J. Phys. Chem. 1991,95, 2404-241 1
2404
Theoretical and Infrared Matrix Isolation Study of 4(3H)-Pyrimidinethione and 3( 2H)-PyrMazlnethione. Tautomerism and Phototautomerism Maciej J. Nowak, Leszek Lapinski, Jan Fulara, Institute of Physics. Polish Academy of Sciences, AI. Lotnikow 32/46, 02-668 Warsaw, Poland
Andrzej I.&,
and Ludwik Adamowicz*
Department of Chemistry, The University of Arizona, Tucson, Arizona 85721 (Received: July 10, 1990; In Final Form: September 14, 1990) IR spectra of 4(3H)-pyrimidinethioneand 3(2H)-pyridazinethioneisolated in argon and nitrogen matrices are reported. The thiol and thione tautomeric forms of 4(3H)-pyrimidinethione were found in matrices in relative concentrations -5:l. IR spectra of gaseous sample showed that the ratio of tautomers in matrices corresponds to the gas-phase tautomeric equilibrium. Only the thione form of 3(2H)-pyridazinethione was observed in matrices. UV-vis irradiation of the matrices caused the conversion of the thione forms of the studied molecules into the thiol forms. The IR spectra of the thione and thiol tautomers were predicted theoretically at SCF/3-21GS level, and a comparison of the predicted and experimentally observed spectra enabled an assignment of most of the observed bands.
Introduction In the present work we report spectroscopical and a b initio quantum mechanical studies on two closely related diazinethiones, 4(3H)-pyrimidinethione and 3(2H)-pyridazinethione. These r\
Y
4(3H)-pyrimidinethione (X = CH, Y = N) 3(2H)-pyridazinethione (X = N, Y = CH) ~
compounds differ by the relative position of the CH and N groups in the aromatic ring. Such a minor structural alternation considerably differentiates the physicochemical properties of diazinethiones and affects their tautomerism. Our interest in diazinethiones stems from their presence in the biological material. The diazinethione moiety can be found in biologically important molecules such as, for example, in 6mercaptopurine (used in cancer chemotherapy') and in various clinically useful drugs.2 Diazinethiones undergo diverse chemical transformations, making them convenient intermediates in the synthesis of various bioorganic compounds. In particular, the sulfur substituent can be easily removed by reductive or oxidative desulfurization, it can be modified to give ring-fused pyrimidines or pyrazines, or it can be displaced by a variety of nucleophile^.^ (1) Landquist, J. K. In Comprehensive Heterocyclic Chemistry; Katritzky, A. R., Rees, Ch. W., Eds.; Pergamon Press: Oxford, 1984; Vol. 1. (2) Hurst, D. T. In An Introduction to the Chemistry and Biochemistry of Pyrimidines, Purines and Pteridines; Wiley: New York, 1980. ( 3 ) Harnden. M. R.; Hurst, D. T. Ausf. J . Chem. 1990,43, 5 5 ; Aust. J . Chem. 1983, 36, 1477; Aust. J . Chem. 1988, 41, 1209.
Diazinethiones may occur in various tautomeric forms in which the protons are located either a t the sulfur atoms (thiols) or nitrogen atoms (thiones). The recent 13CNMR4 and UV5 studies and dipole moment measurements6 performed for pyrimidine-4thione and its N- and S-methylated derivatives dissolved in DMSO, benzene, and water revealed that this compound exists in the thione form with the labile proton attached to the N 3 endocyclic nitrogen atom. The thione form was also ascribed to pyridazine-3-thione following the studies in the crystalline state7#* and in water solutions? The pK, measurements have shown that pyrimidine-4thione occurs in polar solvents as a mixture of 4(3H) and 4(1H) tautomers in a 2:l proportion.lOJl Although no indication of the thiol form has been found in the parent compounds, data exist suggesting that 2-phenylpyrimidine-4-thione may occur in that form in the DMSO solution." The tautomerism of heterocycles is strongly influenced by environmental effects. In particular, it has been showni2-15that the vapor-phase protomeric equilibria for oxo- and mercaptopyridine and -pyrimidines differ from the corresponding equilibria in solutions by factors ranging from lo3 to lo5. In the present work, we estimated the gas-phase tautomerism of pyrimidine-4-thione and pyridazine-3-thione by means of IR spectroscopy and ab initio quantum mechanical calculations. Although condensed-phase tautomerism seems to be more biologically relevant, the gas-phase data constitute a helpful prerequisite for the determination of the solvent equilibria. It has recently been recognized that there exists another factor that may strongly influence the tautomeric equilibria-irradiation by UV light. The spectroscopical experiments performed in our laboratory have shown that the UV irradiation of molecules deposited in inert-gase matrices induces an intramolecular proton transfer. For example, upon UV irradiation with the 313-nm line of the high-pressure mercury lamp, the oxo tautomeric forms of matrix isolated 2( 1H)-pyridone,16 4(3H)-pyrimidone,I7JE 3(4) Barlin, G. B.; Fcnn, M. D. Heterocycles 1986, 24, 1301.
(5) Albert, A.; Barlin, G. B. J . Chem. Soc. 1962, 3129. (6) Mussetta, M.-T.;Selim, M.; Trinh, N . Q.C. R. Hebd. Seances Acad. Sci., Ser. C 1973, 276, 1341. (7) Lautie, A.; Hervieu, J.; Belloc, J. Spectrochim. Acta 1983, 39A, 367. (8) Carlisle, C. H.; Hossain, M. B. Acta Crysfollogr. 1961, 21, 249. (9) Barlin, G. B.; Young, A. C. J . Chem. Soc. B 1971, 1261. (IO) Katritzky, A. R.; Lagowski, J. M. Adu. Heterocycl. Chem. 1963, I , 339. (1 1 ) The Tautomerism of Heterocycles; Adu. Heterocycl. Chem., Suppl. I ; Elguero, J., Marzin, C., Katritzky, A. R., Linda, P., Eds.; Academic Pres: New York. 1976. (12) Levin, E. S.;Rodionowa, G. N . Dokl. Chem. (Engl. Transl.) 1967, 172, 75. (13) Beak, P.; Fry, Jr., F.S.;Lee, J.; Staele, F. J . Am. Chem. Soc. 1976, 98, 171. (14) Beak, P. Acc. Chem. Res. 1979, 10, 186. (1 5 ) Shugar, D.; Psoda, A.; Lnndoldt-Bornstein, submitted.
0022-3654/91/2095-2404$02.50/00 1991 American Chemical Society
The Journal of Physical Chemistry, Vol. 95, No. 6, 1991 2405
Tautomerism and Phototautomerism
TABLE I: Total Electronic and Nuclear Vibration Energies of tbe 4 ( 3 H ) - P y r i m i d i ~ t b mTautomeric Forms' 4s-pmd 4HS-pmd*' 4HS-pmdd -656.872 217 -656.875 048 -656.889 533 SCF/3-2 1G* 0.082 708 0.082682 0.087 115 ZPE/3-2 1G* -660.246 551 -660.248 669 -660.248 224 SCF/DZP -0.975 123 -0.975 233 -0.971 760 MBPT(2)/DZP Contributions to the Relative Stability SCF/3-2 1G* 0.00 45.46 38.03 SCF/ DZP 0.00 4.39 -1.17 0.00 -8.83 -9.12 MBPT(Z)/DZP 0.00 -10.53 -10.59 0.91*ZPE/3-21GS
SCF/3-21G* SCF/DZP
4S-pmd* -656.868 857 0.086 5 1 1 -660.228 248 -0.973 946 54.28 52.45 -5.74 -1.44
0.00
Cumulative Relative Stabilityb -14.97
-20.88
45.27
3.78 3.55
Dipole Moment, D 3.10 2.91
1.22 1.22
9.18 8.82
"The relative stability calculated taking the 4s-pmd form as reference. Energies in hartrees, relative energies in kJ mol-'. "CF/DZP+MBPT(2)/DZP+0.9lCZPE/3-2IG*. CTheproton of the SH bond located close to the H(C5) atom. dThe proton of the SH bond located close to the N(3) atom. TABLE II: Total Electronic and Nuclear Vibration Energies of the 3(2H)-Pyridazinethione Tautomeric Forms'
3s-pdZ 3HS-pdZ -656.850617 -656.826683 SCF/3-2 1G* 0.086 464 0.081 496 ZPE/3-21G* -660.21 1 644 -660.201 012 SCF/DZP -0.977 682 -0.983 652 MBPT(2)/DZP Contributions to the Relative Stability SCF/3-2IGS 0.00 62.8 0.91 *ZPE/3-21GS 0.00 -11.9 SCF/DZP 0.00 27.9 0.00 -15.7 MBPT(Z)/DZP Cumulative Relative Stabilityb 0.00
SCF/3-21G* SCF/DZP
Dipole Moment, D 5.18 4.99
0.3
3.71 3.62
The relative stability calculated taking the 3s-pdz form as reference. Energies in hartrees, relative energies in kJ mol-'. bSCF/ DZP+MBPT(Z)/DZP+O.91 *ZPE/3-21G1. (2H)-pyridazinone,I9 and cytosine20convert to the corresponding hydroxy forms. We also found that a similar photoreaction occurs for a sulfur derivative of oxopyridine, Le., for 2( 1H)-pyridinethione, isolated in the Ar and N2 matrices.21 In the present work we have extended our investigation to a new class of heterocyclic compounds-the diazinethiones.
Computational Methods The structure optimizations for 4(3H)-pyrimidinethione and 3(2H)-pyridazinethione molecules in their thione and thiol (mercapto) tautomeric forms were performed by using the SCF procedure with the standard 3-21G* basis set with the GAUSSIAN 86 program.22 Both molecules were assumed planar. The IR frequencies and intensities were subsequently calculated by using the SCF analytical derivative procedure incorporated in GAUSSIAN 86. The calculated wavenumbers of all the normal modes were (16) Nowak, M. J.; Fulara, J.; Lapinski, L.; Les, A.; Adamowicz. L., manuscript in preparation. (17) N0wak.M. J.; Fulara, J.; Lapinski, L. J . Mol. Srrucr. 1988,175,91. (18) Lapinslu, L.; Fulara, J.; Nowak, M. J. Specrrochim. Acru, Purr A 1990,46,61. (19) Lapinski. L.;Fulara, J.; Czenninski. R.; Nowak, M. J. Specrrochim. Acru, Purr A, in press. (20) Lapinski, L.; Nowak, M.J.; Fulara, J.; Les, A.; Adamowicz, L. J . Phys. Chem.. in press. (21) Nowak, M. J.; Lapinski, L.; Rostkowska, H.;Les, A.; Adamowicz, L. J. Phys. Chem.. in press. (22) GAUSSIAN86. release c; Bimbley. J. S.;Frish, M.; Raghavathan, K.; De Frees, D.; Schlegel, H. B.; Whiteside, R.; Fluder, E.; Seeger, S.;Fox, D. J.; Head-Gordon, M.; Pople, J. A.; CarnegieMellon University: Pittsburgh, 1988.
TABLE III: Contributions to the Relative Energy of the t(lH)-Pyridinethione Tautomeric Forms (kJ mor')
E(thio1) E(thione) -0.84 -12.01
SCF MBPT(2) full (core frozen)' calcns with first-order correlation orbitals' (FOCOs) MBPT(2) -2.96 MBPT(4)-MBPT(2), scaledb 6.62 CCSD-MBPT(2), scaledc 4.28 T(CCSD), scaledd 0.42 0.91ZPE -10.98 cumulative re1 density, kJ mol-' SCF + 0.91ZPE -1 1.82 SCF+MBPT(2) + 0.91ZPE -23.83 SCF+MBPT(2) + MBPT(4) + O.9lZPE -17.21 SCF+MBPT(2) + CCSD+T(CCSD) + 0.91ZPE -19.13 'Seven 1s core orbitals were frozen. b[MBPT(4)'b'o' - MBPT(2)1hio1]/9'h'01 - [MBPT(4)'b'o"C- MBPT(2)*'a"0]/q'h"; tf MBPT(2)x(full)/MBPT(2)x(FOCO), x = thiol, thione. CSubstituteCCSD for MBPT(4) in footnote b. T(CCSD)'h"l/qtb'ol - T(CCSD)'hiOM/Tthione*
scaled down by a single factor of 0.9, which is a commonly used procedure to correct for the anharmonicity of the vibrations.23 The relative stability of the various tautomeric forms has been also estimated at the SCF+MBPT(2) level by using the double f basis set with polarization functions (DZP) located on all atoms (d symmetry functions on carbon, nitrogen, and sulfur; p symmetry functions on hydrogen atoms); see Tables I and 11. The role of the higher order electron correlation effects has been investigated on a model system, i.e., thioformamide, exhibiting the characteristic features of diazinethiones, i.e., the sulfur-carbon double bond and the tautomerizable proton located near the nitrogen atom; see Table 111. The correlated calculations were performed with the first-order correlation orbitals (FOCOS)~'and the singleand double-excitation coupled cluster (CCSD) methods with noniterative inclusion of the triple excitations (Le., CCSD+T(CCSD)).Z5 The theoretically predicted vibrational frequencies enable the analysis of the vibrational modes in terms of the internal coordinates. The internal coordinates were chosen in the way recommended by h l a y et a1.% Those coordinates are listed in Tables (23) Hess, B. A., Jr.; Schaad, L. L.; CBrsky, P.; Zahradnik, R. Chem. Reu. 709. (24) Adamowicz, L.; Bartlett, R. J. J . Chem. Phys. 1987, 86, 6314. Adamowicz, L. J . Phys. Chem. 1989,93, 1780. Adamowicz, L. J . Compur. Chem. 1987, 10, 928. (25) Lee, Y.S.;Kucharski, S.;Bartlett, R. J. J. Chem. Phys. 1984,81. 5906. (26) Pulay, P.; Fogarasi, G.; Pang, F.;Boggs, J. E. J . Am. Chem. SOC. 1979, 101, 2550. 1986,86,
2406 The Journal of Physical Chemistry, Vol. 95, No. 6,I991
Nowak et al.
CHART I
D
3
%
W
U
2
a a
ci L0 A
m
a LS-pmd
C
1,H S- pm d 0.1
S
0.1
4 6
s H'
f
4 A
I
H 4 S - pm d'
4 H S- pmd*
IV and VI11 in the supplementary material (see paragraph at end of paper). The transformation of the force constant matrix from Cartesian coordinates to the internal coordinates was performed, and the potential energy distribution (PED) was calculated for all the normal modes. The frequencies and PEDS of the calculated normal modes together with the theoretically predicted intensities of the corresponding IR bands are given in Tables V, VI, VII, and IX in the supplementary material.
Experimental Section 4(3H)-Pyrimidinethione was prepared in the standard reaction with PISloin boiling pyridinez7 from its oxo analogue, 4(3H)pyrimidinone (purchased from Sigma Chemical Co.). 3(2H)Pyridazinethione was obtained in a similar way from 3(2H)pyridazinone (synthesized as described in ref 28.) The compounds were purified by recrystallization, column chromatography, and vacuum sublimation. Matrices were prepared as described elsewhere.29 In brief, matrices were formed by deposition of vapors of the studied compound mixed with a large excess of matrix gas (argon or nitrogen) on the CsI window mounted to the coldfinger of our continuous-flow helium cryostat. The temperature of the cold CsI window was 6-7 K. Matrix gases of spectral grade were obtained from VEB Technische Gase, Leipzig, GDR. Investigated compounds evaporated from the microovern were placed inside the vacuum chamber of the cryostat and heated up to about 350 K (the temperature was the same for both compounds). Gas-phase measurements were performed in a 12-cm-pathlength quartz cell sealed under vacuum and placed in the thermostat. The cell contained 2 mg of 4(3H)-pyrimidinethione, which totally evaporated when the temperature of the cell reached 483 K. The temperature was constant with accuracy of 0.1 K. Thermal decomposition occurred at temperature higher than 483 K. Infrared absorption spectra were taken on the Perkin-Elmer 580B spectrophotometer. Integral absorbances of the bands were measured by numerical integration. UV-vis irradiation of the matrices was performed by the light from a 200-W high-pressure mercury lamp fitted with a cutoff filter (aqueous solution of K N 0 3 ) transmitting the light with X (27) Duffin, G. F.; Kendall, J. D. J . Chem. S a . 1959,3789. Armarego, W. L.F.J . Chem. Soc. 1965,2778. Koppel, H.C.; Springer, R. H.;Robins,
R. K.; Cheng, C. C. J . Org. Chem. 1%1,26,792. Lardenois, P.; Selim, M.; Selim, M . Bull. Soc. Chim. Fr. 1971, 1858. Mahnvald, R.; Wagner, G. Pharmazie 1982, 37, 257. (28) Overend, W. G.; Wiggins, L. F. J . Chem. Soc. 1947, 239. Homer, R. F.; Gregory, H.; Wiggins, L. F. J . Chem. Soc. 1984, 2191. (29) Szczesniak. M.; Nowak, M. J.; Szczepaniak, K.; Person, W. B.; Shugar, J . Am. Chem. SOC.1983,105, 5970.
0.0
0 .c
.i; - -
3450 3350 3100 3000 2650 2550 c i ' Fipn 1. Infrared absorption spectrum of 4(3H)-pyrimidinethioneisolated in low-temperature matrices in the region 3500-2500 cm-I: (A) argon matrix; (B) the effect of 0.5 h of UV-vis irradiation of argon matrix; (C) nitrogen matrix; (D) the effect of 0.5 h of UV-vis irradiation of nitrogen matrix.
> 330 nm. The time of irradiation was 30 min for 4(3H)-pyrimidinethione and 1 h for 3(2H)-pyridazinethione. Tautomerism 1 . 4(3H)-Pyrimidinethione. 4(3H)-Pyrimidinethione may potentially exist in three tautomeric forms, shown in Scheme I. Since the theoretical calculations predict very large differences between the internal energies of the thione forms with H atom attached to nitrogen in 1 or 3 position (Table I), we consider in this work only the tautomeric equilibria between the thione form with the H atom at nitrogen in the 3 position (4s-pmd) and the thiol form (4Hs-pmd; Chart I). I . I . NH and SH Stretching Modes. The analysis of the spectra of matrix isolated 4(3H)-pyrimidinethione showed the presence of the very characteristic absorptions due to stretching vibrations of the N H and SH groups at 3394 and 2604 cm-' (Ar; Figures la,c and 2). This indicates that in inert-gas matrices both thiol (4HS-pmd) and thione (4s-pmd) tautomers of 4(3H)-pyrimidinethione exist simultaneously. The absorption bands in the IR spectra of both tautomers were separated, in the whole studied range, by using the phototautomeric effect (see next section). We compared those resolved spectra with the results of theoretical normal-mode calculations. The agreement between the experimental and theoretically predicted spectra seems to be sufficiently good to support our conclusion that the spectra were due to the presence of the two tautomeric forms in the matrix. 1.2. ThiollThione Ratio. The relative concentrations of the thiol and thione tautomeric forms of 4(3H)-pyrimidinethione in the matrix may be estimated by using different methods.I8 The most reliable method, in our opinion, is based on the change of IR band intensities during the phototautomeric reaction. In the first version of this method we used the integral intensities of selected individual bands before and after UV irradiation. We observed that after 30 min of UV-vis irradiation of the matrix, each of the bands of 4s-pmd decreased to traces with only 4% of their initial intensity. It meant that 96% of the molecules, initially in the thione form, converted to the thiol form, and hence, since there were no other photoproducts, one might apply the formula'*J [thiol]/[thione] = 0.961/(Pv - I)
(1)
where I is the intensity of a band of the thiol form in the initial
The Journal of Physical Chemistry, Vol. 95, No. 6, 1991 2407
Tautomerism and Phototautomerism
CHART I1
0.0- 1 0.0 I L 3500 3300 2700 2500 cm-' Figure 2. Comparison of NH and SH stretching absorption bands of 4(3H)-pyrimidinethioneobserved (A) in gas phase (483 K) and (B) in an argon matrix. In the gas-phase spectrum the background due to quartz windows is electronically subtracted.
spectrum and PVis the intensity of the same band after irradiation. The integral intensities of the bands at 1565, 1465, 1382, 1294, 1 144, and 667 cm-I in Ar and the corresponding bands in N, matrices were used in this estimation. The average values of the obtained [thiol]/[thione] ratio was 5.1 f 0.3 in Ar and 5.3 f 0.3 in N2. One may also employ another version of the method for estimation of the [thiol]/[thione] ratio using the sums of intensities of all the observed bands (Eobl"P) scaled by the sums of their theoretically predicted intensities (EokAth(thiol)) and applying the formula'8-21
E IcxP(thiol) -=
[thione]
Ath(thione)
- obs
Ob
Oh
Ob
E Ath(thiol) E IcxP(thione)
I?\
\&I
Wgobtained the ratio of the tautomers [thiol]/[thione] = 5.9 and 5.3 (using intensities measured for compounds isolated in Ar and N, matrices, respectively). Although the intensities of individual bands are often not predicted precisely by a SCF/3-21G* calculation and the experimentally measured intensities are perturbed by the matrix environment, the above results are in reasonable agreement with the estimation based on the phototautomeric effect. 1.3. Free Energy Difference. Assuming that the ratio of tautomers observed in the matrix correspond to the equilibrium in the gas from which the matrix was formed (at 350 K, the temperature of the microoven), the free energy difference might be estimated as 4.7 f 0.4 kJ/mol. For the oxygen analogue, 4(3H)-pyrimidinone, the free energy difference between the oxo and hydroxy tautomers was estimated as 2.4 kJ/mol from the gas-phase measurement" and 2.5 kJ/mol from the matrix isolation measurement.I8 Likewise, the results of measurements of the tautomeric equilibria of 2( 1H)-pyridinone performed in the gas phase ([hydroxy]/[oxo] = 2.5 at 403 K)" and in inert-gas matrices ([hydroxy]/[oxo] = 2.6, a t 380 K)16 are in fair agreement. In general, it is not quite clear if the thermodynamic tautomeric equilibrium was completely achieved in the vapors coming from the microoven in the cryostat and how close the frozen equilibria in matrices correspond to those observed in the gas phase. There are some examples showing that the tautomeric equilibria determined from gas-phase measurements agree with the ratio of tautomers observed in matrices. Interestingly, the relative concentrations of tautomers in matrices are not strongly influenced by other factors such as different rates of sublimation of the tautomers.
1.4. Gas-Phase Data. We performed a gas-phase measurement in the 3600-2500-cm-' range of the infrared spectrum for 4(3H)-pyrimidinethione. The fragments of the spectrum taken for a totally evaporated sample at 483 K are presented in Figure 2. The ratio of the intensities of the bands due to the NH (at 3408, 3396 cm-') and SH (at 2608, 2594 cm-I) stretching vibrations I(rNH)/I(rSH) = 3.72 shows that a considerable amount of molecules adopt the thione form in the gas phase at 483 K. From the equation [thiol] PXP(rsH) A(rNH) (3) Ithionel pxp(rNH) A(rSH)
--
---
where PXP(x)is the observed integral absorbance of the band x and A ( x ) is the absolute intensity of the band, we estimated the ratio A(rNH)/A(rSH) = 11.1 for the absorption bands in the Ar matrix. In this estimation, the formerly determined ratio of the tautomers in the matrix was taken into account. Assuming that the A(rNH)/A(rSH)ratio is not much different for the absorptions in the gas phase, we may estimate the ratio of tautomers in gas phase at 483 K as [thiol]/[thione] = 3.0. This corresponds to the free energy difference of 4.4 kJ/mol. The theoretically predicted large differences of the energy of the tautomers of 20.9 kJ/mol (MBPT(2) level, see Table I) seems to be an overestimation. In the oxygen analogue, Le., 4(3H)-pyrimidinone,18 the ratio of concentrations of the hydroxy and the oxo forms in matrices was about 1:2. Comparing the oxo and thio analogues, a shift toward higher relative stabilization of the fully aromatic form can be noticed when oxygen is substituted by the sulfur atom. Similarly, in 2( 1H)-pyridinethioneZ1the [thio]/[thione] ratio was 30:1, while in 2( 1H)-pyridinone the [hydroxy]/[oxo] ratio was about 2.6:1.16 2. 3(2H)-Pyridazinethione. For 3(2H)-pyridazinethione only two non-meso-ionic tautomers may exist. These are the thione (3s-pdz) and the thiol (3HS-pdz) forms (see Chart 11). In the matrix isolation study performed in the present work, only the 3s-pdz form was detected (after deposition on the matrix, Le., before UV-vis irradiation). In the spectral range where the NH and S H stretching bands may be expected, the N H band was found in the Ar and N2 matrix spectra but no trace of the SH absorption was observed (Figure 3a,c), Absorption bands registered in the entire spectral region fit in with the theoretically predicted IR spectrum of the 3s-pdz form (see next section). This spectrum seems to be due to one form (3s-pdz) because during UV-vis irradiation all the bands decreased at the same rate. The lack of the SH stretching band and of other absorptions in the positions where the thiol bands were found after UV-vis irradiation indicates that in the limit of detection of our IR spectroscopy no 3HS-pdz form exists in Ar and N2matrices. Phototautomerism The effect of UV-vis irradiation (A > 330 nm) of matrix-isolated 4(3H)-pyrimidinethione was a decrease of the bands due to the thione form (to their nearly complete disappearance after 30 min of irradiation) and an increase of the bands due to the thiol form (both effects presented in Figures lb,d and 4b,c and in Figure 6b,c in the supplementary material). No new bands appear throughout the entire studied spectral range (4000-250
2408 The Journal of Physical Chemistry, Vol. 95, No. 6, 1991
Nowak et al,
I
B
-0 --L
Cl
W U
z a m a
0
v i m Q
U W
z a
m Q
0
A
v,
m
a
-6 %
0.1 .
0.4
0.0I -L 3450 3350 I
I
0.01
I
1
I
3100 3000
4
0.1
A
0.:
\
0.1
I
0 , o L
1600
J 1400
1200
4 1000 cm-'
2650 2550 cm-l
Figure 3. Infrared absorption spectrum of 3(2H)-pyridazinethione isolated in low-temperature matrices in the region 3500-2500 cm-': (A) argon matrix; (B) the effect of 1 h of UV-vis irradiation of the argon matrix; (C) nitrogen matrix; (D) the effect of 1 h of UV-vis irradiation of the nitrogen matrix.
lA I
L
L
I
I
900 700 500 300 c 6 ' Figure 5. (A) IR absorption spectrum of argon matrix isolated 3(2H)-pyridazinethione (1650-250 cm-I); (B) effect of 1 h of UV-vis irradiation. The black dots indicate the remains of absorptions due to thione form.
1600
1400
1200
1000 cm"
900 700 500 300 crri' Figure 4. (A) IR absorption spectrum of argon matrix isolated 4-
(3H)-pyrimidinethionc (1650-250 cm-'); (B)effect of 0.5 h of UV-vis irradiation of the argon matrix; (C) the difference spectrum (A) minus (B), where UV-vis induced changes in the spectrum are better visualized.
em-'). One hour of UV-vis irradiation (A > 330 nm) of matrix-isolated 3(2H)-pyridazine-thione caused a nearly complete disappearance of the initial IR spectrum (only traces of bands with about 6% of their initial intensities were left). The coloration of the matrix changed from greenish yellow to nearly white. Structural similarity of 3(2H)-pyridazinethione with 4(3H)-pyrimidinethione led to a supposition that for this case we also observed a phototautomeric reaction. In the spectrum of the photoproduct, taken after UV-vis irradiation, the characteristic SH stretching band appeared at 2601 cm-' (Figure 3b,d). The spectrum that appeared after UV-vis irradiation (Figure 5b and Figure 7b in the supplementary material) fits in with the predicted spectrum of the thiol tautomer sufficiently well to support the assignment of the photoproduct to the thiol 3HS-pdz form. In our previous studies concerning phototautomeric reactions of matrix-isolated 4(3H)-pyrimidinoneI8and 3(2H)-pyrida~inone,'~ we observed two simultaneous photoreactions, Le., the photoinduced proton transfer and a ring-opening photoreaction of the oxo form. The open-chain photoproducts were conjugated ketenes (with the characteristic very strong infrared band near 2140-2130 cm-I). In the thio analogues of those compounds, studied in the present work, no bands that could be attributed to thioketenes were observed. That is in agreement with the general tendency of the thiones to photoinduced hydrogen abstraction reactions and almost no tendency to cleavage reactions.30 Infrared Absorption Spectra 1 . General Characteristics. 4(3H)-Pyrimidinethione. We report the IR spectra of Ar and N2matrix-isolated tautomers of (30) Ramamurthy, V. Thiocarbonyl Photochemistry. Org. Photochem. 1985, 7, 231-338.
Tautomerism and Phototautomerism 4(3H)-pyrimidinethione and 3(2H)-pyridazinethione. We assumed that the spectra of matrix isolated species correspond to the spectra that could be obtained for gaseous samples. In Figure 2 we compare the high-frequency region of the spectrum of 4(3H)-pyrimidinethione obtained in the Ar matrix and in the gas phase. In the gas phase, the N H and SH stretching vibration ) 2608, bands are sharp doublets (at 3408, 3396 cm-' ( r N Hand 2594 cm-I (rSH)),an effect that could be attributed to unresolved rotational states. The positions of those bands are very close to the frequencies in the matrix spectrum. The spectrum of matrix-isolated 4(3H)-pyrimidinethione consists of the two sets of bands attributed to the different tautomers, 4S-pmd and 4HS-pmd, coexisting in the matrix. These sets were separated by using the method described in ref 18 based on the change of the spectrum caused by UV-vis irradiation. The spectra obtained before irradiation are shown in Figures la,c and 4a and Figure 6a (supplementary material). Spectra after irradiation are shown in Figures 1b,d and 4b and Figure 6b (supplementary material). The effect of irradiation is illustrated by the difference spectra (Figure 4c,and Figure 6c (supplementary material)) where the positive peaks correspond to 4s-pmd and the negative peaks to 4HS-pmd. As is seen, the changes due to irradiation are not significant since the substrate of the phototautomeric reaction4s-pmd-is a minor tautomer in the matrix. 3(2H)-Pyridazinethione. The spectra of 3(2H)-pyridazinethione obtained in Ar and N2 matrices before irradiation (presented in Figures 3a,c and 5a and Figure 7a (supplementary material)) are assigned to only one tautomeric form, the thione form (3s-pdz). In the case of this molecule, the effect of UV-vis irradiation is much more pronounced. The spectra obtained after irradiation consist of the bands attributed mainly to the second thiol tautomer (3HS-pdz). The weak absorptions due to the remains of 3s-pdz that are left after irradiation are shown by black dots in the figures (Figures 3b,d and 5b and Figure 7b (supplementary material)). The wavenumbers of the maxima of the absorption bands together with their relative intensities are collected in Tables V, VI, VIII, and IX (supplementary material) for all the observed tautomers and compared with the theoretically obtained spectra. The assignment of the experimental bands to the normal modes, given in the tables, is based mainly on the comparison of experimental positions and intensities of the bands with calculated ones. The matrix effect, observed in case of some bands, Le., the shift of the ' bands caused by the change of the matrix (Ar into N2), helped to assign the bands involving the N H deformation, which are very sensitive to the environment. 2. IR Spectra of the Thione Forms. The 3500-3000-~m-~ region: The bands of the N H stretching vibration were found at 3394 (Ar) and 3400 cm-l (Ar) in the spectra of 4s-pmd and 3s-pdz, respectively. The N H stretching band in the spectrum of 2( 1H)-pyridinethione reported in our previous workz1was at 3400 cm-I (Ar). Comparing the spectra of the thio compounds with the spectra of their oxo analogues, we found that the wavenumbers of the N H stretching vibrations are lower when oxygen is replaced by the sulfur atom. In the oxo forms of 4(3H)-pyrimidinone, 3(2H)-pyridazinone, and 2( 1H)-pyridinone, the corresponding bands were at 3428,3426, and 3438 cm-I (Ar), respectively. This characteristic feature was noticed first by Lautie and Novak in their studies of CC14 solutions and crystalline samples of thiocarbonyl heterocycle^.^' The rNH bands are sensitive to the matrix environment. While the frequencies shift down by 6-8 cm-l in the N 2 matrix, the intensities increase in comparison to the Ar matrix. As presented in Tables VI and IX (supplementary material), the relative intensities (scaled as described in the footnotes of the tables) are l .5 times higher in the nitrogen matrix than in the argon matrix. The bands due to the CH stretchings were not observed in the spectra of the thione forms of the studied compounds. The 170&250-cm-' region: The characteristic band of the stretching vibration of the two conjugated double bonds in the (31) Lautie, A.; Novak, A. Chem. Phys. Lori. 1980, 71. 290.
The Journal of Physical Chemistry, Vol. 95, No. 6,1991 2409 ring, due to the 45 mode, is found a t 1607 (Ar) and at 1603 cm-l (Ar) in the spectra of 4s-pmd and 3s-pdz. The wavenumbers of those bands are well predicted theoretically. An intense band due to the N H in-plane bending vibration ( 4 6 ) is predicted by the SCF/3-21G* calculation for both compounds near 1560 cm-', which fits in well with the experimental absorptions attributed to the 4 6 mode (Tables VI and IX (supplementary material)). In the spectra of 3s-pdz, several details of assignment in the region 1250-950 cm-' are not clear. The SCF/3-21G* calculation predicts two relatively strong bands at 1173 (411) and 1149 cm-' (412). In the experimental spectrum, only one very strong band consisting of two components a t 1182 and 1185 cm-l (Ar) is observed. It seems that the two components are due to one of the two theoretically predicted bands. The weaker bands at 1206, 1217, and 1230 cm-' are most possibly due to the second one. At present, a more accurate assignment in this region is not possible. The frequencies of two bands with considerable contributions from the N-N stretching vibration ( 4 1 4 , 4 1 7 ) are strongly underestimated by the SCF/3-21G* calculations, and the assignment of the three bands between 1100 and lo00 cm-' is not quite certain. In the same region (1250-950 an-')of the spectrum of 4S-pmd, two relatively strong bands Q11 and 4 1 2 are predicted, while only one band of a comparable intensity is observed at 1170 cm-l (Ar). Similar discrepancies between the theoretical and experimental spectra in this range were previously discussed in the reported spectrum of 4(3H)-pyrimidinone.I8 The assignment of the infrared absorption bands in the region below 950 cm-l is in our opinion quite reliable for both studied compounds. The predicted frequencies of the out-of-plane vibrations are overestimated as usual in calculations performed for N - h e t e r o ~ y c l e s , ' ~but - ~ ~taking ~ ~ ~ this into consideration the assignment does not seem to lead to ambiguities. The characteristic bands in this region are due to the N-H wagging vibrations (419). Although their predicted frequencies are strongly overestimated (by about 70 cm-I), those bands (placed at 740 and 731 cm-' for 4s-pmd and 3s-pdz) are easily recognized in the experimental spectrum because of their strength and because of their considerable (about 30 cm-I) blue shift in the N 2 matrix (more interaction with the trapped molecules). In the l i t e r a t ~ r e the ~ ~ band , ~ ~ due to the C-S stretching vibration was usually expected in the region 1400-1050 cm-I. An analysis of the theoretical spectra of 4s-pmd and 3S-pdz shows that there is not only one normal mode due to the C-S stretching vibration, but this vibration contributes to several normal modes, for which frequencies are spread from 1200 to 400 cm-I. The C=S stretching vibration is always coupled with in-plane deformational vibrations of the ring. In the spectra of 4s-pmd and 3S-pdz, the bands of relatively high intensity found at 1170 (Ar) and 1182 cm-I (Ar), respectively, were interpreted as being due to 4 1 2 mode. Similar characteristic intense bands are also present in the spectra of other thiocarbonyl heterocycles: 2( 1H)pyridinethioneZ1and 2-thio-, 4-thio-, and 2,4-dithio~racil.~~ The /3 R1 ring in-plane deformational vibration contributes predominantly to the normal mode of this band while the contribution of the C=S stretch is only 10-25%. The C=S stretching contributes also to several other normal modes (Tables VI and IX (supplementary material)). The most considerable contribution (30-40%) is in the case of the band placed near 440-450 cm-I (mode 4 2 4 in case of 4s-pmd and 3S-pdz), where C = S stretching is coupled with the /3 R2 ring in-plane deformational vibration. Similar absorption bands, where the C=S stretching is coupled with the ring deformation (according to calculations), are also observed in other related compound^.^'.^^ Rare Thione Form ( H a t N I ) . The theoretical calculations of the IR absorption spectrum of the rare tautomeric form, the thione with a labile hydrogen attached to nitrogen in position 1 (4s-pmd in Chart I), were also performed. The spectral positions (32) BeIlamy, L. J. Infrared Spectra of Complex Molecules; Chapman and Hall: New York, 1980. ( 3 3 ) Spinner, E. J . Chem. SOC.1960, 1237. (34) Rostkowska, H.; Szczepaniak, K.; Nowak, M. J.; Leszczynski, J.; KuBulat, K.; Person, W. B. J . Am. Chem. Soc., in press.
2410 The Journal of Physical Chemistry, Vol. 95, No. 6, 1991
and the intensities of the absorption bands of 4s-pmd (Table VI (supplementary material)) and 4S-pmd* differed considerably, which would facilitate the identification of isomers. Comparing results of calculations with the experimental spectrum, however, we cannot find any traces of absorptions due to the 4S-pmd* form. 3. IR Spectra of the Thiol (Mercapto) Forms. The IR spectra of the thiol forms of both studied compounds are better predicted theoretically than spectra of the thione form, and hence the assignment seems reliable in most of the spectral regions. The 3100-2600-cm-' range: In this range only very low intensity absorption bands are expected. Contrary to the spectra of the thione form, in thiol tautomer it was possible to detect the absorption bands of C H stretching vibrations (near 3000 cm-I). The characteristic, for the thiol tautomer, bands observed in the IR spectra of 4HS-pmd and 3HS-pdz are due to vibrations of the S H group. The low-intensity bands of SH stretching vibration ( 4 4 ) were found at 2604 and 2601 cm-' (Ar) for the two compounds, respectively. For other related compounds isolated in the argon matrix, the frequencies of corresponding bands were 2610 cm-' for 2-pyridinethio121and 26 15 cm-' for 2-p~rimidinethiol.~~ The 1600-250-cm-' range: Bands of the SH in-plane bending vibrations were found at 894 and 889 cm-' (Ar) for 4HS-pmd (417) and 3HS-pdz (416) (for 2-pyridinethiol and 2-pyrimidinethiol at 88421and 907 cm-' (Ar),35respectively). In nitrogen matrices the bands due to the S H bending modes are blue shifted by 3-4 cm-I. In the range 1200-800 cm-I, the calculated spectra fit in a little worse with the experimental ones than in other regions. In previous studies concerning theoretical interpretation of the IR spectra of heterocycles, there were always some uncertainties in this region. The average intensity of all the bands in the spectra of 4HS-pmd and 3HS-pdz is smaller than for 4s-pmd and 3s-pdz. For 3HS-pdz, the most intense band has the predicted absolute intensity only of 82 km/mol (similarly for 2-pyridinethiol the greater predicted intensity was 86 km/mol). Comparing the experimental spectra of 3s-pdz and the spectra of the product obtained in the photoreaction of 3s-pdz (Figures 3 and 5 , and Figure 7 (supplementary material)), it is easy to notice that the spectrum of the photoproduct consists of the bands with much smaller average intensity. That point, together with the fact that all stronger bands predicted for 3HS-pdz were found in the spectrum of the photoproduct, provides support for our conclusion that, in fact, the photoproduct is 3HS-pdz. 8-20929
Discussion Essential features of the IR matrix isolation and gas-phase spectra were resolved. However, there remain some uncertainities in the assignment that should be clarified in a subsequent investigation, for example, the origin of the peaks in the 1250950-cm-' region corresponding to the thione form of pyrimidine-4-thione. Also, the presence of the rare 4( 1H)-pyrimidinethione tautomer and the S H rotamer (SH directed toward the C5 atom) of 4-mercaptopyrimidine were not clearly established. We observed a tendency of a decreasing thiol/thione ratio in the following sequence: pyridine-2-thione (X = CH, Y = CH), pyrimidine-4-thione (X = CH, Y = N), and pyridazine-3-thione (X = N, Y = CH). Such a tendency is confirmed by the a b initio theoretical calculations and can be rationalized in the following way. The sulfur exocyclic atom is usually a better proton acceptor than the nitrogen endocyclic atom and, therefore, the proton that gives rise to the formation of the thiol tautomeric form in pyridine-2-thione (X = CH, Y = CH) is located predominantly at the sulfur atom. The thiol tautomer is additionally stabilized by the formation of an aromatic bonding structure. On the other hand, the pair of two adjacent endocyclic nitrogen atoms attracts a proton more strongly than daes a single endocyclic sulfur atom. Consequently, the proton that gives rise to the formation of the thione tautomeric form in pyridazine-3-thione (X = N , Y = CH) occupies the position near the nitrogen atom. An intermediate (35) Rostkowska, H.; Nowak, M. J.; Leszczynski, J., manuscript in prep aration.
Nowak et al. situation corresponds to the third case where two nitrogen atoms are separated by a C H group, as in pyrimidineethione (X = CH, Y = N). The proton attraction by the nitrogen moiety is weaker. Such a situation gives rise to both tautomeric forms, Le., thione and thiol in pyrimidine-Cthione. The numerical values of the thiol/thione ratio obtained experimentally significantly differ, however, from the theoretical predictions. It is difficult to recognize a single major contribution to the relative energy that could be responsible for such a discrepancy. Most probably, there are several factors, some of which are discussed as follows: ( I ) Zero-point vibrational energy: To obtain an alternative estimation of the zero-point vibrational contribution to the relative stability of tautomers, we used the experimental frequencies instead of theoretical ones. For a few modes not visible in the experimental spectrum, we still used the theoretical values. The relative ZPE values calculated from the experimental frequencies were not very different (6% and -2% deviation for pyrimidine-4-thione and pyridazine-3-thione, respectively) from the purely theoretical predictions. This indicates that errors in the ZPE calculation should not be a significant source of the discrepancy between the theoretical and experimental estimation of the relative stability of the tautomers. Such a conclusion is additionally supported by our ab initio calculations of the ZPE values performed with the MBPT(2)/6-31G** method on a model compound, Le., thioformamide. The scaled (by 0.91 factor) ZPE contribution to the tautomer relative stability diminished by 2 kJ mol-' (from 10.1 kJ mol-' at the SCF/3-21G* level to 8.0 kJ mol-' at the MBPT(2)/6-31G** level), remaining a nonnegligiblecontribution. ( 2 ) Thermodynamic Equilibrium: Another possible reason for this discrepancy may come from the interpretation of the experimental data. In the present experiment two sudden processes occur-sublimation in the microoven and subsequent freezing in the low-temperature matrix. These processes do not facilitate the thermodynamic equilibration of the matrix deposit. The extent to which thermodynamic equilibrium is reached in the matrix is uncertain. Consequently, the estimation of the relative tautomer stability based on the equilibrium thermodynamics may not be fully justified. In our experiment, the sample is heated up to a temperature close to the thermal decomposition threshold just before it is suddenly deposited in the low-temperature matrix. Under such experimental conditions, the molecules in the sample are probably excited to some higher vibrational states that can hardly be described by the commonly applied harmonic oscillator model. In principle, a more reliable estimation of the relative concentration of tautomers (isomers) should include rotational and vibrational partition functions, as shown by S l a n i ~ ~ a . ~ ~ ( 3 ) Inaccuracies in theoretical calculations: The theoretical ab initio calculations suffer from several drawbacks. First, the molecular geometry was optimized at the S C F level with a relatively poor basis set, and incomplete basis sets were used in the final S C F and MBPT(2) calculations. Second, the higher order electron correlation contribution is neglected. To estimate the importance of this contribution, the following calculations were performed. We chose a small model system, i.e., 2( 1ti)-pyridinethione, where the carbonsulfur double bond and the tautomerizable proton bound to the nitrogen atom are present simultaneously. For this system, we calculate the contribution to the relative stability of the tautomers arising from the second-order and higher order electron correlation effects (see Table 111 and Table X (supplementary material). We used the first-order correlation orbital (FOCO) method and the coupled cluster method (CC) described in detail e l s e ~ h e r e . ~The ~-~~ CC/FOCO method is a unique approach presently available to determine infinite-order electron correlation effects for larger molecular systems. The results for the model system presented in Table I11 and Table X (supplementary material) indicate that the cumulative contribution to the relative stability of the tautomers arising from the electron correlation effects in all orders is important. One may observe that the second-order correlation (36) Slanina, Z. Ado. Quantum Chem. 1981, 13, 89.
2411
J. Phys. Chem. 1991, 95, 241 1-2415 correction is only partially cancelled by the higher order corrections determined in the coupled cluster procedure. In spite of such a cancellation, the cumulative electron correlation contribution is large and amounts to almost 40% of the tautomer relative stability. This seems to suggest that one cannot obtain a correct relative energy of tautomers of 2( 1H)-pyridinethione at the lower orders of the many-body perturbation theory because of cancellations of lower order terms with higher order terms. We conclude that the large contribution to the relative stability of the tautomers arising from the low-order (e.g., from second order) perturbation theory may be considerably reduced by higher order terms. As a consequence, the relative stability estimated with the S C F scaled ZPE MBPT(2) higher order coorelation methods would fall closer to the experimentally derived values than the MBPT(2) result, as is probably true for diazinethiones. However, the inclusion of the higher order correlation terms does not seem to be enough to compensate for the systematic discrepancy between the theoretical and the experimental estimates of the relative stability of the tautomers.
difference was similar (AF 4.5 kJ/mol). 3(2H)-Pyridazinethione adopts exclusively the thione form in inert gas matrices. Upon UV irradiation, the photoinduced isomerization to thione occurs in the matrix isolated samples of 4(3H)-pyrimidinethione and 3(2H)-pyridazinethione. The IR spectra of the thione and thiol tautomers were predicted theoretically at the SCF/3-21G* level, and a comparison of the predicted and experimentally observed spectra gave an assignment of most of the observed bands. The theoretical a b initio evaluation of the thiol/thione concentration ratio falls closer to the experimental estimate when the higher order electron correlation effects are taken into account. However, the discrepancy between the theoretical and experimental estimates of the relative stability of the tautomer's still persists.
Conclusions
Supplementary Material Available: The internal coordinates (Table IV and VII), experimental frequencies, integral intensities, and assignment to the normal modes (Tables V, VI, VIII, IX) for 4S-pmd, 4HS-pmd, 3S-pdz, and 3HS-pdz tautomers; details of the ab initio calculations for 2(1H)-pyridinethione (Table X); IR spectra of N z matrix isolated 4(3H)-pyrimidinethione and 3(2H)-pyridazinethione (Figures 6 and 7) (1 5 pages). Ordering information is given on any current masthead page.
+
+
+
The gas-phase and matrix isolation experiments showed that 4(3H)-pyrimidinethione exists, under conditions where intermolecular interactions are minimized, as a mixture of thiol and thione tautomers. The relative concentration of tautomers, in the gas phase ([thiol]/[thione] 3 at 483 K) and in matrix experiments ([thiol]/[thione] 5 at 350 K), depends on the temperature. In both cases, the experimentally derived value of the free energy
- -
N
Acknowledgment. This study was supported, in its experimental part, by the grants C.P.B.P.Ol.12. and C.P.B.R.ll.05. provided by the Polish Academy of Sciences.
Kinetics of the Oxidation of NADH by Methylene Blue In a Closed System Peter Sevcikt and H. Brian Dunford* Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6C 2C2 (Received: July 16, 1990; In Final Form: October 19, 1990)
The kinetics of anaerobic oxidation of nicotinamide adenine dinucleotide (NADH) by methylene blue was investigated in phosphate and glycine buffers with an excess of NADH in a closed system. The reaction is first order with respect to the concentration of methylene blue. The observed rate constant is pH independent over the pH range of 7.0-10.6 and increases with NADH concentration in a saturated mode. The oxidation process was also investigated under aerobic conditions in a closed system. Applying the steady-state approximation to methylene blue, the rate constant for reoxidation of the reduced form of methylene blue by oxygen was estimated to be 1.62 X IO2 M-' s-I at pH 9.0 at 25 OC. The implications of the present results for NAD' regeneration and oscillatory reactions are noted.
Introduction The aerobic oxidation of nicotinamide adenine dinucleotide (NADH) catalyzed by the enzyme horseradish peroxidase (HRP) is known to exhibit damped oscillations in a closed system.' When this reaction is carried out in an open system in which oxygen and/or NADH are continuously supplied to a well-stirred solution containing HRP, methylene blue (MB'), and 2,4dichlorophenol (DCP), a variety of nonlinear behavior can be observed including bistability2 (even in the absence of MB' and DCP), chaos,3 and sustained oscillations.4 Sustained oscillations started immediately after MB+ was added to a steady-state reaction mixture of NADPH, O2 and lactoperoxidase.5 Analysis and computer simulations of some proposed models and mechanisms causing oscillations with N A D H have been performed.*" However, the mechanistic role played by MB+ in the sustained peroxidase-catalyzed oscillations is not clear and needs to be e l ~ c i d a t e d . ~ According *~J~ to Olsen and Degn,4 the role of MB+ 'Permanent address: Department of Physical Chemistry, Comenius University, 842 15 Bratislava, Czechoslovakia.
must be catalytic since the total amounts of O2 and NADH consumed greatly exceed the amounts of MB' in the solution. Meantime, Burger and FieldIz discovered oscillations during the methylene blue catalyzed oxidation of sodium sulfide by 02. The catalyst and O2oscillate only in a continuous-flow stirred tank reactor (CSTR). Oxygen is present in relatively low concentration; it is essentially consumed in one phase of an oscillation and must ( I ) Yamazaki, I.; Yokota, K.; Nakajima, R. Biochem. Biophys. Res. Commun. 1965, 21. 582. (2) Degn, H. Nature 1968, 217, 1047. (3) Olsen, L. F.; Degn, H. Nature 1977, 267, 177. (4) Olsen, L. F.; Degn,H. Biochim. Biophys. Acra 1978, 523, 321. (5) Nakamura, S.; Yokota, K.; Yamazaki, 1. Nature 1%9, 222, 794. (6) Yokota, K.; Yamazaki, I. Biochemistry 1977, 16, 1913. (7) Olsen, L. F. Biochim. Biophys. Acta 1978, 527, 212. (8) Fedkina, V.; Ataullakhanov, F.; Bronnikova, T. Biophys. Chem. 1984, 19, 259. (9) Aguda, B. D.; Clarke, B. L. J . Chem. Phys. 1987,87, 3461. (10) Aguda, B. D.; Larter, R. J . Am. Chem. SOC.1990, 112, 2167. ( 1 1) Alexandre, S.; Dunford, H. B. Manuscript in preparation. (12) Burger, M.; Field, R. J. Nature 1984, 307, 720.
0022-3654/91/2095-241 1%02.50/0 0 1991 American Chemical Society
