J. Phys. Chem. 1983, 87,367-371
magnetic interactions do not lead to spectral diffusion of saturation and hence the V i line shape shows no sensitivity to this motional process.' At approximately 16 GHz, the z and x turning points will be coincident for the -1/2 nuclear manifold giving rise to a region of the spectrum which will have sensitivity to z-x interconversion Cy-axial motion) quenched. At approximately 30 GHz, the z and y turning points will be coincident for the -1/2 nuclear manifold giving rise to a region of the spectrum where sensitivity to z-y interconversion (x-axial motion) will be quenched. We conclude that utilization of multiple microwave frequencies will make it possible to observe anisotropic motion in greater detail. By positioning the z turning point relative to x and y one can adjust the way in which different motional processes will compete and alter ST-EPR spectra. Qualitative or first-order analysis is possible by comparison of experimental spectra with isotropic reference spectra if the two are recorded at two or three dif-
387
ferent frequencies which are meaningful in the context of the above discussion. Quantitative information can then be obtained with the aid of computer-simulated spectra. Our simulations suggest that, in order to ensure that the motion about the three principal axes is defined correctly, one must determine the relative angle between the diffusion tensor and the magnetic tensor. It may be possible that this restriction can be relaxed if spectra are recorded at a variety of microwave frequencies.
Acknowledgment. This work was supported by grants from the National Institutes of Health GM-07884 and the Muscular Dystrophy Association. A.H.B. and K.B. were recipients of fellowships from the Muscular Dystrophy Association. L.R.D. is a recipient of a Research Career Development Award. Registry No. GAPDH, 9001-50-7; [15N,2H]maleimide, 83803-37-6.
Molecular Orbital Study of the Protonation of DNA Bases Janet E. Del Bene Depafiment of Chemistry. Youngstown State Unlverslty, Youngstown, Ohio 44555 (Received: August 13, 1982)
Ab initio SCF calculationswith the STO-3G basis set have been performed to determine the optimized structures of the neutral and protonated DNA bases, thymine, cytosine, adenine, and guanine. Single-point Hartree-Fock calculations at these geometries have then been carried out with the split-valence 4-31G basis set to obtain the protonation energies. The most favorable protonation sites are O4on the C5 side of the C4=0 group in thymine, N3 in cytosine, N1 in adenine, and N7 in guanine. A relationship exists between the nature of the highest occupied n orbital and the preferred protonation site in each base, but a correlation between n orbital energies and relative protonation energies is not found. Protonation leads to significant geometrical changes in the base, particularly in bond lengths and angles near the protonation site. Charge transfer to the proton occurs and is accompanied by polarization of the electron density of the base toward the protonation site.
Introduction Protonation of nucleic acid bases at various centers plays an important role in certain biochemical processes and has been the subject of extensive experimental studies and theoretical investigations. The first theoretical studies based on ab initio calculations used molecular electrostatic potentials derived from ab initio wave functions for the bases as a means of investigating protonation sites.lr2 The molecular electrostatic potential describes the interaction energy between the unperturbed base and the proton (viewed as an external point charge), the assumption being that the interaction is purely electrostatic. While these studies have provided some insights into protonation, they do not yield values of protonation energies, and they neglect the electron redistribution (charge transfer and polarization) and structural changes which accompany protonation. Two recent ab initio studies of the protonation of selected DNA bases3i4 employed the minimal STO-3G basis set for the calculations, which were per(1)R. Bonaccorsi, A. Pullman, E. Scrocco, and J. Tomasi, Theor. Chim. Acta, 24, 51 (1972). (2) R. Bonaccorsi, E. Scrocco, J. Tomasi, and A. Pullman, Theor. Chim. Acta, 36, 339 (1975). (3) A. Pullman and A. M. Armbruster, Theor. Chim.Acta, 45, 249 (1977). (4)P. G.Mezey, J. J. Ladik, and M. Barry, Theor. Chim.Acta, 54,251 (1980).
formed at either standard or experimental geometries for the bases. However, it has since been demonstrated that this basis set severely overestimates absolute protonation energies and fails to give consistent relative protonation energies.k8 Therefore, it seems appropriate at this time to investigate the protonation of the DNA bases at a higher level of theoretical treatment. In the present study, optimized STO-3G structures for the neutral and protonated DNA bases thymine, cytosine, adenine, and guanine have been determined. For the ions, various isomers in which protonation occurs in the plane of the base have been considered. Single-point Hartree-Fock calculations with the split-valence 4-31G basis set have then been performed at these geometries to evaluate the protonation energies. The results of this study are reported in this paper.
Method of Calculation Protonation energies have been computed as the energies of the reaction B + H+ BH+ as given by HartreeFock calculations with the 4-31G basis setg at optimized
-
(5) H. Umeyama and K. Morokuma, J. Am. Chem. Soc., 98,4400 (1976). (6)J. E.Del Bene, J. Am. Chem. Soc., 100,1673 (1978). (7)J. E.Del Bene, Chem. Phys. Lett., 55,235 (1978). (8) J. E. Del Bene, Chem. Phys. Lett., in press. (9)W. J. Hehre, R. F. Stewart, and J. A. Pople, J. Chem. Phys., 51, 2657 (1969).
0022-3654/83/2087-0367$01.50/00 1983 American Chemical Society
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0
H \1 086
111 221
il
Del Bene
Jll9
7
OiS
H 1, Structure of thymlne.
STO-3G'O geometries. A recent study of the protonation of monosubstituted carbonyl bases has shown that, except for F as a substituent, relative protonation energies can be reasonably well described by this method, although absolute values are overestimated.E The optimized STO3G geometries of the bases and ions have been determined by using gradient optimization techniques."J2 A t convergence, bond distances and bond angles from the final and penultimate optimization cycles were identical with fO.OO1 A and f O . l O , respectively. All calculations in this study were done on an AMDAHL 470 V/5 computer using a version of the Gaussian 80 system of programs.13 Correlation and zero-point vibrational energy corrections also contribute to the protonation energy, but these have been neglected in the present study owing to the size of these systems. Although it is not possible at this time to evaluate the effect of correlation on the protonation energies of DNA bases, some relevant data for smaller systems are available. Correlation lowers the protonation energies of a series of monosubstituted carbonyl bases.8 At fourth-order Mraller-Plesset perturbation theory,14J5 correlation lowers the calculated Hartree-Fock protonation energy of H,C=O by 4.8 kcal/mol and that of H,C=NH by 3.9 kcal/mol.16 Moreover, it appears that trends in protonation energies of related bases are established at the Hartree-Fock level and are not usually altered by the inclusion of correlation.8J6 Of course, the zero-point vibrational correction would also lower the protonation energy of each of the DNA bases. This correction is +9.2 kcal/mol for H,C=O and +9.5 kcal/mol for H,C=NH.16
1.020
H Flgure 2. Structure of cytosine.
Y\
OZC
H
Flgure 3. Structure of adenine.
Results and Discussion The optimized STO-3G structures of the neutral and protonated DNA bases thymine, cytosine, adenine, and
-__ (10)R. Ditchfield, W. J. Hehre, and J. A. Pople, J. Chem. Phys., 54, 724 (1971). (11)P. Pulay, Mol. Phys., 17,197 (1969). (12)H.B.Schlegel, Ph.D. Thesis, Queen's University, Kingston, Ontario, 1975. (13)J. S. Binkley, R. A. Whiteside, R. Krishnan, R. Seeger, D. J. DeFrees, H. B. Schlegel, S. Topiol, L. R. Kahn, and J. A. Pople, Carnegie-Mellon University, Pittsburgh, PA 15213. (14)J. A. Pople, J. S. Binkley, and R. Seeger, Int. J.Quantum Chem. Symp., 10, l(1976). (15)R. Krishnan and J. A. Pople, Int. J. Quantum Chem., 14,91 (1976). ~~. ~, (16)J. E.Del Bene, M. J. Frisch, K. Raghavachari, and J. A. Pople, J. Phys. Chem., 86, 1529 (1982).
H
Flgure 4. Structure of guanine.
guanine are reported in Tables I-IV, and the bases are illustrated in Figures 1-4, respectively. The computed Hartree-Fock 4-31G protonation energies at these geom-
Protonation of DNA Bases
The Journal of Physical Chemistry, Vol. 87, No. 2, 1983 389
TABLE I: Thymine and Protonated Thymines
TABLE 11: Cytosine and Protonated Cytosines base
1-2f 2-3 3-4 4-5 5-6 1-6 1-H 2-0 3-H 4-0 5-c 6-H C-Hb C-HC
1.422 1.420 1.434 1.505 1.323 1.408
1.018 1.219 1.020 1.221 1.522 1.086 1.086 1.088
Rd
6-1-2 1-2-3 2-3-4 3-4-5 4-5-6 5-6-1 1-2-0 2-3-H 3-4-0 4-5-c 5-6-H 6-1-H 5-C-Hb H-C-HC C-C-be
ed
123.5 112.4 127.3 114.2 119.2 123.4 123.6 115.7 120.4 117.2 122.5 119.7 109.2 108.0 128.4
Distances ( A ) 1.353 1.348 1.347 1.353 1.479 1.484 1.509 1.507 1.324 1.323 1.440 1.436 1.026 1.027 1.349 1.349 1.029 1.028 1.210 1.210 1.526 1.526 1.086 1.086 1.086 1.086 1.088 1.088 0.991 0.991 Angles 121.1 119.3 124.4 113.1 119.9 122.2 124.5 117.0 118.8 116.8 124.4 119.0 109.6 108.6 126.8 109.3
(deg) 121.7 119.3 123.7 113.4 119.9 122.0 115.8 119.4 118.2 116.8 124.4 120.8 109.6 108.6 126.8 109.2
1.431 1.453 1.365 1.429 1.364 1.371 1.026 1.208 1.028 1.343 1.524 1.094 1.085 1.088 0.991 124.0 110.9 124.8 120.8 115.9 123.6 126.0 114.8 120.5 121.0 121.2 119.8 110.0 108.4 127.0 109.9
1.436 1.447 1.362 1.430 1.367 1.366 1.026 1.207 1.029 1.343 1.526 1.094 1.083 1.088 0.989 123.9 110.5 125.4 120.8 115.4 124.0 125.5 117.0 112.1 123.4 120.7 119.8 112.0 108.4 126.8 108.9
(I H+ a t 0, o n the N, side of the C,=O group. The in-plane meth 1 hydrogen. The out-of-plane methyl hydrogens. is the 0 - H distance, and e is the C-0-H angle. e The angle between the C-C bond and the bisector of the H-C-H angle. f See Figure 1 for numbering system.
etries are reported in Table V. This study of protonation has been restricted to protonation in the plane of the base, since K protonation is less favorable energeti~ally.'.~J' In the following sections, protonation of the individual bases will be discussed, followed by some observations of trends in geometric and electronic structural changes which accompany protonation. Some comparisons between the results of this study and experimental data will also be made. However, it should be noted that these calculations refer to protonation in the gas phase, whereas experimental data have been obtained in solution. Thymine. In thymine, four distinct protonation sites have been found which correspond to protonation of this base on each side of the two carbonyl groups. The data of Table V show that O4 protonation is preferred to 02. This correlates with the nature of the highest occupied n orbital in thymine, which, although delocalized, is associated primarily with 04. The more favorable protonation site at O4 is on the C5side of the C4=0 group, due in part to a smaller repulsion energy between the added proton and the in-plane hydrogen of the methyl group, compared to the proton and the more acidic imide hydrogen. The computed protonation energy is -21 1.8 kcal/mol. The electrostatic potential results of ref 1 also predict that O4 is the preferred site of protonation, but find only a single minimum at O2 which is symmetrically placed between the two adjacent N-H groups. It is interesting (17)R. Lavery, A. Pullman,and B.Pullman,Theor. Chim. Acta, 50, 67 (1978).
1-2e 2-3 3-4 4-5 5-6 1-6 1-H 2-0 4-N 5-H 6-H N-Ha N - ~ b
RC 6-1-2 1-2-3 2-3-4 3-4-5 4-5-6 5-6-1 1-2-0 3-4-N 4-5-H 5-6-H 6-1-H 4-N-H0 4-N-Hb
ed
H+ at O,(N,)
H+ at 0 , ( N 3 ) H+ at N,
Distances ( A ) 1.445 1.388 1.440 1.335 1.310 1.381 1.471 1.450 1.327 1.344 1.388 1.394 1.020 1.027 1.221 1.358 1.392 1.347 1.080 1.076 1.088 1.090 1.014 1.022 1.013 1.021 0.991
1.379 1.336 1.381 1.450 1.341 1.391 1.028 1.352 1.348 1.080 1.091 1.022 1.021 0.991
Angles (deg) 122.8 120.0 116.8 123.9 117.2 115.6 126.1 123.9 117.0 117.7 120.1 119.6 119.5 120.3 116.9 115.6 120.4 120.4 123.7 124.4 119.9 119.2 119.2 119.1 121.6 121.9 108.8
119.6 124.8 115.0 123.0 118.0 119.6 114.0 115.9 120.3 124.1 121.6 119.4 121.7 106.0
1.432 1.452 1.367 1.439 1.351 1.374 1.024 1.208 1.347 1.077 1.093 1.022
1.021 1.026 123.6 111.7 125.0 118.9 118.1 122.7 125.5 118.9 119.7 121.6 120.1 122.0 120.1 177.8
a The amino hydrogen cis to N,-C,. The amino hydrogen trans t o N3-C,. The 0,-H or N,-H distance. For 0, protonation, the C-0-H angle; for N protonation, the angle between the bisector of the C,-N,-C, angle and the N-H bond. e See Figure 2 for numbering system.
to note that the most favorable protonation site in the thymine molecular plane is the least favorable site for hydrogen bonding of thymine with water. The most stable hydrogen-bonded water-thymine structures are amide wobble dimers, one in the N1-H and O2 region, and the other in the imide region of the water-thymine surface. Owing to the bridging nature of these structures, they are more than 4 kcal/mol more stable than the open hydrogen-bonded water-thymine complex on the C5 side of C4=0, which has a stabilization energy of only -3.9 kcal/mol.18 Cytosine. Protonation of cytosine in the molecular plane may occur at any one of three distinct positions, two at the C2=0 group and one at N3. The data of Table V indicate that N3 is the preferred protonation site, with a protonation energy of -249.2 kcal/mol. Compared to protonation at NB,protonation at O2is less favorable by 7.5 kcal/mol when the proton adds on the N3 side of the C2=0 group and by 20.4 kcal/mol when it adds on the N1 side. Although the highest occupied n orbital of cytosine is highly delocalized over the carbonyl oxygen and NS, it is polarized toward N, so that there is a relation between the nature of this orbital and the preferred protonation site. The protonation energies for both nitrogen and oxygen protonation in cytosine are greater than the oxygen protonation energies of the other pyrimidine base, thymine. In ref 3, both electrostatic potential and ab initio SCF calculations with the STO-3G basis set were reported for the protonation of cytosine. While the electrostatic potential results favor N3as the protonation site by about (18)J. E. Del Bene, J. Chem. Phys., 76, 1058 (1982).
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The Journal of Physical Chemistry, Vol. 87, No. 2, 1983
TABLE IV: Guanine and Protonated Guanines
TABLE 111: Adenine and Protonated Adenines base
1-2d 2-3 3-4 4-5 5-6 1-6 5-7 7-8 8-9 4-9 2-H 6-N 8-H 9-H N-Ha N-H~
RC 6-1-2 1-2-3 2-3-4 3-4-5 4-5-6 5-6-1 4-5-7 5-7-8 7-8-9 8-9-4 9-4-5 1-2-H 5-6-N 7-8-H 8-9-H 6-N-Ha 6-N-Hb oc
H'at N , H+ at N,
H'at N,
Distances ( A ) 1.368 1.413 1.342 1.303 1.370 1.405 1.386 1.389 1.415 1.419 1.357 1.377 1.424 1.413 1.309 1.314 1.400 1.404 1.392 1.370 1.091 1.093 1.383 1.348 1.085 1.088 1.020 1.026 1.014 1.021 1.014 1.022 1.027
1.312 1.390 1.382 1.384 1.420 1.416 1.421 1.307 1.416 1.367 1.095 1.341 1.087 1.025 1.023 1.023 1.027
1.361 1.355 1.352 1.385 1.418 1.360 1.422 1.343 1.347 1.421 1.093 1.375 1.095 1.030 1.017 1.015 1.029
Angles (deg) 115.9 122.8 130.4 125.3 110.5 112.0 126.4 127.0 116.6 118.5 120.2 114.4 111.4 111.7 103.4 103.2 113.8 113.2 106.2 106.7 105.2 105.2 114.3 114.3 121.8 124.3 125.4 125.7 127.1 127.0 119.7 122.6 120.4 118.6 179.2
117.0 126.2 116.9 121.1 118.6 120.2 110.6 103.8 113.2 105.8 106.6 118.7 123.3 126.3 126.6 119.8 120.6 178.9
117.9 129.4 110.2 127.1 117.5 117.9 106.6
1-2f 2-3 3-4 4-5 5-6 1-6 5-7 7-8 8-9 4-9 1-H 2-N 6-0 8-H 9-H N-Hb N-HC
H+at H' at O,(N,Y .I O,(C,) ".
H+ at N,
H' at
base 1.400 1.318 1.411 1.368 1.477 1.455 1.413 1.312 1.398 1.385 1.021 1.391 1.219 1.083 1.021 1.014 1.013
Distances ( A ) 1.409 1.405 1.341 1.346 1.372 1.369 1.420 1.417 1.382 1.380 1 . 3 9 6 1.382 1.428 1.426 1.305 1.305 1.418 1.416 1.364 1.369 1.027 1.028 1.368 1.366 1.353 1.350 1.088 1.088 1.025 1.025 1.019 1.019 1.017 1.018 0.992 0.991
1.357 1.371 1.421 1.356 1.482 1.498 1.410 1.315 1.401 1.377 1.027 1.357 1.208 1.086 1.024 1.020 1.020 1.024
1.399 1.341 1.389 1.363 1.482 1.445 1.409 1.340 1.355 1.411 1.023 1.371 1.216 1.093 1.029 1.017 1.017 1.031
125.2 126.1 110.1 129.9 120.0 108.7 111.4 104.0 112.6 106.4 105.6 115.4 131.4 116.8 126.1 127.5 118.5 122.0
Angles (deg) 122.4 122.5 124.2 124.1 111.9 111.8 128.1 127.9 117.7 117.7 115.7 116.0 110.9 111.3 103.4 103.3 113.7 113.7 1 0 7 . 1 107.2 104.9 104.5 116.6 116.9 123.1 129.6 119.6 1 1 7 . 5 125.7 125.7 126.9 126.8 117.9 118.0 123.0 122.7 109.0 106.7
127.1 119.5 117.8 124.6 121.4 109.6 110.6 104.1 112.7 105.3 107.2 120.9 132.6 115.8 126.3 126.8 121.2 120.8 178.9
125.7 125.8 109.8 129.8 121.4 107.6 107.5 109.1 108.0 109.3 106.2 116.3 129.4 116.1 126.1 125.4 118.3 122.4 180.6
Rd
6-1-2 1-2-3 2-3-4 3-4-5 4-5-6 5-6-1 4-5-1 5-7-8 7-8-9 8-9-4 9-4-5 1-2-N 5-6-0 6-1-H 7 -8-H
108.8 109.2 108.9 106.4 115.1 124.6 125.3 125.4 118.2 122.9 181.9
The amino hyThe amino hydrogen cis to N,-C,. R is the N-H distance, and e is drogen trans t o N,-C,. the angle between the bisector of the C-N-C angle and the N-H bond. See Figure 3 for numbering system.
8-9-H 2-N-Hb 2-N-HC
a
20 kcal/mol, the STO-3G calculations predict only a 2 kcal/mol difference between O2 and N3 protonation energies (-291.5 vs. -293.3 kcal/mol). However, these energies were computed for structures with nonoptimized geometries. When optimized STO-3G geometries are employed, the STO-3G calculations predict that the O2 protonation energy of cytosine is -304.1 kcal/mol, which is 4 kcal/mol greater than the N3protonation energy. These comparisons demonstrate both the geometry dependence of computed protonation energies and the unreliability of the STO-3G basis set for predicting absolute as well as relative protonation energies. The preferred site of protonation in cytosine is not the preferred hydrogen-bonding site. Hydrogen bonding between water and cytosine in the cytosine molecular plane is most favorable in the N,-H and O2 region, where a restricted wobble dimer is formed. A cyclic structure in which the water molecule bridges adjacent N1-H and 0 2 hydrogen-bonding sites is most stable. A slightly less stable dimer is found in the region between O2 and the amino N-H, with preference for a bridging structure between the amino group and N3.19 Adenine. Protonation of adenine in the molecular plane may occur at the three nitrogens, N1, N , and N7. The data of Table V show that protonation of either one of the nitrogens in the six-membered ring (N, or N3) is preferred (19) J. E. Del Bene, J . Comp. Chem., in press.
0'
Y
.
I.
N,
The H' at 0, on the N , side of the C,=O group. The amino hydrogen amino hydrogen cis to C,-N,. The 0 - H or N-H distance. e For 0, trans to C,-N,. protonation, the C-0-H angle; for N protonation, the angle between the bisector of the C-N-C angle and the N-H bond. See Figure 4 for numbering system. a
TABLE V: -
Protonation Energies (kcal/mol)a
thymine H' at O z ( N l ) r b-203.3; H+ at O,(N,), -204.4; H+ at O,(N,), -208.9; H+ at O,(C,), -211.8 cytosine H' at O,(N,), -228.8; H+ at O,(N,), -241.7; H+ at N,, -249.2 adenine H' at N,, -241.1; H' at N,, -240.0; H+ at N,, -232.1 guanine H+ at O,(N,), -220.4; H'at O,(C,), -233.1; H' at N,, -228.5; H' at N,, -244.7
a Hartree-Fock 4-31G energies at optimized STO-3G geometries. H+ at 0, o n the N , side of the C,=O group.
over protonation at N7. The protonation energies are -241.1, -240.0, and -232.1 kcal/mol, respectively. The preference for N1 and N3 protonation is related to the nature of the highest-energy n orbital, which is associated primarily with these atoms. Experimentally, the preferred protonation sites of adenine are N1 and N3, the distinction between the two being difficult, although N1 appears to be slightly preferred.2"-22 Protonation of N3 in adenine (20) L. F. Cavalieri and B. H. Rosenberg, J. Am. Chem. SOC.,79,5352 (1957). (21) G. Zubay, Biochim. Biophys. Acta, 28,644 (1958). (22) J. J. Christensen, J. H. Rytting, and R. M. Izatt, Biochemistry, 9, 4907 (1970).
Protonation of DNA Bases
is also less favorable by 9.2 kcal/mol than N3 protonation in cytosine, in agreement with experimental data.20p21*23 The electrostatic potential results of ref 1 also show N1 and N3 to be the preferred protonation sites. The STO-3G calculations at fixed geometries (ref 4) predict that the N1 protonation energy is -286.6 kcal/mol, which is 13.5 kcal/mol greater than the N3 protonation energy. However, with optimized STO-3G geometries, the preference for N1 is reduced to 4.0 kcal/mol. Guanine. Four distinct protonation sites for guanine have been found. The most favorable is N7, for which the protonation energy is -244.7 kcal/mol. This is 11.6 kcal/mol greater than the protonation energy for O6protonation on the C5 side of the C6=0 group, which is the next favored site. The region between O6and N7 is quite favorable for the approach of a proton. A relationship is once again found between the nature of the highest occupied n orbital, which is associated primarily with N7,and the preferred protonation site. The preference for N7 protonation in guanine is in agreement with experimental dataup% and in contrast to the situation in the other purine base, adenine, where N7 is the least favorable in-plane protonation site. The electrostatic potential results of ref 2 also favor N7 as the protonation site but identify three rather than four minima on the surface, the one of the N1 side of the C6=0 group being absent. The minimal basis STO-3G calculations of ref 4 predict a reversal in the relative protonation energies, with O6 being preferred by 1.1 kcal/mol over N7. Trends. Large structural changes accompany the inplane protonation of DNA bases. In particular, protonation of a carbonyl oxygen results in a significant 0.120.14-A lengthening of the C-0 bond, which indicates a loss of double-bond character. Simultaneously, adjacent C-N and C-C bond lengths decrease 0.06-0.11 A. Within the ring, the internal angle at the carbonyl carbon increases 7-8". Protonation of a nitrogen in a six-membered ring leads to a slight lengthening of about 0.01-0.02 A in one adjacent C-N bond, and a more significant 0.05-0.06-8, lengthening of the other. The corresponding C-N-C angle of the ring increases 6-8". Protonation of N, in the purine bases adenine and guanine leads to a very slight decrease in the C5-N7 bond length and an increase of about 0.03 A in the N7-C8 bond. The C5-N7-C8 angle also increases by about 5". Thus, characteristic changes are found in bond lengths and angles near the protonation site. In addition, other significant structural changes occur, as evident from Tables I-IV. These results indicate that reliable computed protonation energies cannot be obtained if the geometry of the ion is simply the frozen geometry of the base with the addition of H+. For oxygen protonation of a DNA base, the distance from the carbonyl oxygen to the added proton is about 0.99 A, and the C-O-H angle varies from 106" to 110". For nitro en protonation, the N-H distance is approximately 1.03 , and the angle between the bisector of the C-N-C angle and the N-H bond is approximately 180". Thus, the structural data indicate that protonation occurs at a lone pair of electrons on the oxygen or nitrogen. Protonation is accompanied by a major redistribution of electron density, as shown by Mulliken population analyses.% The most prominent change is charge transfer
x
(23)J. J. Christensen,J. H. Ryttina, . - and R. M. Izatt, J.Physic. Chem., 91,2700 (1967). (24)P.D.Lawley, Proc. Chem. SOC.London,290 (1957). (25)A. M. Fiskin and M. Beer, Biochem. J.,1289 (1965).
The Journal of Physical Chemistry, Vol. 87, No. 2, 1983 371
to the proton in the amount of approximately 0.5 e. This occurs through the a electron system of the base and has a stabilizing effect on the ion. Charge transfer is accompanied by a polarization of electron density toward the protonation site, the a electron cloud being readily polarizable. For oxygen protonation, polarization leads to a significant increase of 0.3-0.4 e in the T electron population of the oxygen. This compensates for the loss of oxygen a electron density, with the result that the oxygen experiences a slight increase of less than 0.1 e in total density. This pattern of electron density change has been observed previously in protonated carbonyl compounds.6 For nitrogen protonation, the a electron density at the protonation site also increases 0.3-0.4 e. However, the nitrogens do not lose a electron density in the ion, so that the effect of nitrogen protonation is to increase the total nitrogen electron density by approximately 0.4 e. In the discussion of the protonation of the individual bases, a relationship was noted between the nature of the highest occupied n orbital of a particular base and the preferred protonation site in that base. That is, those atoms which are the major contributors to the highest occupied n orbital are the preferred protonation sites. However, there is not a simple one-to-onerelation between the n orbital energies of the DNA bases and their protonation energies. The absence of such a relationship is not surprising in view of the delocalized nature of these orbitals. In addition, no correlation is evident between the protonation energies and the total or a electron densities in the neutral bases at the potential protonation sites. Conclusions In this study, Hartree-Fock 4-31G calculations at optimized STO-3G geometries have been performed to investigate the protonation of DNA bases. The results support the following statements. 1. The preferred protonation sites in the molecular plane of the DNA bases are O4on the C5 side of the C 4 4 group in thymine, N3 in cytosine, N1 in adenine, and N7 in guanine. These do not coincide with the preferred hydrogen-bonding sites of these bases with water. 2. Those atoms which are the major contributors to the highest occupied n orbital in the neutral DNA bases are the preferred protonation sites. However, a one-to-one relation between the n orbital energies and the protonation energies of these bases does not exist, owing to the delocalized nature of the orbitals. 3. Protonation leads to significant structural changes in the bases, particularly in bond lengths and angles near the protonation site. Hence, reliable computed protonation energies cannot be obtained if the geometry of the base is frozen in the ion as H+ is added. 4. Protonation is characterized by charge transfer to the proton, and a major redistribution of electron density, which becomes polarized toward the protonation site. The neglect of such changes is a major limitation of molecular electrostatic descriptions of protonation.
Acknowledgment. This work was supported in part by research grant GM27955 from the National Institutes of Health, National Institute of General Medical Sciences. The support of NIH and of the Youngstown State University Computer Center is gratefully acknowledged. Registry No. Thymine, 65-71-4; cytosine, 71-30-7; adenine, 73-24-5; guanine, 73-40-5. (26)R. S.Mulliken, J. Chem. Phys., 23, 1833 (1955).
