J. M. FRITSCH, S. V. TATWAWADI, AND R. N. ADAMS
338
dynamic mechanism. Probably, an experimental exclusion of a static quenching mechanism operative in fluid solutions (t,hat might be in addition to the wellestablished dynamic quenching) has not yet been made, since most of the experiments recorded in the literature have been carried out with very intense saturating light flashes. It would be interesting to measure the relative quantum yield of triplet formation in weaker flashes as a function of oxygen pressure. This would be a more crucial test for the existence of nonphosphorescent S .02complexes. We believe that Weiner and Seliger,18 like Rosenberg and ShombertlZ3 erred in reporting oxygendependent triplet lifetimes by confining their analysis
to a later interval of time than in the present work. We showed that oxygen interacts more strongly with the slow component of decay, i.e., Kwm> Keqd. Therefore, the observed decay at later times, when forced into an exponential formalism, will appear faster owning to the increased relative contribution of the dimer. Weiner and Seliger’s report that the lifetime of triplets does not decrease indefinitely with increasing oxygen pressure indeed tends to support the mechanism presented in this paper. Their data on the “saturation” of the collisional decay constant at high oxygen pressure cannot be interpreted on the basis of their own mechanism, especially if a simple exponential decay is a parallel requirement, as they maintain.
Electron Paramagnetic Resonance Studies of Vitamin K and Vitamin E Quinones’
by John M. Fritsch, Shankar V. Tatwawadi, and Ralph N. Adams Department of Chemistry, University of Kansas. Lawrence, Kansas 6 6 0 4 (Received July $26,1966)
A detailed study has been made of the electron paramagnetic resonance (epr) spectra of the anion radicals of vitamin K and E type quinones. In addition, the rates of electron transfer (homogeneous electron exchange) between the anions and the parent compounds were measured via epr line broadening. The epr spectra show that the characteristic side chain of vitamin K type quinones has relatively little effect on over-all chemical reactivity but materially influences the rate of simple chemical reactions such as electron transfer.
Despite intensive study over many years, the exact roles played by biologically important quinones in such processes as blood clotting and oxidative phosphorylation are still unknown. The final solution of such problems clearly will come from in vivo studies. Much remains to be known, however, about the oxidation-reduction characteristics, reactivities, etc., of biologically important quinones at the molecular level. This study is concerned with a detailed description of the electron paramagnetic resonance (epr) spectra of the monoanion radicals of several vitamin The Journal of Physical Chemistry
K and E type quinones, their electron transfer rates (homogeneous electron exchange), and predictions of their rates of reaction in oxidation-reduction processes. The specific compounds investigated are shown in Figure 1.
(1) This work was supported by the Petroleum Research Fund administered by the American Chemical Society and the University of Kansas through its operation of computer facilities. The work wfts initiated while one of the authors (R. N. A.) wm a J. S. Guggenheim Fellow and this support is gratefully acknowledged.
ELECTRON PARAMAGNETIC RESONANCE STUDIES OF VITAMINK
Experimental Section The solutions of the radical ions were obtained by standard electrochemical reduction at a mercuryplated platinum gauze external to the epr cavity.2 The solvents, N,N-dimethylformamide (DMF) and dimethyl sulfoxide (D34SO) were purified by conventional methods. Solutions were 0.5 to 1.0 mM in the various quinones and contained 0.1 M tetraethylammonium perchlorate (TEAP) as supporting electrolyte unless otherwise noted. The epr spectra were obtained with a Varian V-4500 spectrometer with a 6411. magnet and Fieldial attachment. The reduction potentials for radical ion generation were chosen between the oneand two-electron polarographic waves. Half-wave potentials (essentially identical with the formal reduction potentials) for the one-electron process in DMF are listed in Table I.
339
1,4-Naphthoquinone
0
Vitamin K~czo, (2-Methyl-3-phytyl-l,4-naphthoquinone)
0 H3c)@; H3C
0
0
p-Benzoquinone
Duroquinone
in N,N-Dimethylformamidea H3C Compound
1,4Naphthoquinone Vitamin K3 Vitamin K1(lO) p-Benzoquinone Duroquinone Vitamin E quinone
0
OH CH3 I (CH&H&HkH--)
~&C z H~ J & , &H
Table I : Polarographic Half-Wave Potentials
E1/a
L431 CH3
3-
CH3
CH3
0 Vitamin E quinone (a-tocoquinone)
-0.60 f 0.02 -0.69 f0.03 -0.76 f 0.02 -0.48f0.02 -0.76 rf: 0.02 -0.75 f 0.03
0 For the first reduction step, a one-electron process. volts us. a saturated calomel reference electrode.
Vitamin Ks (Menadione; 2-methyl-1, 4-naphthoquinone)
Figure 1. Vitamin quinones and related molecules studied by epr. In
Interpretation of Epr Spectra. Epr spectra of some of the vitamin quinone anions have been reported previously but without complete interpretation of the hyperfine ~plittings.~-*From the structural viewpoint vitamin K3 or menadione may be considered the simplest of the vitamin K quinones. Under moderately high resolution even the vitamin K3 anion spectrum is quite complicated as seen in Figure 2. I n general it was not possible to readily assign a single interpretation of' splitting constants for the spectra of the vitamin K3 :tnd Klclzo,anions. This was also true for vitamin E quinone anion. To establish which combinations of possible coupling constants and line widths could apply, each plausible approximate combination was computer plotted. For each of the spectra studied there was only one combination of coupling constants and line widths which yielded a reasonable approximation to the experimental spectrum. The precision of the coupling constants was then improved by varying the approximate values over small ranges
until optimum fittings of computer and experimental spectra were attained. Unequivocal assignments for the ring protons in positions 5, 6, 7, and 8 of vitamins K3 and Klclzo, and the smaller methyl splittings in vitamin E cannot be made. These were assigned to be in best agreement with couplings predicted by Mclachlan-modified Huckel MO calculations. The parameters used for these calculations are given in the Appendix. The long side chain in vitamin Klclzo, and vitamin E quinone was treated in the MO calculations as a methyl group with a slightly increased inductive effect. This treatment alters the coulomb integral of the carbon to which the side chain is attached as indicated in the Appendix. This approach is justifled by the epr spec(2) R. N. Adams, J . Electroanal. Chem., 8, 151 (1964). (3) S. Blois, Bwchim. Bwphys. Acta, IS, 165 (1955). (4) J. E. Wertz and J. L. Vivo, J . Chem. Phys., 24, 479 (1956). (5) M. Adams, M. 8. Blob, Jr., and R. H. Sands, ibid., 28, 774 (1958). (6) M.5. Blob, Jr., and J. E. Maling, Bwchem. Bwphys. Res. Commun., 3, 132 (1960). (7) Y.Matsunaga, Bull. Chem. SOC.Japan, 33, 1436 (1960). ( 8 ) R. W. Brandon and E. A. C. Lucken, J . C h m . SOC.,4273 (1961).
Volume 71, Number d
January 1967
340
J. M. FRITSCH, S. V. TATWAWADI, AND R. N. ADAMS
Table 11: Proton Coupling Constants for the Vitamin K Semiquinone Radicals 7
Semiquinone
Solvent
1,4-Naphtjhoquinone
Vitamin Ii)*
-
VitaminKtcw)
-
2
DMSO6 DMF EtOH-HzOc Alkd DMSO DMF DMF-lO%HzO E tOH-Hz0’ Alkd DMSO
3.31 3.27 3.23 3.22 2.69@ 2.69@ 2. 80e 3. 01’ 2.94’ 2. 636
DMF
2. 63e
Alkd
2.2’
3
Proton couplings (gauss) a t position numbersa 5 6 7
3.31 3.27 3.23 3.22 2.69 2.69 2.51 2.38 2.40 1.410 1.229 1.398 1,238 1.1
8
0.300 0.26 0.513 0.57 0.22 0.22 0.40 0.64 0.59 0.30
0.633 0.57 0.655 0.57 0.76 0.78 0.69 0.64 0.59 0.74
0.633 0.57 0.655 0.57 0.62 0.61 0.69 0.64 0.59 0.74
0.300 0.26 0.513 0.57 0.37 0.36 0.40 0.64 0.59 0.26
0.30
0.74
0.74
0.26
0.55
0.55
0.55
0.55
a The precision of the coupling constants is ~ t 0 . 0 2 gauss. Data from E. W. Stone and A. H. Maki, J. C h m . Phys., 36, 1944 (1962). A basic alcohol-water mixture, data from G. Vincow and G. K. Fraenkel, ibid., 34, 1333 (1961). d Alkaline aqueous solution with some alcohol or acetone, data from ref 5 and 6. * Methyl proton couplings. 50% v/v alcohol-water buffer, apparent pH 8. Nonequivalent proton couplings a t methylene carbon of side chain. c
f
Figure 2. Epr spectrum of vitamin Ka monoanion in dimethyl sulfoxide.
tra which show that within the present resolution there is no unpaired electron density in the side chain beyond the first methylene group. Blois and Maling earlier predicted and verified that interactions with protons further out on the side chain would be very weak if, indeed, they existed.6 It is very interesting that, with the increased resolution of the present study, it can be shown clearly that the methylene protons of vitamin Klclzo) semiquinone are nonequivalent. This lack of equivalence has also been noted in the nmr spectra of the parent molec~le.~Steric interactions between the phytyl chain and the adjacent 2-methyl group might cause this. However, it is felt more likely due to hydrogen bonding of the ethylenic proton to the carbonyl oxygen in the anion radical. The infrared The J
o u T of ~ ~Physical ~
Chemistry
spectrum indicates any such hydrogen bonding in the parent compound must be weak.I0 In favor of the hydrogen bonding argument for the semiquinone are (1) increased electron charge on the carbonyl oxygen in the anion radical, (2) potential formation of a sixmembered ring, and (3) a decrease in the nonequivalency of the methylene protons with increasing temperature. For even a weak hydrogen bond, a molecular model clearly shows the methylene protons would be nonequivalent due to twisting of the methylene carbon out of the plane of the aromatic system. As far as can be ascertained, the methylene protons of vitamin E semiquinone are equivalent. Tables I1 and I11 summarize all of the coupling constants for the vitamin K and vitamin E type compounds studied and compare them with some previous literature data. The proton couplings at positions five and eight in the naphthoquinone system are markedly solvent dependent. This is also true, to a lesser extent, for positions two and three. These differences in coupling constants for aqueous and DMF DMSO systems are clearly seen in Table 11. All the couplings are essentially invariant between DMF and DMSO. However, the intrinsic line width for the Ks anion is considerably larger in DRIIF. The line width for Klczo) anion is large in both solvents and may reflect asmall ~
(9) C . von Planter, E. Billeter, and M. Kofler, Helv. Chirn. Acta, 42, 1278 (1959). (10) 0. Ister and 0. Wiss in “Vitamins and Hormones,” R. S. Harris, G. F. Marrian, and K. V. Thimann, Ed., Vol. 17,Academic Press, New York, N. Y.,1959, p 62.
ELECTRON PARAMAGNETIC RESONANCE STUDIES OF VITAMINK
Table III : Proton Coupling Constants for Vitamin E and Related Semiquinones4 Solvent system
Semiquinone
pBenzoquinone Duroquinone Vitamin E quinone
Proton coupling, gauss
DMF DMSOb EtOH-H20C DMF Methyl H’s EtOH-H20c Methyl H’s DMF Methyl, position 5 and 6 Methyl, position
2.41 (2.419) (2.368) 1.91 (1.897) 2.21 1.60
3 Methylened
0.81
a The precision of the coupling constants is kO.01 gauss, previous literature data in parenthesis. b Data of Stone and Data of B. Venkataraman, Maki, see footnote b in Table I. B. G. Segal, and G. K. Fraenkel, J . C h m . Phys., 30, 1006 (1959). d For two equivalent methylene protons on side chain a t position 2.
(0.05-0.1 g) unresolved coupling of the ethylenic proton. The point of examining the epr spectra of the vitamin quinone anions in detail was to see if there might be important differences in unpaired electron distribution between members of the series which would reflect differences of chemical reactivity in the aromatic portion of the molecules. It is quite clear that the effects of substitution, even of large side chains, is minimal with respect to the aromatic portion of the 1,P naphthoquinone nucleus. Certainly, positions for chemical reactivity become blocked as one proceeds in substitution from 1,4-naphthoquinone and vitamin Both 340 calculations and the experimental electrochemical and epr results support this view. The conclusions with regard to the K1 vitamin series can be transposed to the K2 compounds since these differ only in the length and nature of isolated double bonds in the side chain. I n the next section, the above conclusions are contrasted to the marked differences in rates of chemical reactions of these compounds where the large side chains do play an important role. Electron Transfer Rates. The electron transfer process of present interest is given for a neutral molecule Ar and its radical anion Are - by the equation
AT.-
+ Ar E Ar + Ar. -
This is the simplest of chemical reactions in which the reactants are identical with the products. (This process is commonly called homogeneous electron exchange. The terminology electron transfer is used here to differentiate it from such processes as spin-spin electron
341
exchange in which radical ions also participate under other conditions.) For the present study Ar represents the parent quinone and Ar - the semiquinone anion radical. Since one of the two species is paramagnetic, the rate constant k,,, can be determined from the epr line broadening as a function of the concentration of the parent quinone.11J2 Table IV lists the values of k,,, determined. A major difficulty in evaluating these rate constants is the heavily overlapped spectra of the vitamin K type molecules. The problem was solved by a computer technique which accounts for merging of lines concurrent with the broadening process.
Table IV : Electron Transfer Rates in N,N-Dimethylformamide Compound
kexoa
1,4Naphthoquinone Vitamin KS Vitamin Kt(%, p-Benzoquinoneb Duroquinoneb Vitamin E quinone In I./mole-see.
b
(4.2 k 0 . 3 ) X (4.0 i 0.5) X ( 1 . 3 & 0.5) X (3.8 A 0.2) X (6.2 k 0.3) X (1.6 f 0 . 3 ) X
108 lo8 108
lo8 lo7 lo7
Data from ref 12.
Contrary to the previous effect on e1ect)rondistribution and reactivities of the aromatic nucleus, in the rate studies a long side chain has a considerably greater effect than a simple methyl substitution. The value of k,,, for the Kl(20, system is one-third that of the K) and vitamin E quinone has a rate constant one-fourth that of duroquinone. From the precision of the k,,, values in Table IV it can be seen that these differences are quite real. The k,,, values can be used to predict the cross reaction rates of compounds in a conventional oxidationreduction reaction via the theory and prediction of Marcus.13 I n the one-electron oxidation-reduction reaction kia
Oxl
+ RedB-+
Redl
+ Ox2
(1)
with an equilibrium constant of Klz and a forward rate of klz, one denotes the individual k,,, values as kll for Oxi/Redl and kzz for Ox2/Redz. If k11 and kzz (11) R. I. Ward and S. I. Weissman, J . Ant. C h a . Soc., 79, 2086 (1957). (12) T.Layloff, T. Miller, R. N. Adams, H. Fah, A. Horsfield, and w. Proctor, Nature, 205, 4969 (1965). (13) R. A. Marcus, J . cha. Phys., 43, 679 (1965).
Volume 71, Number B January 1967
J. M. FRITSCH, S. V. TATWAWADI, AND R. N. ADAMS
342
&s well as K12 are known, according to Marcus, the forward rate of the cross reaction is given by
where the quantity f is given by Inf =
(In K d 2 4‘ In (kllkz2/1022)
(3)
Equation 2 has been found valid for quite a few inorganic oxidation-reduction reactions especially by Sutin and co-workers.l* Rather than predict the values of k12 for the compounds studied herein, it is of more interest to compare the relative ratios of predicted lc12 values. If one considers a series of reductants Red2, Reds, Re&, etc., all reacting with the same 0x1 (or the opposite process of several oxidation species reacting with a common reduction species) then the relative values k12, k13, k14, etc., can be calculated readily via eq 2 and 3. Thus, consider the situation where Red3 is used in eq 1 and assume ka3 equals 4k22for which the equilibrium constant is unchanged. From eq 3, the effect of the change on f is very small but the over-all value of k13 is increased by a factor of 2. This applies to the reactions of vitamin E quinone as compared to duroquinone where the k,,, values are in this ratio and their formal potentials are so close that any difference in equilibrium constants with a common reactant are small. For vitamins K3 and K1czo) the predicted crossreaction rates are more influenced by the relative equilibrium constants since their formal potentials differ somewhat. From these potentials if K3 and K1(20)react with a common reductant, the equilibrium constants should differ by a factor of about 12. This enters into eq 2 but still does not materially affect the value off (frequently close to unity) in eq 3. Hence, it can be predicted that vitamin K3 should react six to seven times faster than vitamin Klczo)with a common reductant. Summary The epr hyperfine spectra show clearly that from the reactivity viewpoint, the aromatic nuclei of the vitamin K type compounds are practically uninfluenced by the nature of the side chain. (This argument cannot be applied indiscriminately to vitamin E quinone
The Journal of P h y 8 k l C h m 6 t r y
because of intramolecular interactions of its side chain with the ring and the further discussion is limited to the K type compounds.) Thus, vitamin K3, or certainly 2,3-dimethyl-l,P-naphthoquinone, is an acceptable substitute for the K1 or K2 vitamin series in terms of intrinsic reactivity. On the other hand, the rates of the simple chemical reactions are affected by the size of the side chain. Two attitudes may be taken with regard to the rate differences. If the differences in rates are considered significant, then they may well play a role in biological electron transport processes. For instance, the exact role of ubiquinone in mitochondrial electron transport is open to question because its oxidation-reduction rate differs somewhat from that of the over-all electron transport rate in the respiratory chain.ls If, alternately, the rate differences, which are indeed small, are considered noninfluential in this sense, then the only biological significant differences in the vitamin K compounds are that the side chains provide lipid solubility or spatial characteristics significant for proper participation in the complexity of oriented in vivo reactions. The latter, of course, may be considered as a type of specific reactivity.
Appendix I n the MO calculations,16the fairly standard value of X = 1.2 was used. The coulomb integral for oxygen 1.26&~and the resonance was taken as LYO = CYC integral for the carbonyl bond was BCO = 1.55P~c.~’ For those carbons to which a methyl is attached, the coulomb integral used was CYCC(M~)= CYC - 0.313~c.’~ For the side chain treated as a methyl group, the coulomb integral of the ring carbon to which the side , chain is attached was taken as CYC(C-R) = CYC hPcc. The value of h was varied between -0.2 and -0.8 in a sequence of calculations employing the other mentioned parameters. A value of - 0.4 was found to fit best, as would be expected from this model.
+
+
(14) R. J. Campion, N. Purdie, and N. Sutin, Inorg. Chem., 3, 1091 (1964). (15) B. Chance in “Biochemistry of Quinones,” R. A. Morton, Ed., Academic Press, New York, N. Y., 1965, Chapter 14. (16) A. D. McLachlan, Mol. Phys., 3, 233 (1960). (17) J. Gendell, J. H. Freed, and G. K. Fraenkel, J. Chem. Phya., 37, 2832 (1962). (18) J. P. Colpa, C. Maclean, and E. L. Mackor, Tetrahedron, 19, Suppl. 2, 65 (1963).
