Photoreduction of Orotic Acid in Aqueous M e d i u m
1199
Flash Photolysis. 11. Photoreduction of Orotic Acid in Aqueous Medium M. A. Herbert and H, E. Johns* The Ontario Cancer Institute, Toronto 5, Canada (Received May 15, 1972) Publication costs assisted by The Ontario Cancer institute
The long-lived transient observed in the flash photolysis of orotic acid has been identified as the hydrogen adduct radical. This radical arises from a biphotonic process in which the first photon is required to produce an orotic acid triplet, while the second photon is absorbed by this triplet. This highly excited molecule then abstracts a hydrogen atom from a water molecule adding the hydrogen atom to the carbonyl oxygen. This adduct may then dissociate (pK 4) leaving the same radical as the electron adduct. The absorption spectrum is the same as the electron adduct spectrum with a peak absorption at 325 nm. The variation of yield with pH has been measured and is higher at neutral pH than at acid pH. The second-order decay rate of the radical has been measured. Possible decay mechanisms have been suggested.
-
The uv photochemistry of orotic acid (uracil-6-carboxylic acid) has been extensively studied in aqueous solution using steady-statel-9 and flash p h o t o l y ~ i s l ~ -techniques. ~3 It has been shown that the dimerization of orotic acid (OA) occurs when an OA molecule in the triplet state interacts with a ground-state molecule. The reaction rates for the dimerization and radiationless deactivation of the triplet state molecule, its energy level, triplet absorption spectrum, the quantum yield for intersystem crossing, and the rate constants for various quenching reactions have been measured. In our flash photolysis studies we have observed a longlived transient absorption in OA under high-intensity flashes.12 A similar species has also been seen by Godfrey and Boag.10 In this paper we will show that the transient species is produced when a triplet orotic acid molecule absorbs a second photon, exciting it to a higher lying energy state. This highly excited molecule can react with a water molecule, abstracting a hydrogen atom, leaving an OH radical. The OH radical may then attack a groundstate orotic acid molecule, adding to it to form the .OH adduct. The absorption signal detected is from both these species. In this paper we present our observations on the properties of the long-lived transient, and the formation and decay of the radicals.
Experimental Section The flash photolysis system used has an output of approximately 6 X 1017 photons (1 yeinstein) in the wavelength range 200-350 nm. The width of the exciting flash is 1.2 psec at half peak height and is 99.9% complete in less than 4 psec. We are able to detect absorption signals as small as 3 X 10-4 using a 45-cm path length (three passes of a 15-cm cell) and a pulsed xenon analyzing lamp. The system is described in greater detail else~here.1~3~5 The orotic acid was from Cyclo Chemical (Grade I) or Mann Research Labs, while the [6-14C]orotic acid was from Amersham Searle Co. Identical experimental results were obtained using the OA in either the stock or recrystallized form. Solutions were mixed freshly for each experiment using water distilled four times in our quartz still. The solutions were deoxygenated by bubbling “prepurified grade” nitrogen (Gas Dynamics, Canadian Anaesthetic Gases) through them for at least 40 min prior to the ex-
periment, and then were forced into the cell under a nitrogen atmosphere and sealed off under a positive nitrogen pressure. Unbuffered solutions had pH values from 4.3 to 5.0 for the concentrations of orotic acid used. Adjustments of the pH were made using HC104 or KOH (Analar Grade, BDH). Thin layer chromatography (TLC) was carried out on thin layer cellulose plates (Machery Nagel and Co.) in a solvent system of 1-propanol-water (70:30 v/v). The chromatograms were then autoradiographed on Kodak No-screen X-ray film for 4 days. The chromatograms were cut up and counted in a Nuclear Chicago liquid scintillation counter using PPO and (CH3)2POPOP16 in p-dioxane fluid. A cold dihydroorotic acid marker was run on the chromatograms and detected using Fink’s reagent test.17a This test involves spraying the chromatogram with 0.5 M NaOH, drying, and then spraying with a solution of 1 g of p-dimethylaminobenzaldehyde in 100 ml of ethanol and 10 ml of concentrated HC1. Compounds with a saturated 5,6 bond react with the sprays to produce a yellow spot on the chromatogram. Uracil, barbituric acid, and isobar(1) A. Haug and P. Douzou, 2.Naturforsch. 6, 20,509 (1965). (2) A. Haug, J. Amer. Chem. Soc., 86, 3381 (1964). (3) E. Sztumpf-Kulikowska, D. Shugar, and J. W. Boag, Photochem. Photobioi., 6,41 (1967). (4) D. W. Whillans and H. E. Johns, Photochem. Photobioi., 9, 323 (1969). (5) D. W. Whillans, M.S. Thesis, University of Toronto, 1968. (6) M. Chariier and C. Helene, Photochem, Photobioi., 6, 501 (1967). (7) C. L. Greenstock, I. H. Brown, J . W. Hunt, and H. E. Johns, Biochem. Biophys. Res. Comrnun., 27,431 (1967). (8) E. Sztumpf and D. Shugar. Photochem. Photobioi., 4, 719 (1965). (9) C. Helene and F. Brun, Photochem. Photobioi., 11, 77 (1970). (10) T. Godfrey and J. Boag, personal communication. (11) M. A. Herbert, J. W. Hunt, and H. E. Johns, Biochem. Biophys. Res. Commun., 33, 643 (1968). (12) M. A. Herbert and H. E. Johns, Photochem. Photobioi., 14, 693 (1971). (13) R. W. Yip, W. D. Riddell, and A. G. Szabo, Can. J . Chem., 48, 987 (1970). (14) (a) W. B. Taylor, J. C. LeBlanc, D. W. Whillans, M. A. Herbert, and H. E. Johns, Rev. Sci. Instrum., 43, 1797 (1972);(b) J. C. LeBlanc, M. A. Herbert, D. W. Whillans, and H. E. Johns, Rev. Sci. instrum., 43, 1814 (1972)). (15) (a) D. W. Whiilans, M. A. Herbert, J. W. Hunt, and H. E. Johns, Biochern. Biophys. Res. Commun., 36, 912 (1969);(b) M. A. Herbert, Ph D. Thesis, University of Toronto, 1972 (16) PPO and (CH3)zPOPOP are scintillators PPO I S 2,5-diphenyloxazole and (CH3)zPOPOPI S p-bis[2-(4-rnethyl-5-phenyloxazolyl)]benP C ~ O
--.I-.
(17) (a) R. M. Fink, R. E. Clive, C. McGaughey, and K, Fink, Anal Chem., 28, 4, (1956):(b) C. L. Greenstock. Trans Faraday SOC., 66, 2541 (1970). The Journal of Physicai Chemistry, Vo/. 77, No. 10, 1973
M. A.
1200
*.Id'
,
----.----
Herberl and H. E. Johns I
Figure 2. Variation of adduct signal (HA)wilh total triple1 signal (HA H T ' ) lor 3 X M orotic acid solution measured at 340 nm. The total triplet signal is used as a measure of flash intensity. The solid line is the regression lit for y = ax" wilh a = 0.8 and b = 1.8. This shows a dependence on the square of t h e flash inlensity.
+
(ai
Figure 1
A ~ s o ~ D 5' ouva ~ 'IC-
a so ,Ion 01 3 x IO-' M
or01 c acid 0 sp ayev dl a sRet.0 xweo 0 1 50 #sec, divison Tne sana 1"e PC "9C""P ,rert,ca ~ . 1". ~. ~ . ". . ~ ~ .. ~ ~. ~ ." a n = 0 1 v -~ 4% ac C",."Pr!
are an equal mix of anion and neutral molecules (triplet pK is 4.5).'2 Equation 1 now is
~
division. X 340 nm). (bl Dc coupled adduct sign'ai shown a t i msecldivision. T h i s corresoonds lo the slow decav in a which appears as a straighl line'(vertica1 gain = 0.01 \j/division. A ~
~~
340 nml.
bituric acid were detected by their uv absorption under a mineralight. Decay of the Transient Signal. Figure la shows the transient signal observed a t a sweep rate of 50 psec/division in an unbuffered aqueous solution of 3 x 10-5 M o r otic acid. The transient absorption consists of a large fast decaying signal from the triplet12 followed by a smaller slower decaying signal from the adduct. The decay of the triplet is fast enough (lifetime is -15 psec) that the ac coupling time constant of the system (1 msec) has little effect on it. However, the slower decaying signal is badly distorted since its lifetime is longer than this time constant. To observe the proper decay of the adduct, we used a Tektronix 1A5 differential amplifier with the internal reference voltage adjusted to cancel out the dc level. The signal was then dc coupled into the oscilloscope. Figure l b is the dc coupled signal shown with a sweep rate of 1 msec/division. Comparison of this trace to that in Figure l a shows that the effect of the ac time constant is to increase the apparent rate of decay of the signal. (Note that the signal is displayed with a vertical gain of 0.1 V/division in Figure l a and 0.01 V/division in Figure lb.) All adduct decay rates were measured from dc coupled traces. Measurement of Triplet Signal. From the traces given in Figure la, we can calculate the peak signal due to the triplet and that due to the adduct. HT', the peak signal above the sloping base line, is a first approximation to the triplet signal. It does not include those triplets which were converted to adduct during the flash. The corrected triplet signal HT is given by cT
HT = H / + - H A
(1)
LA
where tT and C A are the extinction coefficients of the triplet and adduct, respectively, and HA(Figure la) is the absorption due to the adduct. The adduct extinction eA is the sum of the extinctions of the electron adduct (or protonated electron adduct) and the .OH adduct. A t pH 4.5, the value of is about l o 4 M-1 cm-',l"J while tT is 1.2 X lo' M-' cm-1 if we assume that the triplets present The Joumalol PhysicalChernisfry. Vol. 77. NO. 10, 1973
HT = H,'
+ 1.2HA
(2)
We can approximate HT by the sum (HT' + Ha). This approximation reduces the value of HT by less than 5%. an error less than the normal experimental errors encountered.
Results ( a ) Intensity Dependence. Figure 2 shows the variation of the adduct signal HAwith flash intensity. Since we are unable to measure the flash intensity directly, we have used the total triplet signal (HT' + HA)as a measure of the flash intensity. The solid line is the regression fit to the experimental points using the equation = axh
(3)
with a = 0.8 and b = 1.8. Thus within experimental error (4~15%)the yield of adduct depends on the square of the flash intensity and requires ( i ) either the absorption of two photons, or (ii) a triplet-triplet reaction for the production of a n adduct molecule. To separate possibilities i and ii, we measured the relative yield of adduct signal as a function of triplet lifetime. Increasing amounts of the triplet quencher manganese sulfate's (MnSO,) were added to a solution of 2.4 X 10-5 M orotic acid. This increased the triplet decay rate from a value of 3.4 x 104 sec-' (30-psec lifetime) with no added quencher to 11.5 x l W sec-I (8.7 psec) a t a quencher concentration of 1.4 x lO-'M. The results are shown in Figure 3. There is no change in the relative adduct signal size when MnSO. is added as a triplet quencher up to a concentration of 1.4 X lo-' M in MnSO4. Thus the production of adduct must be by mechanism i, that is, a biphotonic absorption. If the adduct were produced through a triplet-triplet reaction the yield would drop by a factor of 2 for each doubling of the decay rate of the triplet. Because the excited singlet lifetime for orotic acid in solution is 5 X 10-11 sec,'Sa and the triplet lifetime is greater than 5 X 10-6 sec, the probability of absorbing a We have measured a rate consIan1 01 5 x 10' M - ' rec ~' lor Ihe quenching 01 Orotic acid triplet signal by MnSO.. ( 1 9 ) (a) J. EiSinger and A. A. Lamola. B i o c h m Biophys. Acta. 240, 299 (181
(1971): (b) M. Charlie,, C . H&ne. Phys.. 66. 700 (1969).
and M. Dourianl. J. Chem.
Photoreduction of Orotic Acid in Aaueous Medium
. . f
,
I
I
[On] = 2 . 4 ~ 1 6 ~ M
1.21.0
e .-.e,
[MnS04]=0 ,p e 0,s c
cP
1201
t
0
-0
k
[MnS04]=1.4x
IC
-
i i
300
second photon during a triplet lifetime is more than lo5 times greater than that for absorption during the excited singlet lifetime. Thus the second photon must be absorbed by the orotic acid molecule in its triplet state. The triplet state energy of orotic acid has been calculated as 2.7 eV from energy transfer13 and phosphorescence measurements,l,6J9b and the absorption of a second photon from the flash would produce an excited molecule with about 6-9 eV of energy. ( b ) Absorption Spectra. To obtain reproducible absorption spectra, it was necessary to use an internal monitor to correct for pulse to pulse variations in the flash intensity. A beam splitter directs part of the analyzing light to a second monochromator-photomultiplier detector which is set at a fixed wavelength providing a reference signal for each flash. The measured signals are then normalized to a constant reference signal correcting for any flash output variation. Figure 4 shows the absorption spectrum of the longlived transient measured 150 psec after the flash in a solution of 5 X 10-5 M orotic acid at pH 1, 4.5, and 8.2. The spectra from the flash photolysis experiments are the result of duplicate points taken every 5 nm and are normalized at their peaks. Although the points are not shown for clarity, they rarely differ by more than &5%. The solid line is Greenstock's spectruml7b of the solvated electron (eaq-) adduct to orotic acid measured at neutral pH using pulse radiolysis. This spectrum is very similar to that measured by Hayon20 for the electron adduct. This spectrum has also been normalized to the adduct spectral peak. The spectra shown are very similar, peaking at 325 nm. At pH 1, the spectrum has a distinct shoulder at 355 nm while at pH 4.5 and 8 there is only a slight shoulder at 345 nm. The similarity of the flash photolysis absorption spectra to the electron adduct spectrum in pulse radiolysis suggests that we have formed a radical with a structure similar to the OA electron adduct. We suggest that we are observing the OA C4 carbonyl radical formed by hydrogen abstraction from a water molecule by the doubly excited OA molecule. When the OA triplet absorbs a second photon it will have about 6-9 eV, sufficient energy to break an H-OH bond in water, an endothermic reaction requiring 5.1 eV.21 The OA molecule adds on the hydrogen atom producing the He adduct radical and leaving an SOH radical in the solution. This .OH radical will attack ground state orotic acid molecules adding to form .OH adducts to OA with a rate constant of 5.2 X lOQ M-1 sec-1.17b At an orotic acid cencentration of 5 X 10-5 M , 63% of the -OHradicals will have reacted with the orotic acid within 3.8 psec, thus the build-in of the .OH adduct absorption will be hidden under the first part of the triplet decay.
350
400
450
500
Wovelenqth(nrn1
Figure 4. Adduct absorption spectra measured at p H 1 , 4.5,and 8. The solid line is the electron adduct spectrum measured by G r e e n ~ t o c k at ' ~ neutral ~ pH by pulse radiolysis.
A t neutral pH, the shoulder on the spectrum at 345 nm is the result of the absorption of the .OH adduct produced. At acid pH, a new shoulder is observed at 355 nm. This absorption may be from the carbonyl radical formed on the carboxyl group. A t acid pH the carboxyl group is uncharged4322 and may abstract an H. from water to form a carbonyl radical which absorbs at 355 nm. At neutral pH, this group is negatively charged and probably does not abstract. Thus, no shoulder is observed at 355 nm. The similarity of the flash spectra to the electron adduct spectrum suggests that the H. addition to OA occurs at a carbonyl group, probably the C4 position.20 Addition of an H. to either the c5 or c6 position produces a spectrum peaking at 345 nm.17b The addition of H. to the C4 carbonyl produces a radical which undergoes deprotonation with a pK of about 4 . l 7 b The deprotonated form is then identical with the electron adduct. This species has a peak absorption coincident with our observed spectra as seen in Figure 4. It has been shown in observations on thymine23 and orotic acidl7b that the peak absorptions of both the electron adduct and the protonated electron adduct occur a t the same wavelength. This observation accounts for the same observed peak position of the spectra measured at pH 1, 4.5, and 8. There is, however, a decrease of about a factor of 2.5 in the peak extinction of the orotic acid electron adduct as it protonates with a pK of about 4.17b Thymine shows a similar sized decrease upon protonation.23 ( c ) p H Dependence. In Figure 5 the adduct signal ( H A ) observed a t 340 nm is plotted against pH from pH 0.5 to 9.5 for a solution of 4 X M orotic acid. As the pH is increased from 0.5, H A decreases from an initial value of 0.06 to a minimum of 0.025 at pH 2.4, then rises to a plateau value of 0.13 above pH 6. The general shape of the curve can be explained as follows. The initial drop occurs as the ground-state orotic acid molecule ionizes from the neutral to anionic form with a pK of 1.8.4,22(Overlapping pK curves cause an apparent shift of the midpoint of the decreasing curve back to a pH of about 1.) As the ground-state molecule ionizes, the in(20) E. Hayon, J . Chem. Phys., 51,4881 (1969). (21) J. A. Ghormley and C. J. Hochanadel, J. Phys. Chem., 75, 40 (1971). (22) E. M. Woollev. R . W. Wilton. and L. G. Heoler. Can. J. Chem.. 48. -, 3249 (1970). (23) (a) L. M. Theard, F. C . Peterson, and L. S. Myers, Jr., J Phys. Chem.. 75. 3815 (1971): (b) L. M . Theard. F. C. Peterson. and R . L. Voigt, Gulf General Atomic Report No GA-10208 (1970) I
The Journal o f f h y s i c a i Chemistry, Vol. 77, No. 10, 1973
1202
M. A.
Herbert and H. E. Johns
41.
L A 3
l
2
3
C
5
6
7
R
S
l
C
Pd
Figure 5. Variation of adduct signal H A with p H . The error bars represent the range of values measured ( A 340 nm).
tersystem crossing decreases by a factor of 2,4 resulting in only one-half the number of triplets, which can absorb a second photon (to form adducts). As the pH is further increased, the signal HA increases approximately fivefold with the midpoint of the increase a t a pH. of 4.3. This increase can be explained by the following two factors. (i) When the triplet absorption spectrum is multiplied by the flash output spectrum and the product integrated, it can be seen that a given number of anion triplets (the triplet has a pK of 4.5)12 will absorb twice as many photons as will an equal number of neutral triplet molecules of orotic acid. Thus, there is a factor of 2 increase in triplet absorption as the triplet ionizes in going from below to above its pK. If we assume that after biphotonic absorption the formation of an adduct is 100% efficient,, the yield of adducts will also increase by a factor of 2. (ii) The additional 2.5-fold increase occurs as the adduct undergoes dissociation of a proton with a pK of about 4.17b The extinction of the dissociated form is greater than that of the protonated form by a factor of 2.5. Together these factors can account for the observed change. ( d ) Decay of the Transient. Figure 6a is a first-order plot (log of absorbance us. time) of the decay of the longlived transient observed a t 340 nm in an unbuffered solution of 4 x 10-5 M orotic acid. Figure 6b is a second-order plot (l/absorbance us. time) of the same data. The points were measured on a photograph of a dc coupled transient signal such as shown in Figure lb. The first-order plot (Figure 6a) is concave upward and cannot be represented by a good straight line. The second-order plot (Figure 6b) is a good straight line. The second-order decay rate constants (2k/t) for orotic acid a t pH 2, 4.5, and 8 are listed in Table I. The results were measured a t wavelengths of 340 and 390 nm, and are the average of a t least five measurements while the error shows the range of values obtained. The extinction coefficient is calculated from the absorption spectra assuming a peak extinction of 1.0 x 104 M-1 cm--1 for pH 817b and 5.1 X 103 M-1 cm-1 for pH 2 (protonated form) and a mix of 50% protonated and 50% dissociated adduct for pH 4.5 giving an adduct extinction of 7.6 X 108 M-1 cm-1. Rased on these values, the decay rate constant, 2k, has been calculated. The observed decay rate constant at 390 nm for all pH values and a t 340 nm (pH 2) is 1.4 X lo9 M - 1 sec-1. Greenstock17b measured a decay rate of 2 X 108 M-1 sec-1 for the .OH adduct to orotic acid, while The Journal of Physical Chemistry, Voi. 77, No. 10, 1973
Figure 6. Decay of the adduct signal at 340 nm. (a) First-order plot of log of absorbance vs. time. The points do not lie on a straight line. ( b ) Second-order plot of l/absorbance vs. time. These data are the same as in part a. The points lie on a straight line of slope 2 k / c = 7 X 1 O5 cm sec- l . TABLE I: Second-Order Decay Rate Constants ( 2 k ) of the Orotic Acid Adduct Signal fh X
IO3,
2 k / e X 10;. cmsec-
pH
M-'cm-'
340
2.0
4.0
(3.4i 0.3)
5.2 7.0
(3.2 i O . 9 )
390
4.5 8.0 2.0
h,nm
4.5
1.8 2.2
(2.5 f 0.9) (7.8 f 3) (6.5 f 2)
8.0
3.0
(4.8
i 2)
2k X 109, M-l
sec-1
(1.4f 0.2) (1.7 k 0.4)
(1.7f 0.5) (1.4 (1.4 (1.4
k 0.4)
*
0.4)
f 0.5)
Theard, et U L . , measured ~~ decay rate constants of about lo9 M - l sec-I for most pyrimidine .OH and eaq- adducts. ( e ) Chromatography. Thin layer chromatography was carried out in an attempt to isolate permanent products produced by the decay of these adducts. Solutions of 8 x M [6-14C]orotic acid were mixed at pH 4.5 (unbuffered) and 1. Samples were irradiated in the flash photolysis apparatus for 0, 5, 10, and 20 flashes and one sample was irradiated in a monochromator at 280 nm by steadystate methods. 1OO-wl volumes were spotted on t,he chromatograms and run in l-PrOH-Hz0 (70:3O v/v) and then autoradiographed for 4 days. Several compounds were spotted as markers, and their R, values (kO.05) measured in this solvent system: dihydroorotic acid (Rf = 0.48), uracil (0.62), barbituric acid (0.32), isobarbituric acid (0.5G). Orotic acid and orotic acid dimers were observed with Rf values of 0.41 and 0.19, respectively. In the unbuffered solutions, a spot running with an Rp of 0.33 increased to a value of 0.41% of the spotted material after 20 flashes, while a small amount of a product with an Rf of about 0.6 was also visible. The product with an Rf of 0.6 was probably uracil caused either by a spontaneous decarboxylation of the orotic acid or a radiationinduced d e c a r b ~ x y l a t i o n .The ~ ~ product running with an Rf of 0.33 may be barbituric acid or possibly orotic acid glycol. SinEe there is no marker for the glycol, its presence can not be ruled out. The glycol is an expected product from the radical decay of the orotic acid OH adducts. Despite repeated attempts the chromatography results with the pH 1 samples were very poor. To avoid dissolving the emulsion on the plate, it was necessary to neutralize the acid before spotting. This left a solution containing (24) D. W. Whillans, personal communication, 1972
1203
Photoreduction of Orotic Acid in Aaueous Medium 0.1 M salt and even repeated desalting procedures could not prevent severe streaking and running of the spots. ( f ) Effect of N20, MnS04, and 0 2 on Yields. Saturation of the orotic acid solution with N2O (by bubbling) had no effect on either the triplet or adduct signals observed. This means that no significant fraction of the observed adduct absorption is produced by the attack of solvated electrons on the orotic acid, ruling out the possibility that we are observing ionization of the orotic acid. Ionization would produce a solvated electron which would attack a ground-state orotic acid, producing an adduct with an extinction of 8500 M - 1 cm-1 1 7 b with a peak absorption at 330 nm. The observed signal would then be the sum of the absorption from the ionized molecule plus the absorption of the electron adduct. The addition of NzO to the solution would block this reaction, reducing the adduct signal seen. The use of a 10-2 M MnS04 solution as an optical filter to absorb photons of wavelengths less than 240 nm did not change the ratio of adduct to triplet signal observed. This means we are not observing a reaction of orotic acid molecules excited by the flash directly from the ground state, SO,to the second excited singlet state, Sz. The adduct signal was very sensitive to the presence of oxygen in the solution. Bubbling the solution with a mixture of 0.5% oxygen and 99.5% nitrogen reduced the lifetime of the adduct to under 50 psec, after which point the transient due to the adduct could not be separated from the triplet signal accurately. This corresponds to an oxygen quenching rate of about 3 X 109 M - 1 sec-1 in agreement with other measured rates for 0 2 attack with orotic acid radicals.17b Discussion and Conclusions The following equations summarize the reactions occurring in the production of the adducts. OA OAs OAs
hv
OAt
OA""
+
-
% __t
+ H2O
OAs
(4)
OAt
(5)
OA
(6)
0.4""
(7)
OAH.
+
5.2 x 10' M - ~ s w - ~
.OH
(8)
#OH OA OAOH. (9) A ground-state orotic acid molecule, OA, absorbs a photon in the flash exciting it to an excited singlet state OAs (eq 4). From the excited singlet state, a fraction of these molecules ( (PIBc) undergo intersystem crossing to become triplets, OAt (eq 5), while the rest decay back to the ground state (eq 6). During the flash, a fraction of the triplets absorb a second photon, producing a highly excited molecule OA** (eq 7 ) which may have up to about 9 eV of excitation energy above the ground-state level. This molecule now interacts with a water molecule to abstract a hydrogen atom forming the H. adduct (eq 8) and leaving a hydroxyl radical (.OH) which can attack a groundstate orotic acid molecule to form the .OH adduct (eq 9). The hydrogen abstraction results in the addition of the hydrogen atom to a ketyl oxygen of the orotic acid, probably a t the C4 position,20 resulting in structure 1, which has a pK of about 4I7b and dissociates to structure 2 at pH values above this pK. Structure 2 is the same as the
-
OH
I
-H+
1
0-
I " 9 H
2
electron adduct produced in pulse radiolysis at neutral pH and explains why the spectra of the adduct made in the flash and the pulse radiolysis electron adduct are the same. The extra electron of the adduct is likely delocalized through the C5, C6, and carboxyl group positions as shown by resonant structure 3. Adam95 has shown that in
3
4
isoorotic acid, structure 4, the extinction coefficient of the electron adduct is considerably less than that for orotic acid. Since isoorotic has less resonant structure than orotic acid, he concludes that the higher extinction in the orotic acid radical indicates a larger degree of resonance for the orotic acid electron adduct radical. Delocalization of the unpaired electron onto the carboxyl or carbonyl group in orotic acid has been suggested to explain observed esr (electron spin resonance) signals.26 In the uracil anion it has been suggested that the delocalization is onto the CZ carbonyl group.26 Esr studies of the free radicals produced by Ha and .OH attack on uracil and t h ~ m i n e indi~~,~~ cate that there is conjugation between a t least two resonant forms of the radical. The study of y-irradiated 5nitro-6-methyluracil crystals29 shows that the H . has added on to the oxygen on position 4 and the free electron is conjugated between the c6 position and the C4 position. Huttermann reports esr evidence for resonant structures in orotic acid similar to 3. He has also detected the addition of an H - on the oxygen in the carboxyl g r o ~ p . ~ O The hydroxyl radical produced (eq 8) attacks at the 5 or 6 position. This produces a spectrum peaking around 345 nm17b which is added to the electron adduct spectrum producing the shoulder observed a t 355 nm. The decay mechanism of the observed radical species can only be suggested at this time. At neutral pH, the reaction of the electron adduct with an -OH adduct might eliminate an OH- and leave two ground-state orotic acid molecules. The reaction of two .OH adducts could disproportionate to produce orotic acid glycol and orotic acid. This product may be the spot observed with an Rf of 0.33. At acid pH, the reaction of two protonated electron adducts could eliminate hydrogen, leaving two orotic acid molecules, while the reaction of the He adduct with an .OH adduct could eliminate a molecule of water, leaving two orotic acid molecules. G. E. Adarns, C. L. Greenstock, J. J. van Hemrnen, and R. L. Wilson. Radiaf. Res.. 49. 85 (1972). J. K. Dohrmann and R. Livingston, J . Amer Chem. SOC., 93, 5363 (1971). J. N. Herak and W. Gordy, Proc. Nat. Acad. Sci. U.S., 54, 1287 (1965). C. Nicolau, M. McMillan, and R. 0. C. Norman, Biochim. Biophys. Acta, 174, 413 (1969). W. Snipesand 6.Benson,J. Chem. Phys., 48,4666 (1968). J. Huttermann. J. F. Ward. and L. S. Mvers. Jr.. J . Phvs. Chem.. 74,4022 (1970)
The Journalof Physicai Chemistry, Vol. 77, No. 10, 1973
George M. Wyman and Bizhan M. Zarnegar
1204
It has been reported that an excited orotic acid reacts with acrylonitrile to form the 'acrylonitrile radical.9 The first step is almost surely a hydrogen abstraction reaction, but the authors report that they could not observe dihydroorotic acid, the expected product from disproportionation of two H. adducts, when the H. adduct is formed a t the 5, 6 bond positions. This would suggest that in their system, the hydrogen abstraction does not add a
hydrogen atom to the' C6 or Cg positions, but rather to a carbonyl oxygen as we suggest. Acknowledgments. The authors take pleasure in acknowledging the financial support of the Medical Research Council of Canada and the National Cancer Institute of Canada. We are indebted to Drs. J. W. Hunt, T. Penner, and D. W. Whillans for helpful discussions.
Excited State Chemistry of lndigoid Dyes. 11. The Interaction of Thio- and Selenoindigo Dyes with Hydroxylic Compounds and Its Implications on the Photostability of Indigo' George M. Wyrnan" and Bizhan M. Zarnegar Department of Chemistry, University of North Carolina, Chapel Hili, North Carolina 27514
(Received November 6, 1972)
Publication costs assisted by the U.S. Army Research Office-Durham
The fluorescence and the photoisomerization of trans-thioindigo is quenched by phenol and p-nitropheno1 at the diffusion-controlled rate. The effectiveness of ethanol, cy-trifluoroethanol, and a,a'-hexafluoro2-propanol as fluorescence quenchers ranges from 2.5 to 58% of the diffusion-limited value and increases with increasing acidity of the alcohol. The fluorescence quenching rates of selenoindigo and several dyes related to thioindigo with one or more of these hydroxy compounds were also determined. The results support the idea that an intramolecular proton transfer in the excited (Si)state is responsible for the unique photostability and lack of fluorescence exhibited by indigo and its ring-substituted derivatives.
It is now generally recognized that indigo (Ia) and its ring-substituted derivatives exist only as the trans isomers2,3 and that, in contrast with other indigoid dyes,4*5 they will not undergo photochemical trans cis isomerization,2q6 nor do they exhibit any appreciable fluorescence.' When it was observed that the N,N'-dimethyl-8,9 and N,N'-diacetyl derivatives2.10 undergo photoisomerization, the photostability of the parent compound was attributed to hydrogen bonding.2 Although the existence of hydrogen bonds in indigo in its ground state is now firmly established from crystallographic data,3 this does not provide a wholly satisfactory explanation for the photostability, since the energy required for exciting the molecule to its Si state (ca. 48 kcal) is far greater than the stabilization that may be expected from the formation of two typical hydrogen bonds (ea. 10 kcal). In a recent communication an alternative explanation was proposed by one of us for this remarkable photostability,ll suggesting that a fast proton transfer occurs in the Si state (as represented by eq 1) and that this takes precedence over the other excited state processes (fluorescence and cis-trans isomerization) normally encountered in of this type. The greatly enhanced (ca. 1O6-fo1d) acidity of Ar-NH groups and the similarly increased basicity of Ar-CO functions in the SI state is well known12 proton transfer phenomena have and such been reported for other compounds that are hydrogen
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The Journalof Physical Chemistry, Vol. 77, No. 70, 1973
7
I
Ia, X = NH Ib, X = S IC, X = S, 6,6'-diethoxyId, X = S, 6,6-di-n-hexoxyIe, X = S e
$.
0-H
bonded in the ground state, e.g., derivatives of salicylic acid,l3 o-hydroxyazines,l4 and some hydro~vbenzazoles.~~ (1) Presented in part at the V I International Conference on Photochemistry, Bordeaux, Sept, 1971. (2) w. R, Brode, E, 0, pearson, and G . M, wyman,J. A ~ Chem. ~ ~ SOC.,76, 1034 (1954). ( 3 ) H. V. Eller, Bull. SOC. Chim. Fr., 106, 1444 (1955); E. A. Gribova, Kristallografiya, 1, 53 (1956). (4) G. M. Wyman and W. R. Brode, J. Amer. Chem. Soc., 73, 1487
(1951).
.
