2025
COMMUNICATIONS TO THE EDITOR
April 20, 1959
Reaction of I with potassium t-butoxide slurried in cyclohexene gave a 29% yield of allene (strong absorption at 2005 and 1995 cm.-l). After distillation and chromatography on Florisil the product was still slightly impure. Found: C, 87.80; H, 11.32. By analogy with I1 structure VI1 would be assigned t o the allene. VI1
Experiments with olefin mixtures are in progress t o determine the reactivity of t h e intermediate as compared with CBr2,*CC12,9and CHCOOEt.lo
crude coenzyme preparation led t o an enhanced formation of 11. In addition, it was observed (Table I) t h a t the specific radioactivity of I1 formed de novo by the extract from A . gossypii is approximately the same as t h a t of added 6,7-dimethyl-Sribityllumazine-2-C14, indicating t h a t t h e added I is converted directly t o 11, and does not merely stimulate the transformation of another compound in the crude extract t o the vitamin. TABLE I THEENZYMATIC INCORPORATIOS OF
RIBOFLAVIS All reaction vessels contained 40 prnoles of sodium pyruvate, 40 pmoles of .-\TP, 10 mg. uf crude coenzymes (.\rinour), particles a n d supernatant from centrifuged A , gossypii sonicate, volume adjusted tu 3.1 1111. with buffer mixturc :it PH 6.9. Incubated in air in \Vnrburg flasks with slinking DEPARTMEKT OF CHEMISTRY OF MICHIGAN H. D. HARTZLERfor 14.5 hours a t 30". Riboflavin was separated from 6,;UNIVERSITY ditnethyl-8-ribit)lluniazine a n d purified by column and A N NARBOR,MICH. paper chromatography to constant specific RECEIVEDFEBRUARY 9, 1957 Riboflavin was estimated by measurement o f the light absorption in 0.l.V S a O H a t 450 mp, 6 , 7 - D I M E T H Y L - 8 - R I B I T Y L L ~ M A Z 1 S E - ' 7 - c 1ISTO ~
( 8 ) P . S. Skell and A. Y . Garner, THISJ O U R N A L ,78, 5430 (1956). (9) W. van E. Doering and W. A . Henderson, Jr., i b i d . , 80, 5274 (1958). (IO) P. S . Skell and R . M ,E t t e r , Chcm. a n d Ind.,621 (1958).
T H E CONVERSION OF 6,7-DIMETHYL-&RIBITYLLUMAZINE (6,7-DIMETHYL-&RIBITYL-2,4[ 1H,3H]PTERIDINEDIONE) T O RIBOFLAVIN BY EXTRACTS
OF 'ASHBYA GOSSYPII
Sir: In pievious studies i t was shown t h a t the addition of formate-C14 or other known labeled precursors of riboflavin' t o intact cells of Ashbya gossypii led t o a specific radioactivity in isolated 6,7-dimet hyl-8-ribityllumazine (6,7-dimet hyl-8ribityl-2,4 [lH,3H]pteridinedione) (I) higher than t h a t of riboflavin (11) in the early time periods of incubation.2 These findings suggest t h a t I is not a biological degradation product of 11, b u t is a probable intermediate on t h e pathway of biosynthesis of t h e vitamin. The demonstration of t h e biological conversion of I t o I1 would provide more direct evidence t h a t this substance is on t h e path of biogenesis of riboflavin from simpler precursot compounds. Attempts to obtain riboflavin synthesis from I with washed intact cell suspensions of A . gossypii were unsuccessful. This is due presumably t o t h e inability of the compound to penetrate the cells. That a permeability barrier may indeed be responsible for the lack of success in these experiments is suggested by the observation that incubation of washed cells of -4.gossypii with added riboflavin2-CI4 for 24 hours under aerobic conditions did not lead to incorporation of radioactivity into intracellular I I.
R I
k
I1
R = Ribityl However, addition of I to a reaction mixture containing cell-free extracts of d4, gossypii in the presence of pyruvate, adenosine triphosphate, and a (1) G . W. E. Plaut, 1.B i d . C h e m . , ¶OS, 513 (1954). (2) G. F. Maley and G. W. E . Plaut, Federation Proc., 17,268 (1958); J . B i d . Chcm., 934, 641 (1959). (3) C. F, Mnley and C , W. E,P l n u t , unpubliebed obsorvntiom,
Control
Experimental
None 0.174 0.513 0.039
...
1 97 0.1R9 0.613 0.124 7590
...
GlOOO
.
ri7'7011
Compound added or isolated
G,'i-Diniet2z~l-S-ribityllum~zirle, arltlecl, pmoles Riboflavin, initial, pmoles 14.5 hours, pmoles Increase, pmoles Total radioactivity, c . p . n i . formed, c.p.m./pinole 6,T-Dimethyl-8-ribit~llurnnzi11c, ndtletl. c.p.m./pmole
, ,
The conversion of I t o I1 can be visualized t o occur by way of the addition of two two-carbon compounds, e.g., acetyl-Co-I, or possibly a fourcarbon fragment (e.g., acetoin or diacetyl) t o the methyl groups of the lumazine derivative. Such a mechanism would be consistent with the pattern of labeling of the aromatic ring of riboflavin observed in experiments with intact cells of A . gossypii.4 In view of its structural relationship to the pterins it is possible that I could be a precursor of other pterins, e.g., folic acid, as well as of the flavins. (4) G. W. E. Plaut, J. B i d C/ie,?r.,211, I l l (1954). ( 5 ) Fellow of t h e American Heart Association. (6) Senior Research Fellow U.S.P.H S. (SF261). These studies were aided b y fund from t h e Williams IVaterman Fund and the National Institutes of Health (H3891).
SEW YORKDEPARTMENT OF HEALTH XLBASY,SEW TORK GLADYS F. h1.4LEY6 G . \V, E. P L A U T ~ LABORATORY FOR T H E S T U D Y O F HEREDITARY A N D h f E T A R O L I C DISORDERS USIVERSITY O F UTAH COLLEGE OF hfEDICISC SALTLAKECITY,UTAH RECEIVED FEBRUARY 21, 1959
NEW REACTION OF RECOIL HYDROGEN ATOMS WITH ALKENES'
Sir: Studies of the reaction of recoil tritium atoms, such as produced by the He3(n,p)T process, with gaseous alkanes, have shown t h a t "hot" hydrogen atoms can react efficiently by a simple displacement t o form H T and the labeled form of t h e (1) Work oupportsd by United Statan Atoplio Energy Commission.
TABLE I RELATIVE YIELDSOF RADIOACTWE PRODUCTS (NORMALIZED TO 100 FOR ACTIVITY I N P A R E T T I I Y D R O C A R B O N ) Molccules produced by mechanism ticscribed are boldface. 3Iytlrocnrl~oris:Ire rcpresrntcrl by :ipl)ropriate symbols: tlius butane; /=\ cis-butrnc, etc.
/v
Am
+
Tritiated products
_-___
HP CIL / // A A A/ >1 L Y . 1~ / - ‘/~ >.~~ //A A/“a A=/ /W 143 13 3 41 0 41 31 0 lO(1 7 4 0 0 0 n /\/ 171 11 1 4 o 60 46 o 12 io0 1:; n o (i /”\ 191 15 I 5 o 69 58 o 14 1.7 ino n n n (I 116 9 0 0 0 21 0 0 10 0 0 IOC 0 0 0 /V\ 1T8 D 0 33 0 42 0 0 4 0 0 0 21 101) 0 +A=/ 184 1.3 3 0 0 23 0 0 22 2 r, 100 n n so /V 235 15 15 5 6 1 o o n o O 0 (1 o 100 500 14 o 50 0 75 44 o 101)“ /W He(l:50) 8 o 8 o n IO 160 .i o 01 ( 5 0 : l ) n 46 n 49 o loo 4 0 0 4 0 0 Yield of butene-1 activity in this run reduced by order of magnitude compared to run cliiitaining no inntlerator. IIydrocarbon
>=
/w\
+
/v +
a
alkane.2r3,4Only small amounts of labeled degradation products, apparently formed by displacement of alkyl groups instead of hydrogen atoms, are formed. The present work on the reaction of recoil tritium with alkenes indicates the existence of an additional reaction mechanism. .4s with alkanes (see Table I, butane) about 60-S0yO of the tritium is found in H T and the labeled parent molecules. However, appreciable yields (10-20%,) of labeled degradation products are also found. Table I shows that the yield pattern of these unsaturated fragments varies in a very specific manner with the alkene reagent. The following mechanism for this “fragmentation” reaction is postulated. A tritium atom, having some residual recoil energy, adds on to the double bond of the alkene, forming a “hot” radical. This radical then decomposes by cleaving a carboncarbon bond without rearrangement, to form a smaller radical and an olefin. Because the tritium atom carries excess energy, it will attack the double bond indiscriminantly and form approximately equal amounts of both possible “hot” radicals. CII3CHzCHzCH=CH2 507, C H ~ C H ~ C H Z C H T C--+ H~* CH3CHzCFIZ. CHT=CH?
+4 L)
+
50% CH3CH?CHzCH*CH2T + CH3CHz’ CH2=CTICII?T
+
For example, the tritium atom will react with pentene-1 as shown, t o give similar amounts of tritiated propylene and ethylene. From pentene2, however, the same reaction will yield butene-1 and propylene. This mechanism is in accord with results on the decomposition of deuterated butyl radicals a t 500°.5 Since addition of a hydrogen atom t o a double bond is exothermic, t h e reaction probably could proceed at thermal energies. However, in the presence of an excess of helium as a moderator, the yields of t h e labeled fragments, as well as the yield of the parent molecule, are reduced greatly. On the other hand, these products are unaffected by the presence of a small amount of oxygen as (2) M. El-Sayed and R. Wolfgang, THIS J O U R N A L , 79, 3286 (1957) (3) hl. El-Sayed, P. Estrup and R . Wolfgang. J. P h y s . C h e m . , 62, 1356 (1958). (4) D. Urch and R. Wolfgang, unpublished work. (5) J. McNesby, D. Drew and A. Gordon, J . Chcm. P h y s . , 24, 1260 (1956).
scavenger. Therefore, this reaction to form labeled fragments apparently also takes place before the recoil tritium reaches thermal energies. Thus, while this is not a “hot-atom” mechanism occurring only a t very high kinetic energies, it may be termed “epithrrmal” to denote that it competes effectively only a t energies above the thermal range. This mechanism accounts for the results so far at hand, some of ivhich are sliown in Table I. II’ork to demonstrate the further predictions of this rriotlel is continuing. The authors wish to thank Profcssor IT. noering for his interest and criticism. CHEMISTRY DEPARTMEST YALE UNIVERSITY S E W IIAVES, C O X N E C T I C K r
11.iVIn RIC17ARD
L.
s.
GRCIX \vC>I.FC.ASG
R E C E I V EFEBRL-.XRY D 11, 1050 COENZYME Q.
VIII. STRUCTURE STUDIES O N A PLANT QUINONE
Sir: We have isolated from alfalfa a quinone having coenzyme Q-like activity’; structural data support I, 2,3-dimethyl-5- 13’-methyl-2’-butenyl-oktakis(3’-methyl-2’-butenylene) ]-benzoquinone.
’
C H a G H CH;’
CH, 1
(C,H,CH=CC€I,),H
I. r = 9 11. n = 3
0
Crane and L e ~ t e risolated ~,~ from alfalfa a quinone (Q-254), m.p. 42-43”, which appeared t o be a trimethylbenzoquinone with a fourth substituent of nine mono-unsaturated isoprenoid units. Kofler reported4 a substituted benzoquinone, 1n.p. 48-49”, from alfalfa. of Q-254 and The coenzyme Q-like studiesj-8 on “unidentified factors” in alfalfa in ( I ) Kindly tePted by D r . 1:. L. Crane, Cniver,ity uf Texas, Austin ( 2 ) F. L . Crane a n d R. L. Lester, P h n f P h y r z o i . , 33, (Suppl.) V I 1 (1958). (:1) F. L. Crane, ibid., in press. [ A ) M. Kofler, “Jubilee Volume, Emil Barell,” F. IIoffmaniiLaRoche and Co., L t d . , Basel, 19-16. ( 5 ) M , G. Vavick, A . Werty a n d A. R. Remmerer, Polrilry Scr., 32, 433 (1953). (6) H. M. Scott, II. Fisher a n d J. 51. Snyder, i b i d . , 32, 5 X (1953). (7) C. H. Hill, R. L. Borcbers, C. \V.Ackerson a n d P. E. Xfussehl, ibid., 32, 775 (1953). (8) B. March, J. Biely and S. P. Touchburn, i b i d . , 34, 968 (1955).
