J, phys.
1378
ct".
1882, 86, 1378-1386
Llnear Dlchrolm Studies of Nucleic Acld Bases In Stretched Poly(Vlny1 Alcohol) Film. Molecular Orientation and Electronlc Transltlon Moment Dlrectlons Yukio Matwoka and k n g l Nordin' Insthie of phvsicel Chemkby, chelmers UnhwsW of Teohndogy. S-4 12 96 &thenburg, Swsdsn (Re&&:
June 9, 1981)
UV linear dichroism of thymine, uracil, cytosine, cytidine, guanine, guanosine, and adenine partially oriented in stretched poly(viny1 alcohol) f i i was measured in the 215-320-nm region to determine the directions of electronic transition moments. lR dichroism was measured in the same stretched f i i in the region 1900-1400 cm-' to obtain information about the molecular orientation tensor ((cosZi cos Z j ) ] . The first ?M* transitions of thymine (266nm) and uracil (260nm) were found to be polarized at about -31O and -1l0, respectively, with respect to the Nl-C4line toward the N3 atom, and the first (268nm) and second (240nm)transitions of cytosine directed at 25' and 6O. The first (280nm) and second (248nm) transitions of guanine were obtained at 4 O and - 8 8 O relative to the N3-C6line toward the N1 atom, and the second (263nm) and third (235nm) transitions of adenine directed at 9 O and -75O. The results are in reasonably good agreement with crystal studies.
Introduction The importance of getting information about the directions of the electronic transition moments of nucleic acid bases has been accentuated in the studies of linear1s2 and circular dichroism3 and photochemistry' of nucleic acids. The experimental methods so far have been (1) polarized reflectance spectra of single crystal~,~~O (2) polarized absorption spectra of single cry~tals,l'-'~ (3) fluorescence polarization,7J"1B (4) polarized spectra of molecules oriented in stretched f i l m ~ , ~and * ~(5) l spectra of molecules oriented by external electric fields.22 Methods 1and 2 can give absolute directions of transition moments in crystals of known structure and low symmetry, but electxonic interactions between the same chromophoric species in the crystal can introduce considerable ambiguity.'s2 This problem of interaction can be eliminated by dilution in a host crystal or in a glassy matrix of, for instance, ethylene glycol/water16-18 or 2-propanol/iso~ e n t a n e .The ~ molecules do not have any preferential orientation in the glass but can be photoselected by excitation with polarized radiation, and the resulting fluorescence polarization can give relative directions of (1) (a) Hofrichter, J.; Eaton, W. A. Annu. Reu. Biophys. Bioeng. 1976, of the Linear and Magnetic Cir5,511. (b) Thuhup, E. W. cular Dichroism of Planar Organic Molecules";Springer-Verlag: Heiderberg, 1980. (2) NordBn, B. Appl. Spectrosc. Reu. 1978, 14, 157. (3) Buch, C. A.; Brahma, J. In 'Phyuicochemical Propertiesof Nucleic Acids"; Ducheene, J., Ed.;Academic Prees: London, 1973; Vol. 2, p 147. (4) Daniels, M. In 'Photochemistry and Photobiology of Nucleic Acids"; Wang, S. Y., Ed.;Academic Prees: New York, 1978; Vol. 1, p 23. (5) Clark, L. B., unpublished results (1972) quoted in ref 4. (6) Anex, B. G.; Fucaloro, A. A.; Dutta-Ahmed, A. J. Phys. Chem. 1976, 79, 2636. (7) C a b , P. R.; S i m p n , W. T. J. Am. Chem. SOC.1970,92, 3593. (8) Callis, P. R.; Fanconi,B.; Simpeon, W. T. J . Am. Chem. Soc. 1971, 93, 6679. (9) Clark,L. B. J. Am. Chem. SOC.1977,99,3934. (10) Chen, H. H.; Clark, L. B. J. Chem. Phys. 1973,58,2593. (11) Stewart, R. F.; Davideon, N. J. Chem. Phys. 1965,39,256. (12) Eaton, W. A.; Lewis, T. P. J. Chem. Phys. 1970,63, 2164. (13) Lewis, T. P.; Eaton, W. A. J. Am. Chem. SOC.1971, 93, 2054. (14) Stewart, R. F.;Jensen, L. H. J. Chem. Phys. 1964, 40, 2071. Jpn. 1971, 44, 938. (15) T&, M.; Tanaka, J. Bull. Chem. SOC. (16) Callis, P. R.; Rosa,E. J.; Sipeon, W. T. J. Am. Chem. Soe. 1964, 86, 2292. (17) Wileon, R. W.; Callis, P. R. J. Phys. Chem. 1976,80,2280. (18) Cab,P. R. Chem. Phys. Lett. 1979,61,563. (19) Tohara, A.; H~akawa,A. Y. Chem. Phys. Lett. 1980, 75, 146. (20) Fucaloro, A. F.; Forster, L. S. J. Am. Chem. SOC.1971,93,6443. (21) Bott, C. C.; Kurucsev, T. In 'Molecular Optical Dichroism and Chemical Applications of Polarized Spectroecopy"; Nordh, B., Ed.; Lund University Press: Lund, Sweden, 1977; p 81. (22) Seibold, K.; Labhart, H. Biopolymers 1971, 10, 2063.
''hm
0022-3654/82/2086-1378$01.25/0
transition moments.23 Generally, f i i dichroism can provide directions of transition moments, when the molecular orientation is known, or information on molecular orientation, when the moment directions of specific transitions are known.2 Although the technique of film dichroism is very direct, there have been only a few attempts to use it for studying transition moment directions of nucleic acid bases. This seems to stem from the difficulty of estimating the orientation of molecules with low symmetry in the stretched film. In the first film dichroism study of nucleic acid bases by Fucaloro and Forster,20the solute orientation in poly(vinyl alcohol) (PVA) was assumed to obey the Tanizaki orientation model,% and relative transition moment directions were derived.20i2s However, since the Tanizaki model is valid only for rod-shaped molecules, but not for planar molecules, the result is ambiguous and the experimental spectra must be reinterpreted in terms of another, proper orientation model.2s Another source of error in earlier works is the concept of "orientation axis". For symmetric and elongated molecules, it is usually easy to estimate such an axis (the long axis) which has the highest tendency to become aligned pardel to the direction given by the orientation constraint. For small, unsymmetrical molecules, however, where the orientation may be determined in a complex way by various peripheral substituents of the molecule, it may no longer be meaningful to speak about an orientation axis.2 Thus,the direction "z", which diagonalizes the orientation tensor of the present molecules, is not necessarily related to any maximum in the orientation distribution function. The transition moment directions in cytosine and cytidine were recently discussed by Bott and Kurucsev21on the basis of W and IR linear dichroism spectra in oriented films;however, orientation and moment directionsof the other nucleic acid bases have still not been investigated. The primary object of this paper is to study the orientation of the DNA bases in poly(viny1 alcohol) matrix and to determine the electronic transition moment directions. For this purpoee the linear dichroism was measured in both the IR (1900-1400 cm-') and UV (215-320 nm) regions. (23) Albrecht, A. C. h o g . React. Kinet. 1970,5, 301. Jpn. 1969,32,75; 1965,38,1798. (24) Tanizaki, Y. Bull. Chem. SOC. (25) Fucaloro, A. F.; Forster, L. S. Spectrochim. Acta, Part A 1974, 30, 883. (26) (a) Thulatrup, E. W.; Michl, J.; Eggers,J. H. J. Phys. Chem. 1970, 74, 3868. (b) Michl, J.; Thulstrup, E. W.; Eggers, J. H. Ibid. 1970, 74, 3878. (c) Thulatrup, E. W.; Michl, J. Ibid. 1980, 84, 82.
0 1982 American Chemical Society
The Journal of Physical Chemistry, Vol. 86,No. 8, 1982 1379
Transition Moments of Nucleic AcM Bases
A general expression for the reduced dichroism of a lowsymmetrical planar molecule was used for the analysis.2 By the aid of the IR dichroism, a molecular coordinate system was first determined providing a diagonal orientation tensor. The directions of electronic transition momenta are fmally compared with results from single-crystal studies and MO calculations.27-30 Experimental Section Materials and Preparation of Sample Film. Thymine and cytosine obtained from Merck Chemical Co. and uracil, cytidine, adenine, guanine, and guanosine from Sigma Chemical Co. were used without further purification. The sample films were obtained as follows. A 10% PVA solution was prepared by dissolving PVA powder (Elvanol 71-30 from E. I. Du Pont de Nemours Co.) in distilled water. The PVA solution was mixed with a suitable quantity of a solution of the sample (ca. 5 X lo9 M), and the r e s d t i i solution was poured onto a horizontal glass plate and kept for 3 days in a ventilated, dust-free place. The reference film was prepared from the same PVA solution under identical conditions. The solubility of guanine in PVA was increased by addition of a small amount of hydrochloric acid (to a pH still above 6). That guanine was present in neutral form was checked from the IR spectrum in the film. The film thickness was varied between and cm to allow study of IR and UV linear dichroism at varied solute concentrations. Apparatus. UV dichroic spectra were measured on a Cary 219 spectrophotometer supplemented with a rotatable Glan air-space polarizer preceding the sample in the light path. Differentially measured linear dichroism was recorded on a Jasco 5-500 spectropolarimeter for exact checking of the UV dichroism.2 IR dichroic spectra were recorded on a Nicolet MX-1 Fourier-transform spectrometer. The transmittances TI,and T,, of sample f i i , and TI,,and TI,of reference film, were measured, with light to the dipolarized parallel (11) and perpendicular (I) rection of stretch, providing the absorbances A, = log Tr,,/T,, and A, = log Tr,/T,. The stretch ratio R,of the film is defined as before.24 Basic Equations for the Analysis of Linear Dichroism. We consider low-symmetrical planar molecules and assume that the distribution of molecules is uniform around the polymer stretching direction (uniaxial stretching of a matrix f i ) . Then the isotropic absorbance ( A 3 is given by Ai, = (A,, 2A,)/3. We shall further assume that any solvent effect on transition energies or moment directions is independent of orientation. As we shall see, there is experimental evidence that this assumption is justified. Let x ’y ’z’ be an arbitrarily chosen orthogonal coordinate system associated with the molecular framework. The direction cosine of the i’th axis with respect to the stretching direction Z is represented by cos Zi’. For the interpretation of the linear dichroism of an assembly of identical molecules, the orientation is characterized by the orientation tensor:
+
i
(COS* 22’)
22’cos ZY’)
(COS
22‘ cos Z X ‘ )
1
22’cos ZX’, (cosZ y ‘ cos ZX‘) (COS Z y ‘ cos 2 x 9 (COS’ 2 x 9 (COS
(cos 2 2 ’ cos Z y ’ ) (cos2 z y ‘ )
(COS
If two axes y’and z’are chosen arbitrarily in the molecular (27) Berthod, H.;Giessner-Prettre, C.; Pullman, A. Znt. J . Quantum Chem. 1967,1, 123. (28) Hug, W.;Tinoco, I., Jr. J. Am. Chem. SOC.1973, 95,2803. (29) Nagata, C.;Imamura, A.; Fujita, H. Adu. Biophys. 1973, 4 , 1. (30) Srivastava, S.K.; Mishra, P. C. Znt. J. Quantum Chem. 1980,18, 827.
plane and if only transitions with moments in the molecular plane are observed, the reduced dichroism LD’ E LD/Ai, = 3(A,,- A,)/(All + 2AJ can be expressed by (Appendix A) LD’ = 3/22((3(cos2 Zy’) - 1) sin2 0’ + @(COS2 22’) 1) cos28’ + 6 (cos Zy ’ COS Zz ’) sin 8’ COS 6’)
= 3(S,,,, sin2 e’ + S,,,, cos2 e’
+ Sytztsin e’
cos e’) (1)
where 8’ is the angle between the transition moment and the z’axis, and Syryt,Szlzl,and S, are order parameters: syy (3(C0S2zy’) - 1 ) / 2
s,,,, = (3(COS2
’)
- 1)/ 2
SYlZ‘= 3 (cos z y ’ cos zz ’)
If we rotate the system x ’y’z ’around the x ’axis an angle a into a new system xyz (x = x ? , eq 1can be rewritten as follows: LDr = 3(S, sin2 (0’ - CY) + S,, cos2 (0’ - CY)+ S,, sin (e’ - CY)cos (6’ - a)) = 3(Sy, sin2 e
+ S,, cos2 e + S,,
sin e cos 6)
(2)
where 6 = 8’ - a is the angle between transition moment and the z axis (e’ and CY are taken to be measured counterclockwise relative to the z’axis). It is easy to see (Appendix B) that the angle a,at which zy! of eq 2 disappears and the orientation tensor becomes diagonal, corresponds to extrema in Szzand S., The axes of the diagonal system are labeled so that the order parameters S,,, S, and S,, fulfill S,, 1 Syr 1 Sz,. Only two order parameters are independent, since S,, + S,, + S,, = 0. In the diagonal system xyz, eq 2 reduces to LDr = 3(S,, sin2 0 S,, cos2 e) (3)
+
Results UV Linear Dichroism. Figure 1shows linear dichroism (LD) and calculated isotropic spectra (Abo)of each compound, together with the wavelength dependence of the reduced dichroism (LD’). The isotropic spectra are very All comsimilar to those obtained in aqueous pounds show positive linear dichroism in the studied wavelength region as expected from the fact that the absorption is dominated by in-plane polarized transitions and that the shapes of the molecules make them behave more like disks than like rods (S,,, S,, both positive).2 Theoretically, the transitions in planar molecules can be polarized along any direction parallel to the molecular plane or exactly perpendicular to it.2 Any existing out-of-plane (n-r*) transition in the region 220-300 nm of nucleic acid bases, however, must be very weak4 and is therefore ignored in the following, which means that we can directly apply eq 2. As seen from eq 2, the reduced dichroism as a function of wavelength will be flat over isolated absorption bands only if 8 is constant (S,,, S,,, and S,, are, of course, independent of wavelength). LD’ can therefore provide a test of whether different electronic transitions are present in the wavelength region. Thus, a look at the LD’ curves and the isotropic spectra directly reveals the presence of two transitions (designated I and 11) in the spectra of thymine and uracil, and three transitions (designatedI-111) in the spectra of cytosine, cytidine, guanine, guanosine, and adenine, in the region above 215 nm. The apparent (31) Voet, D.;Gratzer, W. B.; Cox, R. A.; Doty, P. Biopolymers 1963,
1, 193.
1380
The Journal of Physical Chemistry, Vol. 86, No. 8, 1982
Matsuoka and Nord6n
%T
1oc 9c 80
7c 6C
80
70 34
c3
60
02
220
240
260
280
a nm
300
50
i
i
I
1800
I1 1600
40
c m-'
%T
moo,
%T 00 30 10
-
'0
Flgure 1. Linear dlchrolsm (- -), calculated isotroplc spectra (-), and reduced dichroism (0)of (a) thymine at R, = 3.9, (b) wadi at R, = 3.2, (c) cytoslne at R, = 3.9, (d) cytidtne at R, = 5.5, (e) guanine at R = 5.5, (f) guanosine at R ,= 5.5, and (9) adenine at R = 3.9 in PVA films.
10
,
TABLE I: Apparent Band Positions in the Isotropic Spectra of Nucleic Acid Bases apparent band positions, nm compd
I
I1
thymine uracil cytosine cytidine guanine guanosine adenine
266 260 268 274 280 282 -270
< 240 < 230 240 240 248 255 263
I11 1800
< 230 < 230 < 225 < 230
< 245
wavelength positions of these transitions are summarized in Table I. The possibility of hidden transitions in the spectrum of adenine will be discussed later. The different behavior of the LIY curves of the nucleosides cytidine and guanosine, as compared to cytosine and guanine, can be due to a different, more complex orientation distribution of the unsymmetric moleculea and/or to changed transition moment directions by the substitution of the ribose group. IR Dichroic Spectra. Most nucleic acid bases possess assignable in-plane vibrational bands in the IR region.
1600
cm-' Figure 2. Infrared dichroic spectra (Tl,/Trl,(-)and T , / T , , (---) of (a) thymine at R, = 4.3, (b) uracil at R , = 6.5, (c) cytosine at R , = 6.5, (d) guanine at R, = 6.5, and (e) adenlne at R, = 6.5 in PVA films.
Figure 2 shows typical dichroic spectra observed in the 1850-1450-cm-' region, where the absorption of PVA itself is not excessive. The background absorption due to the matrix (PVA) has been subtracted in the presented spectra. Regarding the main features, the spectra are very similar to those observed in the solid (32) (a) Tsuboi, M.; Kyogoku, Y.; Shimanouchi, T. Biochim. Biophys. Acta 1962,55,1. (b) Kataura, T.; Morikawa, K.; Tsuboi, M.; Kyogoku, Y.; Seno, T.; Nishimura, S.Biopolymers 1971, 10, 681.
Transition Moments of Nucleic Acid Bases
Flgure 9. Dependence of thymine and (b) cytosine.
UT
on R , for UV and I R bands of (a)
The 1705- and 1670-cm-' bands of thymine (Figure 2a) correspond to the C2=0 and C4=0 stretching vibrat i o n ~ .The ~ ~fact ~ that the reduced dichroism of the 1705-cm-' band (0.33) is larger than that of the 1670-cm-' band (0.24) shows that the transition moment direction of the former band, i.e., the C,--O bond, lies closer to the diagonalizing direction z (the orientation axis) than the latter transition (the C 4 4 bond). In the IR spectrum of uracil (Figure 2b), the band a t 1710 cm-' also contains contributions from the C2=0 stretching and N3-H bending vibrations whereas the band at 1675 cm-l mainly corresponds to the C4=0 stretching vibrati0n.w LIY was 0.22 for the 1710-cm-' band and 0.41 for the 1675-cm-' band. Cytoeine shows a C f l stretching vibration at 1626 cm-l (LIY = 0.39) and a N - C stretching vibration at 1500 cm-' (LDr = 0.36)33t3"(see Figure 24. The IR spectrum of guanine (Figure 2d) shows two bands at 1710 and 1640 cm-', corresponding to C f l stretching and NH2 bending vibrations, The LD' value of the former band (0.03) was much smaller than that of the latter one (0.30). Adenine shows two bands a t 1650 and 1598 cm-' (Figure 2e), which can be assigned to NH2 bending (LDr = 0.43) and C-C stretching (LD' = 0.21) vibrations, respecti~ely.~~~ As expected from the inefficient orientation in the matrix, both the UV and IR LIY values were of much smaller magnitudes than those observed in crystals. However, repeated measurements under the same conditions showed a good reproducibility of the observed values. The dependence of LD' on the stretch ratio was also measured in an attempt to estimate limiting values of the reduced dichroism. Two examples are shown in Figure 3. At R, = 4.3 and 6.5, IR and UV linear dichroism refer to the same film. The experimental uncertainty is indicated by error bars. Apart from a constant factor, the dependences of the reduced LR and W dichroism on the stretch ratio were (33) Tsuboi, M.; Takahashi, S.;Harada, 1. In "PhysicochemicalProperties of Nucleic Acids"; Duchesne, J., Ed.;Academic Prese: London, 1973; Vol. 2, p 91 and references cited therein. (34) (a) Teuboi, M. Appl. Spectrosc. Reo. 1969,3,45. (b) Tmoco, I., Jr.; Holcomb, D. N. Annu. Reo. Phye. Chem. 1964,15, 371. (35) Kyogoku, Y.; Higuchi, S.;Tsuboi, M. Spectrochim.Acta, Part A 1967,23, 969.
The Journal of Physical Chemtstry, Vol. 86,No. 8, 1982 1381
the same within the experimental uncertainty (for example, the LD' vs. R,curves of the 1626-cm-I and 240-nm bands of cytosine overlap each other if the first one is multiplied by 1.7). This can be seen as strong evidence that any matrix-olute interactions have very little influence on the effective transition moment directions. Determination of Order Parameters and Transition Moment Directions. Since the studied IR bands are inplane vibrations, we can use eq 2 for the calculation of the order parameters. There are three independent order parameters S,, S,,, and S in this equation. These can in principle be determinerfrom measurements on three vibrations with different 8' values, which is equivalent to determining the diagonalizing angle a and the two independent order parameters of the diagonal orientation tensor (Appendix B). Unfortunately, it was here only possible to exploit two IR bands with different 8' values, and in order to still be able to characterize the orientation we shall now consider the limits of the order parameters in relation to the observed dichroism. The maximum and minimum reduced dichroisms observed among all UV and IR transitions can, as seen from eq 3, be used to obtain upper and lower limits of S,, and S,,: S, I f/3LDrmin< f/3LDrmmIS,,
(4)
We have found it reasonable to base this estimate on reduced dichroism values extrapolated to R;' = 0. For example, in the case of thymine the approximate values 0.60 for the 1705-cm-' band and 0.41 for the 1670-cm-' band were obtained (Figure 3a). As seen from Figure 4a, where (S,,,S,) can be anywhere within the shaded area, these limits provide fairly little information about the orientation of thymine. Similar results are found for the other bases, except that the two-ring molecules have limits a little further toward the corner (1,-0.5) of perfect orientation. However, although the relative magnitudes of S,, and S,, are very difficult to estimate since they should sensitively depend on the form of the molecules and on substituents, experience from other planar molecules of similar size can give a pretty good idea about the magnitude of S,,. Comparison with other studies thus indicates that S,, is less than ca. -0.30. In S,,), we can see from Figure 4a fact, since S,, = -(S,, that the limit of S,, is -0.32. Let us therefore test some S,, values between this limit and the lower theoretical one (-0.50), by insertion into eq 2, to calculate the corresponding values of the order parameters S,, and S,. For thymine, in Figure 4b, we take the arbitrary z'axis to be the N3-C6 line and rotate the yz system to find the angle a at which S,, vanishes. For example, since the angles 8'1705 and 8'167,) are known (72' and -63' relative to the z' axis),3swe have 0.60 = 3(S,, sin2 72' + S,, cos2 72' + S,, sin 72O cos 72')
+
0.41 = 3(Syysin2 (-63')
+ S,,
cos2 (-63") + S,, sin (-63') cos (-63'))
from eq 2 (a= ) ' 0 and the observations LDrITw= 0.60 and LD'1670 = 0.41. The order parameters S ,, and S,, determined from these equations are displayed in Figure 4b at four different S,, values. The result of the same calculations at various rotational angles a (0' < a I180') is also shown in Figure 4b. As expected (Appendix B) S,, becomes zero at the same time as S,, is a maximum (and S, a minimum), for example, at a = 14' if S,. = -0.50, or a = 18O if S,, = -0.455, and so on. The diagonal system xyz and corresponding order parameters (Sz,,SyJ are given in
1382
The Journal of Physical Chemistry, Vol. 86, No. 8, 1982
Matsuoka and Norden
a
TABLE 11: Transition Moment Directions in Thymine, Uracil, and Derivatives ~
(Ai
Y
P
compd 0.5 .......
::.::,::-.
szz
1.0
.... .... ... .:.. .. .. . .
I
f
transitions
wavelength, nm
~
~~~~
orientation," deg
thymine
I
uracil
I
1-methylthymine 1-methyluracil
I I I I
Experimental 266 -31 ?: 6 or 51 % 20 260 7 + 2 or -11 % 3 270 -20 275 -19 270 -9 276 0 or 7
thymine
I I I I I
Theoreticalb 267 156 257 169 258 171 250 -8 244 179
uracil
~~
ref this work this work 5 11 5 12 27 30 27 28, 29 30
Angles measured from the N,-C, line toward the N, atom. Italic values are the most probable directions (see Discussion). MO calculations (SCF-CI,27SCF-MOCI,a.m variable electronegativity PPP and CNDO/s-CI"). Szz:Q.3?4
0 3: ................
.... ....
a+
.......... 90b
-03-
0 3-
522-0.323
.....+............. ..__
/..............
CY-,
........... ,
O -03-
9
/
.w ................
......
.',.%
,y0.40
,,30.25 -010
.....;.....
..-.
CC)sxx - 0.400
-
0.40
red0.25 10.10 SZZ
1
- 0.15-
..
0
-
....... .....-........... E.-..-' .
-0.15-
5%
TABLE 111: Transition Moment Directions in Cytosine wavetransi- length, tions nm
I
268
I1
240
I I1 I I1 I I1
265 230 270 227
I I1 I I1 I I1
302 243 263 213 295 233
-0.10
¶ob-'-.
........ / ..........................
0.10
Flgure 4. (a) Experimentally estimated upper and lower limits of S and S , and diagonal (S,,S,) values of thymine (0)for S, = -0.503 (A), -0.455 (B), -0.400 (C), and -0.325 (D). Consistent electronic transition moment directions (douMeheaded arrows) in the correspondlng diagonal system xyzare shown for the reaspective S,vakres. (b) The chosen z'axis and calculated , S (-) and S , (- -) curves 0 to 180' and for thymine. Calculation accordlng to eq 2 from a = ' diagonal system at S,, = 0.
-
Figure 4a for the respective S,, values. The estimated diagonal order parameters Syyand S,, now allow any electronic transition moment angle 0 to be determined according to eq 3. Thus, for transition I of thymine, we have from the observed LDr2= 0.51 = 3(0.126 sin2 01 + 0.374 cos2 0,) which gives lt9,l = 65' relative to the z axis. This result is also shown in Figure 4a, together with the directions obtained with S,, = -0.455 (B), -0.400 (C), and-0.325 (D). The same procedure was applied to the other bases, and the result is shown in Figures 5 (cytosine and uracil), 6 (guanine), and 7 (adenine). In the case of uracil, the point CSZz,S,,,)was off the orientation triangle (Figure 5a) for any S,, 1-0.253 and S,, 5-0.312, so -0.312 < S,, < -0.253. The point (0.217,0.070) (S,, = -0.287) was then taken as representative for uracil, which is also consistent with the values of thymine and cytosine. The S,, values of guanine
ref
orientation," deg Experimental 25 i 3 o r - 4 6 f 4 6 +4 o r - 2 7 + 3 12i 3 between - 11 and 9 142 1 -5i 3 35 + 1 4 o r - 2 8 + 7 1 5 + 2 0 o r - 6 2 16 Theoreticalb 68 177
this work 7 13 21
27
18
28
24 65 124
30
" See footnote a , Table 11.
See footnote b , Table 11.
and adenine should, according to the shape of these molecules, be smaller than the pyrimidines. From the upper value of cytosine, S, = -0.370, we shall preliminarily assume S,, I-0.40. By experience, S,, = -0.50 is never reached in PVA, and comparison with S,, values observed recently for g-aminoacridinium+ (-0.464), acridone (-0.452), and 9-amino-lO-methylacridinium+(-0.416)36 suggests that the interval that we assume for the purines (-0.455 5 S,, 5-0.400) is well justified. Discussion Our value of the transition moment angles are in Tables 11-V compared with corresponding data from single-crystal studies and MO calculations. As shown in the tables, there are two possibilities for each transition. However, one direction can usually be discarded after considering the result in relation to the single-crystal data or fluorescence polarization data. Thymine and Uracil. The present results only give evidence for a single transition (I) in the first absorption band of thymine and uracil. This result is supported by the polarized fluorescence data of Wilson et al." and (36) Matsuoka, Y.;NordBn, B. Chem. Phys. Lett. 1982, 85, 302.
Transition Moments of Nucleic AcM Bases
The Jouml of Fhyslcal Chemistty, Vol. 86, No. 8, 1982 1383
TABLE IV: Transition Moment Directions in Guanine and Derivatives wavelength, compd transitions nm guanine
Experimental 280 248 270 230 278 253 294 24 1
I I1 I I1 I I1 I
O-ethylguanine
guanine hydrochloride
I1 I I1 I I1 0 Angles measured from the N,-C, line toward the N, See footnote b, Table 11. sion). guanine
SYYr
orientation: deg
ref
423or-61i4 31 * 3 o r - 8 8 i 4 -14t 5or44i 5 - 8 5 2 1 0 o r - 6 5 2 10 -4* 3 -75* 3 2 - 82
this work
8 9 9
Theore ticalb 288 27 110 243 36 265 30 158 261 62 atom. Italic values are the most probable directions (see Discus-
a
I
z
- 0.25 -
-0.5-
5
b
b
szz
SYZ
szz
(A) Sxx:-0.455
so' -0.3 '*-
~ 0
- 0.3 - 0.025
-0.6
*,.
