ELSEVIER

29 November 1996

Chemical Physics Letters 262 (1996) 789-796

CHEMICAL PHYSICS LETTERS

Multiple site proton affinities of methylated nucleic acid bases

Johan Smets a Linda Houben a Kristien Schoone a Guido Maes a,l L u d w i k A d a m o w i c z b

a Department of Chemistry, Unioersity ofLeuven, Celestijnenlaan 20OF, B-3001 Heverlee, Belgium b Department of Chemistry, University of Arizona, Tucson, AZ 85721. USA

Received 20 August 1996; in f'mal form 16 September 1996

Abstract

Proton affinities of nucleic acid base molecules methylated at the site where the sugar connects to the base in the DNA helix are calculated by the use of ab initio methods. Geometry optimizations and frequency calculations are performed at the HF/6-31G(d) level, while the final energies and proton affinities are calculated with the MP2/6-31 l(2d,2p) method. The proton affinities are reported for temperatures of 0 and 298.15 K. The proton affinity values for the most basic protonation sites are: 891.5 kJ/mol for 1-methyluracil, 982.8 kJ/mol for l-methylcytosine, 961.3 kJ/mol for 9-methyladenine and 980.5 kJ/mol for 9-methylguanine. The calculated proton affinity values of the singly methylated nucleic acid bases are in agreement with the experimental values for the sugarylated bases, the nucleosides.

I. Introduction

Protonation is one of the simplest chemical reac- tions. The proton affinity of a basic center in a molecule is defined as the negative of the enthalpy change associated with the gas phase protonation reaction M + H ÷ ~ MH + at this center. Relative proton affinities are most often determined by mass spectroscopic measurement of the equilibrium con- stant for the proton-exchange gas phase reaction MH ÷ + B ~ M + B H + [1-5]. On the other hand, absolute proton affinities can be obtained from ion- ization thresholds for the MH ~ MH++ e- reaction [3].

It has been demonstrated in a number of studies that theoretical calculations can yield quantitative

i Senior Research Associate of the Belgian NFWO.

results for absolute proton affinities [6-14]. The so-called G2 and more economical G2(MP2) and G2(MP2,SVP) methods yield proton affinities with an accuracy of 10 kJ/mol [6,7,10,11].

The transferability of these gas phase proton affinities to solution is, however, limited due to solvation effects. These effects are often demon- strated with the classic example of aliphatic amines, whose relative basicities totally differ depending on the environment [15].

Proton affinities are used to predict the H-bond properties and the products of protonation reactions in the gas phase and solutions [16-32]. A number of empirical and theoretical correlations have been de- veloped in which the proton affinities are related to various properties of systems with hydrogen bonds [22-32]. In particular, relations with the charges at the protonation centers [30], the core ionization po- tentials [31 ], the H-bond enthalpies and IR frequency shifts [24,27,29,32] induced by H-bond formation

0009-2614/96/$12.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S 0 0 0 9 - 2 6 1 4 ( 9 6 ) 0 1 1 5 1 - 7

790 J. Smets et a l . / Chemical Physics Letters 262 (1996) 789-796

have been proposed. These correlations demonstrate that the proton affinity of the basic H-bond interac- tion site can be used as a measure of the ability of the site to form an H-bond. Moreover, in IR matrix isolation or solution studies of molecules with multi- ple basic H-bond interaction centers, proton affinity values can be used to assign particular complex absorptions to the most probable type of H-bonded complex of the molecule with other systems [24,26,32]. The proton affinities obtained in the pre- sent work will be used in a future interpretation of the complicated IR spectra of matrix-isolated H- bonded complexes of nucleic acid bases. These ma- trix-isolation IR studies are in progress.

The hydrogen-bond properties of nucleic acid bases are important because they govern the repro- duction and transcription processes of DNA in nature [33,34]. In the DNA double helix structure, the Wat- son-Crick nucleic base dimers involve either two or three hydrogen bonds. In such circumstances, not only the most basic site is directly involved in the formation of a hydrogen bond, but other weaker H-bond centers also participate. The H-bond network becomes even more diverse and extended when en- zymatic reactions and enzyme-nucleic acid base in- teractions occur.

The nucleic acid bases uracil, cytosine, adenine and guanine possess multiple basic interaction cen- ters. For example, cytosine has four proton-accepting interaction sites: two lone pairs at the C202 group, and the lone pairs at the N 3 and C a N 4 centers. When relatively large differences exist between the proton affinities of the different interaction sites in the molecule, it is impossible to obtain experimental proton affinity values for the weaker interaction sites. Furthermore, from mass spectroscopy experiments only the proton affinity value but not the protonation site is obtained. For these above two reasons theoret- ical calculations, if sufficiently reliable, can comple- ment the experimental data.

Surprisingly, only a few papers have reported on theoretically calculated proton affinities of the nu- cleic acid bases [8,14,15,19]. The methods employed used a low level of theory and have included only the semi-empirical AM I approach and the ab initio H F / 4 - 3 1 G / / S T O - 3 G approach. There has been no report so far on proton affinities for methylated nucleic acid base analogues, although these seem to

be the most relevant systems to study in relation to the H-bonding properties of nucleic bases in DNA, particularly when the methyl group is placed in the position where the sugar moiety connects to the base. Calculations for such systems are presented in this Letter.

2. Methodology

The theoretical method employed in the present calculations is, by necessity, more approximate than the most extended methods applied to study smaller systems. All the molecular structures of the cations and neutral systems used in the study have been optimized at the Hartree-Fock (HF) level with the 6-31G(d) basis set. These were followed by vibra- tional frequency calculations performed to verify whether true minima of the potential energy surface were obtained and to determine the temperature con- tribution to the proton affinities. The calculated vi- brational frequencies were scaled by the commonly used single scaling factor of 0.8929 [35]. Next, sin- gle-point second order many body perturbation (MP2) calculations were performed with the 6-311G(2d,2p) basis set for all the HF/6-31G(d) optimized struc- tures. This level of theory is denoted MP2/6- 311(2d,2p)/ /HF/6-31G(d) in the tables and the further discussion. The MP2 scheme is not expected to yield an accuracy of 10 kJ /mol in the proton-af- finity estimations as obtained with the G2 (MP2,SVP), G2 (MP2) or G2 methods, but this approach is, at present, the best method still feasible for systems with multiple protonation sites such as those considered in this work; e.g. 9-methylguanine has as many as 5 protonation sites. The ab initio calculations were performed with the GAUSSIAN 92 and GAUSSIAN 94 [35] packages and from the total energies of the neutral and cation systems (these include the electronic and nuclear vibrational ener- gies) the proton affinity values are calculated at 0 and 298 K. The corrections for the zero-point vibra- tional and temperature-dependent contribution to the enthalpy were calculated based on the scaled HF/6- 31G(d) frequencies and were taken from the GAUSSIAN output.

J. Smets et al . / Chemical Physics Letters 262 (1996) 789-796 791

3. Results and discussion

Table 1 lists the proton affinity values for all the protonation centers in 1-methyluracil, 1-methylcyto- sine, 9-methyladenine and 9-methylguanine. Table 2 tabulates the most important Muiliken charges, inter- nuclear distances and Vx, + vibrational frequencies of the most stable protonation products. On request, these parameters can be obtained for all the other protonation products.

3.1. 1-Methyluracil

1-Methyluracil has 4 lone electron pairs which can react with a proton, and all these pairs are located on the two oxygen atoms. Fig. la illustrates the two most stable protonation products of 1-meth- yluracil.

The most favorable protonation site is the oxygen lone pair located on the C 5 side of the C,O, group. This result is consistent with the RHF/4-31G single point calculations of Del Bene for thymine [8]. The

obtained proton affinity at this site is 891.5 kJ/mol, which is more than for the other lone pair on the same oxygen atom by 12 kJ/mol, and more than for the protonations at the oxygen lone pairs of the C202 group by 40 to 50 kJ/mol. The experimental proton affinity values for uracil, thymine and thymi- dine are 870, 874 and 941 kJ/mol , respectively [4,5,19], and our result lies between these two exper- imental values, but definitely closer to the thymine value.

The protonation at the oxygen lone pair located on the C 5 side in the C,O, group is accompanied by an increase in the Ca0 * distance by 0.10 A, and this elongation is similar for protonations at all four protonation sites. The equilibrium O - H bond length for the incoming proton is about 0.95 ,~ (this dis- tance is probably underestimated at the RHF level) and the angle between the O4H bond and the C404 bond is 114 °. The absorption band for the new Vo, + mode is predicted at 3604 cm -~. The Mul- liken population analysis shows that the protonation at the C 5 side of O a is accompanied by a charge flow towards the protonated oxygen, resulting in a

Table 1 Proton affinity values (kJ/mol) for I-methyluracil, l-methylcytosine, 9-methyladenine and 9-methylguanine calculated at 0 and 298.15 K

Method

1-methyluracil C202; Niside C202; N 3 side C404; N 3 side C404; C5 side

RHF OK 858.8 863.4 893.2 906.0 MP2 OK 840.7 845.2 873.7 885.5 MP2 298K 846.2 850.4 879.5 891.5

l-methylcytosine C202; Ni side C202; N 3 side N 3 C,tN 4

RHF OK 969.1 1007.3 1001.8 845.2 MP2 OK 941.8 976.3 971.7 851.3 MP2 298K 948. I 982.8 977.8 857.2

9-methyladenine N t N 3 C6N6 N7

RHF OK 981.0 973.5 880.0 952.7 MP2 OK 955.3 945.0 874.2 928.4 MP2 298K 961.3 951.6 879.8 933.7

9-methylguanine C2N 2 N3 C606; N x side C606; N 7 side N 7

RHF OK 801.5 923.1 945.8 981.8 999.2 MP2 OK 817.7 902.1 913.2 947.3 975.0 MP2 298K 823.2 907.0 917.0 951.2 980.5

Ab initio energy values from RHF and MP2/6-31 IG(2d,2p) single point calculation of structures optimized at the RHF/6-31G(d) level. Zero-point vibrational energies were obtained from frequencies calculated at the RHF/6-31G(d) level.

792 J. Smets et a l . / Chemical Physics Letters 262 (1996) 789-796

Table 2 Specific Mulliken charges (e), distances (,~) and vibrational frequencies (cm- 1 ) for the most stable protonation products of 1-methyluracil, 9-methyladenine and 9-methylguanine and the two most stable protonation products of 1-methylcytosine

Characteristic 1 -methyluracil 1 -methycytosine 1 -methylcytosine 9-methyladenine 9-methylguanine 04; C~ side 02; N 3 side N 3 N~ N 7

Mulliken charges C202 -0 .507(+0.051) -0.441 (+0.131) -0 .469(+0.103) C404 orC4N 4 -0 .656( -0 .033) -0 .246(+0.090) -0 .246(+0.090) N 3 -0.595(+0.006) -0 .370(+0.231) -0 .498(+0.259) -0 .622(+0.007) N z - 0.326 ( + 0.060) C6N 6 orC606 -0 .267(+0.079) -0 .488(+0.052) N 7 -0 .532(+0.020) -0 .286(+0.221)

Distances C202 1.177(-0.019) 1.298(+0.099) 1.180(-0.019) N3C 4 1.334(+0.039) 1.341 (+0.046) C404 orC4N 4 1.290(+0.096) 1.314(+0.036) 1.314(+0.036) NtC 6 1.357 ( + 0.031) NTC 8 1.283 ( + 0.002) 1.306 ( + 0.027) CrN 6 orCrO 6 1.317(-0.029) 1.187(-0.007) X-H" 0.954 0.955 1.001 1.000 1.000

Frequencies ~xH + 4036 4019 3811, 3802 3826 3844

Mulliken charges from the single point RHF/6-31 l G(2d,2p) calculations and distances and frequencies from RHF/6-31G(d) calculations of optimized geometries. The differences with the non-protonated base are indicated in brackets.

charge increase of 0.13 e at C404 and a charge decrease of 0.05 e at C202.

3.2. 1-Methylcytosine

1-Methylcytosine possesses four possible protona- tion sites: the two lone pairs on the C202 group, one lone pair on the N 3 a tom and one lone pair on the N 4

atom of the amino group. Fig. lb shows the two most stable protonation products of 1-methylcyto- sine.

The oxygen lone pair on the N 3 side of the C202 group is the preferred protonation site, this protona- tion being associated with an enthalpy change of 982.8 kJ/mol. This is a remarkable result since most other studies obtain the N 3 a t o m as the most likely protonation site [8]. However, the difference between the protonation enthalpies at 02 and N 3 is only 5 kJ/mol. The protonation enthalpies at the other two sites, i.e. at the oxygen lone pair on the N 1 side of the C202 group and at the lone pair on CaN, ~, are smaller by 35 and 125 kJ/mol than the protonation at the most preferred protonation site, respectively.

This demonstrates, as expected, that protonation at the N 4 a tom is less likely, but with a proton affinity value of 857.2 kJ /mol at room temperature for this site, the formation of a relatively strong H-bond at this site is still possible.

Protonation at the oxygen lone pair on the N 3 side of the C202 group elongates the C202 bond dis- tance with 0.10 ,~. The OH + bond length is found to be 0.95 ,~ and the angle with respect to the C202 bond is 111 °. The vibrational frequency for the VoH+ vibration is predicted to be around 3588 cm-1. The protonation at the N~ atom shows an increase in the NaC 4 bond by 0.05 A, but the bond elongation is the largest for protonation at N 4, where the C 4 N 4 bond increases by 0.13 A, reflecting the change f rom sp 2 tO sp 3 hybridization. The protonation at N 3 gives rise to a new vibrational absorption of the /"NH+ mode around 3400 cm-1. The normal mode analysis of this vibration shows some vibrational coupling with the vS(NH2) mode. The Mulliken population analy- sis of the protonation products shows a decrease in atomic charge at all the protonation centers with respect to the neutral cytosine. The largest changes

J. Smets et al. / Chemical Physics Letters 262 (1996) 789-796 793

(a)

~ C

. C ~ e"C"

I ~,~...,~

(b) c_~ ,,,o N

I ~i C

3

~ r'

I q~Cr

(c) o , . . i °

i

I .: .. ,/- Lc'_' 3 i~.~ N

"oF'

I !

i

l ~ ~-.~. ~

I I

I

~..~0

I"

i i \

(~' " ~L., ~;--o

" ~ ~- ,.

Fig. 1. The two most stable protonation products of I-methy|uracil (a), l-methylcytosine (b), 9-methyladenine (c) and 9-methylguanine (d).

794 J. Smets et a l . / Chemical Physics Letters 262 (1996) 789-796

in the electron population can be seen at the N 3 and N 4 atoms, and they are 0.23 and 0.32 e, respec- tively, when protonation occurs at these atoms.

3.3. 9-Methyladenine

For 9-methyladenine, there are four different in- teraction channels with a proton, and all of these are interactions with the lone pairs on nitrogen atoms. Fig. lc illustrates the two most stable protonation products of 9-methyladenine.

The most favorable protonation site is the N~ atom and the corresponding calculated proton affin- ity is 961.3 kJ/mol at room temperature. The proton affinities at the other N atomic centers are only slightly smaller for the protonation in the plane of the molecule, i.e. smaller by 10 kJ/mol for protona- tion at N 3 and smaller by 28 kJ/mol for protonation at N 7. However, they are larger for the out-of-the- plane protonation at C6N6, the difference being 81 kJ/mol. These results show that the strongest H- bonds will be formed on the pyrimidine ring of adenine with the H-bond formed in the plane of the molecule.

Protonation at N~ is accompanied by a bond elongation of the NIC 6 distance of 0.03 ~, and a decrease in the C2N 3 and N6C 6 bond distances by 0.04 and 0.03 A, respectively. The N~H ÷ bond distance is estimated to be 1.00 A and the angle with respect to N~C 2 is 117 °. The u~H absorption is located around 3420 cm - l . The Mulliken population analysis shows a decrease in the atomic charges at all the protonation centers when protonation takes place. The charge decreases at the N 1, N3, N 6 and N 7

atoms when 9-methyladenine is protonated at N] are 0.26, 0.08, 0.08 and 0.02 e, respectively.

3.4. 9-Methylguanine

With 9-methylguanine, the number of protonation centers has increased up to 5: the lone pair on the N 2

atom of the amino group, the lone pair on N 3, two lone pairs on 06 and one lone pair on N 7. Fig. ld illustrates the two most stable protonation product of 9-methylguanine.

The most stable protonation product is formed at the N 7 atom. This protonation is accompanied by an enthalpy change of 980.5 kJ/mol at room tempera-

ture. The energy differences with the other protona- tion products are rather large, being 157 kJ/mol for protonation at the amino group, 73 kJ /mol for pro- tonation at the N 3 atom, 63 kJ /mol for protonation at the 06 atom involving the lone pair on the N~ side and 19 kJ /mol for protonation of the 06 atom involving the lone pair on the N 7 side. These results indicate that the strongest interaction centers are situated in the purine part of the molecule at N 7 and at 06 (the lone pair on the N 7 side), away from the interaction centers involved in the formation of the normal Watson-Crick base pairing. This opposes the result obtained for 9-methyladenine, where the largest proton affinity values are found for the centers in- volved in the Watson-Crick base-pair formation.

Protonation at the N 7 atom elongates the N7C 8 bond distance by °0.030 .~ and reduces the C606 distance by 0.007 A and the NzC 2 distance by 0.036 • ~. The calculated N - H + bond distance is 1.00 ~. The formation of this bond gives rise to a new IR absorption which is predicted at around 3430 cm - j . As in the case of 1-methylcytosine and 9-methyl- adenine, the Mulliken population analysis for 9- methylguanine shows a relatively large decrease of the charge at the protonation center. For example, protonation at N 7 results in an atomic charge de- crease of 0.22 e at the N 7 a tom.

4. Discussion

4.1. Protonation sites

The ab initio calculations identify the following protonation sites as the most favorable for the N- methylated nucleic acid bases: the O 4 lone pair on the C 5 side in 1-methyluracil; two energetically close lying protonation sites, the 02 lone pair on the N 3 side and the N 3 atom in l-methylcytosine, the N~ atom in 9-methyladenine and the N 7 atom in 9-meth- ylguanine with the corresponding proton affinities of 891.5, 982.8, 977.8, 961.3 and 980.5 kJ/mol, re- spectively. These proton affinities for the N-methyl- ated nucleic acid bases are systematically lower by about 50 kJ/mol than the ab initio calculated affini- ties of Del Bene for the 'bare' nucleic acid bases [8]. The difference can be attributed to the contributions of the electron correlation and the zero-point vibra-

J. Smets et al . / Chemical Physics Letters 262 (1996) 789-796 795

tionai energy which were not accounted for in the Del Bene studies [8].

It has to be noted that for 9-methylguanine and 1-methyluracil, the site with the highest proton affin- ity is not the one involved in the H-bonding patterns of the Watson-Crick base pairs. The lone pairs on the amino groups in 1-methylcytosine, 9-methyl- adenine and 9-methylguanine are capable of forming relatively strong H-bonds, since these sites have proton affinities comparable to ammonia (853,5 kJ/mol) [3].

4.2. Accuracy

Acknowledgements

The cooperation between the research groups of Leuven and Tucson was supported by the NATO International Collaborative Grant INT-9313268. K. Schoone acknowledges the support of the Belgian IWT. L. Adamowicz and J. Smets acknowledge the support from the Office of Health and Environmental Research, Office of Energy Research and the Depart- ment of Energy (Grant No. DEFG0393ER61605). G. Maes acknowledges the Belgian NFWO for a perma- nent research fellowship.

The calculated proton affinity values are between 20 and 30 kJ/mol larger than the experimental values for the non-methylated bases [4,5,19]. They match very well the experimental values of Greco et ai. for cytidine and guanosine, 977.8 and 981.2 kJ/mol [5]. The calculated proton affinity for 9- methyladenine is, however, 15 kJ/mol lower than the proton affinity of adenosine (975.7 kJ/mol) and the calculated proton affinity for 1-methyluracil is 50 kJ/mol lower than for thymidine (941.0 kJ/mol) [5]. The proton affinities obtained in this work for the N-methylated nucleic acid base derivatives are between the experimentally obtained values for the sugarylated and the non-sugarylated nucleic acid bases. The consistently larger proton affinities of the sugarylated nucleic acid bases can be explained by the electron-donating properties of the sugar sub- stituent and by the influence of H-bonding interac- tions between the sugar and the base. Therefore, we can conclude that the ab initio proton affinities are in quite good agreement with the experimental findings since the methyl group, which is less electron donat- ing than the sugar substituent, will induce a smaller increase in the proton affinity of the base than the sugar moiety. However, the difference between the calculated proton affinity of 1-methyluracil and the experimental proton affinity of thymidine raises doubts about the experimental value for thymidine. This conclusion is also supported by an examination of the differences between the proton affinities for the nucleotides and the nucleic acid bases, where a rather constant difference of 30 kJ/mol is observed, except for thymine where the difference is as large as 70 kJ/mol.

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