4466
J. Phys. Chem. 1995, 99, 4466-4470
Transition Moments of 2'-Deoxyadenosine Leigh B. Clark Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0359 Received: January 4, 1995@
Polarized reflection spectra from two faces of single crystals of 2'-deoxyadenosine have been measured (600140 nm). Absorption curves obtained by Kramers-Kronig analysis of the reflection spectra are consistent with the set of transition moments reported in a similar study of 9-methyladenine and 6-methylaminopurine. The present results for six observed transitions are as follows: I (f= 0.09, B = 67", /z = 274 nm); I1 (f= 0.18, 8 = 35", II = 254 nm); I11 (f= 0.25, 8 = - 4 5 O , /z = 213 nm); IV (f= 0.11, 8 = 15O, II = 202 nm); V (f= 0.33, 8 = 64", ,I= 189 nm); VI1 (f= 0.28, 8 = 6", ,I= 159 nm). [The in-plane angle, 8, is measured from the C4-Cs reference axis toward c6. Transition VI of the earlier study was not observed.] The shifts in transition moment directions arising from crystal field induced exciton mixing in 2'-deoxyadenosine are calculated to be small (1 1" or less), and therefore the results derived from the crystal spectra using an oriented gas approach appear to be fairly representative of the "free" adenine chromophore. Furthermore, it is concluded that the presence (or absence) of a deoxyribose group has little effect on the spectral parameters of the adenine chromophore.
Introduction
Knowledge of the experimental transition moment directions of the purine and pyrimidine bases finds use in the interpretation of the optical properties of nucleic acids and other polymers and in the refinement of theories of the electronic structure of complex, heterocyclic molecules. Much of the available transition moment data for these chromophores has been obtained from optical studies of single or linear dichroism studies of stretched Although an appreciable body of data is now available from the study of crystal systems, there are still questions regarding the confident application of the crystal results in the predictions of polymer properties. In this regard, intermolecular interactions in crystals lead to intensity shifts and corresponding changes of the apparent transition moment directions. Attempts at treating the interactions arising from exciton mixing (transition dipole terms)* and the terms arising from permanent electric dipoles have been made. Very recently, Sreerama, Woody, and Callis have performed an extensive theoretical analysis of the combined effects of dipole terms involving both permanent dipole and transition dipole moments on the state mixing in crystals of 9-methyladenine and N6-methyladenine (6-methylaminopurine).lo An understanding of these effects is necessary in order to extract from crystal spectra transition moment information pertaining to the free chromophore. It is the latter for which specific information is needed. The present work was undertaken to provide data for a third crystal system containing the adenine chromophore. The crystal structure of 2'-deoxyadenosine is, of course, different from that of the two systems containing the adenine chromophore that were studied previously: 9-methyladenine (9-MA) and 6-methylaminopurine (6-MAP). Changes in the crystal environment might lead to differences relative to the earlier results, and a further test of the methods of Sreerama, Woody, and Callislo might then be possible. A second motivation was to determine the effects, if any, of the presence of the deoxyribose group bonded directly to the chromophore. For example, does the deoxyribose group with its bulk and array of dipoles alter the @
Abstract published in Advance ACS Absrracrs, March 1, 1995.
transition moment directions of the base chromophore in any significant fashion? Are the transition moment directions obtained from single crystal studies of, for example, 9-methyladenine applicable to adenosine, deoxyadenosine, or to nucleotide versions of the chromophore? The recently published results for guanosine indicate that there is little effect indeed.6 Another example of this result would be an important corroboration of this conclusion. In this paper we present polarized, ultraviolet spectra taken from single crystals of 2'-deoxyadenosine. The absorption curves have been derived from experimental polarized reflection spectra through Kramers-Kronig analysis, and these spectra are compared with the results obtained from an earlier study of 9-methyladenine and 6-(methylamino)purine crystal^.^.^ One of the several spectra obtained is for radiation polarized close to perpendicular (18") to the plane of the adenine chromophore, and this spectrum provides some information regarding transition intensity polarized normal to the molecular plane.
Crystal Structure and Procedures Single crystals of 2'-deoxyadenosine were easily grown by slowly cooling hot, saturated solutions (-48 h) or by slow evaporation. The crystals obtained were prisms that were several millimeters across and up to 10 mm long. The crystals were elongated along the c axis and showed very good cleavage (with a razor or scalpel blade) parallel to the b axis. The planes of the two adenine chromophores of the unit cell lie close (18") to the cleavage plane which has Miller indices (201). Indexing of the crystal planes was performed goniometrically by measuring interfacial angles. The structure of 2'-deoxyadenosine has been presented by Watson, Sutor, and Tollin as monoclinic, space group P22 with two molecules per unit cell.I1 A projection of a representative molecule onto the (201) cleavage plane is shown in Figure 1. The relationship of this plane to the crystal morphology is shown in Figure 2. Data were collected from freshly cleaved surfaces of the (201) plane. In addition, spectra were measured from a plane cut perpendicular to the (201) plane and parallel to the b axis. This prepared face had Miller indices close to
0022-365419512099-4466$09.00/0 0 1995 American Chemical Society
Transition Moments of 2'-Deoxyadenosine
J. Phys. Chem., Vol. 99, No. 13, 1995 4467
Ref
,
Wavelength (nm) 18
6pO
,
,
4qO
2010
250
3O :
10 L
I
, :1
If0
1O:
2'-deoxyadenosme
Y Y
14
,
12
ac cut face 2 -
0
0
Figure 3. Reflection spectra of 2'-deoxyadenosine for radiation polarized parallel the b axis and nc-plane of both (201) and (26 o 5 ) faces.
Figure 1. Projection of a representative 2'-deoxyadenosine molecule onto the (201) crystal plane. The disposition of the other molecule in the unit cell can be obtained by a 180" rotation about the b axis. Both molecules are spectroscopically equivalent on the (201j face. The arrow displays the DeVoe-Tinoco convention for transition moment directions.
f
ac (201)
~ ' ~ " ' ' ~ " ' ' " " ~ ' " " ' " " " ' ' ' '
Wavelength (nm) I
I
?:o,
I
I
1
,
200 1 , I 180 I I
I
I
, ,
160 150 I 140 I
1
I
130
30000
,- 25000
-i5s
20000
v
5 .w
15000
Q
' 8
10000
v)
5000
0
Frequency (1 O3 cm") Figure 4. Absorption spectra derived by Kramers-Kronig analysis
of the reflection spectra shown in Figure 3.
cut (26 0 5) *-.
-.
Y
Figure 2. Crystal form of 2'-deoxyadenosine. (top) A typical crystal grown by slowly cooling saturated, aqueous solutions is shown. (bottom) View down the b axis showing the cut (26 0 5) plane (dashed line).
(26 0 5) and was cut and polished with the diamond blade of an ultramicrotome. The technique for this procedure has been described before.6J2 Spectra were measured immediately after polishing. Each crystal face yields two orthogonally polarized spectra, viz. one polarized parallel to the b axis and another polarized perpendicular to the b axis (or parallel to the ac plane). Since the b axis spectrum of the cut face was identical to the b axis spectrum obtained from the (201) cleavage plane, we conclude that no spectrally apparent disturbance of the lattice was created by the polishing process. The spectrometers used to collect the reflection data and the Kramers-Kronig transformation procedure have been described
b e f ~ r e . ~The J ~ solution spectrum was obtained with a nitrogenflushed Cary 15 spectrophotometer using trimethylphosphate solvent (Aldrich Chemical Co.) in 0.1 mm pathlength absorption cells. Spectra
(201) Face. The reflection spectra polarized along the b axis and ac plane taken from the (201) cleavage plane along with the ac-plane polarized spectrum of the polished face are shown in Figure 3. The corresponding absorption spectra derived by Kramers-Kronig analyses are shown in Figure 4. The spectra from the (201) face of 2'-deoxyadenosine resemble those from the (100) face of 9-meth~ladenine.~This resemblance is not surprising since the disposition or orientation of the adenine chromophores relative to the principal optical axes of these faces are very similar in the two structures. Analysis of the spectra is complicated owing to the overlapping of absorption bands throughout the region examined and the consequent ambiguity in resolving individual band components. The detailed analysis of similar data in the earlier work on 9-methyladenine and 6-(methy1amino)purineled to a mutally consistent set of transition moments for eight electronic transitions for the adenine chromophore. This derived, model spectrum when applied within the context of the oriented gas model was consistent in reproducing the observed spectra taken
4468 J. Phys. Chem., Vol. 99, No. 13, 1995
Clark
TABLE 1: Comparison of Model Spectrum for Deoxyadenosine and 9-Methyladenine ~~
deoxy A
9-MAa
P
v (cm-')
I
35 200 36 400 37 500 38 500 37 500 47 000 49 000 53 700 58 000 63 000 68 000
I1 I11 IV V
VI' VI1 VIIIf
Ad (cm-I)
v (cm-')
1100 1200 1200 6000 4500 4500 4500 5000 6000 7000 9000
34800 35 800 36500 36500 39400 47 000 49500 52800 58 000 63 000 70 000
0.016 0.09
83
0.068 0.18 0.25 0.11 0.30
25 -45 15 72 -45 6 -45
0.10 0.23 0.10
fb
8' (deg)
0.012 0.008 0.004}
0.066 0.18 0.25 0.11 0.33 0.07 0.28 0.10
0'09
67 35 -45 15 64 -45 6 -35
Ad (cm-') 750 900 1200 4000 4000 4500 5000 5500 7000 7000 8000
Taken from ref 5. The oscillator strength for the first vibronic component of transition I was incorrect in Table 3 of ref 5. The correct value is shown here. Three-dimensional oscillator strengths. Transition moment direction according to the DeVoe-Tinoco convention. See Figure 1. Full width at half maximum used for Gaussian construction of spectra. Included here but observed only in 6-MAP spectra. Somewhat uncertain. funcertain. 35000 T3yo,,3p?,
5
30000
25000
7
g
20000
Y
.-6
15000
,
,2;0
Wavelength (nm) , , , 2 y 0 ,-,I;? , l ~ o , I p o'PO, ,
,
1
yo
,
i
I
g
10000
v)
a
a
5000
o1.""""'"""'''''"~ 30
40
50
60
70
80
Frequency (lo3 cm-') Figure 5. Reconstructed crystal absorption spectra for (201). The spectra shown are obtained by projecting the model adenine spectrum onto the crystal axes and then Gaussianizing the result according to the parameters given in Table 1. The curves here are to be compared with the corresponding experimental curves of Figure 4. from both of those crystal systems. All of the observed strong bands were consistent with in-plane polarized transitions. It is of interest therefore to see if transition moments of the model spectrum derived in the earlier work are consistent with the spectra obtained here for 2'-deoxyadenosine. With this proposition in mind, the model spectrum for the adenine chromophore obtained earlier from crystal spectra of 9-MA and 6-MAP were used as the starting point. Transition moment vectors of the adenine model spectrum were projected onto the principal optical axes of the (201) face; the component oscillators strengths were evaluated and Gaussianized. Predicted crystal spectra were then obtained simply by summing the individual band components. Finally, the input parameters were varied in order to obtain a reasonably close fit with the experimental deoxyadenosine curves. The spectra computed in the above fashion for the (201) plane are shown in Figure 5. In order to match better the appearance of I, this transition was divided into a vibronic progression. The bulk of intensity for I, however, is represented by a transition centered on the band. Only modest changes in transition moment directions and oscillator strengths (relative to the earlier work) were necessary in order to obtain the fit shown. Table 1 compares the earlier model parameters with those used in fitting the present spectra. The largest apparent angular shift in the transition moment directions is the change of 16" for band I. These results not only support those obtained earlier
for the adenine chromophore but also suggest that deoxyribose substitution does not lead to any large distortions of the optical properties of the basic chromophore. Although this outcome was not unexpected, we felt it was valuable to establish the result by experimental demonstration. Cut Face. The spectrum polarized perpendicular to the b axis on the polished (26 0 5) face shows a generally increasing absorption background with several shoulders. The shoulders occur at or near energies of the in-plane polarized transitions and may arise in part from the modest projection of those transitions vectors onto this axis. Although the angle between the plane of the adenine moieties and the (26 0 5) face is 18", projections of in-plane polarized transitions onto the ac-plane axis range from a maximum of 10% for transitions polarized along the long molecular axis to essentially 0% for transitions polarized along the short molecular axis. We have attempted to remove the components of the in-plane polarized transitions from this spectrum so as to obtain a residue arising from transition intensity polarized normal to the adenine plane. The model spectrum was projected onto the ac axis of this face, and the resulting components were Gaussianized and subtracted from the experimental spectrum. The resulting residue curve is shown in Figure 6. In the region of transition I there is no residue. This result is consistent with the notion that I is, in fact, polarized close to the molecular plane. The appearance of the first positive feature near 41 kK suggests either that I1 has a substantial out-of-plane character or that this feature marks the appearance of a band polarized normal to the adenine planes. If this feature is a component of 11, then the effective, out-of-plane cant of I1 amounts to 10". This value seems large in view of the approximately 1" out-of-plane shift predicted for this transition from crystal mixing calculations (vide infra). If this feature is a distinct transition, then its oscillator strength amounts to -0.01. The increasing diffuse absorption along this axis suggests that there may be considerable congestion building toward higher energy for transitions with polarizations normal to the molecular plane. There have been reports of weak nn* transitions to the red of the lowest energy ZJG*band. In particular, the phosphorescence excitation study of adenosine given by Daniels et a1.14 assign two origins at 304 nm and 331 nm to nn* transitions. The suggested oscillator strengths of these bands were of the order of and thus their observation by reflection spectroscopy is out of the question. Recent INDO/S calculations1° for 9-methyladenine place the lowest two m* transition at 285 and 275 nm with oscillator strengths of 0.003 and 0.006. Again, such bands would be difficult to observe with the reflection
Transition Moments of 2'-Deoxyadenosine
J. Phys. Chem., Vol. 99, No. 13, 1995 4469
Wavelength (nm)
Wavelength (nm)
25000
20000
1
------Ac 20000
- -
r
c
iz
-E
c
'
.-+ E E 0
9
15000
10000
u)
I
~
5000
300 270250
350 '
"
'
1
~
'
1
180
220 200 ~
I
~
'I l'
I
'
I
160 150 '
I
'
I
I
I
140 '
I
2'-deoxyadenosine
i
i
......
randomized crystal spectra
30
40
50
60
70
Frequency (1 O3 cm")
Figure 7. Solution and randomized crystal spectra of deoxyadenosine. The solid curve is for solution in trimethylphosphate solvent, and the broken curve is the "randomized" crystal spectrum.
technique owing to their weakness. However, the same calculations position a stronger nn* band at 243 nm (f= 0.02), and this wavelength is nearly that of the 40.9 kK (244 nm,fX 0.01) feature in the residue spectrum (Figure 6). Given the above evidence, this feature is tentatively assigned to an nn* transition. Two more nn* bands are predicted to occur near 49 kK with a combined oscillator strength of 0.024, and this intensity is just about that which is observed in the residue spectrum between 46 and 50 kK. When crystal field mixing of states is treated using the N O / S calculations mentioned above,1° serious mixing of m* and nn* transitions is predicted. The extent of mixing is such that the nature of some m* transitions changes to nn*. Even so, unless such a transition was somewhat removed from the much stronger nn* bands, the in-plane polarized character would probably not be distinguishable. The intensity polarized normal to the molecular plane would still appear in, for example, the residue spectrum. In this sense, the residue spectrum does reflect perpendicularly polarized intensity irrespective of the mixing of states. Solution Spectrum. Finally, it is of interest to compare the crystal spectra with the spectrum obtained from 2'-deoxyadenosine dissolved in trimethylphosphate. For this comparison we have simply summed the spectra of the three orthogonal axes and divided by three to effectively randomize the result. The resulting spectrum is presented in Figure 7 along with the actual solution spectrum. Except for the apparent difference in the energy separation between I and 11, the two spectra are very nearly the same. The similarity in overall oscillator strengths between the two spectra is evidence that crystal forces are not seriously distorting the electronic states by creating large hypo- or hyperchromatic effects or radical energy shifts.
crystal face is examined. The result is that the dichroic ratios of transitions deviate from oriented gas values, and the apparent transition moments deduced from data from different crystal faces will themselves in general vary. The natural question to ask is, how representative of the free molecule are transition moments deduced from crystal spectra. Previous work2 has suggested that angular changes of the direction of transition moments rarely exceeds 5"-10". In w>.at follows we will appraise the magnitudes of these exciton mixing effects by developing (through trial and error) a model free molecule spectrum that leads to the observed intensity distribution of the (201) face when exciton mixing is treated. Crystal wave functions and energies are obtained by diagonalizing the usual Hamiltonian matrixL5with elements
where the Eli are transition energies, 6i, is the Kronecker 6, and qi is the linear combination of one site exciton functions for the jth excited state and the Tth irreducible representation of the crystal factor group. For deoxyadenosine with two molecules per unit cell there are two such linear combinations
where the subscripts refer to the sites and
n
where n and s refer to cells and sites, repectively. Here Wi,, is a single site exciton function where the molecule at site s of cell n is excited to the jth excited state and all other molecules are in their ground state.
Crystal Field Effects Intermolecular interactions in the crystal cause the zeroth order (oriented gas) crystal states to mix. The mixing coefficients depend on dipole-dipole lattice sums which in turn are dependent on the direction of the exciton wave vector, k. Since the direction of the wave vector of the optically accessible crystal states must match the direction of the normal to the crystal face, the state mixing will vary depending on which
Site 2 is obtained from site 1 by a Cz rotation about an axis parallel to b and a translation parallel to b, i.e., 1 = (x,y,z)and 2 = (-x,y+l/2,-z). Hence the "-"factor group combination corresponds to an Au crystal state and is polarized perpendicular to the b axis of any crystal face parallel to b. On the other hand, the combination corresponds to a B, crystal state and is polarized parallel to the b axis.
"+"
4470 J. Phys. Chem., Vol. 99, No. 13, 1995 TABL 2: Inner Dipole-Dipole Lattice Sums (cm-l/ *P
B
Clark
fr
TABLE 3: Comparison of Models Based on Oriented Gas and Exciton Mixing oriented gas“
site combinations
1.1 aa
bb cc ac
2752 58 --5375 573
exciton mixingb
1.2
f
8 (deg)
f
8 (deg)
-1516 -960 31 -47
0.09 0.18 0.25 0.11 0.33 0.07 0.28 0.10
67 35 -45 15 64 -45 6 -35
0.05 0.16 0.21 0.05 0.48 0.09 0.35 0.10
78 32 -45 20 64 -45 11 -35
mn refer to the equivalent and inequivalent site combinations 1,l or 1,2. The sums for vector combinations a b and bc equal zero. Also 2,2 = 1,l; 1,2 = 2,l for all axis combinations.
The Hamiltonian is a sum of all individual molecule Hamiltonians plus all intermolecular electrostatic interactions in the crystal, V. A separate diagonalization must be carried through for each crystal axis. Matrix elements can be approximated by fiist expanding V in a multipole series and retaining the leading dipole-dipole term. This term involves lattice sums of transition moments as well as sums involving transition moment and permanent electric dipole moments. Since little is known about the permanent dipole moments of the ground and excited states of deoxyadenosine, we are forced to ignore such terms. Nevertheless, the results of such limited exciton mixing calculations are still useful in appraising the magnitudes of the effects involved. Dipole-dipole lattice sums were evaluated following the E w a l d - K ~ r n f e l d ~recipe. ~ ? ~ ~ In this procedure each lattice sum is expressed as a sum of two terms: an inner sum, $,: and a macroscopic term
where YO is the volume of the unit cell and the dj’ is thejth transition dipole on site r. Any general lattice sum can be expressed in terms of lattice sums of unit vectors directed along the three crystallographic axes, and we give in Table 2 the unit inner lattice sums for dipoles directed along the a, b, and c axes. The appropriate macroscopic term must be added in any particular case. The above approximate calculations were carried out for the model adenine spectrum that was used to fit the (201) spectra in the previous section. The model was then varied in a trial and error fashion until the predicted component oscillator strengths agreed with the components that were obtained when the oriented gas model was fit to the experimental (201) spectra. A reasonable fit was obtained with little trouble, and only modest calculated changes in apparent transition moment directions were needed to accomplish this task. The new, adjusted model “free molecule” spectrum that satisfactorily produces the observed (201) spectra is given in Table 3 along with the model spectrum obtained in the oriented gas analysis for comparison. The apparent shift of 11” for band I is the largest change, and most are appreciably less. Conclusion
The set of transition moments needed to fit the crystal spectra of 2’-deoxyadenosine is close to that obtained earlier for the adenine chromophore. The directions of the two lowest energy transitions are modestly affected (-16” and +loo for I and 11,
A@ (deg) -11
+3 0 -5 0 0 -5 0
”Parameters used to fit (201)spectra assuming the oriented gas model. Input parameters that lead to the observed spectra when exciton mixing is considered. Difference owed to crystal field mixing.
respectively). No change in their intensities is observed. These results suggests that the presence of a deoxyribose group has little influence on the transition moments of the parent chromophore, and as a result we conclude that optical parameters evaluated for the individual bases or methylated bases are applicable to the chromophores in more complicated assembages like polynucleotides and nucleic acids. Calculation of the approximate effects of exciton mixing again appear to suggest that only modest changes arise from intermolecular interactions in the crystal and that the transition moments obtained from crystal spectra using the oriented gas analysis are reasonable representations of the actual moments. 2’-Deoxyadenosine is the third molecule containing the adenine chromophore (after 9-methyladenine and 6-methylaminopurine) for which a significant number of transitions have been characterized. The fact that a “single set” of transition parameters is consistent with the crystal spectra of all three systems reinforces the notions presented above. It must be recognized that the analysis given here of the spectrum of such a complex chromophore as adenine must surely miss a number of weaker bands in the spectrum. Many such weaker bands must occur in the energy range studied here. That the absorption intensity in all these spectra can be reasonably reconstructed with eight principal transition moment vectors suggests that the main absorption bands are being accounted for adequately. References and Notes (1)Callis, P.R. Ann. Rev. Phys. Chem. 1983, 34, 329. (2)Zaloudek, F.;Novros, J. S.; Clark, L. B. J . Am. Chem. SOC.1985, 107, 7344. (3) Novros, J. S.;Clark, L. B. J . Chem. Phys. 1986, 90, 5666. (4)Clark, L. B. J . Phys. Chem. 1989, 93, 5345. (5) Clark, L.B. J . Phys. Chem. 1990, 94, 2873. (6) Clark, L.B. J . Am. Chem. SOC. 1994, 116, 5265. (7) Matsuoka, Y.; Norden, B. J . Phys. Chem. 1982, 86,1378. (8) Fucaloro, A.; Forster, L. S. J. Am. Chem. SOC.1971, 93, 6443. (9)Holmen, A.; Broo, A.; Albinsson, B. J . Phys. Chem. 1994,98,4998. (10) Sreerama, N.; Woody, R. W.; Callis, P. R. J . Phys. Chem. 1994, 98, 10397. (11) Watson, D. G.;Sutor, D. J.; Tollin, P. Acra Crysrallogr. 1965, 19, 111.
(12)Xu,S.; Clark, L. B. J . Am. Chem. SOC.1994, 116, 9227. (13) Campbell, B. F.;Clark, L. B. J . Am. Chem. SOC.1989, 11, 8131. (14) Daniels, M.; Ballini, J.-P.; Graslund, A,; Rupprecht, A,; Asbrink, L. Biophys. Chem. 1988, 30, 225. (15) Craig, D. P.;Walmsley, S. H. Excitons in Molecular Crystals: Theory and Applications; W. A. Benjamin, Inc.: New York, 1968. (16) Komfeld, H. Z . Phys. 1924, 22, 27. (17)Clark, L. B.; Philpott, M. R. J . Chem. Phys. 1970, 55, 3790. JP9500293
