J. Phys. Chem. 1983, 8 7 , 275-280
Figure 2. They correspond to different temperatures (120 and 143 “C) of the condensed halogen at the top of the sample tube. Not surprisingly, the absorbance around 400 nm increases with the temperature of the condensed halogen, but more important, for each curve there is a maximum above 400 nm, while there is none for the I,/ CsC1-LiC1 melt. This increased absorption at longer wavelengths is entirely consistent with the notion that 12C1has a peak at 437 nm and is present at relatively greater concentration in the I,-ICl/CsCl-LiCl system because of the inhibitory effect of IC1 on the disproportionation of I,Cl-. The position of the maximum shifts from -415 to -405 nm (Figure 2) with an increase in the temperature of the condensed halogen. At these temperatures the condensed phase may be a solution of I, and IC1. If so, the partial pressures of both vapors would be greater at the higher temperature. The shift of the maximum to a shorter wavelength can be explained by assuming that the increase in [IClf] is proportionately greater than the increase in [12Cl-]. Based on the Raman results, one can assume that [IC] is negligible in this melt. Because of this evidence that 12C1-in the melts has an absorption maximum at a wavelength greater than 415 nm, the earlier speculation that the spectrum of the 12/CsC1LiCl melt in the 400-nm region is a superposition of the spectra of 1,Cl- and 1,- gains support. The invariance in the shapes of the spectral curves with increasing I, pres-
275
sure, as well as the near constancy of IlI2/IlM in the Raman spectra when melt temperature or I, pressure are varied in the 12/CsC1-LiC1 system, signifies that equilibria 5, 6, and 7 are coupled so as to keep [IC]/ [I,Cl-] approximately constant. The reason for this is not evident from the data presently available.
Conclusions The symmetric stretching vibrations of 13-, a higher polyiodide, 12Cl-,and IC1,- were observed in the Raman spectra of the several melts which were the object of this study. Where comparisons were possible, the visible-ultraviolet absorption spectra of these polyhalides in the molten salts agreed with the spectra of the same species in aqueous solution. In general, the chemistry of the POlyhalides in melts seems to parallel their behavior in aqueous solution. Acknowledgment. We thank Dr. Victor A. Maroni for his encouragement and helpful suggestions. W.C.C. is grateful to the Division of Educational Programs of Argonne National Laboratory for an appointment as Faculty Research Participant for the summer of 1981. This work was supported by the US.Department of Energy/Office of Basic Energy Science/Division of Materials Science. Registry No. KI, 7681-11-0;LiI, 10377-51-2;CsCl, 7647-17-8; LiCl, 7447-41-8; 12, 7553-56-2;ICI, 7790-99-0; Is-, 14900-04-0;I2Cl-, 17705-05-4;IC12-, 14522-79-3.
Perturbation of the Hydrogen-Bond Equilibrium in Nucleic Bases. An Infrared Study R. Buchet and C. Sandorfy’ Gpartement de Chimie, Universit6 de Montrtkl, Montr6al. Ou6bec, Canada H3C 3V1 (Received: July 30, 1982: I n Final Form: September 29, 1982)
The ways in which the hydrogen-bond equilibrium can be perturbed in the 1-cyclohexyluracil(CU) dimer and in the l-cyclohexyluracil/9-ethyladenine(EA) complex have been examined by infrared and proton NMR spectroscopy. The perturbers used were high concentrations of halofluorocarbon anesthetics containing an acidic hydrogen like chloroform,halothane, methoxyflurane,and enflurane and barbiturates like phenobarbital (PB). It is shown that these perturbers are capable of opening a large proportion of the hydrogen bonds present in these base pairs. The effect of phenobarbital is particularly spectacular. The possible biological importance of these observations is stressed.
A large part of the forces holding nucleic base pairs together in DNA comes from hydrogen bonding (H bonding). Hence, any process that leads to the separation of the two helices such as normal or abnormal cell division (cancer) must comprise in its initial stages the dissociation (“opening”)of at least a part of these H bonds. Therefore, it appeared to us worthwhile to study the way in which they are affected by external agents in the hope that a way can be found in which the H bonds in the various nucleic acid pairs can be selectively affected. In this preliminary account only two model systems will be examined: the self-associated cyclohexyluracil dimer and the 9-ethyladenine/cyclohexyluracilpair (Figure 1). These were chosen for their relatively high solubility in organic solvents. Compounds having anesthetic potency have been used as perturbing agents. The reason for this choice is the observation, made earlier in this laboratory, that anesthetics “open” H bonds of the O-H...O, N0022-3654ra3/20a7-0275$0 i.5oro
H.-.N, N-H-..O=C types (and others).’+ This was shown, among others, for general anesthetics like dichloromethane, chloroform, halothane (CF3CHC1Br), methoxyflurane (CH30CF3CHC12), and enflurane (CF2HOCF,CHFCl), for local anesthetics like benzocaine, procaine, and lidocaine, and for barbiturates like phenobarbital or pent~barbital.~In the present study the (1)T. Di Paolo and C. Sandorfy, J. Med. Chem., 17,809 (1974). (2)R. Massuda and C. Sandorfy, Can. J . Chem., 55, 3211 (1977). (3)G.Trudeau, K. C. Cole, R. Massuda, and C. Sandorfy, Can. J. Chem., 56, 1681 (1978). (4)C.Sandorfy, Anesthesiology, 48,357 (1978). (5)G.Trudeau, M.Guerin, J. M. Dumas, P. Dupuis, and C. Sandorfy in ’Topics in Current Chemistry”, Vol. 93,Springer-Verlag, West Berlin, 1980,pp 91-125. (6)C. Sandorfy in “Progress in Anesthesiology”, Vol. 2,B. R. Fink, Ed., Raven Press, New York, 1980,pp 353. (7) M. Guerin, J. M. Dumas, and C. Sandorfy, Can. J.Chem., 58,2080 (1980).
0 1983 American Chemical Society
278
Buchet and Sandorfy
The Journal of Physical Chemistry, Vol. 87, No. 2, 1983
I
- + 3500
3400
33C0
3232
CM-'
Flgure 2. (. .) in a
-
Part of the infrared spectrum of 0.02 M cyclohexyluracil: mixture of 25 % CHC13 and 75 % CCI,; (- - -) in CHCI,.
Figure 1. Chemical structures of 1-cyclohexyluracil (CU), 9-ethyladenine (EA), and phenobarbital (PB).
above-mentioned halogenated anesthetics and phenobarbital have been used as H-bond "breakers". This does not mean, however, that the results presented here concern in any way the mechanism of anesthesia; simply the Hbond-breaking ability of certain anesthetics is exploited. The methods used were infrared and proton NMR spectroscopy. Most information that we have been able to obtain is connected with NH2 and NH stretching vibrations and with the H-bond shift of the proton signal in NMR. Experimental Section A.C. spectrograde chloroform was shaken with sulfuric acid in order to rid it of its alcohol content. After decantation it was dried on MgSO,, distilled, and kept in darkness. Diglyme (2-methoxyethyl ether) from the Aldrich Chemical Co. was dried with sodium, distilled, and kept in a glovebox under dry nitrogen atmosphere. All manipulations have been carried out in a glovebox. A.C. spectrograde CC14 and CH2C12were used without further purification. 9-Ethyladenine and 1-cyclohexyluracilfrom Vega Biochemicals were lyophilized in chloroform, dried under vacuum, and kept in a refrigerator. All deuterated products came from Merck, Frosst Canada, Inc. Their purity was given as 99% by the company. The anesthetics, halothane (CF3CHC1Br,Hoechst Pharmaceuticals), methoxyflurane (CH30CF2CHC12,Abbott Laboratories), enflurane (CHFC1CF20CF2H,Ohio Medical Products), and phenobarbital (May and Baker) were used without further purification. The infrared spectra were measured on a Perkin-Elmer Model 621 instrument. Sodium chloride cells were used with length of 1.148 mm for the region from 4000 to 3000 cm-' and 0.108 for the 2000-1500-~m-~ region. The resolution was of the order of 2 cm-'. The 'H NMR spectra were recorded by means of a WH-90 Brucker spectrometer operating at 90 MHz in the Fourier transform mode. CDC13was used, unless otherwise stated, as solvent as well as internal lock. The chemical shifts reported are in 6 (ppm) downfield from Me4Si. the concentrations of the solutions of nucleic bases were of the order of 0.02 M. Results Self-Associated Dimer of I-Cyclohexyluracil. The NH stretching region of the IR spectrum of cyclohexyluracil
3'
I
3500
3400
3300
3200
CM-'
Part of the infrared spectrum of 0.02 M cyclohexyluracil: (. .) in a 5040% mlxtwe of methoxyflwane (CH30CF2C!iC12)and CCI,; (- - -) in methoxyflurane. Figure 3.
(CU) is shown in Figure 2. The two bands seen at 3400 and 3200 cm-' correspond to the free and H-bonded v(NH) vibrations, respectively.8 Since CU is not sufficiently soluble in CCl,, we prepared a 0.02 M solution in a mixture of 25% CHC1, and 75% CCl,. This was compared with a solution in 100% CHC13. The relative intensities of the bands at 3400 and 3200 cm-' undergo spectacular changes: as the concentration of chloroform increases, the intensity of the free v(NH) band increases and that of the associated v(NH) decreases. The same phenomenon was observed previously on similar systems and it is entirely general: in chloroform the free/associated equilibrium is tilted in favor of less association. It has been shown2s5that the acidic hydrogen of chloroform is the most important factor in bringing about this change: a part of the N-H.. .O=C is replaced by C-H...O=C type H bonds between chloroform and a CU molecule. (It is possible that a part of the NH groups of CU enters N-H. .N type H bonds; also chloroform might form C-H.-.N type H bonds; there is no real evidence for this in our spectra, however.) Enflurane and dichloromethane behave similarly, only their perturbing effects are somewhat less pronounced. The case of methoxyflurane is more complicated (Figure 3). For a 1:l mixture of CC14and methoxyflurane our 0.02 M solution gave three bands, at 3400,3340, and 3200 cm-l, respectively. In pure methoxyflurane the 3200-cm-' band decreases in intensity, the new band at 3340 cm-' increases, and the free band at 3400 cm-' remains approximately unchanged. The 3340-cm-' band must then correspond to a species more weakly associated than the self-dimer. One could think about the formation of a H bond between the NH group of CU and the CH,0CF2 group of methoxyflurane although the adjacent CF2 group is expected to render the oxygen lone pairs less available for proton acceptance than in ordinary ethers. This could occur a
(8)Y. Kyogoku, R. C.Lord, and A. Rich, J. Am. Chem. SOC.,89,496 (1967).
Hydrogen-Bond Equilibrium in Nucleic Bases
The Journal of Physical Chemistry, Vol. 87,No. 2, 1983 277
7
7
8 .3500
3300,
3500
3100
3300
3100
CM-'
CM -
75
IO0
50
( % CDC13) Flgure 5. Variatlon of the NH and CH(5) proton chemical shifts of cyclohexyluracll (9.4 and 7.2 ppm, respectively) with CDCI, concentration in mlxed CDCI,-CCI, solutlons. Concentration of CU: (-) 0.2 and (- - -) 0.1 M.
3500
3300
3100
(CM-') Flgure 4. Intensity changes of the v(NH) bands of cyclohexyluracil. a: (-) the solvent, 40% CHCI, 60% dlglyme; (---) 0.2 M cyclahexylwacil. b: (-) the s o h " 80% CHCI, 20% dlglyme; (---) 0.2 M CU. c: (-) the solvent, 40% CCI, 60% diglyme; (---): 0.2 M CU.
+
+
+
-
concomitantly with C-H. .O=C bond formation. This interpretation is compatible with the insensitivity of the free band in this case: the equilibrium would be between a more and a less associated species, not a free species. The opening of a H bond is, however, involved in every case, for all the halocarbon anesthetics that we have examined. In order to examine conditions when the solvent is a proton acceptor, we dissolved CU in diglyme (Zmethoxyethyl ether) and then added CC4, CH2C12,or CHC13 to the solution. The band at 3400 cm-l is weak for these solutions showing that most of the NH groups are engaged in either CU-CU or, more likely, CU-solvent H bonding. When CHC1, is added to the solution, the free band becomes much stronger and the association band loses intensity (Figure 4a-c). CH2C12behaved in a way similar to CHC13 but influenced the H-bond equilibrium to a lesser extent. This can be linked to the lesser acidity of CH2C12. CC14had no noticeable effect on the spectra showing the very moderate perturbation effected by halocarbons containing no acidic hydrogen. (At low temperatures, however, much stronger perturbations are 0bserved.l) In all these experiments the concentration of CU was kept constant at 0.2 M. The lH NMR spectra confirm the results obtained in IR. It is well-known that the chemical shift of the NH proton yields information on H bonding in nucleic bases.*ll So we have measured the NMR spectra in mixtures of CC14and CDCl,. With increasing CDCl, concentration the NH proton band shifts to higher fields showing that more and more CU-CU H bonds are dissociated. Figure 5 illustrates this result. The dashed curve represents the (9) L. Katz, J.Mol. Biol., 44, 279 (1969). (IO) L. Katz and S. Penman, J. Mol. Biol., 16, 220 (1966). (11) I. Morishima, T. Inubushi, T. Yonezawa, and Y. Kyogoku, J.Am. Chem. Soc., 99, 4299 (1977).
2400
2300
2200
2100
CM-' Flgure 6. CD stretching band of pure CDCI, (- - -) and of a 0.1 M solution of g-ethyladenine In CDCI, (-).
variation of the chemical shifts of the NH proton (9.4 ppm) and of the CH(5) proton (7.25 ppm) at a CU concentration of 0.1 M. The solid curve applies to the same bands at 0.2 M. An increase in CU concentration favors association; consequently the NH signal shifts to lower fields. (This is why the curve related to the 0.2 M solution is above the one for 0.1 M.) The CH(5) signals (the horizontal lines at the bottom of the figure) are practically unaffected by either the CU or the CDC13concentration and the other CH are even less affected. Similar results have been obtained with CD2C12,except that this molecule is a less efficient H-bond breaker than CDC13. 9-Ethyladeninel Cyclohexyluracil Dimer. First we have studied the association between the self-associated 9ethyladenine (EA) dimer and chloroform which has some importance from the point of view of the problems treated in this paper. Adenine possesses four tertiary nitrogens which are eligible proton acceptors. The bands of the primary amino group of adenine do not seem to be affected by chloroform. In order to ascertain the existence of H-bond formation between adenine and chloroform, we followed the variation of the CD stretching band of CDC13with increasing adenine concentration. Deuterated chloroform had to be used to avoid interference from other CH groups. In Figure 6, the dashed line corresponds to pure CDC13and the solid
278
The Journal of Physical Chemistry, Vol. 87,No. 2, 1983
Buchet and Sandorfy
TABLE I: Proton Chemical Shifts of the Pair 9-Ethvladenine ( E A ) and 1-Cvclohexvluracil ( C U P protons
6
6,'
ib
12.11 8.36 7.94 7.27 7.18 6.42 5.75 5.66 4.40 4.32 4.24 4.16 1.97 1.89 1.64 1.56 1.48
11.02 8.41 7.94 7.30 7.21 6.19 5.78 5.69 4.39 4.31 4.23 4.15 1.97 1.88 1.62 1.55 1.46
A=6,-6,
-1.09 0.05 0.03 0.03 -0.23 0.03 0.03 -0.01 -0.01 -0.01 -0.01
-0.01 -0.02 -0.01 -0.02
a The chemical shifts are given in ppm downfield from tetramethylsilane. The concentration of each nucleic base 50% CDCI, + 5 0 % CC1,. 100% CDC1,. was 0 . 0 2 5 M.
line to a solution containing 0.1 M EA in CDCl3 The latter exhibits a fairly broad shoulder at about 2220 cm-' overlapping the main CD band at 2250 cm-'. This is not a band of EA, so we have to assign it to a CCl,D*..N(tert) type H bond. The 'H NMR spectra confirm this assignment. The chemical shift of the CH proton of CHC1, shifts regularly to lower fields as the concentration of EA increases. (It is at 7.260 ppm for pure CHC1, and 7.277 ppm with 0.1 M EA). It is to be noted that, unlike EA, CU does not produce a shoulder at the C-D band of CDC1,. The variation of the NMR shift is also less significant. All this shows that chloroform has a much greater tendency to form H bonds with EA than with CU. The H bonds in the CU/EA dimer are expected to bear a close resemblance to those of the Watson-Crick pair adenine/uracil. These have been studied by several authors*" and we have no reason to question the assignments made by them. Instead we shall concentrate on the way of opening these H bonds. The spectrum containing 0.025 M CU and 0.025 M of EA is presented in Figure 7. For the dashed-line spectrum the solvent was a 1:l mixture of CCl., and CDC1,; for the solid curve it was pure CDC13. Of prime interest to use are the asymmetrical and symmetrical stretching vibrations of the NH2 group.8 They are located at 3527 (as)and 3416 (8) cm-l for the free group and at 3485 and 3312 cm-I for the H-bonded one. A part of the intensity of the 3416-cm-' band is known to be due to the secondary NH band of CU; both bands are free bands, however. With more chloroform in the solvent the intensity of the free bands increases and that of the associated bands decreases. So here again we encounter the
36CO
3500
3400
33CO ICM-')
3200
1100
303i
Figure 7. Part of the infrared spectrum of an equimolar (0.025 M) solution of g-ethyladenine and cyciohexyluracii: (-) In CDCI,; (- - -) In a 5 0 4 0 % mixture of CDCI, and CCI,.
phenomenon described above: in the presence of the breaker (the anesthetic) many of the H bonds dissociate. A decrease in the concentration of chloroform favors EA/CU pair formation. The interactions which compete with EA/CU dimer formation are almost certainly CC1,H. .O=C for CU and CCl3H.-.N for EA; probably both of these occur. A look at the 1800-1600-cm-' region of the spectrum confirms the above interpretation. An intense band at 1688 cm-' is due to the stretching motion of the CU carbonyls. At the given concentration (0.025 M) it is not sensitive to a change from HNH...O=C to CCl3H...O=C . In both cases the H-bond shift is slight and the overall envelope of the band is not seriously affected by the change. Significant changes are observed, however, in the relative intensities of the free and associated in-plane NH2 bending bands which are at 1629 and 1640 cm-', respectivelya8 (It is well-known that, while the NH (or OH) stretching bands shift to lower frequencies upon H-bond formation, the opposite is true for the bending bands.) As the concentration of CHC1, increases, the band at 1640 cm-' loses and the one at 1629 cm-' gains intensity. This is entirely in line with the behavior of the stretching bands, yielding additional evidence for the perturbation of the H bonds within the EA/CU base pair by chloroform. The results which we obtained with other anesthetics, dichloromethane, halothane, methoxyflurane, and enflurane, were closely similar for the NH2 stretching as well as bending bands. The perturbation of the H-bond equilibrium in nucleic base pairs can be demonstrated equally well by NMR measurements. When CU is added to the solution,the NH signal of CU and the NH2 signal of EA shift, so that the formation of the EA/CU complex can be followed. The H bonds within the latter are known to be stronger than those in the respective self-associated dimers. We examined the evolution of the spectrum of a solution of 0.025
-
TABLE 11: Some Significative Values of the Proton Chemical Shift of the Pairs 9-Ethyladenine ( E A ) and 1-Cyclohexyluracil (CU) in Different Mixtures of Solvent%
protons solvents
NH ( C U )
H2 (EA)
H8 ( E A )
H 5 (CU)
NH, ( E A )
9 0 % halothaneb 75% halothaneb 50% halothane 7 5 % methoxyfluraneb 50% methoxyflurane 100% CD,Cl, 50% CD,C1, + 50% CCI,
11.39 11.74 12.11 11.87 12.45 11.1 12.1
8.33 8.36 8.35 8.31 8.35 8.33 8.28
7.90 7.92 7.92 7.92 7.94 7.91 7.90
7.24 7.27 7.25 7.27 7.25 7.25 7.27
6.42 6.67 6.24 6.42
a See Table I. necessary.
1 0 % deuterated cyclohexane was added as internal lock. The solution was completed with CC1, when
The Journal of Physical Chemistry, Vol. 87, No. 2, 1983 279
Hydrogen-Bond Equilibrium in Nucleic Bases
11 8
I
3500
I
3300
3400
3200
3100
3000
I
(CM- )
Flgurs 8. (-) Part of the Infrared spectrum of the Q-ethyladenlne/ phenobarbltal complex. (- -) Spectrum of a 1:1 physical mixture of the two components. Both in Nujol.
-
M EA and CU using, as the solvent, different mixtures of CCl., and an anesthetic (Tables I and 11). Both the NH (CU) and NH, (EA) peaks shift to higher fields when the quantity of the added anesthetic increases, thereby demonstrating the dissociation of many of the H bonds in the EA/CU pair. For halothane only the NH band can be observed, the NH2 band being obscured by interference from the solvent, but the NH band can be followed in all cases. Both signals can be followed for CDCIBand CD2C12. Barbiturates In this last section the effect of barbiturates on the EA/CU dimer is examined. It is known that certain barbiturates form selective H bonds with adenine derivatives but do not form complexes with uracil derivatives.12 Numerous crystallographic works demonstrated the existence of adenine/barbiturate complexes.'*16 These investigations were conducted with the aim of locating the receptor site for barbiturates. The purpose of the present work has been to ascertain if barbiturates are capable of dissociating the H bonds within the EA/CU pair. Phenobarbital (PB).has been chosen as a representative barbiturate. Assignments of the IR and Raman bands of PB were made by several authors in a variety of experimental conditions and polymorphic forms."-22 First a chloroform solution was prepared containing 0.025 M EA and 0.025 M CU. Then 0.025 M PB was added to the solution. A precipitate was formed and filtered. The IR spectra of both the filtrate and the precipitate were taken. The filtrate did not contain any significant amounts of either EA or PB; it only showed the bands of CU. The spectrum of the precipitate is shown in Figure 8. In order to ascertain that this spectrum belongs to an EA/PB complex, we prepared this complex (12)Y.Kyogoku, R. C. Lord, and A. Rich, Nature (London),218,69 (1968). (13)D.Voet, J. Am. Chem. Soc., 94,8213 (1972). (14)S.H.Kim and A. Rich, Proc. Natl. Acad. Sci. U.S.A., 60,402 (1968). (15)G. L. Gartland and B. M. Craven, Acta Crystallogr., Sect. B, 30, 980 (1974). (16) D. Voet and R. Rich, J.Am. Chem. SOC.,94,5888 (1972). (17)Z.Goenachea, J.Anal. Chem., 218,416 (1966). (18)R. J. Mesley, and R. L. Clements, J. Pharm. Pharmacol., 20,349 (1968). (19)R.J. Mesley, R. L. Clementa, B. Flaherty, and K. Goodhead, J. Pharm. Pharmacol., 20,329 (1968). (20)R. J. Mesley, Spectrochim. Acta, Part A, 26, 1427 (1970). (21)A. J. Barnes, M. A. Stuckey, W. J. Orville-Thomas, L. Le Gall, and J. Lauransan, J.Mol. Struct., 56, 1 (1979). (22) A. J. Barnes, L. Le Gall, and J. Lauransan, J. Mol. Struct., 56, 15, 29 (1979).
3400
3300
3200
3100
(CM-') Flgurs 9. (-) Part of the I R spectrum of the 1:l complex of Ndeuterated phenobarbital and Q-ethyladenine. (- - -) Spectrum of the nondeuterated complex. Both in Nujol mulls.
directly from a chloroform solution containing 0.025 M EA and 0.025 M PB but no CU. The spectrum that we obtained was identical with the spectrum given in Figure 8. This proves that it belongs to the EA/PB complex and that PB has separated EA from CU by dissociating the H bonds between EA and CU replacing them by others. The dashed line in Figure 8 represents a 1:l physical mixture of EA and PB. It is practically the sum of the spectra of the two components. The spectrum of the complex (solid line) has an intense band at 3400 cm-l which does not belong to either EA or PB. In order to assign this band, we deuterated PB and prepared the complex EA/ deuterated PB. The comparison with the nondeuterated complex (Figure 9) shows that the bands at 3270,3220, and 3180 cm-l belong to the amino group of the PB moiety in the complex since they are absent from the spectrum of the deuterated complex. The new band at 3360 cm-' is probably an NHD band. It should be mentioned that the deuterated complex was prepared from a mixture of CDC13 and D20,saturated with D20. This was necessary to keep PB deuterated. It had the drawback that a part of the EA molecules became deuterated in the amino group; it does not prevent one, however, from assigning the bands. Finally, the bands at 3400 and 3320 cm-' belong to the v(NH,) vibrations of the EA moiety and those at 3270,3220, and 3180 cm-' to the PB moiety in the complex. The high frequency at 3400 cm-' may be explained if one of the hydrogens of the NH2 group in EA was only weakly H bonded or not at all. These spectra are, however, insufficient to determine the structure of the complex. Our results are in agreement with those of Kyogoku et al.,12 who have shown that the H bonds between barbiturates and adenine are stronger than those formed between CU and adenine and that there are no H bonds between CU and the barbiturates. Thus we have shown that the EA/CU complex is dissociated by PB.
Discussion and Conclusions The base pair chosen for the spectroscopic study presented in this paper does not occur in biological media. Even if it did, for direct biological relevance it should be considered in its natural context as part of a nucleotide. Since, however, it contains H bonds similar to those which keep the Watson-Crick nucleic base pairs together, it can be considered as a useful model for studying the way in which these vital H bonds can be opened. For perturbing the H bonds we used two kinds of agents: potent general anesthetics containing an acidic hydrogen and barbiturates. These were chosen since their H-bond-breaking ability was
J. Phys. Chem. 1983,87,280-289
280
previously demonstrated in this laboratory. However, the concentrations used were much higher than in clinical practice and for this and other obvious reasons the results presented in this paper do not involve any connection between anesthetic action and the dissociation of H bonds in nucleic base pairs. The intensity changes of the free and associated infrared NH stretching bands and H-bond changes in the NMR chemical shift show conclusively that the H-bond equilibrium in the base pair is strongly perturbed by the anesthetics, many of the H bonds being dissociated. As to the potent general anesthetics, chloroform, halothane, methoxyflurane, enflurance, the perturbation goes mainly through the establishment of an equilibrium of the type2f~23~24 N-H**.:O=C or N-H**.:N-C
+ H-CCl, * C=O:***H-CCl, + NH + H-CC13 * C=N:*..H-CC13 + NH
(23) P. Hobza, F. Mulder, and C. Sandorfy, J . Am. Chem. SOC., 103, 1360 (19811. (24) P. Hobza, F. Mulder, and C. Sandorfy, J. Am. Chem. Soc., 104, 925 (1982).
For phenobarbital our results clearly show that the EA/CU complex is destroyed in favor of an EA/PB complex, which implies the dissociation of the H bonds in the former and the formation of new ones in the latter. This work is meant to be the beginning of thorough investigations on the H bonds in the isolated Watson-Crick base pairs, as well as in a molecular environment approximating in vivo conditions. It is hoped that a scheme will be found making it possible to selectively interfere with these vital H bonds. This could open new perspectives. Acknowledgment. We are indebted to Professor Jacques Weber from the University of Geneva, Switzerland, for making our collaboration possible. We thank Dr. R. Denis for stimulating discussions on anesthetic molecules and Mr. Robert Mayer for assistance in measuring the NMR spectra. Financial assistance from the Natural Sciences and Engineering Research Council of Canada and the Minist6re de 1'Education du Quebec are gratefully acknowledged as well as an I. W. Killam memorial scholarship by the Canada Council to C.S. Registry NO.CU, 712-43-6;EA, 2715-68-6; PB, 50-06-6; CHC13, 67-66-3; CHFC1CF20CF2Hl 13838-16-9; CHzC12, 75-09-2; CCld, 56-23-5; CF3CHC1Br, 151-67-7.
Lowest Excited Singlet State of Hydrogen-Bonded Methyl Salicylate LouAnn Helmbrook, Jonathan E. Kenny,+ Bryan E. Kohler,' and Gary W. Scott* Department of chemistry, Weskyan Unlversity, MMietown, Connecticut 06457 (Received: August 2, 1982; In Final Form: September 23, 1982)
The dual fluorescence shown by methyl salicylate in solution and the room temperature vapor phase has been observed for methyl salicylate seeded in a supersonic helium jet. Under these conditions low rotational and vibrational temperatures are attained, making it possible to measure highly resolved excitation spectra for each of the emission components. This paper reports the measurement of these excitation spectra together with emission spectra and emission decay kinetics as a function of excitation wavelength for both methyl salicylate-h and methyl salicylated in a supersonic jet. The measured spectra are analyzed to determine the nature of the excited states responsible for the dual emission.
1. Introduction
It has been known for many years that the internally hydrogen-bonded methyl salicylate molecule displays dual fluorescence in solution as well as in low-pressuregasses.'-3 Following established usage, these different fluorescence spectra are designated blue (maximum emission intensity at 440 nm) and UV (maximum emission intensity at 335 nm). Recently we reported that this dual emission could also be observed under the low-temperature isolated molecule conditions that obtain in a free jet.4 This is in contrast to the situation in the low-temperature condensed phase where only the blue emission is observed: indicating that the two forms of the molecule responsible for the two different emission spectra do not equilibrate during the expansion. This has allowed us to obtain well-resolved excitation spectra for both hydrogen-bonded species. These spectra together with studies of the fluorescence Present address: Department of Chemistry, Tufts University, Medford, MA 02155. Permanent address: Department of Chemistry, University of California a t Riverside, Riverside, CA 92521.
*
0022-3654/83/2087-0280$0 1.50/0
decay kinetics as a function of excitation and detection conditions afford further insight into the electronic states and potential surfaces for both hydrogen-bonded species. The measured excitation spectra, first reported in our preliminary communication,4together with the kinetic data are presented in detail, analyzed, and interpreted in this paper. Although the dual fluorescence of methyl salicylate was initially thought to originate from an excited-state equilibrium between tautomeric forms of the molecule which are related by proton t r a n ~ f e r ,there ~ , ~ is now conclusive evidence that it arises from an equilibrium in the ground state between two different forms of the molecule that do not equilibrate on the excited-state ~ u r f a c e . ~ - Studies '~ (1) J. K. Marsh, J. Chem. SOC.,126, 418 (1924). (2) A. Weller, Z. Elecktrochem., 60, 1144 (1956). (3) A. Weller, Prog. React. Kinet.,1, 188 (1961). (4) L. A. Heimbrook, J. E. Kenny, B. E. Kohler, and G. W. Scott, J. Chem. Phys., 75, 5201 (1981). (5) J. Goodman and L. E. Brus, J. Am. Chem. Soc., 100, 7422 (1978). (6) K. Sandros, Acta Chem. Scand., Ser. A, 30, 761 (1976). (7) K. K. Smith and K. J. Kaufmann, J.Phys. Chem., 82,2286 (1978). (8) W. Klopffer and G. Kaufmann, J . Lumin., 20, 283 (1979).
0 1983 American Chemical Society
