Theoretical Study of the Photophysics of 8-Vinylguanine, an

Jul 18, 2016 - Paving the way for the application of the algebraic-diagrammatic construction scheme of second-order (ADC(2)) to systems based on the g...
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Theoretical Study of the Photophysics of 8‑Vinylguanine, an Isomorphic Fluorescent Analogue of Guanine Michał A. Kochman,*,† Martina Pola,†,‡ 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 ‡ Institut für Theoretische Physik, Universität Hamburg, Jungiusstraße 9, 20355 Hamburg, Germany § Department of Chemistry and Physics, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada S Supporting Information *

ABSTRACT: Paving the way for the application of the algebraicdiagrammatic construction scheme of second-order (ADC(2)) to systems based on the guanine chromophore, we demonstrate the this excited-state electronic structure method provides a realistic description of the photochemistry of 9H-guanine, in close agreement with the benchmark provided by the CASPT2 method. We then proceed to apply the ADC(2) method to the photochemistry of 8-vinylguanine (8vG), a minimally modified analogue of guanine which, unlike the naturally occurring nucleobase, displays intense fluorescence, indicative of a much longer-lived excited electronic state. The emissive electronic state of 8vG is identified as an ππ*-type intramolecular charge transfer (ICT) state, in which a charge of roughly −0.2 e is transferred from the guanine moiety onto the vinyl substituent. The main radiationless deactivation pathway competing with fluorescence is predicted to involve the molecule leaving the minimum on the ICT ππ* state, and reaching a region of the S1 adiabatic state where it resembles the La ππ* state of unmodified 9H-guanine. The topology of the La ππ* region of the S1 state favors subsequent internal conversion at a crossing seam with the ground electronic state. The sensitivity of this process to environment polarity may explain the experimentally observed fluorescence quenching of 8vG upon incorporation in single- and double-stranded DNA.

1. INTRODUCTION Following the absorption of a photon in the UV range, individual molecules of the five canonical nucleobases of natural DNA/RNA efficiently undergo nonradiative deactivation to the ground state on subpicosecond to picosecond time scales.1−3 These ultrafast nonradiative deactivation processes kinetically out-compete fluorescence emission, and as a consequence, all five canonical nucleobases are practically nonfluorescent. The fluorescence quantum yields of oligonucleotides and DNA/ RNA duplexes are likewise low,4,5 although more complex excited-state intermediates are involved.6−9 The near lack of appreciable intrinsic fluorescence from natural DNA/RNA means that fluorescence spectroscopic studies usually rely on fluorescent markers or labels introduced into the system of interest. Several classes of such probes are in use. The first consists of fluorophores that bind noncovalently to DNA/RNA, such as ethidium bromide (EtBr) and 4′,6-diamidino-2phenylindole (DAPI). The second comprises common fluorophores such as pyrene and fluorescein linked covalently to the target DNA/RNA molecule. Although well-suited to some applications, probes of both these types have the inherent disadvantage of being bulky external attachments to DNA/ RNA. As a consequence, they are generally fairly insensitive to the internal structure of the DNA/RNA molecule and, what is © XXXX American Chemical Society

more, they may significantly perturb its structure and/or reactivity.10 A different approach to fluorescent marking is represented by fluorescent nucleobase analogues11−14 (FBAs): functionalized, cyclized, or otherwise chemically modified analogues of natural nucleobases which exhibit fluorescence. Labeling with a FBA involves the replacement of one or more instances of a natural nucleobase in the target DNA/RNA molecule with its fluorescent analogue. Compared to the externally attached fluorophores, the FBAs offer some important advantages. Many (the so-called isomorphic FBAs) are very similar to their natural counterparts in terms of overall dimensions, structure, and ability to form Watson−Crick base pairs, so that the replacement does not introduce a significant structural perturbation into the target DNA/RNA molecule. What is more, the fluorescence spectra of some FBAs are responsive to specific properties of their environment through enhancement or quenching of emission and/or wavelength shifts in the fluorescence spectra. Examples include FBAs which report on involvement in base pairing,15−17 the presence of an abasic site Received: May 10, 2016 Revised: June 20, 2016

A

DOI: 10.1021/acs.jpca.6b04723 J. Phys. Chem. A XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry A opposite to the FBA,18 the amount of steric freedom available to the FBA,19 or environment polarity.19−21 One fruitful design strategy for fluorescent analogues of the purine bases adenine and guanine has been to substitute these molecules at position C8 with conjugating groups,20,22−34 leading, among others, to the subject of the present study: 8vinylguanine (8vG), whose structure is shown in Figure 1. The

modification controls also the accessibility of its radiationless deactivation pathways. The accuracy of the computational methodology used in the present work was tested extensively through comparison to higher-level theoretical methods and, where possible, experimental data.

2. COMPUTATIONAL METHODS The conformational and tautomeric preference of 8vG is analyzed in section 2 of the Supporting Information, where it is shown that the predominant isomer of 8vG under experimentally relevant conditions is the syn-keto-1H form illustrated in Figure 1b. In what follows, we take into consideration only this isomer. 2.1. Electronic Structure Methods. The singlet ground states of 9H-guanine and 8vG were described using secondorder Møller−Plesset perturbation theory (MP2), whereas their excited electronic states were calculated using the algebraicdiagrammatic construction scheme of second order (ADC(2)).43,44 For the sake of brevity, from now on we collectively refer to this combination as MP2/ADC(2). As a preliminary for the application of the MP2/ADC(2) combination to the molecules under investigation, its accuracy was verified by benchmarking against the higher-level electronic structure methods equations-of-motion coupled cluster with single and double excitations (EOM-CCSD) and complete active space second-order perturbation theory (CASPT2). This was necessitated by the fact that the ADC(2) method is known to have errors of up to a few tenths of an electronvolt for the relative energies of locations on excited-state PESs.45 The same is true for the related approximate coupled-cluster singles-anddoubles (CC2) method,46 which has been also shown to systematically underestimate vertical excitation energies into nπ*-type states of nucleobases relative to higher-level methods.47,48 The discussion of the benchmark calculations is relegated to section 1 of the Supporting Information. The good agreement with these benchmarks leads us to expect that MP2/ ADC(2) provides a realistic description of both 9H-guanine and 8vG and therefore represents a good choice of electronic structure method for the purposes of this study. Ground-state MP249,50 and excited-state ADC(2)51,52 calculations were performed within the computational chemistry software package Turbomole version 6.3.1,53 taking advantage of the frozen core and resolution of the identity approximations. The cc-pVTZ basis set54 was applied in combination with the default auxiliary basis set55 for cc-pVTZ. Except where noted otherwise (see below), calculations were performed in vacuo. When reporting excited-state dipole moments calculated using the MP2/ADC(2) method, we refer always to the orbital-relaxed dipole moments. Because of the localized nature of the cc-pVTZ basis set, the Rydberg-type excited states of 8vG are not detected when this basis set is used. A separate calculation of the vertical excitation spectrum of 8vG was therefore carried out with the aug-ccpVTZ basis set, at the ground-state equilibrium geometry optimized with the cc-pVTZ basis set. The fluorescence of 8vG is strongly (9- to 12-fold) quenched upon incorporation into single-stranded DNA oligonucleotides, and further upon duplex formation, relative to the fluorescence of its deoxyribonucleoside form in aqueous solution,35 suggesting an environment-dependent quenching mechanism. Meanwhile, the present simulations (sections 3.2 and 3.3) indicate that the fluorescent electronic state of 8vG has significant intramolecular charge transfer (ICT) character. As

Figure 1. Structures of 9H-guanine and fluorescent analogue 8vinylguanine (8vG). In experiments, 8vG was characterized in deoxyribonucleoside form, as the free base is poorly soluble in water.35

FBA 8vG was synthesized by Nadler and co-workers as an isomorphic fluorescent analogue of guanine, and it was reported to combine several properties that are favorable for application in studies of DNA structure.35,36 The replacement of guanine with 8vG in double-stranded DNA results in only a minor destabilization of the duplex.35 In fact, 8vG is similar enough to guanine that its nucleotide form may be introduced into DNA by various DNA polymerases.37 In aqueous solution, the deoxyribonucleoside form of 8vG shows intense fluorescence (quantum yield ΦF = 0.72 ± 0.03, emission maximum λmax em = 400 nm) following the irradiation of its lowest absorption band near 305 nm.35 The intensity of fluorescence is found to be reduced on incorporation into oligonucleotides, and further on the formation of B-form duplexes.35 Its fluorescence spectrum also displays slight wavelength shifts due to involvement in guanine quadruplex structures.35 The environment sensitivity of the fluorescence spectrum 8vG provides the basis for its application as a probe of DNA structure, provided that its response to specific conditions can be calibrated through a reference experiment. As pointed out in a recent review by Matsika,38 at present 8vG still awaits a theoretical examination, whereas only relatively limited information is available on the photochemistry of its adenine-based counterpart, 8-vinyladenine. The excited electronic states of this latter compound have been investigated by Kenfack et al.39 and later by Kodali et al.,40 but its potential energy surfaces (PESs), which could explain the mechanism of its fluorescence, have not been explored. Motivated by the ongoing effort toward the development and investigation of fluorescent analogues of purine bases, the present study aims to construct a comprehensive theoretical picture of the photophysics of 8vG, with special regard to the control of optical properties by structural modification. In particular, we hope to explain why 8vG has a long excited-state lifetime and is highly fluorescent, whereas the excited-state lifetime of 9H-guanine photoexcited in the UV range is believed to be very short (subpicosecond41,42). The rest of the paper is organized as follows. First, we outline the calculations that were performed to characterize the optical properties of 8vG. We then discuss the role of structural modification in controlling the character and energy ordering of the excited electronic states of 8vG in comparison to unmodified 9H-guanine. Finally, we consider how structural B

DOI: 10.1021/acs.jpca.6b04723 J. Phys. Chem. A XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry A ICT states of organic molecules are often found to interact strongly with polar environments due to their typically large electric dipole moments,56,57 the possibility presents itself that it is the reduced environment polarity experienced by 8vG in single- and double-stranded DNA that is responsible for the fluorescence quenching. We have therefore examined the influence of environment polarity by applying the conductorlike screening model58,59 (COSMO) implemented in Turbomole 7.0,60 an implicit solvation model whereby the molecule is placed in a cavity within a polarizable continuum. Due to the unavailability of analytic gradients at the MP2/ADC(2) level in combination with COSMO, the computational cost of including of solvent effects in geometry optimizations would have been prohibitively high, and therefore solvation effects were included only in single point calculations at geometries optimized in vacuo. Aqueous solvation was represented by setting the dielectric constant of the environment as ε = 78.4 and the refractive index as n = 1.34. The effect of environment polarity was also investigated by varying ε in the range from 1 to 80 while keeping the other parameters defining the environment fixed. (Higher values of ε represent increasing environment polarity.) Nonequilibrium solvation was imposed when the vertical excitation and fluorescence emission energies were calculated (which is to say, the slow part of the reaction field was equilibrated with the initial state, and the fast electronic part of the reaction field was in equilibrium with the target state). On the other hand, in potential energy surface (PES) scans, the reaction field was always equilibrated with the S1 adiabatic state, and the energies of the other states were obtained using the nonequilibrium approach. The cavity around the molecule was constructed using the standard solvent accessible surface scheme using the optimized atomic COSMO radii (rC = 2.00 Å, rN = 1.83 Å, rO = 1.72 Å, rH = 1.30 Å) and a solvent radius of rsolv = 1.30 Å. Program defaults were used for the remaining adjustable parameters. 2.2. Characterization of Excited Electronic States. A convenient means to analyze the nature of an excited electronic state is the electron density difference map (EDDM), which is defined simply as the difference of the electron density of the excited state and that of the ground state at the same nuclear geometry. Thus, an EDDM illustrates the redistribution of electron density upon photoexcitation. For each relevant structure located at the MP2/ADC(2) level, we generated EDDMs for its lowest few excited electronic states. Moreover, it turns out that the lowest vertical excited state of 8vG involves a substantial amount of intramolecular charge transfer (ICT) from the guanine moiety onto the vinyl group, and we have therefore analyzed quantitatively the redistribution of electron density in its low-lying electronic excited states. This was carried out as follows. A Voronoi−Dirichlet tesselation was performed on the space around the nuclei. This procedure assigns to each atom (which in this context is considered to be a point positioned at the nucleus) a Voronoi−Dirichlet polyhedron (VDP) that contains all points whose distance to that atom is not greater than the distance to any other atom. In this manner, space is partitioned completely and disjointly into VDPs associated with individual atoms. (The partitioning is not disjoint in the rigorous sense, because the surface of each polyhedron is shared with the neighboring polyhedra, but this fine point can be ignored here.) As an illustration, Figure 2 shows the Voronoi−Dirichlet tesselation of space around the atoms of the 8vG molecule.

Figure 2. Schematic illustration of the Voronoi−Dirichlet tesselation of space around the nuclei of the 8vG molecule, within a cubic cell of side length 14 Å. The green lines are the edges of the VDPs associated with the constituent atoms.

The VDP of an atom may be interpreted as the volume of space taken up by the atom within the molecule, and by extension, the electronic charge contained in the VDP may be assigned as belonging to that atom. Unlike some other space partitioning schemes such as Bader analysis,61 the Voronoi− Dirichlet tesselation depends only the nuclear geometry and therefore enables comparison between the charge distributions of different electronic states. Due to program limitations, it was only possible to analyze electron densities from calculations performed in vacuo using Turbomole version 6.3.1. The analysis was carried out with the use of the program bader.62−66 2.3. Exploration of Ground- and Excited-State Potential Energy Surfaces. Ground-state equilibrium geometries of 9H-guanine and 8vG were optimized in internal coordinates, and with no symmetry constraints. Likewise, equilibrium geometries on the respective S1 PESs of these compounds were located and optimized. All resulting structures were confirmed to correspond to potential energy minima by calculating numerically the normal modes of the molecule. We furthermore investigated the nonradiative deactivation pathways of 8vG. Because the internal conversion of singlet excited states of nucleobases to the vibrationally excited ground state predominantly takes place at, or near, conical intersections with the ground state, we searched for minima on the crossing seam (MXSs) between the S1 and S0 states. As a digression, we note here that the MP2/ADC(2) combination has some shortcomings for the description of S1/S0 crossings. Due to its single-reference nature, it is unable to provide a formally correct description of crossings with the ground state, where the ground state will have a strong multireference character. A related problem is that it fails to describe the true (conical) topology of S1/S0 crossings and instead predicts linear S1/S0 crossings,67 a flaw that it shares with certain other methods that do not treat the two intersecting states on an equal footing.68 Nevertheless, it is generally found to correctly predict the locations of crossings with the ground state, in the sense that in the vicinity of a crossing, the calculated energy gap between the S1 and S0 states becomes small. Because in the present work, we are mainly concerned with barriers on excited-state potential surfaces of 8vG along pathways leading from the excited-state C

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useful for the purpose of mapping out PESs along a reaction path. It should be emphasized that although the starting and end points of a LIIC path are minimum-energy geometries, the LIIC path does not, in general, coincide with the minimum energy path (MEP) connecting these structures. Thus, any potential energy barrier along the LIIC path is, in general, higher than or equal to the potential barrier along the MEP.

minimum to the low-energy MXSs, and less with the description of the MXSs themselves, we consider the MP2/ ADC(2) combination adequate for our purposes. The geometries of the MXSs were optimized using the penalty function method of Ciminelli et al.69 Within that method, the geometry optimization proceeds by minimizing the penalty function f(R) defined as f (R) =

⎡ ⎛ E (R) − E0(R) ⎞2 ⎤ E1(R) + E0(R) + c1c 2 2 ln⎢1 + ⎜ 1 ⎟⎥ 2 c2 ⎝ ⎠ ⎥⎦ ⎣⎢

3. RESULTS AND DISCUSSION 3.1. Ground-State Equilibrium Geometries. The ground-state equilibrium geometries (S0-min) of 9H-guanine and 8vG are presented in Figure 3a,b, respectively. We find that

where R denotes the molecular geometry, and E1(R) and E0(R) are the energies of the S1 and S0 adiabatic states, respectively. The role of the first term is to minimize the average of the energies of the S1 and S0 states, whereas the second term minimizes the energy difference between these states. The parameter c1 controls the relative weight of these two goals, and c2 controls the “rate” at which the optimization approaches the crossing seam. We adopted the values recommended in the work just cited: c1 = 5 (kcal/mol)−1 and c2 = 5 kcal/mol. The minimization itself was carried out in Cartesian coordinates using the limited-memory BFGS algorithm. We adopted a three-pronged approach to generate candidate initial geometries for the MXS optimizations. First, we applied various structural deformations, such as various ring puckering modes, and torsions around the C8−C11 and C11−C12 bonds. We also sought and optimized counterparts of the known S1/S0 MXSs of unmodified 9H-guanine. Lastly, we propagated nonadiabatic molecular dynamics trajectories of photoexcited 8vG. Here, the goal was only to produce lowenergy geometries close to the S1/S0 crossing seam; it was not the intention to model any real-world physical situation. The trajectories were simulated with the use of the implementation of the trajectory surface hopping (TSH) method available in the software package Newton-X.70−72 To reduce the computational cost of these simulations, the basis set size was reduced to def2-SV(P).73 The initial conditions (sets of atomic positions and velocities) for the TSH simulations were sampled from the Wigner quantum harmonic oscillator distributions for the respective S0-min structures, such that each of the 20 lowest vibrational modes was in the first excited (v = 1) state. Adiabatic states from S1 to S3 were included in the linear expansion of the electronic wave function. The initially occupied adiabatic state was the S1 state, and its initial population was set to unity. Under these conditions, the molecule has a large amount of vibrational energy and can overcome potential barriers of several tenths of an electronvolt on the excited-state potential energy surfaces to reach lowenergy segments on the S1/S0 crossing seam. Whenever the energy gap between the S1 and S0 decreased to below 0.5 eV, the simulated trajectory was terminated and the final geometry was used as a candidate structure for the MXS optimization. To map out the topology of the ground- and excited-state PESs of 8vG between the excited-state equilibrium geometry of the emissive state at one end and the S1/S0 MXSs on the other, we generated paths between these structures through linear interpolations in internal coordinates (LIIC). The internal coordinate system was set up with the help of the define utility of Turbomole. The ground- and excited-state PESs were subsequently scanned along the LIIC paths through singlepoint calculations. The LIIC depends on the internal coordinate system used to carry out the interpolation and is therefore a somewhat arbitrary construct, but nevertheless

Figure 3. Ground-state equilibrium (S0-min) geometries of 9Hguanine and its analogue 8vG. Below each structure is a representation of its electric dipole moment vector. Selected bond lengths are given in units of Å.

the S0-min structure of 9H-guanine is near-planar except for the slightly pyramidalized amino group nitrogen, in agreement with previous studies which reported geometry optimizations of this molecule at the Hartree−Fock,74 MP275 and DFT76 levels of electronic structure theory. The electric dipole moment vector of 9H-guanine is relatively small in magnitude (6.23 D) and roughly parallel to the C4−C5 bond (Figure 1 for atom numbering). The purine moiety of 8vG adopts a geometry very similar to that of 9H-guanine, which suggests that vinyl substitution at position C8 does not significantly influence its electronic structure in the ground state. The differences in corresponding equilibrium bond lengths are largest (but still minor) for the C5−N7, N7−C8, and C8−N9 bonds, and marginally small for the bonds comprising the six-membered ring of either molecule. The vinyl group eclipses nitrogen N7, with an N7−C8−C11−C12 torsion angle of 0.03°. As with the unmodified nucleobase, the electric dipole moment of 8vG is small (5.84 D) and aligned roughly along the C4−C5 bond. 3.2. Structures of Excited Electronic States. The vertical excitation spectra of 9H-guanine and 8vG are characterized in Table 1. We first compare the vertical excitation spectrum of 8vG in the gas phase to that of the unmodified nucleobase and subsequently discuss the effect of aqueous solvation on the calculated vertical excitation energies. In line with previous CASPT277 and high-level coupled-cluster47 (equation of motion excitation energy coupled-cluster including perturbative triple excitations) calculations, we find that the lowest vertical excitation of 9H-guanine is into the ππ* state conventionally labeled the La state. The S1 (La ππ*) state is closely followed by the S2 (nOπ*) state, which involves electron density transfer from the lone electron pair on the oxygen atom onto the conjugated π-bonding system of the molecule. The secondlowest excited state of the ππ* type is the S3 (Lb ππ*) state. D

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Table 1. Vertical Excitation Energies (ΔE), Oscillator Strengths ( f), and Electric Dipole Moment Magnitudes (μ) of 9HGuanine and 8vG at Their Ground-State Equilibrium (S0-min) Geometriesa compound

ΔE, eV

f

μ, D

(La ππ*) (nOπ*) (Lb ππ*) (nOπ*/nNπ*)

5.063 5.339 5.600 6.117

0.190 0.001 0.297