5672
J . Phys. Chem. 1989. 93, 5612-5678
6. Raman excitation profiles of the drug in solution and bound to calf thymus D N A were obtained. There were no changes in the Raman frequencies upon interaction with nucleic acids, only a decrease in intensity. Acknowledgment. W e acknowledge the help of H. Hatfield, who wrote the normal-coordinate analysis programs for use on an IBM-PC. We also thank the National Science Foundation
(CHE-8510614) for partial support of this work. Registry No. Mitoxantrone, 65271-80-9. Supplementary Material Available: Tabular listing of the results of normal-coordinate analysis of 1,4-diaminoanthraquinone including molecular geometry, symmetry coordinates, and force constants ( 1 1 pages). Ordering information is given on any current masthead page.
Ultraviolet Raman Excitation Profiles for the Nucleotides and for the Nucleic Acid Duplexes Poly(rA)-Poly(rU) and Poly(dG-dC) Joseph R. Perno, Christine A. Grygon, and Thomas G. Spiro* Department of Chemistry, Princeton University, Princeton, New Jersey 08544- 1009 (Received: November 29, 1988)
Excitation profiles (EP’s) for resonance Raman (RR) bands of deoxynucleotide monophosphates dUMP, dCMP, dGMP, and dAMP (U = uridine, C = cytidine, G = guanosine, A = adenosine) have been obtained with excitation at wavelengths between 300 and 192 nm by using a H2-Raman-shifted YAG laser. The EP’s have been corrected for the preresonance enhancement of the internal standard, sulfate. They show a characteristic two-banded appearance, associated with the 260and -2200-nm absorption bands of the purine and pyrimidine bases. Different RR bands are selected for maximum enhancement in the low- and high-energy regions, reflecting substantial differences in the FranckCondon products for the different electronic transitions. The dCMP EP’s are uniquely flat and weak in the long-wavelength region. For dCMP and dAMP, there is evidence of vibronic enhancement of specific RR modes at intermediate wavelengths. The EP’s show considerably more structure than do the absorption spectra, revealing the underlying electronic transitions. For dUMP, dAMP, and dGMP, there is evidence for enhancement via a weak T-T* transition on the low-energy side of the -260-nm absorption band. EP‘s are also reported for the nucleic acid duplexes poly(rA)-poly(rU) and poly(dG-dC) and for the helical single-stranded poly(rA). The main effect, relative to the mononucleotides, is Raman hypochromism, associated with the absorption hypochromism of the major -260 and -200-nm bands. In addition a distinct blue shift is observed for the strong -260-nm electronic transition of the nucleotides, and the lower energy transitions are seen more clearly in the polymers. There is no evidence, however, for enhancement of out-of-plane modes via the n-n* transitions which may also be located in this spectral region.
-
Introduction
It has recently become possible to obtain resonance Raman spectra of nucleic acid bases with excitation in the deep ultraviolet region using the H2-Raman-shifted pulsed Nd:YAG laser.’-4 Because of the high sensitivity and selectivity afforded by the resonance Raman effect, the technique has potential for probing local structure in nucleic acids. Survey spectra of the nucleotides have been published and likely enhancement mechanisms have been discussed.’-2 In addition UVRR spectra have been reported for the nucleic acid duplexes poly(dA-dT) and p ~ l y ( d G - d C ) . ~ Frequency and intensity effects associated with base pairing and stacking have been analyzed. Kubasek et al.3 published low-resolution excitation profiles for the nucleotides and discussed enhancements for the electronic transitions near 260 nm relative to those near 200 nm, especially with reference to previous inferences based on preresonance Raman enhancements I n the present study we have obtained more detailed and extended profiles and have taken into account the preresonance enhancement of the internal standard, sulfate ion,5 via absolute intensity measurements. The excitation profile features are discussed in relation to the purine and pyrimidine excited states. I n addition we report excitation profiles for the polynucleotide duplexes poly(rA)-poly(rU) and poly(dG-dC), and for poly(rA), a stacked single-stranded helix. The most noticeable effect, relative to the nucleotides, is a substantial reduction in intensity, a t all wavelengths, attributable to electronic hypochromism due to base stacking. In addition, a distinct blue shift of the -260-nm excitation profile peak can be seen for both A and U in poly(dA)-poly(rU), as well as for A in poly(rA) and *Author to whom correspondence should be addressed
0022-3654/89/2093-5672$01.50/0
for G in poly(dG-dC). There is also augmentation of A, U, and G peaks a t longer wavelengths, -280 nm, which are attributed to weak A--H* transitions of these residues. N o evidence for n--H* enhancement of out-of-plane modes has been found. Experimental Section
The 5’-monophosphates of deoxycytidine, deoxyadenosine, deoxyguanosine, and deoxyuridine were purchased from Sigma. Poly(rA)-poly(rU), poly(dC-dC), and poly(rA) were obtained from Pharmacia Molecular Biologicals. Solutions of the mononucleotides and poly(rA) (-3 m M in base) and the polynucleotides (1.5-2.2 m M in base pairs) were prepared in 50 m M phosphate buffer (pH 7.0) containing 0.3 M sodium sulfate, added as an internal standard. The concentrations of all species in solution were determined spectrophotometrically with a Hewlett-Packard 8450A diode array spectrophotometer, using molar extinction coefficients of Doty and co-workers.6 Deep U V absorption spectra were obtained with a Cary 118 UV-vis spectrophotometer which was flushed with N2 for -4 h before data acquisition. The Z form of poly(dG-dC) was prepared by dissolving the polymer in a 4 M NaCl solution with 50 m M phosphate buffer.’ ( 1 ) Ziegler, L. D.; Hudson, B.; Strommen, D. P.; Peticolas, W. L. Biopolymers 1984, 23, 2067. ( 2 ) Fodor. S. P. A.; Rava, R. P.; Hays, T. R.; Spiro, T. G . J . A m . Chem. Soc. 1985, 107, 1520. ( 3 ) Kubasek, W. L.; Hudson, B.; Peticolas, W. L. Pror. Natl. Acad. Sci.
LSA 1985, 64, 451, ( 4 ) Fodor, S. P. A,; Spiro, T. G . J . A m . Chem. SOC.1986, 108. 3198. ( 5 ) Fodor, S. P. A.; Copeland, R. A.; Grygon, C. A.; Spiro, T. G . J . A m . .-
Chem. S O ~ . i n,press. ( 6 ) Voet, D.; Gratzer. W. B.; Cox, R . A.; Doty, P. Biopolymers 1968. I . 193.
0 1989 American Chemical Society
Excitation Profiles for Some Nucleotides
The Journal of Physical Chemistry, Vol. 93, N o . 15, 1989 5673
TABLE 1: Assignments for Nucleotide RR Bands Included in the Excitation Profile Measurements Y,
cm-l
cm-l
780 792
Br6
1230 1394 1476 1628
1208 1386 1410 1631
1652 1528 1294 783
:Y 1660 u: 1562 not given glycoside 771 Br6
682 1363 1485 1580 1678
604 1308 1501 1602 1751
730 724 1339 1356 1482 1562 1580 1677
Kk uf, or
ut
Y:
ut
PEDc
u!b
Kk ut
+ Br5 + Y:
A - 1476 cm-' B - 1394 cm-l C-1628 cm-l
dUMP N1C2 (14) + N I R (IO) + CSC4 (IO) N1C6 (9) N3C4 (8) -6C6H (23) C2N3 (15) -6N3H (48) - C40 (27) -NlC2 (38) C2N3 (17) N3C4 (22) N1C2 (21) + C6C5 (20) -NlC6 (19)
+
+ + + +
dCMP C20 (67) - C2N3 (15) -N3C4 (38) - N1C2 (14) N1C6 (28) + C5C6 (19) N I R (18) - C4N (14) - C4C5C6 (13)
dGMP Br6 + BrS 6N7C8N9 (15) + 6C5N7C8 (15) ufb -C8N( (26) - N1C6 (25) + N7C5 (16) Kk + u: 6C8H (40) - N9C8 (32) + C8N7 (21) vf -C4N3 (30) + C5C4 (24) - N7C5 (16) U C ~ C60 (48) -C5C6 (21) + C5C4 (11) + 6 N l H (11) - hr1C6 (IO) Br6
I A
assignmentb
ucalcd",
c
dAMP 6N7C8N9 (19) - N9R (14) 6C5N7C8 (12) + C4N9C8 (11) -N7C5 (39) C8N7 (12) -6C2H (29) - N9C8 (19) 6C8H (15) C5C4 (48) - C4N3 (31)
D 180
200
220
+
'Ab initio calculation of frequencies for the nucleic acid bases of uracil, cytosine, guanine, and adenine.'O bNotation with reference to the modes of the six- and five-membered rings.I0 cPotential energy distribution for 1methyl derivatives of uracil and cytosine and 9-methyl derivatives of guanine and adenine;)O % contribution from the major stretching and bending internal coordinate contributors indicated in parentheses.
Raman spectra were obtained with excitation wavelengths from 299 to 192 nm, produced by H,-shifting the frequency-doubled (532 nm), -tripled (355 nm), and -quadrupled (266 nm) output of a Nd:YAG laser as described previously.8 Front scattered light was collected from a flowing free stream of sample with quartz collection optics and a 1.25-m single monochromator (3600 grooves/mm holographic grating) and a solar blind photomultiplier (Hamamatsu R166UH). Spectra were recorded with 0.5 A/s increments and 1 s integration time at a spectral resolution of 8 cm-'. Mononucleotide spectra were the sum of 3-6 scans, and polynucleotide spectra were summed over 10-1 5 scans. Absolute Raman cross sections were determined from the peak height ratio of the sample and internal standard Raman bands by using the equation9
260
280
300
EXCITATION WAVELENGTH (nm)
Figure 1. Excitation profiles for the indicated RR bands of aqueous dGMP. I ""V"
] A t
NH.
+
+
240
6000
-1
\L
E - 1652 cm-I C - 782 cm-' D - 1528 cm-l
'1
dR
,
4000
2000
0 180
200
220
240
260
280
3 0
280
300
EXCITATION WAVELENGTH (rim)
Figure 2. Excitation profiles for dCMP.
A
dGMP A -1580 cm-1 13 - 1485 c m l C-1678 c m l D - 1363 em I
6000
=.
In[ ~0 - us]4: Is vo - vn
un=us--
where u, and us are the Raman cross sections for the sample and standard (981-cm-' band of sulfate), I, and I, are the peak intensities, u, and us are the vibrational frequencies for the sample and standard Raman bands, respectively, u,, is the laser excitation frequency, and C, and C, are the molar concentrations of the sample and standard, respectively. Self-absorption corrections* were negligible for the frequency regions examined. Cross-section values represent the average of several trials and carry error estimates of f10-15% (20% for 192-nm excitation). Variations in grating and P M T response as a function of wavelength were evaluated previously (see ref 5 ) and found to be (7) (a) Pohl, F. M.; Ranade, A.; Stockburger, M. Biochem. Biophys. Acta 1973, 335, 8 5 . (b) Thamann, T.J.; Lord, R.C.; Wang, A . H. T.; Rich, A. Nucleic Acids Res. 1981, 9, 5443. (c) Benevides, J. M.; Thomas, G. J., Jr. Nucleic Acids Res. 1983, 16, 5147. (8) Fodor, S. P. A,; Rava, R. P.; Copeland, R. A.; Spiro, T. G. J . Raman Spectrosc. 1986, 17, 471. (9) Dudik, J. M.; Johnson, C. R.; Asher, S. A. J . Chem. Phys. 1985,82,
1732.
180
200
220
240
260
EXClTA TlON WAVELENGTH (nm)
Figure 3. Excitation profiles for d G M P .
insignificant (
