4-Aminobenzimidazoleâ1-Methylthymine: A Model for Investigating

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4-Aminobenzimidazole 1-Methylthymine: A Model for Investigating Hoogsteen Base-Pairing between Adenine and Thymine Yevgeniy Nosenko,† Maksim Kunitski,† Tina Stark,‡ Michael G€obel,‡ Pilarisetty Tarakeshwar,§ and Bernhard Brutschy*,† †

Institut f€ur Physikalische und Theoretische Chemie and ‡Institut f€ur Organische Chemie und Chemische Biologie, Goethe-Universit€at, Max-von-Laue-Straße 7, 60438 Frankfurt/Main, Germany § Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, United States

bS Supporting Information ABSTRACT: We report the infrared spectrum of the 4-aminobenzimidazole 1-methylthymine (4ABI:1MT) heterodimer, detected by femtosecond multiphoton ionization. Based on calculations of both the harmonic and the anharmonic frequencies, the observed vibrational spectrum is assigned to a structure that mimics the Hoogsteen base pairing of adenine and thymine. A notable observation made in the course of this study is that there is a significant imbalance in the observed strengths of the H-bonds. While the N 3 3 3 H N bond reveals a large red shift of >700 cm 1 for the NH stretch frequency, the N H 3 3 3 O bond is characterized by only a 50 cm 1 shift. The importance of this observation in the formation of Hoogsteen duplexes by thymine-based oligonucleotides is discussed.

1. INTRODUCTION Complementary pairing of the nucleic acid bases provides replication and transcription of the genetic code in living cells. This biological relevance stimulates the ongoing interest in nucleic acid base interactions from the molecular level. In this respect, studying isolated base pairs is invaluable for understanding the mechanisms that underlie the functionality of DNA. Base recognition relies upon specific triple H-bonding between guanine (G) and cytosine (C) and double H-bonding between adenine (A) and thymine (T). Aside from the most common Watson Crick1 (WC) base pairing motif, several other motifs have been observed in DNA triple helices, RNA molecules,2 and in the interactions of DNA with drugs and proteins.3,4 In Figure 1, the biologically relevant base pairing patterns of the A T base pair are shown. Though not as widely investigated as the Watson Crick isomer, the Hoogsteen (HG) isomer is of particular interest because it has been experimentally observed that replication by human DNA polymerase-ι occurs by HG base-pairing.5 Apart from it being observed in the crystal structure of 9-methyladenine 1-methylthymine (9MA 1MT),6 recent NMR studies have shown that contrary to the widely perceived notion, HG base pairs are widely prevalent in A T rich DNA.7 Though they are observed only as transient conformers for very short intervals of time, their presence seems to indicate that they might play a hitherto unknown role in dictating the properties and functions of DNA.7 Several theoretical studies of A T and 9MA 1MT in the past indicated that geometries possessing HG base pairing are energetically the most r 2011 American Chemical Society

stable ones or are isoenergetic to a number of other structures localized on the theoretical potential energy hypersurface.8 Experimentally, only a single isomer of A T, has been detected and characterized under the isolated conditions of a molecular beam using the double-resonance IR/R2PI ionization spectroscopy.9 This conformer which is neither the biorelevant nor the lowest energy structure exhibits two H-bonds. The first one corresponds to a bond between the thymine imino (N1 H) and a ring nitrogen (N1 or N7) atom of A (Figure 1) and the second one to a bond between the amino group of A and the oxygen of the thymine carbonyl (C2dO) group. In order to prevent such biologically irrelevant conformers from being formed under supersonic jet conditions, one has to block the irrelevant H-bonding sites. Methylation of the glycosidic nitrogen atoms is a useful strategy in this context as evidenced by recent work on the vibrational spectroscopy of hydrates and dimers of A10 and T.11 However, blocking of the HG binding site of A by methylation does not enable the detection of the naturally occurring WC base pairs.9 Despite the fact that the canonical WC form of the guanine-cytosine base pair was observed and vibrationally characterized in photoionization experiments,12 it is intriguing to note that the biorelevant isomers of A T base pairs have not been detected up to now. Special Issue: Pavel Hobza Festschrift Received: June 14, 2011 Revised: August 15, 2011 Published: September 07, 2011 11403

dx.doi.org/10.1021/jp205575w | J. Phys. Chem. A 2011, 115, 11403–11411

The Journal of Physical Chemistry A

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Scheme 1. Synthesis of 1H-1,3-Benzodiazol-4-amine: 3-Nitrobenzene-1,2-diamine was Reacted with Formic Acid and Subsequently Hydrogenated

oligonucleotides based purely on 4ABI do not form stable helices with (dT)n in aqueous solution. We used thymine methylated in its N1 position because this is a favorite H-bonding site of the T nucleobase,11,17 and, as already mentioned, this must be blocked to investigate biologically relevant complexes of thymine. From the methodological point of view, we circumvented the problem of the ultrashort lifetime of the excited states by using femtosecond multiphoton ionization (fsMPI) to detect IR absorption.22 The major drawback of this method is the lack of isomer selectivity due to the spectrally broad fs-MPI cross section at the typical UV wavelength of 267 nm. However, this disadvantage is more than compensated by its ability to effectively ionize molecules with short intermediate excited state lifetimes such as nucleic acid bases. Figure 1. Base pairing abilities of the molecules under study: 4-amine1H-benzimidazole (4ABI) and 1-methylthymine (1MT) in comparison to adenosine (A) and thymidine (T), which may form Watson Crick (WC), Hoogsteen (HG), and the corresponding reverse (rWC and rHG) isomeric base pairs.

Thus, it is useful to examine simpler chemical models of A and T capable of exhibiting biologically relevant base pairing. The use of mimics of nucleic acids base pairs is additionally motivated by the fact that the resonant two-photon ionization (R2PI) of A T with nanosecond laser pulses, utilized in a typical IR/R2PI depletion experiment, is not effective because of the extremely short excited state lifetime of the A T base pair, lying in the hundred femtosecond range.13,14 Hence, model base pairs with chromophores possessing longer excited state lifetimes have attracted considerable interest.15 18 In the context of the present work we mention here the double-resonance UV/R2PI studies by the Leutwyler group on T17 and 9MA.18 The binding sites of these nucleobases have been probed by 2-pyridone (2PY) and evidence for the formation of two antiparallel H-bonds have been given. Moreover, Frey et al. have reported the IR/R2PI spectrum of the 2-aminopyridine 2-pyridone (2AP 2PY) heterodimer, which is a structural mimic of the A T WC base pair.16 Though there have been several theoretical studies of the energies of base pairs exhibiting both Watson Crick and Hoogsteen motifs, to the best of our knowledge, neither theoretical nor experimental investigations have been carried out on the vibrational pattern of an isolated Hoogsteen base pair. In the current study, we investigate the base pairing between 1-methylthymine (1MT) and 1H-1,3-benzodiazol-4-amine (or 4-amine-1H-benzimidazole, 4ABI). The 4ABI molecule possesses only the HG docking site of adenine and does not allow for WC base pairing. 4ABI is known for its mutagenic properties.19,20 Interestingly, a replacement of A by 4ABI in oligonucleotides destabilizes the helical structures.21 Thus, in singly substituted helices, the 4ABI base is arranged in a WC-type fashion to T, however, without H-bonding. For this reason,

2. EXPERIMENTAL AND COMPUTATIONAL SECTION 2.1. Chemicals. All reagents were obtained from commercial suppliers and were used without further purification. Analytical instrumentation included the following: NMR, Bruker DPX 250 (1H: 250 MHz; 13C: 63 MHz); elemental analysis, Elementar vario micro cube; melting points (uncorrected), Kofler hot-plate microscope; FTIR, Perkin-Elmer 1600 series. 4-Nitro-1H-1,3-benzodiazole.23. 3-Nitrobenzene-1,2-diamine (500 mg; 3.26 mmol) was dissolved in formic acid (10 mL) and refluxed under argon for 7 h. After cooling to room temperature, the reaction mixture was neutralized with satd NaHCO3 solution. The product precipitated as a yellow solid and was recrystallized from methylenechloride/n-hexane (522 mg; 98%). 1 H NMR (250 MHz, DMSO-d6): δ = 13.37 (bs, 1H, NH), 8.46 (m, 1H, aryl-H), 8.16 (d, J = 8 Hz, 2H, aryl-H), 7.42 (t, J = 8 Hz, 1H, aryl-H) ppm. 13C NMR (63 MHz, DMSO-d6): δ = 165.6, 145.2, 133.6, 128.0, 126.3, 121.0, 118.7 ppm. Melting point: 247 249 C (ref: 248 149 C).24 IR: ν (KBr) = 3448 (w), 3097 (m), 2974 (m), 2924 (m), 2775 (m), 1636 (m), 1583 (w), 1532 (s), 1490 (s), 1423 (m), 1366 (s), 1339 (s), 1293 (m), 1268 (s), 1184 (w), 1162 (w), 1127 (m), 1065 (w), 944 (m), 859 (w), 798 (w), 736 (m), 623 (w) cm 1. Elem. anal. C7H5N3O2 (163.13): C, 51.54; H, 3.09; N, 25.76. Found: C, 51.26; H, 3.06; N, 25.64. 1H-1,3-Benzodiazol-4-amine (Scheme 1). 4-Nitro-1H-1,3-benzodiazole (100 mg; 0.6 mmol) was dissolved in ethyl acetate/ ethanol 5:2 (35 mL). Palladium on activated charcoal (33 mg; 30 wt %) was added and the dark suspension was stirred overnight at room temperature under a H2 atmosphere. After filtration over Celite and washing with ethyl acetate, the solution was concentrated in vacuo. After crystallization from methylenechloride/ n-hexane colorless needles were obtained (61 mg; 76%). 1 H NMR (250 MHz, DMSO-d6): δ = 12.08 (bs, 1H, NH), 7.98 (s, 1H, aryl-H), 6.87 (t, J = 7.5 Hz, 1H, aryl-H), 6.73 (m, 1H, aryl-H), 6.34 (d, J = 7.5 Hz, 1H, aryl-H), 5.16 (s, 2 H, NH2) ppm. 13 C NMR (63 MHz, acetone-d6): δ = 142.7, 140.6, 125.3, 124.2, 113.6, 106.6, 102.8 ppm. IR: ν (KBr) = 3397 (s), 3288 (s), 3192 (s), 11404

dx.doi.org/10.1021/jp205575w |J. Phys. Chem. A 2011, 115, 11403–11411

The Journal of Physical Chemistry A 3091 (s), 3006 (s), 2909 (s), 2815 (s), 1718 (w), 1626 (m), 1599 (s), 1516 (m), 1479 (m), 1456 (m), 1418 (m), 1366 (m), 1319 (s), 1290 (m), 1252 (m), 1114 (m), 1016 (m), 933 (m), 862 (m), 786 (m), 739 (m), 631 (m) cm 1. Melting point: 107 108 C. Elem. anal. C7H7N3 (133.15): C, 63.14; H, 5.30; N, 31.56. Found: C, 62.91; H, 5.17; N, 31.79. The 1-methylthymine (1MT, colorless, polycrystalline, Sigma) sample was used without further purification. 2.2. Experimental Section. The IR/fsMPI experiments were carried out as described previously.10 Briefly, the base pairs were formed in a supersonic expansion of helium (4.6 grade, stagnation pressure 3 bar) seeded with the thermally evaporated (140 C) bases. For this purpose, we used a pulsed, high temperature modified General Valve (Series 9) nozzle, operated at 10 Hz. The resulting cluster distributions were analyzed in a time-of-flight mass spectrometer after their ionization by fs-MPI using the third harmonic (267 nm, 10 μJ/pulse) of the fundamental output (800 nm, 260 fs autocorrelation) of a Ti:Sapphire CPA laser system. Temperature, nozzle opening duration, and laser-to-nozzle delay were the parameters that were adjusted so that the trimer signals in the mass spectra were minimized close to the limit of detectability. Thus, eventual spectral contamination by fragmentation of larger complexes could be effectively avoided. For the same purpose, carrier gas impurities, in particular, water vapor, were frozen out in a copper coil cooled by liquid nitrogen. The UV beam was split into two, called the signal and the reference beam. The former was spatially overlapped with the counter-propagating IR beam from a seeded optical parametric oscillator (OPO) based on LiNbO3 crystals (τpulse = 6 ns, ΔE = 0.2 cm 1), with the IR pulse preceding the UV pulse by about 80 ns. Here, the double resonance depletion effect is caused by different photoionization efficiencies for cold and vibrationally excited species. The IR/fsMPI signal was obtained as normalized ratio of the IR affected and the reference ion signal. Calibration of the IR frequency was accomplished by means of a wavemeter (ATOS LM-007) with which we measured the red and green wavelengths used for generating the difference frequency as seed radiation for the IR-OPO. The final accuracy thus achieved was (1 cm 1 for the tuning range of 2750 3750 cm 1. The spectra were not corrected for the IR intensity. The IR-OPO provided typically pulse energy of 2 mJ except for the interval of 3460 3510 cm 1, where the laser intensity was reduced due to absorption of the nonlinear crystals.25 At each spectral point, the integrated time-of-flight signals corresponding to the selected ion channels were averaged over 200 laser shots. 2.3. Calculations. Quantum chemical calculations on different conformers of the 4ABI-1MT complex were carried out at both the second order Møller Plesset (MP2) and density functional (DFT) level of theory using the 6-31+G* basis set. The latter calculations were carried out using the Becke gradient-corrected exchange and Lee Yang Parr correlation functional with three parameters (B3LYP) functional. Full geometry optimizations were followed by the evaluation of the harmonic vibrational frequencies.26 A recent study by Leutwyler and co-workers16 indicate that harmonic vibrational frequency calculations obtained using the nonlocal exchange-correlation functional of Perdew and Wang (PW91) yields better agreement with the experiment. Hence, geometry optimizations and the evaluation of the vibrational frequencies were also carried out at the PW91/ 6-31+G* and PW91/6-311++G** levels of theory. The calculated interaction energies were corrected for basis-set superposition

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Figure 2. IR/sfMPI spectra of the (a) 4ABI, (b) 4ABI 1MT, and (c) 1MT.

error using the counterpoise method.27 The zero-point corrected interaction energies were calculated using the zero-point energies obtained at the harmonic level of theory. In general, the experimental spectrum is assigned using suitably scaled harmonic vibrational frequencies. Apart from problems in obtaining uniform and transferable scaling factors, such an assignment yields no information on the contribution of overtone and combination transition frequencies. Given the importance of such modes in the spectrum of a strongly H-bonded system,26 we also calculated the anharmonic frequencies at both the B3LYP/6-31+G* and PW91/6-31+G* levels of theory. The anharmonic calculations involved numerical differentiation to obtain the cubic force constants and were computationally very arduous for the size of the systems studied. The cubic force constants calculated at the B3LYP/6-31+G* level of theory were also used to carry out the anharmonic corrections of the harmonic frequencies calculated at the MP2/6-31+G* level of theory. In our earlier investigations on the interaction of π systems with a water dimer,28 we had employed the “atoms in molecules” (AIM) method,28 30 to analyze the strength of different types of hydrogen bonds. Basically, the method involves the evaluation of H-bond strengths using a correlation between the local electronic kinetic energy and the local electronic potential energy.31 To distinguish the nature of different hydrogen bonds in the 4ABI 1MT dimers, we evaluated their strengths using this AIM methodology.

3. RESULTS The IR spectrum of jet-cooled 1-methylthymine was investigated previously.11,32 It is shown in Figure 2c. The distinct band observed at 3443 cm 1 has been attributed to the ν(N3 H) stretching vibration. Calculation of the harmonic vibrational frequencies carried out by us indicate that this mode appears at 3593 cm 1 (B3LYP/6-31+G*), 3518 cm 1 (PW91/6-311++G**), or 3587 cm 1 (MP2/6-31+G*). While it is well-known that suitable scaling of the harmonic frequencies yields good agreement with the experimental frequencies, it is useful to note that the calculated anharmonic frequency for this mode at the B3LYP/6-31+G* level is 3423 cm 1. 11405



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Table 1. Calculated Structures and Relative Energies of Different Conformers of the 4ABI 1MT Heterodimera

All energies are in kcal/mol, distances are in Å, and angles are in degrees (). ΔE and ΔEB represents the supermolecular interaction energy without and with basis set superposition error (BSSE) correction. ΔE0 represents the zero point vibrational energy (ZPVE) corrected ΔEB. a

There have been few investigations of the vibrational spectra of 4ABI. The emphasis of a combined theoretical and experimental study was to investigate the tautomeric equilibrium of 4-NH2 benzimidazole / 7-NH2 benzimidazole.33 Based on a comparison of the calculated energies and experimental spectra, it was concluded that the 4-NH2 tautomer appears as the most abundant form in a low temperature Ar matrix, but the 7-NH2 form is sufficiently abundant to yield its vibrational signature. The origin of a large deviation between theoretically predicted and experimentally determined frequencies of the NH2 wag mode was attributed to the presence of the 7-NH2 tautomer.33 In this study of 4ABI in the Ar matrix the bands appearing in the IR spectrum at 3517, 3506, and 3423 cm 1 have been assigned to the νaNH2, νN1 H, and νsNH2 modes of 4-NH2 benzimidazole, respectively. The experimental IR spectrum of the 4ABI monomer measured by us in a supersonic beam contains three bands at 3529, 3518, and 3427 cm 1 (Figure 2a). Our calculated B3LYP/6-31+G* anharmonic frequencies for these modes are 3496, 3483, and 3435 cm 1. Interestingly, the difference between the νaNH2 and νN1 H modes is nearly the same in both experiment and theory. While a comparison of the 4ABI monomer spectrum with that of the heterodimer containing 1MT can facilitate the assignment of the latter, it is important to note that an unambiguous assignment of all the bands in the dimer is to a large extent dictated by the level of theory used to obtain the vibrational frequencies. More details on the performance of various theoretical methods on the assignment of vibrational modes of hydrogen bonded dimers can be found in a very recent paper on the formic acid dimer.34 If 4ABI is singly H-bonded via its imidazolic N1 H group, the two amino group stretches cannot be involved in binding (Figure 1) and must remain unperturbed in the dimer spectrum. However, both of these modes must change their frequencies when the amino group is involved in the H-bonding leaving only the νN1 H stretch intact like in the spectrum of the 4ABI monomer. It is useful to examine the similarities and differences in the structures and energies of various possible isomers obtained using different theoretical methods before we discuss the experimental spectrum of the 4ABI:1MT heterodimer. Structures, in which 4ABI is singly H-bonded through the imidazolic N1 H are expected to be higher in energy because they enable the formation of only a single H-bond. On the other hand, the 4ABI 1MT heterodimer can display either a HG structure or a reverse HG (rHG) structure when both the thymine N H and aminobenzimidazole NH2 are involved in the formation of H-bonds. It is interesting to note that irrespective of the

calculational method (DFT or MP2) being used, the calculated interaction energies are nearly similar (Table 1). While the calculated ZPVE corrected B3LYP (ΔE0) energies seem to indicate that the HG is more stable than the rHG structure by a small energy difference of 0.2 kcal/mol, the corresponding MP2 energies indicate that they are isoenergetic. The similar interactions energies obtained at both the B3LYP and MP2 levels seem to indicate that unlike π-bonded complexes, the B3LYP level is more than adequate in yielding reasonable estimates of the interaction energies and as will be shown subsequently, the vibrational frequencies of H-bonded base pairs. It is interesting to note that H-bond formation is dramatically enhanced at the PW91/6-311++G** level of theory. This can be noted from the smaller intermolecular bond distances and larger interaction energy. Similar observations were made in a recent study of the formic acid dimer. There it was found that with the same basis set, the PW91 functional yields an intermolecular O H O distance of 2.583 Å as compared to the experimental electron diffraction value of 2.703 Å.34 Furthermore, it was also noted that there was an overestimation of the infrared intensity for the ν18(C H stretch) band.34 On the other hand, the B3LYP and the more recent B971 functional were found to yield results which are in better agreement with experiment values.34 The IR/fsMPI spectrum of the 4ABI 1MT base pair is displayed in Figure 2b. The calculated harmonic frequencies of both the HG and rHG conformers in this frequency region are displayed in Figure 3 for comparison. The free N3 H stretch of the 1MT molecule (3443 cm 1, Figure 2c) is clearly missing in the spectrum of the heterodimer, which indicates that it is H-bonded in the dimer. Thus, the corresponding π-stacked isomers can be ruled out from consideration, because only a slightly perturbed νN3 H mode would otherwise be observed in the spectrum, which is not the case. The band at 3518 cm 1 is present in both the 4ABI monomer and the heterodimer spectrum. Based on an analysis of the calculated anharmonic B3LYP/6-31+G* frequency of this mode which appears at 3483 cm 1 in the 4ABI monomer and at ∼3487 cm 1 in both the HG and rHG conformers of the 4ABI:1MT dimer, the band at 3518 cm 1 can be unequivocally assigned to the unperturbed, or free, imidazolic NH group (νN1 Hf, see Figure 1). It is useful to note that this mode appears at 3481 and 3482 cm 1 in the anharmonic MP2/6-31+G* calculations. This indicates that the amino-group of 4ABI should be involved in H-bond formation. As a consequence, the asymmetric and symmetric stretchings of the amino group (νaNH2 and νsNH2) of 4ABI are assigned to the bands at 3529 and 3427 cm 1, respectively (Figure 2a). These 11406



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Figure 3. Calculated harmonic vibrational spectra of two conformers of 4ABI 1MT at different levels of theory: (A, B) B3LYP/6-31+G* (rHG, HG), (C, D) PW91/6-311++G** (HG, rHG).

frequency values are red-shifted with respect to that of adenine (3569 and 3452 cm 1)35 by 40 and 25 cm 1, respectively, indicating a weakening of the amino bonds in 4ABI as compared to those in adenine, caused by the missing of the two additional nitrogen atoms in the six-membered ring. This also correlates with a stronger basicity of the amino group in 4ABI as compared to adenine.36 In fact, the frequencies of the amino group stretches of 4ABI are closer to the corresponding frequencies of aniline (3508, 3421 cm 1).37 Except for some small deviations in the frequencies of the hydrogen-bonded stretches, the anharmonic spectra shown in Figure 4 are in good agreement with the experimental results shown in Figure 2. Interestingly, Zierkiewicz et al., have shown that the B3LYP functional yields the anharmonic spectra of the adenine monomer in remarkable agreement with experiment.38 The calculated harmonic (Figure 3) and anharmonic (Figure 4) spectra of both the HG and rHG isomers reveal that they have identical IR signatures. It is interesting to note that calculations carried out at the MP2 level of theory also yield harmonic and anharmonic spectra (Figure 5) similar to those obtained at the B3LYP level of theory. This together with the nearly equal energies implies that they would be indistinguishable in the IR/fsMPI experiment. Based on a comparison of the calculated and experimental spectra together with a visualization of the individual modes, the following assignment can be made. The bands at 3526 and 3377 cm 1 correspond to the free amino group (νNH2f) and the H-bonded amino group (νNH2hb) stretch vibrations, respectively. Namely, the corresponding normal coordinates mainly involve the free and H-bonded NH

Figure 4. Calculated anharmonic (B3LYP/6-31+G*) vibrational spectra (with harmonic intensities) of two conformers of 4ABI 1MT and of the 4ABI and 1MT monomers.

shoulders of the amino-group, respectively. The group of bands in the region 2950 3050 cm 1 is due to the overlapping stretch modes of the CH and CH3 groups, an assignment that is also 11407



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Table 2. Observed Vibrational Frequencies and Assignment for the 4ABI 1MT Base Pair and the Base Monomers 1MTa

4ABI

4ABI 1MT

(A) νNH2a

3529 3518

assignment

3526 3518

(A) νNH2f (A) νN1Hf (T) νN3Hf

3443

(A) νNH2s

3427 3377

(A) νNH2hb

3016 2985

2986

2969

2968

νCH, νCH3

2944 2830 e2750

(T) νNHhb

Ref 11, “a”, “s”, “f” and “hb” superscripts at the vibrational modes denote their asymmetric, symmetric, free, and H-bonded character or localization, respectively.

a

Figure 5. Calculated MP2/6-31+G* (A) anharmonic and (B) harmonic vibrational spectra (all with harmonic intensities) of two conformers of 4ABI 1MT.

significantly weaker than the N H 3 3 3 N bond. The νaNH2 and νsNH2 modes of the 4ABI monomer transform upon base pairing into the νNH2f and the νNH2hb vibrations with red shifts of only 3 and 50 cm 1, respectively. The moderate red shifts tell us that the νNH2f and the νNH2hb modes preserve to some extent their nonlocal character, that is, resemble the asymmetric and symmetric modes in 4ABI, respectively. However, taking into account the different intensities and the broadening of the H-bonded amino group vibrations (3526 and 3377 cm 1, Figure 2b), we prefer to term the modes of the amino group as “free” and “H-bonded”. All the observed vibrational frequencies and the corresponding assignments are listed in Table 2 and some of them are graphically displayed in Figure 6.

supported by a comparison with the IR spectrum of the 1MT monomer (Figure 2c). The broad, irregularly shaped IR absorption with an onset at 2870 cm 1, extending beyond the low frequency limit of our IR spectrometer (2740 cm 1), must then be due to the H-bonded NH stretch of 1MT. Relative to the free NH stretch of the jet-cooled 1MT monomer (3443 cm 1), this corresponds to a red shift of more than 700 cm 1. This indicates a fairly strong H-bond. It should be noted that calculations already of the harmonic spectra reveal that there is a dramatic enhancement of the CH stretch overlapped with the NH stretch at around 3080 cm 1 in Figure 3a,b, indicating generally a breakdown of the harmonic approximation.39 The complex shape of this band at ≈2750, assigned to the strongly H-bonded νNHhb mode, should rather be attributed to the anharmonic coupling with low frequency intermolecular modes as well as with the overtones and combinations of the vibrations from the fingerprint region, like CH/NH bendings. Two of the overtones of the dimer which appear in the 2850 2900 cm 1 region in our calculations, are due to the IR active ring C C stretch of 1MT and the ring CH bend vibration of 4ABM. As in case of isolated aniline, where the interaction of the overtone of 1600 cm 1 with the symmetric NH2 stretch leads to a new band at around 3200 cm 1, the coupling of these overtones with the H-bonded N3 H stretch of 1MT could lead to a significantly broadened band at around 2800 cm 1. According to the observed shifts the H-bonding between the carbonyl group of 1MT and the amino group of 4ABI must be

4. DISCUSSION A shorter intermolecular bond length and an enhanced linear intermolecular bond angle reflect a stronger H-bond. The different strengths of the H-bonds in the 4ABI 1MT dimer can be noted in the geometries (Table 1). The calculated values of the N 3 3 3 H and the H 3 3 3 O distances are 1.839 and 2.042 Å, respectively. The H-bonding angle of the shorter bond is also closer to π: 174.6 vs 163.5. Accordingly, the H-bonding induced NH bond elongations correlate well with the observed red-shifts. The calculated amino and thymine’s NH bonds become thus longer by 0.005 and 0.027 Å, respectively. To assess the relevance of the 4ABI 1MT as a mimic system; it is furthermore interesting to compare the H-bonding geometries of the base pair under study with those of 9MA 1MT. For the data in Table 3 the structures were optimized at the same level of theory (B3LYP/6-31+G*). While the N 3 3 3 H distance of the HG isomers of the two different base pairs is the same within the calculation accuracy, the H 3 3 3 O separation is by 0.09 Å larger in the case of the 4ABI 1MT dimer, that is, 2.042 versus 1.951 Å. This correlates also with values of the bond angle. The calculated structures of the rHG isomers of both 4ABI 1MT and 9MA 1MT reveal only slightly stronger N 3 3 3 H N bonds and systematically weaker the N H 3 3 3 O ones than those of the corresponding HG forms, indicating a weaker H-bonding acceptor ability of the C2dO carbonyl group compared to the C4dO one. Hence, the HG (and rHG) isomer of the 11408



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Figure 6. Graphical display of the different H-bonded and free NH stretch modes in the HG conformer of 4ABI 1MT at the MP2/6-31+G* level. The numbers correspond to the anharmonic frequencies. The mode descriptions defined in Table 2 are enclosed in parentheses. The displacement vectors are depicted so that their middle points are coincident with the corresponding atoms.

Table 3. H-Bonding Distances (in Å) and Angles (in Degrees) of 9MA 1MT in Different Isomeric Structures Calculated at the B3LYP/6-31+G* Level of Theory 9MA 1MT

H3 3 3N — NH 3 3 3 N H3 3 3O

— NH 3 3 3 O

WC

HG

rHG

1.871

1.839

1.836

178.3

176.9

176.9

1.922

1.951

1.979

174.3

170.5

170.8

9MA 1MT base pair is expected to possess a stronger amino 3 3 3 carbonyl H-bond than the 4ABI 1MT dimer. Interestingly, the geometry differences between the HG forms of the 9MA 1MT and 4ABI 1MT dimers (Tables 1 and 3) are larger than between the HG and WC isomers of the 9MA 1MT base pair (Table 3). As was mentioned before, we calculated the H-bond energies at the B3LYP/6-31+G* level using the AIM method. The H-bond energies for the NH 3 3 3 N bond are 9.11 (HG) and 9.18 (rHG) kcal/mol, and for the NH2 3 3 3 O bond are 5.40 (HG) and 4.96 (rHG) kcal/mol, respectively. This data is in good agreement with the observed H-bond geometries and indicates that there are some differences between HG and rHG conformers, a fact which can also be noted in the calculated red-shifts and intensities. For comparison sake, we also calculated the H-bond energies for the corresponding HG and rHG conformers in the 9MA 1MT complex. The energies for the NH 3 3 3 N bond are 9.12 (HG) and 9.19 (rHG) kcal/mol and for the NH2 3 3 3 O H-bond are 6.46 (HG) and 6.04 (rHG) kcal/mol, respectively. Thus, while the energies for the NH 3 3 3 N bond are similar for both 9MA 1MT and 4ABI 1MT complexes, the NH2 3 3 3 O bond are markedly stronger in 9MA 1MT. This seems

to indicate that a 1MT nitrogen (N1) adjacent to the C2dO acceptor in 1MT (rHG conformers) leads to a weaker NH2 3 3 3 O bond (See Figure 1, for atom numbering). This correlates well with the fact that both WC and HG base pairing of A T are experimentally observed within a DNA double helix, whereas no H-bonding was detected between 4ABI and thymine in DNA. The N 3 3 3 H N bonds are comparably strong in all considered base pairs.16,40,41 Hence, it is the relatively smaller energy of the N H 3 3 3 O H-bond, observed for the isolated 4ABI 1MT dimer, which explains why 4ABI in the DNA double helix is incapable of H-bonding with thymine. In a double helix the H-bonding between the nucleobases is perturbed by the sugar phosphate backbone constraints. The bases are therefore twisted with respect to each other by an angle of a few to a few tens degrees dependent on the sequence and particular type of the helix.42 Additionally, the water environment also destabilizes the helix, so that the average stabilization energy for DNA in aqueous solution is 0.7/ 1.6 kcal/mol per H-bond,43 which is substantially smaller than for the isolated base pairs.8,44,45 We should also keep in mind that the HG base pairing motif in DNA, the only available for the 4ABI base, is weaker than the WC base pairing as the latter has been chosen by nature. Under these circumstances, the weak N H 3 3 3 O bond is either easily broken or not formed at all. Although strong, the N 3 3 3 H N bond alone does not keep the oligonucleotide strands together presumably for the following reason. The bases can rotate around the N 3 3 3 H N bond. This in turn provides a better access of the solvent to the carbonyl and the amino group, which are good H-bonding acceptors. The H-bonding interaction between surrounding water molecules and the above acceptors should further destabilize the double helix. The π-stacking interaction important for a single strand structure in addition to the N 3 3 3 H N bonds should not be strong enough to keep the double helix intact. 11409



5. CONCLUSION The 4ABI 1MT base pair, a possible mimic of Hoogsteentype base pairing between A and T, was investigated by the IR/ fsMPI spectroscopy and quantum chemistry calculations. The vibrational spectra indicate that only the planar doubly H-bonded structures mimicking the HG (or rHG) base pairing of A T were detected by multiphoton ionization using 260 fs at 267 nm pulses and IR ion depletion spectroscopy. In particular, no evidence of π-stacked or T-shaped geometries were found. For the HG and rHG isomers a similar stability and great similarity of the vibrational spectra was found theoretically so that they cannot be distinguished in the IR/fsMPI spectrum. It is possible that both forms contribute with overlapping vibrational bands. To the best of our knowledge, a vibrational spectrum of a base pair mimicking the HG interaction between A and T is reported for the first time. The dimer is characterized by the strong N 3 3 3 H N bond manifested by a significant red shift of the NH stretch frequency of g700 cm 1. In contrast, the N H 3 3 3 O bond was found to be substantially weaker, being characterized by a red shift of only 50 cm 1 in the amino-donor vibration. According to our calculations this bond has to be considerably stronger in the A T base pair. The imbalance in the strengths of the H-bonds in the 4ABI 1MT base pair can explain why single strands based on 4ABI do not form stable helices with single strands of thymine. The weaker H-bonds can be broken by backbone induced tension. And one H-bond per base pair is probably not enough to keep the strands together in aqueous solution. ’ ASSOCIATED CONTENT

bS

Supporting Information. Calculated anharmonic vibrational frequencies of two planar conformers of 4ABI-1MT at different levels of theory: (A, B) B3LYP/6-31+G* (HG, rHG), (C, D) PW91/6-31+G* (HG, rHG). This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

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