; [ a ]+34.9' ~ (acetone) 222 mp (log E 4.03); Anujol max 2.77, 2.98 p (OH), 5.73, 5.78 p (acetylated side chain), 6.05 p (unsaturated ketone) ; Found: C, 65.66; H, 7.051. (111) possessed about 60% of the activity4 of hydrocortisone acetate by the one-day oral mouse liver glycogen assay. Bromination of (11) with two moles of bromine' followed by dehydrohalogenation with collidine afforded the dienone (IV) [m.p. ca. 237"; [ Q ] D +100.9' (acetone); X ~ ~ ~239 o HmF (log E 4.19); I (2 hr.) 310 mp (4.06), 262.5 mp (4.18); A='': - -. 0L 2.92, 3.02 I.( (OH), 5.74, 5.82 p (acetylated side 2 3 4 5 678910 20 40 60 80 chain), 6.0 p (unsaturated ketone), 6.12, 6.21 p (di30 50 70 90 ene system); Found: C, 65.66; H, 6.741 and the Irradiation time, hours. isomeric dienone (V) [m.p. ca. 20806; [ a ] D +lO6" (acetone), 281 rnp (log E 4.40), 2.85, Fig. 1.-G values for ethanol, acetic acid and acetaldehyde production as a function of radiation dose. 3.06 p (OH), 5.70, 5.81 p (acetylated side chain), 6.12, 6.18 p (conjugated dienone system); Found: Hydrogen peroxide is formed in water equiliC, 65.86; H, 6.991. 1-Dehydro-9a-fluorohydro- brated with ethylene-oxygen mixtures under atmoscortisone acetate (IV) possessed about 25 times the pheric pressure a t an initial rate corresponding to a activity of hydrocortisone acetate in the mouse G of 20 (*Energy dissipitation in the solution was liver glycogen assay and in the rat systemic granu- calculated from the rate of oxidation of ferrous ion loma inhibition test.4 I t is, therefore, the most in the Fricke dosimeter using a G value of 15.5). potent glucocorticoid known. It is of interest to These G values for hydrogen peroxide production note that enhanced glucocorticoid activity was increase with increasing gas pressure, rising to an reported recently* for 1-dehydtocortisone and 1- order of magnitude comparable to the aldehyde dehydrohydrocortisone acetate, which possess the values a t 120 p.s.i. same chromophoric system as (IV). These G values contrast with those obtained with CHZOAC most organic materials in aqueous solution, which ,I are an order of magnitude lower (Weiss2). Deco I, Double bond between c4-C~. tailed studies to elucidate the nature of the processes leading to this chain utilization of oxygen 11, H a t C5 formulated as "cy" 111, Double bond between CI-C~; are in progress. ~
H a t C5 formulated as "0". IV, Double bonds between C1-G and CrC5. V,Double bonds between C4-C; and C6-Ci.
(5) W. F. McGuckin and E. C. Kendall, THIS J O U R N A L , 74, 5811 (1952). (H) Taken on a micro hot-stage m.p. apparatus. (7) See f o r instance C. Djerassi and C. R. Scholz, THISJOURNAL, 69, 2404 (1947). (8) H . L. Herzog, A. Nobile, S. Tolksdorf, W . Charney, E. B. Hershberg, P. I.. Pearlman and M . M. Pechet, Science, 121, 176 (1955).
,
(1) E . J. Henley, Nucleonics, 11, no. 10,41-43 (1953). (2) J. Weiss, Chem. & I n d , 18,358-9 (1955).
DEPARTMENT O F CHEMICAL ENGINEERING ERNESTJ. HENLEY COLUMBIA UNIVERSITY NEW YORK27, N. Y. JANE P. SCHWARTZ RECEIVED APRIL27, 1955 A NEW PTERIDINE I N URINE REQUIRED FOR T H E GROWTH O F T H E PROTOZOON
CRITHIDIA FASCICULATA
Sir:
R. F. HIRSCHMANN Work carried out jointly in these Laboratories R. MILLER R. E. BEYLER and the Haskins Laboratories1 established that the L. H. SARETT Trypanosomid flagellate Crithidia fasciculata (HerM . TISHLER petomonas culicidarum) could be grown in a chemiRECEIVED MAY7, 195j cally-defined medium in the presence of a "high"
MERCK& Co., INC. CHEMICAL DIVISION RAHWAY, N J .
EFFECT O F IONIZING RADIATION ON AQUEOUS ETHYLENE OXYGEN SOLUTIONS
Sir: In the course of our studies on the effects of ionizing radiations on hydrocarbons in aqueous solution we have noted oxidation of ethylene along with hydrolysis to ethanol. This oxidation leads to a production of acetaldehyde with G values as high as 60 when solutions equilibrated with 1-1 ethyleneoxygen mixtures under a pressure of 120 p s i .
amount of pteroylglutamic acid (PGA), 1 ylassay tube. It was then found that the amount of PGA required for growth of C. fasciculata was markedly spared by a variety of natural materials including certain liver fractions and human urine (adult males), or by certain 2-amino-4-hydroxy-6substitued pteridines. These relationships are illustrated in Table I . By procedures of adsorption and solvent distribu(1) J . Cowperthwaite, M. M. Weber, L. Packer and S. H . Hutner. Ann. N . Y.Acad. Sci., 6 6 , 9 7 2 (1953).
3168
C'OMXZUNICATIONS TO TIIE
EDITOR
L'ol. 77
tion about 20 Ing. of a solid was isolated from 4000 clines, a molecular weight of 282 was calculated liters of urine that supported half-maximum growth from the ultraviolet absorption. The infrared o f C. f(tcsc-icdntn at a concentration o f 0.03 milli- spectrum measured on a KRr pellet showed strong ~iiic~ograiiis per 1111. T h e 1 i ; i n i c I.)iopterin is sug- bands at X50-~;3~4X, 3 3 10, 2975-29.30, 1695-1680, gested for this substance. The material precipi- 1538, 1490, 1418, 1370, 1295, 121.5, 1173, 1130, tated as pale yellow spheres from hot water and 1063 and 823 cm. -- I . decomposed without melting at 250-2SOO. Tt was Riopteriti titrated with periodate consumed 1.7 osidation equivalents per molecular weight optically active, [ c Y ] ~ ~- D3 0 (0.1 S HCl; C, 0.4). and l.,i I t was insoluble in the conirnori organic solvents, of Z37 at PH 3 and 8.5, respectively. Formaldeslightly soluble in water, soluble in dilute acid and hyde, formic acid and ammonia were not detected alkali. Analysis calculated for C S H ~ ~:S C ~, O ~in the oxidation mixtures. Upon adjusting the -I.i.(i; H, 4.64; ?;, 29.5; mol. wt., 237. Found on oxidation mixtures to pH 5 , yellow precipitates two separate samples, C, 41.8,4 i . G ; H, 5.00, 4.S1; came down, and the ultraviolet absorption spectra of these indicated that the material from the pH N,29.:3; S, negative. The ultraviolet absorption spectrum of biopterin 2 osidatjon was 2-amino-4-hydroxy-6-formylpteriw a s typical of 3-aniino-4-hydroxy-alkylpteridinesdine and the material from the pH 8.5 oxidation Thus with maxima a t 2.54 mp and 305 mfi in 0.1 -Y was 2-amin0-4-hydroxy-6-carboxypteridine. NaOH. Assuming biopterin has the same molec- biopterin appears to consume about two inore periodate oxidation equivalents in alkali h y the ular extinction as 2-atnino-4-hydroxy-alkylpteripteridine aldehyde being oxidized to the correTABLE I sponding acid. From these data atid studies on THE EFFECT O F PGA O N THE GROWTHRESPOXSEOF models it is concluded the structure of biopterin is Crilhidinf~isrirzilntnTO CRINE OR TO CERTAIN 6-SUBSTITTTED 2-ainino-4-hydrox?-~(1 ,?-dihydroxypropyl)-pteridPTERIDISES ine. PO.< Additions t o medium-- -The microbiological response to biopterin is 10 100 1000 now obtained in the absence of added PG-k3 Additions per tube (2.a ml.) Sone m; my m-r -_ final strength mediiim" ~. .. . Optical Density The involvement of PGA4in the metabolism of C. 0.02 0 03 0 11 0 88 .fasciculnzta was demonstrated indirectly, however, .\one 0.001 ml. urine o n2 o 0-1 by adding an inhibitory amount of 2,4-diamino-50.01 mi. urine 00,j 0.98 p-chloro-phenoxy-A-ethylpyrimidine (I) to the cul10.7 0 1 nil. uritie ture medium. Under these conditions growth was obtained by adding sufficient PGA to counteract I only if biopterin was present (Table 2). The data of Tables I and I1 illustrate that C. fusciculata required both PGA and a pteridine for growth, implying that these structurally-related substances OH have independent metabolic functions for this R = CH2011" 1 0 in; 0 09 0.25 0.65 0.87 organism. IOOin-; n.07 0.58 0.81 0.88 We are indebted to Mrs. L. H . Smith for some of Test conditions were those described by Xathan and the microbiological assays and to Dr. S. H. Hutner, Cowperthwaite2 except that the adenosine was replaced by Haskins Laboratories, New York, N. 1 ' . for his 10Oy adenine, guanine and uracil per tube and the assay was carried out in slanted test tubes rather than flasks. suggestions concerning the culturing of the test Microbiological findings with other pteridines tested with organism.
-
~
. I
10 m y PGA in the medium were as follows: When R = H , OH or COOH, no activity a t l0y/tube, when R = CHO or CHs, 0.3 to 0.1 as much activity as when R = CHlOH (see table). 2-Amino-4-hydroxy-5formyl-A-methyltetrahydropteridine, the methylpteridine moiety of leucovorin, was inactive a t 10yltube.
TABLE I1 TOXICIT'i PYRIMIDIXE
Rir~pterin
. . . . 10 m:
I00 In., ...
..
100 m y 100m?
OF
2,4-DIAMIS@-~~-~-CHl,oROPHESOXY-6-ETHYL-
GROWTH OF Crifhidici REVERSAL BY PGA"
FOR
PGA
10 m7 100 m: 10 in-, 100mr
0 03 .4:3 .46
0.02 .02
.n:3
.. .. .49
.09 .49 .iA
.oo
0.01
0.03
.. ..
..
.. .. .04 .38
.. ..
.. ..
.03 a Tert conditions :is i n footnote t o T:il,le 1 ; 2.,5 ml. final volume .4j
-.
( 2 ) H. A. Nathan M p d . , 86, I I7 ( I 9 X )
xiid
.I
NUTRITION A K D PHYSIOLOGY SECTION E. L. PATTERSOX H. P . BROQUIST RESEARCH DIVISION ALBERTA M. ALBRECHT M. H . VON SALTZA LEDERLELABORATORIES E. L. R. STOKSTAD PEARL RIVER,S E T V Y O R K RECEIVED MARCH24, 1955
COMPANY ~ a s c i c u . l n t n : AMERICAS CYASAMID
2,4-Diamino-j-p-chloropheno~y-6-ethylpyrimidine (I) 0 l Y 3.7 10 7 Optical Density
... .... ....
(3) Within t h e past year the test organism apparently "lost" its nutritional requirement for preformed folic acid. Factors responsible for this change are unknown. (4) ADnExDGM.-After this Communication was submitted for publication, t h e Editors informed us t h a t Forrest and Mitchell have isolated and characterized what appears t o be t h e same pteridine from wild type Drosophila.
Ci,n.iiprtIiwiiitc, P r o r . .Snr. E.rpi!.
Bioi.
BOND ORDER-LENGTH
RELATIONSHIPS
Sir: The importance of establishing reliable bond order-length relationships for as many atom-pairs as possible is generally recognized. There are several ways of defining bond order (w). Examples are the original method suggested by Pauling, Brockway and Beach, and the more recent method of molecular orbitals. Bond order length curves have been usually constructed by plotting
