Article Cite This: J. Phys. Chem. A XXXX, XXX, XXX-XXX
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Mechanism Underlying the Nucleobase-Distinguishing Ability of Benzopyridopyrimidine (BPP) Michał A. Kochman,*,† Andrzej Bil,‡ and R. J. Dwayne Miller†,¶ †
Max Planck Institute for the Structure and Dynamics of Matter and Hamburg Centre for Ultrafast Imaging, Bldg. 99 (CFEL), Luruper Chaussee 149, 22761 Hamburg, Germany ‡ Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383 Wrocław, Poland ¶ Department of Chemistry and Physics, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada S Supporting Information *
ABSTRACT: Benzopyridopyrimidine (BPP) is a fluorescent nucleobase analogue capable of forming base pairs with adenine (A) and guanine (G) at different sites. When incorporated into oligodeoxynucleotides, it is capable of differentiating between the two purine nucleobases by virtue of the fact that its fluorescence is largely quenched when it is base-paired to guanine, whereas base-pairing to adenine causes only a slight reduction of the fluorescence quantum yield. In the present article, the photophysics of BPP is investigated through computer simulations. BPP is found to be a good charge acceptor, as demonstrated by its positive and appreciably large electron affinity. The selective quenching process is attributed to charge transfer (CT) from the purine nucleobase, which is predicted to be efficient in the BPP-G base pair, but essentially inoperative in the BPP-A base pair. The CT process owes its high selectivity to a combination of two factors: the ionization potential of guanine is lower than that of adenine, and less obviously, the site occupied by guanine enables a greater stabilization of the CT state through electrostatic interactions than the one occupied by adenine. The case of BPP illustrates that molecular recognition via hydrogen bonding can enhance the selectivity of photoinduced CT processes.
1. BACKGROUND The incorporation of modified nucleobases into DNA/RNA provides access to a range of chemical and optical functionalities, which are not available with only the five canonical nucleobases. Examples taken from the toolbox of molecular biology include unnatural base pairs,1−3 fluorescent base analogues (FBAs),4−7 and radiosensitizing agents.8−12 Interestingly, modified nucleobases may also have played a key role in the early evolution of life: within the framework of the “RNA world” model of abiogenesis,13−16 according to which all modern life originated from self-replicating RNA, it has been hypothesized that modified nucleobases were involved in the RNA catalysis of reactions, which are unlikely to have been feasible with catalysts based on the canonical nucleobases alone.17−23 The present study is concerned with the photophysics of the FBA benzopyridopyrimidine (BPP), designed by Okamoto and co-workers,24,25 which represents a proof-of-concept for the typing of A/G single-nucleotide polymorphisms (SNPs) by means of fluorescence spectroscopy. In terms of molecular structure (see Figure 1c), BPP belongs to a class of sizeexpanded fluorescent cytosine analogues based on pyridopyrimidine26 (F, Figure 1b). Some other FBAs of this class27−29 (though not F itself) are also known to be able to distinguish between being paired with adenine and guanine, but as they are larger than BPP, they do not lend themselves as well to © XXXX American Chemical Society
Figure 1. (a) Cytosine (C) and its fluorescent size-expanded analogues, (b) pyridopyrimidine (F) and (c) benzopyridopyrimidine (BPP).
computer simulations. Several other classes of modified bases have also been shown to be able to report, by various means, on the identity of the opposite base.30−40 As illustrated in Figure 2, the extended Watson−Crick face of BPP features a thymine-like and a cytosine-like motif, allowing it to form base pairs with both adenine (Figure 2a) and guanine (Figure 2b) in their respective canonical tautomers.24 Okamoto and co-workers24 have measured the melting temperatures of Received: August 21, 2017 Revised: October 4, 2017 Published: October 6, 2017 A
DOI: 10.1021/acs.jpca.7b08334 J. Phys. Chem. A XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry A
Figure 2. Base pairing modes of BPP with (a) A and (b) G, as inferred by Okamoto and co-workers from 15N NMR measurements.24
Figure 3. Possible structures adopted by the BPP-1MeG base pair. (a) In what we refer to as the distorted Watson−Crick geometry, 1MeG adopts an anti conformation. Due to the bulky methyl substituent, it may “slide” outside the base stack or it may be wedged between BPP and the flanking base pair. (b) In the Hoogsteen structure, 1MeG adopts a syn conformation, while BPP is presumably protonated at nitrogen N3 so as to form a second hydrogen bond with 1MeG. The two forms of the base pair may coexist in thermodynamic equilibrium with one another.
in a moderate degree of quenching, such that thymine and cytosine are difficult to distinguish from one another, or from adenine. Lastly, it was found that when BPP was positioned in the duplex opposite 1-methylguanine (1MeG), with which it cannot form a normal Watson−Crick base pair, fluorescence was partially quenched with reference to the single strand (to a quantum yield of 0.0126), but less so than with the unmodified guanine. It was inferred that base pairing between BPP and guanine plays a key role in the mechanism of quenching. We note here that by analogy to the case of cytosine,41 when BPP is positioned opposite 1MeG, a fraction, possibly even the majority, of molecules may exist in the form of a Hoogsteen base pair in which BPP is protonated at nitrogen N3 (see Figure 3a,b for explanation). It is not known whether the protonated form of BPP is fluorescent. Consequently, the quenching that does occur when BPP is positioned opposite 1MeG may be partially due to the formation of the Hoogsteen base pair. The observed pattern of fluorescence quenching implies that a rapid and efficient radiationless decay mechanism of the fluorescent state of BPP takes place in the BPP-G base pair, but not in the BPP-A base pair. Guanine has the lowest vertical ionization potential (VIP) from among the canonical nucleobases,42 while BPP can be expected to possess a relatively high vertical electron affinity (VEA) owing to its extended π-bonding system. In combination, these factors point toward the hypothesis that the mechanism in question involves a photoinduced, interstrand charge transfer (CT) process from guanine to BPP. However, the hypothesis that guanine quenches the fluorescence of BPP via a G → BPP CT process does not, on its own, explain why the quenching process is
oligodeoxynucleotides (ODNs) in which the pyrimidine base in a single T-A or C-G base pair was replaced with BPP. Either replacement was observed to cause only a minor change of the melting temperature. This was taken to indicate that the BPP-A and BPP-G base pairs are about as stable as the T-A and C-G base pairs, respectively, even though there is a considerable size mismatch between BPP and the pyrimidine base that it replaces. By recording the fluorescence spectra of BPP incorporated into a single-stranded ODN, and the same ODN duplexed with complementary ODNs containing canonical DNA nucleobases and 1-methylguanine opposite BPP, Okamoto et al.24 have demonstrated that the fluorescence quantum yield of BPP is sensitive to the identity of the opposite base. The kinetics of fluorescence decay was analyzed in terms of a triexponential decay function, I(t) = ∑3i=1αi exp(−t/τi). Incorporated into the single-stranded ODN, BPP displayed a moderately high quantum yield of fluorescence of 0.0409. When duplexed to the complementary ODN containing adenine opposite BPP, the fluorescence quantum yield of BPP was reduced only slightly (to 0.0352). In turn, when BPP was positioned opposite guanine, its fluorescence was almost completely suppressed (to a quantum yield of 0.0018), and the fluorescence decay kinetics became dominated by the shortest-lifetime component α1 with τ1 = 37 ps, indicating a rapid and efficient quenching process. This effect forms the basis of the potential application of BPP for the typing of A/G SNPs. Unfortunately, however, the fluorescence of BPP is partially quenched also when it is not paired with guanine, but is flanked by a C-G base pair. This limits the applicability of BPP to sequences in which the A/G SNP site is flanked by two A-T base pairs. Placing BPP opposite a pyrimidine base results B
DOI: 10.1021/acs.jpca.7b08334 J. Phys. Chem. A XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry A
ab initio electronic structure simulations. In particular, the energetics of the interbase CT processes was explored first by considering the electron-attached (i.e., anionic) states of the isolated BPP molecule and, second, by explicitly calculating the excited electronic states of the BPP-A and BPP-G base pairs. The latter approach naturally takes into account intermolecular interactions within the base pairs, which, as we will demonstrate, is crucial for understanding the high selectivity of the quenching process. The rest of the article is organized as follows. First, we describe the setup of the simulations and assess the level of accuracy they are expected to achieve for the systems under investigation. We then examine the excited and electronattached states of the isolated BPP molecule. Finally, we turn our attention to the charge transfer states of the base pairs of BPP and consider the question of the selectivity of the interstrand CT process.
highly selective, which is to say, why base-pairing to adenine causes only a slight reduction of the quantum yield of emission. In order to see why, let us make the very crude approximation that the purine → BPP CT process involves the transfer of exactly a single electron between otherwise uninteracting isolated molecules, with no relaxation of nuclear geometries. Then, the energy cost of charge transfer is given by the difference of the VIP of the purine nucleobase and the VEA of BPP. ΔE = VIP(X ) − VEA(BPP)
where X = A or G
The VIP of adenine is only around 0.3 eV higher than that of guanine.42 Thus, if the VIP of the purine base is taken as a measure of its charge-donating ability, then a purine → BPP CT state in the base pair would be expected to lie only slightly higher in energy in the BPP-A base pair than in the BPP-G base pair. If that were the case, appreciable quenching of BPP fluorescence via interstrand CT would occur also when BPP is base-paired to adenine. This reasoning suggests that some interaction, which is not accounted for in the simplistic isolated-molecules model, stabilizes the CT state in the BPP-G base pair more strongly than in the BPP-A base pair, thereby contributing to the selectivity of the CT process. The findings of Okamoto and co-workers,24 and especially the observation that base pairing between BPP and guanine is required for efficient quenching, specifically point to an interaction between BPP and its purine partner. Indeed, previous theoretical studies of the natural base pairs A-T and C-G have demonstrated that the energies of interbase CT states of base pairs can be sensitive to the mode of hydrogen bonding between the nucleobases;43−47 presumably, a similar effect is at work in the case of BPP, leading to higher selectivity of quenching than would be implied by the IP values of adenine and guanine. We are aware of only a single previous attempt to identify the origins of the base-distinguishing ability of BPP and related FBAs: a recent review paper by Seio and co-workers48 has examined the dependence of the fluorescent quantum yield of a related FBA, pyrimidopyrimidoindole (PPI), on the identity of the opposite base. In the spirit of Janak’s theorem,49,50 the energy of the Kohn−Sham HOMO of the base positioned opposite PPI was taken as a measure of its electron-donating ability and, hence, of the efficiency of quenching via interstrand nucleobase → PPI CT. The strong quenching of PPI fluorescence by an opposite guanine was attributed to an G → PPI CT process. Regarding the cases where PPI was positioned opposite a nucleobase other than guanine, however, it was noted that the fluorescence intensity could not be explained with reference to the electron-donating ability of that nucleobase, indicating that in these cases the fluorescence quantum yield was controlled by other factors. In turn, Liu and co-workers51,52 reported density functional theory (DFT) and time-dependent density functional theory (TDDFT) simulations of the optical properties of several FBAs with multiple hydrogen-bonding modes, including BPP; however, the mechanism of the base-distinguishing ability of BPP and similar analogues was not addressed in those studies. Clearly, then, the resolution of the selective quenching processes of BPP and related FBAs requires a more realistic theoretical model than merely a comparison of isolatedmolecule properties such as ionization potentials and electron affinities, or orbital energies. In order to achieve a better understanding of BPP and its base pairs, in the present study the photophysics of these systems was investigated by means of
2. COMPUTATIONAL METHODS For the sake of brevity, in the main body of the present article, we will present only a skeletal outline of the computational methodology. The detailed description of the simulation setup, and a discussion of its accuracy, is relegated to the Supporting Information. Calculations at the DFT level indicate that N8−H tautomer illustrated in Figure 1 is by far the lowest-energy tautomeric form of BPP. This finding is in agreement with the structures of the BPP-A and BPP-G base pairs as determined by Okamoto et al.24 using 15N NMR spectroscopic measurements (see Figure 2). Hence, in what follows, we take into account only the N8− H tautomer. 2.1. Excited Electronic States of BPP. The ground-state equilibrium geometry of BPP was optimized using Møller− Plesset second-order perturbation theory (MP2) as implemented in the program Gaussian09.53 Planar symmetry was imposed. Here and elsewhere, except when noted otherwise, the diffuse-augmented basis set jun-cc-pVDZ54 was applied. All calculations were performed in vacuo. The vertical excitation spectrum of BPP was calculated using the method of equation of motion coupled cluster including single and double excitations (EOM-CCSD), within Molpro.55 What is more, a geometry optimization was performed at the EOM-CCSD/cc-pVDZ level of theory in order to locate the emissive minimum on the potential energy surface (PES) of the S1 state. This excited-state optimization was performed within the program PSI4.56,57 2.2. Electron-Attached States of BPP. We assumed, as a working hypothesis, that guanine-to-BPP CT plays a role in the base-discriminating ability of BPP. As the first step in modeling that process, we characterized the charge-accepting ability of the isolated BPP molecule by calculating its electron-attached (i.e., anionic) states. A two-pronged approach was adopted: first, the VEAs of the lowest few electron-attached states of BPP were computed with the use of the EOM-CCSD method in combination with the continuum orbital technique of Stanton and Gauss.58 In a second, independent, calculation the VEA for the lowest electron-attached state was obtained using the CCSD(T) method.59 The two coupled cluster calculations were carried out within Molpro55 and ORCA,60 respectively. 2.3. Base Pairs of BPP. As the second step in modeling the mechanism of operation of BPP, we examined the process of quenching by charge transfer in its base pairs. At this stage, in order to keep the simulations tractable, we resorted to the MP2 C
DOI: 10.1021/acs.jpca.7b08334 J. Phys. Chem. A XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry A method for the ground state in combination with the algebraic−diagrammatic construction scheme of second order61 (ADC(2)) for the excited states. In what follows, we will collectively refer to this combination of methods as MP2/ ADC(2). These calculations we performed within Turbomole.62 2.4. Analysis of Electronic Structures. In order to quantify the redistribution of electron density in the excited, and electron-attached, electronic states of BPP and its base pairs, we used the technique outlined in ref 63, which relies on a Voronoi−Dirichlet tesselation of space around the atomic nuclei. What is more, for the excited electronic states of the base pairs, we calculated values of the charge transfer excitation length (DCT) as defined by Le Bahers and co-workers.64
Table 1. Vertical Excitation Energies (ΔE), Oscillator Strengths (f), and Electric Dipole Moment Magnitudes (μ) of BPPa state
ΔE (eV)
f
μ (D)
1 1A′ 2 A′ (ππ*) 3 1A′ (ππ*)
4.149 4.515
0.137 0.003
7.42 7.86 7.32
4 1A′ (ππ*)
5.200
0.107
7.27
1 1A″ (nπ*)
4.872
