Exploration of the Biological Micro-Surrounding Effect on the Excited

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Exploration of the Biological Micro-Surrounding Effect on the Excited States of the Size-Expanded Fluorescent Base x-Cytosine in DNA Laibin Zhang,† Xiaohua Chen,† Haiying Liu,† Li Han,† Robert I. Cukier,‡ and Yuxiang Bu*,† The Center for Modeling & Simulation Chemistry, Institute of Theoretical Chemistry, Shandong UniVersity, Jinan, 250100, P. R. China, and Department of Chemistry, Michigan State UniVersity, East Lansing, Michigan 48823 ReceiVed: December 12, 2009; ReVised Manuscript ReceiVed: February 4, 2010

We present the results of a detailed and systematic computational investigation into the excited-state properties of the fluorescent cytosine analogue x-cytosine (xC). Also examined were the influences of hydration, linking to deoxyribose, base pairing with guanine (G), and base stacking on its absorption and emission processes. The calculated excitation and emission energies agree well with the experimentally measured data. It was found that hydration, linking to deoxyribose, and base pairing with G have a hyperchromic effect on the excitation maximum of xC. The linking sugar will red-shift the fluorescence emission of xC by 7 nm, while hydration and base pairing with G, on the contrary, results in a blue-shift of the fluorescence emission by 8 and 9 nm, respectively. In addition, hydration of xCG will further blue-shift the fluorescence emission of xC by about 17 nm. Furthermore, the fluorescence quantum yield of xC would be increased after hydration, linking to deoxyribose, and base pairing with G. When sandwiched by two identical natural bases, a significant decrease of the oscillator strength as well as a red-shift of the dipole-allowed transition with respect to free xC is observed in all cases. The fluorescence quantum yield of xC was expected to be lowered in the stacked complexes due to a static quenching mechanism. 1. Introduction The design and synthesis of modified versions of DNA is an area that is receiving increasing attention during the past several decades.1 The reasons for this are numerous, and the modification strategies span from the backbone modification2-4 to the base modification.5-16 To date, much attention has been paid to the base modification strategy, primarily motivated by the fact that novel base analogues have potential applications in biotechnology and medicinal chemistry, as well as in material science. An important target in this field is looking for fluorescent base analogues that can be incorporated into oligonucleotides (ODNs) as quasi-intrinsic probes for the investigation of structures and dynamics as well as the kinetics of interactions between DNA/RNA and other molecules. Nowadays, nucleotides possessing various fluorophores have been explored, some even commercially, for incorporation into ODNs for biophysical and biochemical studies.17,18 For instance, the nucleobase 2-aminopurine has been used as a site-specific probe of nucleic acid structure and dynamics5-8 due to the environmental specificity of its quantum yield. The cytosine analogue, pyrrolocytosine, whose fluorescence yield is sensitive to the local structure of the biomolecule when incorporated into a nucleic acid, is used as a fluorescence probe in experimental studies of nucleic acid dynamics.9 Coleman and co-workers reported the synthesis of C-linked coumarin deoxyriboside, which can serve as a fluorescent probe of DNA dynamics.10 Similarly, Saito and co-workers have produced a number of nucleobases with attractive fluorescent properties by fusing benzo- and naphtho-ring systems to hydrogen bonding aromatic * To whom the correspondence should be addressed. E-mail:byx@ sdu.edu.cn. † Shandong University. ‡ Michigan State University.

rings.11 Clearly, these investigations have provided some useful information for creation and design of the unnatural nucleobases. During the past few years, Kool and co-workers have synthesized a range of size-expanded nucleobases,13-15 primarily to see how these modifications affect the Watson-Crick pairing but ultimately to expand the genetic alphabet. These sizeexpanded base analogues were found to be able to form stable DNA-like structures and to have smaller energy gaps between the highest occupied molecular orbitals (HOMOs) and the lowest unoccupied molecular orbitals (LUMOs) than the natural ones, suggesting that they could enlarge the genetic alphabet and also have potential application in biotechnologies.19,20 We are interested in these newly designed bases because they are fluorescent, a property making them candidates as basic probes of biological mechanisms and interactions and as biotechnological reporters, such as for a specific DNA sequence. To apply the fluorescence of these size-expanded base analogues in a directed and useful manner, it is necessary to obtain the excitedstate properties of the size-expanded base analogues and the influences of the environments. Ab initio quantum chemistry stands to play an important role in unraveling the complicated electronic structure of ODNs, although, for systems containing multiple nucleotides, ab initio calculations are primarily limited to time-dependent density functional theory (TD-DFT). Recently, a theoretical study of the absorption spectra of x-bases was made by Varsano et al.21 and the photophysical characters of the y-bases and yy-bases were studied by the current group.22 In this Article, we focus our attention on one of the sizeexpanded base analogues, x-cytosine (xC), because it is the most fluorescent one in the x-bases, for which the fluorescence quantum yield was reported to be 0.52.13d Most importantly, it was demonstrated that a DNA polymerase is able to read the chemical information stored in xC and that the full replication machinery of E. coli is able to recognize the sequence encoded by xC correctly and efficiently,23 which means that it is a more

10.1021/jp9117503  2010 American Chemical Society Published on Web 02/22/2010

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TABLE 1: Vertical Transition Energies (E), Absorption Wavelengths (λ), Oscillator Strengths, and State Assignments for xC Predicted at the TD-B3LYP/6-311++G(d,p) Level states

E/eV

λ/nm

f

state assignmenta

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10

3.85 4.35 4.67 4.73 5.06 5.14 5.37 5.47 5.57 5.60

322 285 265 262 245 241 231 227 223 222

0.073 0.001 0.004 0.020 0.012 0.082 0.002 0.001 0.080 0.064

πHπ*L(87%) nH-1π*L(67%) - πH-2π*L(23%) nH-3π*L(73%) - πH-2π*L(19%) πH-2π*L(37%) + nH-1π*L(21%) + πHπ*L+1(20%) + nH-3π*L(14%) πHσ*L+2(77%) + πHπ*L+1(15%) πH-4π*L(38%) - πHπ*L+1(24%) + πHσ*L+2(16%) πHσ*L+3(83%) - πHπ*L+4(13%) nH-1π*L(68%) - πH-2π*L(14%) πHπ*L+5(30%) + πH-4π*L(21%) + πH-2π*L(20%) πHσ*L+4(73%) + πHσ*L+3(12%)

a

The transitions are described in terms of the nature of the initial and final states and major contributing levels to these states, and contributions larger than 10% are given.

promising candidate for direct usefulness. It is well-known that the photophysical properties of nucleic acids are very complicated and influenced by a variety of factors. An in-depth understanding of the excited-state properties and effects of perturbations induced by the biologic environment, the water solvent, linking to deoxyribose, base pairing, and base stacking is helpful in finding ways for its direct usefulness of its fluorescence. Furthermore, it may help in the design and synthesis of new fluorescent base analogues. Therefore, in this Article, we present here a detailed and systemic computational study on the properties of xC with the aim of gaining more insight into the properties of the excited states by investigating the dependence of absorption and emission spectra on the biologic environments mentioned above. We hope our theoretical predictions are helpful in understanding the photophyscical properties of xDNA ODNs and explaining experimental data in the future. 2. Computational Details All electronic structure calculations were performed with the help of the Gaussian 0324 suite of programs and a locally developed version of it. The 6-311++G(d, p) basis set was used in all calculations until otherwise specified. The ground-state geometries were optimized using both the HF and DFT/B3LYP25,26 methods, while the corresponding geometries of them in the first singlet ππ* state (lowest-energy) were determined employing the CIS27 method without symmetry restraints imposed. These states were confirmed to be potential energy minima by frequency calculations at their respective optimized geometries, as no imaginary frequencies were found. The ground-state geometries of the stacked configurations 5′R-xC-R3′ (R ) A, G, C, and T, respectively) were constructed from the average structures of two segments of B-form xDNA duplexes generated by molecular dynamics simulations using AMBER 8.0.28 Simulation details were given in the Supporting Information. On the basis of the average structures obtained from simulations, the nucleobases were replaced with those obtained from optimization at the B3LYP/6-311++G(d,p) level with Cs symmetry imposed and the phosphates and sugar units were removed and all bases terminated with hydrogens. Vertical transition energies to the low-lying singlet states were computed using the TD-DFT method. The UV-vis spectra were calculated with the help of the SWizard program, revision 4.5,29 using the Gaussian model. 3. Results and Discussion 3.1. Photophysical Properties of Isolated xC and Effects of Water Hydration. The vertical transition energies, absorption wavelengths, oscillator strengths, and state assignments to

Figure 1. The optimized structures of xC, the xCG base pair, and their hydrated complexes (xC-(H2O)4 and (xCG)-(H2O)6). The scheme numbering of the atoms is given for discussion.

the 10 lowest singlet transitions calculated at the TD-B3LYP/ 6-311++G(d,p) level were given in Table 1, while Figures 2 and 3 depict the molecular orbitals involved in the six lowest transitions and the absorption spectra, respectively. As shown in Figure 3, our calculations reveal that there are two absorption bands in the ultraviolet region 200-400 nm with the peaks located at 322 and 220 nm, respectively. It should be mentioned that the absorption bands in Figure 3 are prevalent due to the ππ* transitions (bright states), since the nπ* states and the Rydberg-like states (dark states) have very small oscillator strengths and therefore cannot be detected experimentally. A close analysis of the 322 nm transition shows that it is mainly attributed to the promotion of one electron from the HOMO to the LUMO (87%). The topologies of these frontier orbitals are sketched in Figure 2, which reveals that the first electronic transition was ππ* in nature with both orbitals delocalized over the entire molecule. This is different from yC, for which the lowest electronic singlet transition was calculated to be an nOπ* (where nO denotes the O-centered lone pair orbital) state and mainly dominated by the configuration H-1 f L at the TDB3LYP/6-31+G(d,p) level.22a Compared with the calculated value 3.35 eV by Varsano et al.,21 the reported transition energy (3.85 eV, 322 nm) of the S1 state here is in good agreement

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Figure 2. Molecular orbitals involved in the six lowest electronic singlet transitions calculated at the TD-B3LYP/6-311++G(d,p) level. The arrows indicate the main contributions of the six lowest electronic singlet transitions. Orbital energies are given in parentheses.

Figure 3. Absorption spectra of xC, xC-(H2O)4, and dxC in the ultraviolet region 200-400 nm determined at the TD-B3LYP/6311++G(d,p) level. The half-bandwidths were taken to be equal to 3000 cm-1.

with the experimental absorption maximum (330 nm) of free dxC nucleoside in methanol.13d Furthermore, the excitation maximum of xC is considerably red-shifted compared with those of natural nucleobases, allowing it to be selectively excited in the presence of the other bases. Following the excitation maximum, three strong transitions (ππ*) near 4.73 (S4), 5.14 (S6), and 5.57 (S9) eV are located with the third one having the strongest intensity (see Table 1). Experimentally, it was reported that there is a more intense absorption band below 250 nm for dxC in methanol, though the precise position of the this band is not given.13d On the basis of our calculations, this strong absorption band observed experimentally must be related to the strongest absorption band (220 nm) of xC in the gas phase shown in Figure 3. As shown in Table 1 and Figure 2, there are three nπ* transitions among the 10 lowest transitions in the gas phase with the first (S2) and third (S8) ones mainly localized at the carbonyl group (termed as nOπ*) and the second one (S3)

Zhang et al. mainly localized at the N3 atom (termed as nNπ*). All nπ* states have relatively small oscillator strengths less than 0.005, and thus, direct transitions to these states are difficult to characterize experimentally, especially when they overlap with strong ππ* transitions. The first πσ* state (S5) is mainly dominated by the amino and C1bH1b groups, and it is 1.21 eV above the lowest ππ* state. It should be noted that the πσ* type states play a pivotal role in the photochemistry of aromatic molecules (including DNA bases) because the πσ* states have repulsive potential energy functions, which can intersect not only the potential energy functions of the ππ* and nπ* excited states but also the ground state.30 A direct comparison with experiment is not possible because experimental data about the nπ* and πσ* states are not available at the moment because of the inaccessibility of these two kinds of transitions. Since DNA bases are expected to be planar in the helical environments and our calculations do predict that the xCG base pair has a planar ground-state geometry, the nature of the lowlying excited states of xC with Cs symmetry was also calculated and the corresponding data were compiled in Table S1 in the Supporting Information. A comparison between the results of xC in the C1 and Cs symmetries reveals that the geometric changes have little effect on the absorption spectrum of xC: the state order stays still, and the vertical transition energies are nearly unchanged (with the changes within 0.03 eV). This conclusion can also be drawn by examining Figure 3, from which one can find that the spectra of xC in both the C1 and Cs symmetries are nearly superposed. Water plays an important role in the structure and function of nucleic acids. It has been found that water molecules can increase the stacking interaction in base pairs31 and the stability of some minor tautomers of the DNA bases in the ground state.32 Furthermore, it was found that the degree of hydration has a significant influence on the excited-state structural nonplanarity of guanine.33 Therefore, the hydration effects on the photophysical properties of xC were also considered. Four water molecules were used for the hydration of xC, and the hydrated complex was termed as xC-(H2O)4 (Figure 1). On the basis of the B3LYP/6-311++G(d,p) optimized ground-state geometry, the vertical transition energies of xC-(H2O)4 were calculated at the TD-B3LYP/6-311++G(d,p) level, and the absorption spectrum was shown in Figure 3, from which one can find that the excitation maximum was blue-shifted by about 12 nm. This blue-shift can be simply explained by comparing the Kohn-Sham energy difference between the molecular orbitals that define this transition. As shown in Figure 4a, hydration decreased the energies of both the HOMO and LUMO with the HOMO being decreased more than the LUMO; therefore, the excitation maximum of xC is blue-shifted after hydration. In contrast, the strongest absorption band was slightly red-shifted by about 3 nm. Furthermore, the intensities of both the excitation maximum and the strongest absorption band are increased with the strongest absorption band being increased to a greater extent (Figure 3). That is to say, hydration has a hyperchromic effect on the absorption of xC. In order to investigate the fluorescence emission, the geometry of the first excited singlet state was optimized at the CIS/6311++G(d,p) level and the optimized structure is shown in Figure S3 of the Supporting Information. The fully optimized geometry, characterized by all positive frequencies, is nonplanar, and the nonplanarity is mainly caused by the pyramidalization of the amino group. The difference between 360° and the summation of bond angles at an atom is a measure of the pyramidalization of that atom (termed as pyramidalization

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Figure 4. Molecular orbitals involved in the lowest transitions of the studied complexes: (a) the results were calculated using the TD-B3LYP method; (b) the results were calculated using the TD-CAM-B3LYP method. The orbital energies, excitation energies, oscillator strengths, and configuration weights are also given.

degree). Calculations reveal that the amino group pyramidalization degree of xC in the S1 state is 12.8°. Therefore, compared with the value 7.7° in the S0 state, the amino group pyramidalization degree is increased. As found earlier for other bases,22,33 the inversion of single and double bonds is also observed in the S1 geometry of xC. It was found that the N1-C2, N3-C4, C5-C6, C1b-C2b, and C3b-C4b bonds are elongated, while the C2-N3, C4-C5, and C6-N1 bonds are shortened. The most significant change is a large elongation of the C5-C6 bond by about 0.076 Å. Similarly, in the S1 state of the xC-(H2O)4 cluster, the biggest change in bond lengths of the xC component is observed for C5-C6, which was elongated by about 0.072 Å after electronic relaxation. Furthermore, it is interesting to note that hydration makes the S1 geometry more planar, since the amino group pyramidalization degree of the xC component in the S1 state of xC-(H2O)4 is calculated only to be 0.5°. On the basis of the optimized S1 state geometry, the S1 f S0 transition energy corresponding to the fluorescence emission was calculated using the TD-B3LYP method. The fluorescence of xC would be expected to occur around 359 nm, very close to the reported value (388 nm) for free dxC nucleoside in methanol solution.13d Recently, Mercha´n and Serrano-Andre´s studied the nonradiative deactivation pathways of excited C and concluded that the lowest ππ* excited state is most likely to decay directly to the ground state through a S1/S0 conical intersection without a nπ* intermediate state.34 As xC is more fluorescence-active than natural C, it can be concluded that, if there is a similar conical intersection that links the ππ* state of xC to the ground state, the barrier between the (ππ*)min and the conical intersection must be considerably larger than the 2.5 kcal/mol recommended by Mercha´n and Serrano-Andre´s for C.34 Hydration was found to blue-shift the fluorescence of xC by 8 nm, since the emission of the xC-(H2O)4 was predicted to be around 351 nm at the TDB3LYP/6-311++G(d,p) level. Meanwhile, the corresponding intensity will be enhanced, since the oscillator strength is increased by 13% after water hydration (see Table 5). 3.2. Effects of Binding to Deoxyribose and Base Pairing with Guanine. In this section, the influences of the linking sugar and base pairing with guanine on the photophysical properties

TABLE 2: Vertical Transition Energies (E), Absorption Wavelengths (λ), Oscillator Strengths, and State Assignments for dxC Predicted at the TD-B3LYP/6-311++G(d,p) Level state

E/eV

λ/nm

f

assignment

Exp.a (nm)

S1 S2 S3 S4 S5 S6

3.84 4.42 4.72 4.75 4.99 5.05

323 281 263 261 249 246

0.099 0.001 0.027 0.004 0.000 0.013

ππ* nπ* ππ* nπ* πσ* ππ*

330

a

See ref 13d.

of xC are investigated. It should be noted that, in natural DNA/ RNA, the deoxyribose is attached to the N1 position of pyrimidines, while, in xDNA, the deoxyribose is attached to the C4b position of xC.13d Vertical transition energies, oscillator strengths, and state assignments to the low-lying excited singlet states for dxC calculated at the TD-B3LYP/6-311++G(d,p) level are given in Table 2, and the absorption spectrum is shown in Figure 3. The first transition is mainly dominated by the configuration H f L (87%), and the orbitals involved are shown in Figure 4a. As depicted by Figure 4a, the orbitals involved in the lowest transition of dxC are mainly localized on the xC zone and are in fact identical to those involved in the S1 transition of isolated xC. Furthermore, though binding to deoxyribose increases the energy levels of both the HOMO and LUMO, as shown in Figure 4a, the H-L gap is nearly unchanged. Therefore, the excitation energy is hardly changed, but the intensity is increased, since the oscillator strength is increased by 36% after linking to deoxyribose. It was reported that, for natural C, the oscillator strength of the first transition is increased by 50% after binding to deoxyribose.35 Therefore, the extent of oscillator strength increasing when linking to deoxyribose is decreased after fusion of a benzene ring into the C molecule. The strongest absorption band is red-shifted by 5 nm. Overall, linking to deoxyribose has a hyperchromic effect in the low energy region and a hypochromic effect in the high energy region on the absorption of xC (Figure 3). In order to investigate the influence of the linking sugar on the fluorescence of xC, the first singlet

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TABLE 3: Vertical Transition Energies (E), Oscillator Strengths (f), and Assignments to the Lowest Singlet Transition and Fluorescence Emission of the xCG Base Pair Predicted at the TD-CAM-B3LYP/6-311++G(d,p) Level isolated xCa

xCG base pair TD-CAM-B3LYP first transition fluorescenceb

E/eV

f

assignment

E/eV

f

assignment

4.28 3.79

0.113 0.122

ππ* on xC ππ* on xC

4.17 (4.16) 3.70 (3.70)

0.088 (0.082) 0.100 (0.096)

ππ* ππ*

a The transition energies for the individual xC in the complex geometry are given in parentheses. b The S1 geometry of xCG was optimized at the CIS/6-311G(d,p) level.

TABLE 4: Vertical Transition Energies (E), Oscillator Strengths (f), and Assignments to the Lowest Singlet Transition and Fluorescence Emission of the (xCG)-(H2O)6 Cluster Predicted at the TD-CAM-B3LYP/6-311++G(d,p) Level isolated xCa

(xCG)-(H2O)6 TD-CAM-B3LYP first transition fluorescenceb

E/eV

f

assignment

E/eV

f

assignment

4.35 3.89

0.112 0.120

ππ* on xC ππ* on xC

4.17 (4.09) 3.70 (3.70)

0.088 (0.080) 0.100 (0.092)

ππ* ππ*

a The transition energies for the xC component in the (xCG)-(H2O)6 cluster are given parentheses. b The S1 geometry of (xCG)-(H2O)6 was optimized at the CIS/6-311G(d,p) level.

excited state of dxC was fully optimized at the CIS/6311++G(d,p) level. As the first transition is in fact a localized one, the geometry changes are similar to those found in the S1 geometry of isolated xC. On the basis of the S1 geometry, the fluorescence emission was calculated using the TD-B3LYP method. The fluorescence maximum of dxC is expected to occur around 366 nm, and the associated oscillator strength is 0.114. Thus, it can be concluded that linking to deoxyribose will result in a red-shift of the fluorescence emission by 7 nm and an increase of the corresponding oscillator strength by 34% (see Table 5). Next, we turn to analyze the effect of the WC hydrogen bonding on the photophysical properties of xC. It is well-known that, although the TD-B3LYP method can be used to accurately predict energies and transition dipole moments for valence states, it grossly underpredicts the energies of charge-transfer states.36 Therefore, the vertical transition energies of the low-lying excited singlet states for the xCG base pair were calculated using the TD-DFT method with the CAM-B3LYP37 functional proposed by Yanai et al. recently to overcome the limitations. The CAM-B3LYP functional has been demonstrated to provide good results for intermolecular charge-transfer transitions.37-39 The TD-CAM-B3LYP calculations in this paper were performed with the help of a locally developed version of Gaussian 0324 programs into which the CAM-B3LYP has been coded. In order to make comparisons with the xCG base pair, the vertical excitation energies of isolated xC were recalculated with the TD-CAM-B3LYP method and the results are listed in Table 3. An examination of Table 3 reveals that the excitation energy of the lowest transition of isolated xC calculated by the TDCAM-B3LYP method is larger than that obtained by the TDB3LYP method, in agreement with those found for natural bases.39 For xCG, the TD-CAM-B3LYP method reveals that the first transition is essentially identical to the first transition of xC alone and the orbitals involved are the same orbitals as those involved in the first transition of isolated xC (Figure 4b). Compared with isolated xC, the transition energy of this transition is blue-shifted (0.11 eV) and the oscillator strength is increased by 28%. This is different from that of yC, for which base pairing with G was found to have little effect on the lowest ππ* transition.22a The blue-shift observed here can be simply explained by comparing the Kohn-Sham energy difference between the xC-localized electronic level that defines the ππ*

transition. As shown in Figure 4b, the energy difference between the two orbitals involved is 7.10 eV for xCG, which is 0.13 eV larger than the HOMO-LUMO gap of isolated xC. Note that the orbitals involved in the lowest transition are identical to those involved in the first transition of isolated xC. The shift essentially accounts for the energy blue-shift found in the excitation energy. Thus, it can be concluded that hydrogen bonding to G will result in a blue-shift on the absorption maximum of xC and an increase of the corresponding oscillator strength (hyperchromic effect). Hydration effects on the xCG base pair were also considered. As shown in Figure 1, six water molecules were used to hydrate the xCG base pair (termed as (xCG)-(H2O)6) and the water binding mode was according to that observed for the natural GC base pair through molecular dynamic simulations.40 Because the (xCG)-(H2O)6 cluster is quite large, the 6-31+G(d,p) basis set was used to optimize the ground-state geometry and the 6-311++G(d,p) basis set was used to calculate the transition energies. Compared with the nonhydrated planar xCG, the ground state of (xCG)-(H2O)6 optimized at the B3LYP/631+G(d,p) level is highly nonplanar, similar to that observed for the hydrated natural GC base pair.41,42 On the basis of the ground-state geometry, the vertical transition energies to the low-lying transitions are calculated using the TD-CAM-B3LYP method and the results are compiled in Table 4. The first excited state S0 f S1 transition is predicted to be ππ* in nature, and the orbitals involved resemble those involved in the first transition of isolated xC (Figure 4b). As shown in Table 4, the vertical transition energy to the lowest ππ* transition is further blue-shifted after hydration. In detail, the transition energy of the lowest ππ* state is blue-shifted to 4.35 eV at the TD-CAMB3LYP/6-311++G(d,p) level, which is 0.18 eV larger than that of isolated xC. To analyze the effect of the WC hydrogen bonding on the fluorescence of xC, the S1 (ππ*) states the xCG and (xCG)-(H2O)6 were fully optimized at the CIS/6-311G(d,p) level and the optimized structures are shown in Figure S3 of the Supporting Information. The fully optimized structures, characterized by all positive frequencies, are not planar. Since the first transitions are in fact local transitions, the geometry changes in the S1 structures are mainly on the xC component. On the basis of the S1 geometries, the fluorescence emission was calculated using the TD-CAM-B3LYP method. For xCG,

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TABLE 5: Vertical Transition Energies (E, nm) and Oscillator Strengths (f) to the Lowest Transitions and Fluorescence Emissions of the Studied Complexesa isolated xC xC-(H2O)4 S1 (E/f)

dxC

xCG

322/0.073 310/0.083 323/0.099 290/0.113 (297/0.088)b fluorescence 359/0.085 351/0.096 366/0.114 327/0.122 (E/f) (335/0.100)b

(xCG)-(H2O)6 284/0.112 319/0.120

a For isolated xC, xC-(H2O)4, and dxC, the values were calculated at the TD-B3LYP/6-311++G(d,p) level, while those for xCG and (xCG)-(H2O)6 were calculated at the TD-CAM-B3LYP/ 6-311++G(d,p) level. b The corresponding results calculated at the TD-CAM-B3LYP/6-311++G(d,p) level were given in parentheses.

the calculations reveal that the fluorescence maximum is blueshifted and the intensity is increased compared with isolated xC. In detail, the TD-CAM-B3LYP method reveals that the fluorescence emission was blue-shifted by 0.09 eV (9 nm), and the corresponding oscillator strength was increased by 22%. These results are different from those reported previously for another cytosine analogue, pyrrolocytosine,43 whose fluorescence emission was red-shifted and the associated oscillator strength was nearly unchanged after pairing with G. In the case of the (xCG)-(H2O)6 cluster, the fluorescence is calculated to be 0.19 eV (17 nm) blue-shifted compared with that of isolated xC at the TD-CAM-B3LYP/6-311++G(d,p) level. Furthermore, the correlated oscillator strength is 20% larger than that of free xC. Table 5 summarizes the transition energies and the corresponding oscillator strengths to the lowest transitions and the fluorescence processes of isolated xC, xC-(H2O)4, dxC, xCG, and (xCG)-(H2O)6, from which one can find that the oscillator strengths associated with fluorescence of xC-(H2O)4, dxC, xCG, and (xCG)-(H2O)6 were larger than that of isolated xC, ranging from 13 to 34%. As known, the rate constant (kF) for the fluorescent process is proportional to the oscillator strength by eqs 1 and 2, and the fluorescence quantum yield is defined by eq 3 (see next section). Therefore, the relations imply that the increase in the oscillator strength (by 13-34%) corresponding to the fluorescent transition of xC induced by water hydration, linking to deoxyribose, and base pairing with G may result in an enhancement in the fluorescence quantum yield. 3.3. Effects of Base Stacking. In this section, the effects of base stacking on the photophysical properties of xC will be considered. Here, we mainly consider the effects of two natural bases sandwiching xC. The studied stacked 5′R-xC-R3′ (R ) A, G, C, and T, respectively) complexes were shown in Figure 5. Obviously, it would be better to consider the influences of the bases in the opposite strand. However, here we only consider the stacking effects of the neighboring bases in the same strand due to the computational restrictions and this strategy has been successfully used to investigate the stacking effects of some other base analogues.43-46 Since there is little experimental information on the geometries of xDNA duplexes, two fragments of B-form xDNA duplexes were designed and simulated by molecular dynamics (simulation details were given in the Supporting Information). On the basis of the average structures obtained from simulation, the nucleobases were replaced with those obtained from optimization at the B3LYP/6-311++G(d,p) level with Cs symmetry imposed and the phosphates and sugar units were removed and all bases terminated with hydrogens. 5′ A-xC-A3′ Trimer. The calculated excitation wavelength and oscillator strength to the lowest singlet transition of the 5′AxC-A3′ trimer are given in Figure 6. The MOs that participate

Figure 5. Stacked complexes studied in this paper. These configurations were constructed from the average structures of two segments of B-form xDNA duplexes by replacing the nucleobases with those obtained from optimization at the B3LYP/6-311++G(d,p) level with Cs symmetry imposed, and the phosphates and sugar units were removed and all bases terminated with hydrogens.

Figure 6. Molecular orbitals involved in the lowest transitions of the 5′ A-xC-A3′ and 5′G-xC-G3′ trimers. The orbital energies, excitation energies, oscillator strengths, and the configuration weights calculated at the TD-CAM-B3LYP/6-311++G(d,p) level are also given.

in the electronic transition in this trimer are HOMO-1, HOMO, and LUMO. In the HOMO-1, the one-electronic density is delocalized over the three bases, while that in the HOMO is delocalized on the 5′xC-A3′ pair. On the contrary, the oneelectronic density in the LUMO is mostly localized on the xC component. The spreading of molecular orbitals across more than one nucleobase in base-stacked structures is well-known; for instance, when two guanine moieties are adjacent in a basestacked environment, the HOMO has a component on both bases and is lower in energy than the HOMO of a free guanine.47

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The electronic structure of the 5′A-xC-A3′ trimer (shown in Figure 6) can be seen as a composite of the 5′A-xC3′ and 5′xCA3′ dimers, in that its lowest transition resembles dimer transitions. The HOMO-1 and LUMO resemble the HOMO and LUMO of the 5′A-xC3′ dimer, respectively (see Figure S4 in the Supporting Information). As depicted by Figure 6, the S0 f S1 transition involves two contributions: HOMO-1 f LUMO (55%) and HOMO f LUMO (36%). Thus, this electronic transition has partial charge-transfer character. The excitation energy and oscillator strength are calculated to be 4.06 eV and 0.044, respectively, which indicates that it is an xC-like transition. The low oscillator strength represents only 50% of that associated with free xC and probably results from the strong mixing of the MOs in the ground state. Because of the strong π-orbital overlap in the ground state, the fluorescence quantum yield of xC would be lowered by a static quenching mechanism with a reduction of the radiative rate constant in the 5′A-xC-A3′ trimer. 5′ G-xC-G3′ Trimer. Figure 6 also shows the excitation wavelength and oscillator strength to the lowest singlet transition of 5′G-xC-G3′ trimer. The MOs that participate in the electronic transition in this trimer are HOMO-2, HOMO-1, HOMO, and LUMO. The one-electronic density in the HOMO and HOMO-1 MOs is delocalized over the three bases, while that in the HOMO-2 is delocalized on the 5′G-xC3′ pair. On the contrary, the one-electronic density in the LUMO is localized primarily on the xC component. HOMO-2, HOMO-1, and LUMO resemble HOMO-1, HOMO, and LUMO of the 5′G-xC3′ dimer, respectively, while the HOMO resembles the HOMO of the 5′ xC-G3′ dimer (see Figure S5 in the Supporting Information). Therefore, it seems that the upper G (5-site) has stronger influences on the photophysics of xC while the lower G (3site) has relatively small influences. As depicted by Figure 6, the S0 f S1 transition is ππ* in nature and is an xC-like transition, which involves three contributions: HOMO-1 f LUMO (43%), HOMO f LUMO (28%), and HOMO-2 f LUMO (23%). Thus, this electronic transition also has partial charge-transfer character. The excitation energy was calculated to be 4.12 eV with an associated oscillator strength of 0.031, which is only 35% of that associated with free xC, indicating that there is a larger hypochromic effect for the xC-like transition in this trimer. Therefore, the fluorescence quantum yield of xC would be lowered by a static quenching mechanism with a reduction of the radiative rate constant in the 5′G-xC-G3′ trimer due to the strong π-orbital overlap in the ground state. 5′ C-xC-C3′ Trimer. The calculated excitation wavelength and oscillator strength to the lowest singlet transition of the 5′CxC-C3′ trimer are given in Figure 7. The MOs that participate in the lowest electronic transition in this trimer are HOMO-1, HOMO, and LUMO. The one-electronic density in the highest occupied MOs is mainly delocalized over the 5′xC-C3′ pair. On the contrary, in the LUMO, the one-electronic density is mostly localized on the xC component and it resembles that found for the 5′xC-C3′ dimer (see Figure S6 in the Supporting Information). The S0 f S1 transition, which is an xC-like transition on the basis of transition energy (4.12 eV) and oscillator strength (0.055), involves two contributions: HOMO f LUMO (78%) and HOMO-1 f LUMO (14%). Therefore, this electronic transition also has partial charge-transfer character. The calculated oscillator strength represents only 63% of that associated with free xC, and probably results from the strong mixing of the MOs in the ground state. Because of the strong π-orbital overlap in the ground state, the fluorescence quantum yield of

Zhang et al.

Figure 7. Molecular orbitals involved in the lowest transitions of the 5′ C-xC-C3′ and 5′T-xC-T3′ trimers. The orbital energies, excitation energies, oscillator strengths, and the configuration weights calculated at the TD-CAM-B3LYP/6-311++G(d,p) level are also given.

xC would be lowered by a static quenching mechanism with a reduction of the radiative rate constant in the 5′C-xC-C3′ trimer. 5′ T-xC-T3′ Trimer. The calculated excitation wavelength and oscillator strength to the lowest singlet transition of the 5′T-xCT3′ trimer are also shown in Figure 7. The MOs that participate in the lowest electronic transition in this trimer are the HOMO and LUMO. The one-electronic density in both the HOMO and the LUMO is mainly localized on the xC. The HOMO resembles that found for the 5′xC-T3′ dimer, and the LUMO can be seen as the composition of the LUMOs of the 5′T-xC3′ and 5′xC-T3′ dimers (see Figure S7 in the Supporting Information). The S0 f S1 transition is dominated by the HOMO f LUMO (89%) configuration and is an xC-like transition. Because the oneelectronic density has a small distribution on the cytosine components in both the HOMO and LUMO, this electronic transition also has partial charge-transfer character. The excitation energy of this state was calculated to be 4.17 eV. As was seen with the other trimers, the stacking interaction leads to a reduction (51%) of the oscillator strength due to the mixing of the MOs in the ground state, which in turn will lead to a reduction of the fluorescence quantum yield of xC by a static quenching mechanism with a reduction of the radiative rate constant. As discussed above, the first transitions are of ππ* character and are the xC-like transitions in all of the studied stacked complexes. The excitation energies are slightly red-shifted (by 0.0-0.11 eV) depending upon the stacking configurations. However, although π and π* orbitals of xC are always involved, orbitals with coefficients on more than one base are frequently involved. The change in orbitals that are involved is associated with a large change in the oscillator strengths for the first transitions. As shown in Figures 6 and 7, the oscillator strengths are decreased by 37 to 65% compared with that of isolated xC and probably results from the mixing of the MOs in the ground state. This hypochromicity is a typical effect of base stacking.48 In summary, a significant decrease of the oscillator strength as well as a red-shift of the dipole-allowed transition with respect to free xC is observed in all of the studied stacked complexes. To analyze the effect of base stacking on the fluorescence of xC, the S1 states of the stacking configurations should be

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optimized, but this is precluded with the current implementations of the TD-DFT and CIS methods. However, on the basis of the discussions above and the results reported for other base analogues,44-46 the oscillator strength of the fluorescent transition would be greatly reduced as occurred in the absorption process in the stacking complexes due to the strong π-orbital overlap. Because the oscillator strength (f) is directly related to the rate constant (KF) of the fluorescence process by eqs 1 and 2:49,50

D)

3he2 f 8π2me ν

(1)

where D is the square of the transition dipole moment, e is the charge on an electron, and me is the mass of an electron. The rate constant (KF) is defined as

Acknowledgment. This work was supported by NSFC (20633060, 20573063, and 20973101), NCET, and Virt Lab for Comput Chem & SCC of CNIC-CAS, MCBILIN at MSU, and HPCC at SDU. Supporting Information Available: Molecular dynamic simulation details; a table of vertical transition energies, absorption wavelengths, oscillator strengths, and state assignments to the low-lying singlet transitions for xC with Cs symmetry; S1 structures of xC, xCG, and their hydrated complexes; and figures containing molecular orbitals, excitation energies, oscillator strengths, and main contributions to the lowest singlet transitions of stacked 5′R-xC3′ and 5′xC-R3′ (R ) A, G, C, and T, respectively) dimers. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes

KF )

-3 -1

1 64π n〈ν 〉 D 4πε0 3hc3 4

(2)

where ε0 is the permittivity of free space, n is the refractive index of the medium, ν is the frequency of the fluorescence transition, h is Plank’s constant, and c is the speed of light in a vacuum. Moreover, the fluorescence quantum yield is defined as

φF )

kF kF + kNR

(3)

where kNR stands for the rate constant of the relevant nonradiative processes. Clearly, if kNR is unchanged, the quantum yield φF is determined by kF. Therefore, this relation implies that reduction of oscillator strength corresponding to the fluorescent transition of xC by base stacking with natural bases may result in a reduction in its fluorescence quantum yield (a static quenching mechanism). 4. Conclusions In this paper, theoretical calculations have been performed to characterize the excited-state properties of a fluorescent sizeexpanded base analogue x-cytosine (xC). The influences of the environments including hydration, linking to deoxyribose, base pairing with G, and base stacking were also considered. Some interesting phenomena and characters have been observed. For isolated xC, the nature of the low-lying singlet transitions are discussed and the calculated electronic absorption peaks and emission maximum are in good agreement with reported experimental values. Water hydration and hydrogen bonding to G are demonstrated to have a hyperchromic effect on the excitation maximum of xC. It was found that hydration blueshifts the excitation maximum and fluorescence by 12 and 8 nm, respectively. Similarly, base pairing with G blue-shifts the fluorescence of xC by 0.09 eV (9 nm), and hydration of xCG blue-shifts the fluorescence of xC further (by 0.19 eV, 17 nm). Furthermore, the fluorescent quantum yield would be increased after hydration, linking to deoxyribose, and base pairing with G. In the case of the stacked configurations, a significant decrease of the oscillator strength as well as a red-shift of the dipole-allowed transition with respect to free xC is observed in all cases. Because of the strong mixing of the molecular orbitals, the fluorescence quantum yield of xC is expected to be lowered in the stacked complexes due to a static quenching mechanism.

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