Excited State Electronic Structures of 5,10-Methenyltetrahydrofolate

Article pubs.acs.org/JPCA

Excited State Electronic Structures of 5,10-Methenyltetrahydrofolate and 5,10-Methylenetetrahydrofolate Determined by Stark Spectroscopy Raymond F. Pauszek, III, Goutham Kodali,‡ and Robert J. Stanley* Department of Chemistry, Temple University, Philadelphia, Pennsylvania 19122, United States S Supporting Information *

ABSTRACT: Folates are ubiquitous cofactors that participate in a wide variety of critical biological processes. 5,10-Methenyltetrahydrofolate and its photodegradation product 5,10methylenetetrahydrofolate are both associated with the light-driven DNA repair protein DNA photolyase and its homologues (e.g., cryptochromes). The excited state electronic properties of these folate molecules have been studied here using Stark spectroscopy and complementary quantum calculations. The tetrahydrofolates have relatively large difference dipole moments (ca. 6−8 Debye) and difference polarizabilities (ca. 100 Å3). This extensive excited state charge redistribution appears to be due largely to the pendant p-aminobenzoic acid group, which helps shuttle charge over the entirety of the molecule. Simple calculations based on the experimental difference dipole moments suggest that tetrahydrofolates should have large two photon cross sections sufficient to enable two photon microscopy to selectively detect and follow folate-containing proteins both in vitro and in vivo.

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photolyase (PL).3 This cofactor harvests blue light which is transferred via FRET4,5 to the catalytic cofactor flavin adenine dinucleotide (FAD or FADH•) to generate the two-electron reduced state, FADH−.6−8 The UV−visible absorption spectrum of PL-bound methenylTHF shows a strong absorption band centered at 384 nm. This absorption feature is considerably red-shifted from that in aqueous solution, which has a maximum at approximately 350 nm.8 This large solvatochromic shift is indicative of extensive charge redistribution upon optical excitation. THF consists of a pterin moiety linked with a paraaminobenzoic acid group (PABA) and one or more glutamate residues. In many folate-dependent biological processes reactivity is achieved by modification to N(5) or N(10). The molecules studied in this work, shown in Figure 1, are 5,10methenyltetrahydrofolate (methenylTHF) and 5,10-methylenetetrahydrofolate (methyleneTHF). The latter was thought to be the photodecomposition product of the former based on early experiments on PL that showed that this cofactor could be selectively removed from the protein by irradiation with high intensity ultraviolet light.4,9 It has only recently been shown that the photodegradation product of this process was indeed methyleneTHF, which has a blue-shifted absorption spectrum centered at approximately 300 nm.8 Interestingly, this photoreduction process only occurs in the presence of the conserved tryptophan triad of photolyase.

INTRODUCTION Tetrahydrofolate (THF) and its derivatives are important for a variety of cellular metabolic processes in which folates act as one-carbon group donors or acceptors. Such folate-dependent processes include DNA methylation, purine and pyrimidine synthesis, amino acid synthesis, and remethylation of homocysteine to methionine, among others.1,2 THF is also an important photobiological cofactor. 5,10-methenylTHF (Figure 1, top) serves as the antenna chromophore in many organisms for the light-activated DNA repair enzyme DNA

Special Issue: A. W. Castleman, Jr. Festschrift Figure 1. Structures of 5,10-methenyltetrahydrofolate (top) and 5,10methylenetetrahydrofolate (bottom). The pterin ring numbering is shown in red. © 2014 American Chemical Society

Received: January 31, 2014 Revised: May 5, 2014 Published: May 9, 2014 8320

dx.doi.org/10.1021/jp501143u | J. Phys. Chem. A 2014, 118, 8320−8328

The Journal of Physical Chemistry A

Article

⎧ ε(ν)̃ Bχ d ⎛ ε(ν)̃ ⎞ Δε(ν)̃ = (fc |F |⃗ )2 ⎨A χ + ⎟ ⎜ 15ch dν ̃ ⎝ ν ̃ ⎠ ν̃ ν̃ ⎩

The pterin moiety has also been used as the backbone structure for a number of fluorescent nucleobase analogues (FBAs). The spectral characteristics of these FBAs have been shown to be sensitive to local environment and are, therefore, useful probes of dynamical processes involving nucleic acids.10−12 The tetrahydrofolates discussed here are structurally similar to these FBAs, and the effects of the electron-rich paminobenzoic acid moiety to the pterin ring on excited state electronic charge redistribution may provide useful information for the design of novel pterin-based analogues incorporating electron donating groups. The ubiquity of folates in cellular processes and pathologies,13 and their rich photochemistry and photophysics in solution14−18 or from interaction with a protein matrix19 suggests that a detailed knowledge of the electro-optical properties of these molecules will be useful in understanding both biological and nonphotobiological processes involving these chromophores. The electric-field induced shifts of the absorption features may be a useful probe of local electrostatic environments in which folates are bound, similar to experiments performed on the active sites of several proteins using vibrational Stark spectroscopy.20−23

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+

d2 ⎛ ε(ν)̃ ⎞⎫ ⎜ ⎟⎬ 30c 2h2 dν 2̃ ⎝ ν ̃ ⎠⎭ Cχ

(1)

where |F⃗| is the magnitude of the applied electric field in V/m, h is Planck’s constant, and c is the speed of light; fc is the local field factor due to the enhancement of the applied electric field by the solvent cavity, and was calculated25 to be 1.6 for both methenylTHF and methyleneTHF in ethanol26 and 8 M LiCl.27 The Aχ coefficient, which represents the transition polarizability and higher order terms, is generally negligible for an immobilized sample. The Bχ coefficient is related to the → →

→ →

→ →

difference polarizability tensor (Δ α 0n = α n − α 0) of the molecule, Bχ =

→ 5 → TrΔα0n + (3 cos2 χ − 1) 2 → → ⎛3 1 → → ⎞ × ⎜ m⃗ 0n ·Δα0n·m⃗ 0n − TrΔα0n⎟ ⎝2 ⎠ 2

(2) → →

where m⃗ 0n is the transition dipole moment for S0 → Sn. TrΔα 0n → →

and m⃗ 0n·Δα 0n·m⃗ 0n are the trace of the difference polarizability tensor and the projection of the difference polarizability along the transition dipole moment, respectively. The Cχ term is related to the difference dipole (Δμ⃗0n = μ⃗n − μ⃗0) and ζ0n A the angle between Δμ⃗ 0n and m⃗ 0n,

MATERIALS AND METHODS

Materials and Sample Preparation. Ethanol (EtOH) and LiCl were purchased from Fisher Scientific. MethenylTHF and methyleneTHF were purchased from Schircks Laboratories. All reagents were used as received. The solid folate powder was dissolved in the appropriate solvents with the aid of sonication. EtOH and 8 M LiCl were used as solvents for methenylTHF, while methyleneTHF was not appreciably soluble in organic solvents that are suitable for low temperature spectroscopy. Sample concentrations were in the range of 200−400 μM. Because of the known decomposition of methyleneTHF in acidic or neutral solutions,8 concentrated NaOH was added to the LiCl solution to a final concentration of 100 mM, pH 13. To investigate the effect of pH on the spectra of methyleneTHF, experiments were also carried out with methyleneTHF in 8 M LiCl, without adjusting the pH. These samples were flash frozen within 10 min of preparing the solution to avoid appreciable degradation of the folate. Stark cuvettes were constructed using ITO-coated boroaluminosilicate glass slides (Delta Technologies No. CB-90INS107) and 55 μm kapton spacers. The capacitance of the resulting solution-filled optical capacitor was about 300 pF. Typical field strengths were ∼3 × 105 V/cm. These same cuvettes were used to obtain the absorption spectra. All low temperature spectra presented in this work are corrected for increased concentration relative to room temperature measurements due to solvent contraction upon freezing. Stark Spectroscopy and Data Analysis. The experimental setup of the Stark spectrometer has been discussed in detail previously. Stark spectra were analyzed according to the procedure of Liptay,20,24 in which the Stark spectrum of an isotropic sample is described by a linear combination of the zeroth, first, and second derivatives of the energy weighted extinction coefficient ε(ṽ)/ṽ, weighted by the coefficients Aχ, Bχ, and Cχ:

Cχ = |Δμ0⃗ n |2 {5 + (3 cos2 χ − 1)(3 cos2 ζA0n − 1)}

(3)

The angle χ between the polarization of the incident light and the applied electric field F⃗ is varied by rotating the sample with respect to the incident light. Spectra taken with a minimum of two values of χ are needed in order to solve for the Bχ and Cχ coefficients. The low temperature absorption spectrum was fitted to a sum of Gaussian functions and the Stark spectra, measured at two values of χ, were fitted simultaneously to eq 1 as described in detail previously.28 The uncertainties of the fitted parameters were estimated by a Monte Carlo simulation of 200 iterations in which the initial parameters are varied by 50% from the starting conditions and fit to the data. Parameters and errors are reported as the mean results of this simulation and the standard deviations, respectively. TD-DFT and Finite-Field Calculations. The initial coordinates of methenylTHF were obtained from the crystal structure of E. coli photolyase.29 Folate-pendant glutamate residues were deleted. The geometry was optimized at the DFT/B3LYP/6-31+G(d,p) level of theory. To form methyleneTHF a hydrogen atom was added to the carbon bridging N(5)−N(10) of the methylTHF optimized geometry. This structure was optimized at the same level of theory. These geometries were used for all further calculations. Excitation energies, ground state dipole moments, and transition dipole moments were calculated at the TD-DFT/B3LYP/6-31+G(d,p) level of theory, and difference dipole moments were calculated using the finite-field method.30 Calculations were performed in polarizable continuum models31 (PCM, ethanol, and water for methenylTHF and water for methyleneTHF) using Gaussian 09.32 All structures were edited and visualized using ChemCraft software, v 1.7 (http://www.chemcraftprog. 8321



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com) except for Figure 4, for which Chimera v 1.7 (UCSF) was used.

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RESULTS Absorption SpectraQualitative Results. The low temperature and room temperature absorption spectra of methenylTHF in ethanol and 8 M LiCl (pH 6) are shown in Figure 2a and Figure 2c. For both room temperature and low

Figure 3. Room and low temperature absorption (a, c) and Stark spectra (b, d) of methyleneTHF in 8 M LiCl at pH 13 and pH 6, respectively.

the ∼10 nm red-shift upon cooling is larger than the shift observed for methenylTHF. Stark SpectraQualitative Results. The Stark spectra for methenylTHF in EtOH and 8 M LiCl (pH 6) are shown in Figure 2b and Figure 2d for multiple angles of χ. The spectra have been normalized to an external field of 106 V/cm for comparison. The decrease in signal amplitude with increasing values of χ indicate that the value of ζ01 A is smaller than the magic angle, 54.7°. The spectra in both solvents show evidence of a vibronic progression, with spacings of 1398 ± 190 cm−1 in EtOH and 1476 ± 116 cm−1 in 8 M LiCl. These frequencies roughly correspond to the ground state vibrational frequencies of methenylTHF measured by Schelvis’ group using resonance Raman spectroscopy.33 In that study the strongest vibronic band appears at 1638 cm−1 with satellite bands at 1520 and 1502 cm−1. It should be noted that the Stark spectra were measured in equal energy steps of 135 cm−1 so vibronic resolution on the level of the RR experiment is not expected. However, the ca. 1400 cm−1 progression obtained here suggests that the excited state nuclear configuration is relatively unchanged compared to the ground state, as is consistent with the RR result. The Stark spectra of methyleneTHF in 8 M LiCl at pH 13 and pH 6 are shown in Figure 3b and 3d for multiple angles of χ. Again, the decrease in amplitude with increasing χ indicates that ζ01 A is less than the magic angle. The spectra do not show resolved vibronic features, indicating that the vibronic progressions that form the spectrum are due to low frequency vibrations and/or that the individual vibronic bands are much

Figure 2. Room and low temperature absorption spectra (a, c) and Stark spectra (b, d) of methenylTHF in ethanol and 8 M LiCl, respectively.

temperature spectra, a ∼10 nm bathochromic shift is observed for methenylTHF going from 8 M LiCl (pH 6) to EtOH. This relatively large solvatochromic shift indicates that the Δμ⃗01 for methenylTHF is large. A 5 nm red-shift is also observed upon cooling to 77 K in both cases, probably due to the loss of populated higher excited vibronic levels. The apparent maximum extinction of methenylTHF at 298 K in EtOH is nearly identical to that for 8 M LiCl (pH 6) (∼21 300 M−1 cm−1 vs ∼21 000 M −1 cm −1 , respectively). At 77 K, methenylTHF has ∼10% greater extinction in EtOH, 25 400 M−1 cm−1 vs 22 600 M−1 cm−1. The low temperature and room temperature absorption spectra of methyleneTHF in 8 M LiCl at pH 13 and pH 6 are shown in Figure 3a and 3c, respectively. The spectra are considerably blue-shifted compared to methenylTHF, with peak maxima at 306 nm at 298 K and 315 nm at 77 K for solutions at pH 13. At lower pH, these bands are slightly blueshifted to 300 nm at 298 K and 310 nm at 77 K. In both cases, 8322



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Figure 4. Optimized geometries of methenylTHF (a, b) and methyleneTHF (c, d) calculated at the B3LYP/6-31+G(d,p) level of theory. The structures on the left are shown viewing the x−y plane, while the structures on the right are rotated 90°, viewing the x−z plane. Calculated ground and excited state permanent dipole moments are shown as cyan and red arrows, respectively.

broader than those for methenylTHF. To our knowledge there is no resonance Raman data for comparison. Data collection at wavelengths below 300 nm was not possible because of low light intensity in this region due to absorption by the ITO/glass slides. TD-DFT and Finite-Field CalculationsResults. The ground state optimized geometries and permanent ground/ excited state dipole moments of methenylTHF and methyleneTHF are shown in Figure 4. The ground state geometry of methenylTHF using B3LYP/6-311+G(d,p) level of theory in both ethanol and water as PCM solvent is planar (in the x−y plane). The computed ground state dipole moments of μ⃗c0 were 14.16 and 14.36 D, respectively, directed along the long (x) axis of the molecule (see Figure 4) . TD-DFT at B3LYP/631+G(d,p) level of theory was used to calculate lowest optically accessible transitions using the ground state optimized geometry. The lowest ππ* transition, assigned as S0 → S1 (S01) is observed at 3.2948 eV (376.31 nm) with oscillator strengths of about 0.85 in both solvents, in good agreement with experimental absorption spectra. The transition dipole moment for the S01 transition points in the same direction as the ground state dipole moment with dipole strength of |m⃗ c01| = 26.84 D. This transition is primarily HOMO → LUMO character with electron density moving from pterin moiety of the methenylTHF to the p-amino benzoic acid (see Supporting Information Figure S1). The excited state dipoles for methenylTHF μ⃗c1 are 23.23 and 23.63 D in ethanol and water, respectively. The calculated difference dipole moments are Δμ⃗c01 = 9.26 and 9.66 D, respectively, about 50% larger than our experimental result (see later). However, the finite-field method yields Δμ⃗FF 01 = 7.81 and 8.12 D, which are in better agreement with the experimental values. The difference density (Figure 5a) shows that the net change in electron density occurs mostly on the pterin moiety of the molecule. Specifically the C(4) carbonyl oxygen, C(4a) carbon, and N(8) show net loss of electron density while N(5), N(10), and the PABA moiety gains electron density. The optimized ground state geometry for methyleneTHF at the B3LYP/6-311+G(d,p) level of theory is nonplanar and is bent between the pterin and PABA groups, forming a 24° angle with respect to the pterin (x−y) plane. The ground state dipole moment is directed roughly parallel with the plane of the pterin ring system with a magnitude of m⃗ c0 = 13.48 D. TD-DFT

Figure 5. Difference electron density maps of methenylTHF (a) and methyleneTHF (b) for the lowest optically active transition. Increased electron density is indicated by red, and decreased electron density is indicated by blue.

calculations showed that the lowest two optical transitions are at 3.93 eV (315.49 nm) and 4.197 eV (295.35 nm), respectively, with oscillator strengths of 0.005 and 0.816. Both transitions are π−π* character in nature. The lowest ππ* is mainly a HOMO → LUMO transition (S01) with significant charge transfer character (not shown). The second ππ* transition (see Supporting Information Figure S2) has contributions from HOMO−1 → LUMO (0.68) and HOMO → LUMO+2 (0.11) and is assigned as S02. The transition dipole moments for both of these transitions are in the plane of the p-aminobenzoic acid with dipole strengths of |μ⃗c01| = 0.13 c and |m⃗ 02 | = 20.17 D, respectively. The lowest optically accessible state is therefore assigned as the S02 (ππ*) transition. The excited state dipole for this optically accessible state of methyleneTHF is μ⃗ c2 = 22.35 D and is roughly in the same direction as ground state dipole moment. The difference dipole moment is Δμ⃗ c02 = 9.96 D, in good agreement with the experimental value. The finite-field approach gives Δμ⃗ FF 02 = 6.05 D which is underestimated by ∼2 D compared to the 8323



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experimental value. The difference density (Figure 5b) shows that most of the electronic reorganization upon optical excitation takes place on the p-aminobenzoic acid, while the out of plane pterin is relatively unperturbed upon optical excitation. Quantitative Analysis of Folate Stark Spectra. Representative simultaneous fits to the low temperature absorption and Stark spectra of methenylTHF and methyleneTHF are shown in Figure 6 and Figure 7, respectively.

Figure 7. Representative simultaneous fits to the absorption and Stark spectra of methyleneTHF in 8 M LiCl, pH 13.

± 3° for methenylTHF in EtOH and 8 M LiCl, respectively, and ζ02 A of 13 ± 5° and 0.002 ± 0.004° for methyleneTHF in aqueous solution at pH 13 and 6, respectively. These small ζ0n A indicate that charge redistribution occurs primarily along the direction of the transition dipole moment upon excitation in both molecules As methenylTHF is relatively planar, Δμ⃗ is expected to lie within the plane of the molecule. Referring to Figure 8, the angle between the computed transition dipole and difference dipole is less than 1°, with Δμ⃗c01 lying slightly out of the x−y plane. Under these assumptions, there are two possible directions for |Δμ⃗X01| which are shown as the solid arrows in Figure 8. The transition dipole moment of methyleneTHF points from N(5) to N(1) and has a significant z component (relative to the p-aminobenzoic acid, x−y plane) due to the bent geometry of the molecule. In this case, Δμ⃗ c02 is rotated 10° (clockwise in the x−y plane) from the transition dipole moment, and also has a significant z component. The estimated direction (projection onto the x−y plane) of the experimentally measured Δμ⃗ X02 is shown in Figure 9. In all cases, there is a considerable increase in the polarizability of the molecule upon excitation. The values for

Figure 6. Representative simultaneous fits to the absorption and Stark spectra of methenylTHF in 8 M LiCl.

These results are gathered in Table 1. As expected from the observed solvatochromic shifts, the values for the difference dipole moments of methenylTHF are large. For methenylTHF the experimental |Δμ⃗ X01| = 6.87 ± 0.12 D in EtOH and |Δμ⃗X01| = 6.31 ± 0.01 D in 8 M LiCl (pH 6). The smaller error for the aqueous solvent is likely due to the use of Stark spectra at only two values of χ, which decreases the degrees of freedom for the nonlinear fit. The smaller value of |Δμ⃗X01| in aqueous solution compared to EtOH suggests that the water cavity polarizes the ground state electronic structure in a configuration that is similar to its excited state than is the case for ethanol. The degree of solvent organization around methenylTHF in the ground state is expected to be more extensive for polar aqueous solutions compared to ethanol due to the positive charge of the molecule. The fitted difference dipole moment of methyleneTHF in aqueous solvent is larger in magnitude than methenylTHF, with a |Δμ⃗ X02| = 8.00 ± 0.12 D in 8 M LiCl at pH 13 and 8.16 ± 0.12 at pH 6. Also in agreement with the qualitative analysis are the low values of ζ01 A of 14 ± 3° and 16

→ →

TrΔα 0n were found to be 145 ± 12 Å3, 82 ± 0.4 Å3, 146 ± 14 Å3, and 105 ± 2 Å3 for methenylTHF in EtOH and 8 M LiCl (pH 6) and methyleneTHF in 8 M LiCl at pH 13 and 6, → →

respectively. The projection of the TrΔα 0n onto the transition → →

dipole moments are very similar to the TrΔα 0n itself, 8324



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Table 1 molecule

solvent

methenylTHF (n = 1)

EtOH 8 M LiCl, pH 6 8 M LiCl, pH 13 8 M LiCl, pH 6

methyleneTHF (n = 2) a

→ →

α 0n (Å3)a

TrΔ

145 ± 12 82 ± 0.4 146 ± 14 105 ± 2

→ →

m⃗ 0n Δα 0n m⃗ 0n (Å3)a 134 ± 17 74 ± 0.5 113 ± 37 82 ± 5

|Δμ0⃗ Xn |(D)a

ζ0n A (deg)

± ± ± ±

14 ± 3 16 ± 3 13 ± 5 0.01 ± 0.01

6.87 6.31 8.00 8.16

0.12 0.01 0.12 0.12

f = 1.6.

For 6MI, σ2P = 2.5 Goeppert−Mayer (GM), as measured by Woodbury.36 We have measured the TPA cross section for 6MAP, a fluorescent adenine analogue and a pteridone, by direct two photon excitation and found that σ2P = 3.4 GM.37 Using the difference dipoles obtained herein, we estimate that σ2P for these folates may be as high as 75 GM, well above that for pterins without the PABA group. Thus, this group of folates and their analogues will make very useful optical probes for imaging by two photon microscopies.38 Along these lines, the large difference dipole moments and difference polarizabilities of methenylTHF and methyleneTHF suggest that these molecules may be a useful scaffold for nonlinear optical (NLO) applications.39−41 NLO-active chromophores require a large hyperpolarizability β0 which can be estimated by42

Figure 8. Possible orientations of the difference dipole moment of methenylTHF in ethanol (green) and 8 M LiCl (blue). The calculated transition dipole moment is also shown (red).

β0 = 6Δμ ⃗

m⃗ 2 E2

The β0 were calculated to be 80 × 10−30 and 36 × 10−30 esu for methenylTHF in EtOH and 8 M LiCl and 18 × 10−30 and 24 × 10−30 esu for methyleneTHF in 8 M LiCl pH 13 and 6, respectively. These values are small compared to biological molecules useful for NLO applications, such as retinal, which has a β0 of 150−300 × 10−30 esu in organic solvents.43 However, derivatization of the molecule may increase the hyperpolarizability of the molecule, as has been shown for the donor−π−acceptor flavin dyad azobenzylflavin, which has a β0 of 720 × 10−30.28 These Stark spectra afford a way to potentially probe the internal electric fields of folate-binding proteins. For example, 5,10-methenylTHF is the antenna cofactor of DNA photolyase in many organisms. The direction of the transition dipole moment of methenylTHF in E. coli photolyase was calculated by Heelis in 1997 using semiempirical methods. He found that m⃗ 01 points roughly from N(5) to N(8) of the pteridine ring.5 However, the calculated difference dipole moment of 0.14− 0.45 D was much smaller than determined here, not surprising given the large size of folate and the computational tools available at the time. The absorption spectrum of the PL-bound chromophore is red-shifted by ∼15 nm compared to methenylTHF in ethanol. This observation suggests that the lowered polarity of the binding site may not be the sole reason for this shift. According to the crystal structure of E. coli PL, the cofactor is bound near the surface of the protein and may be solvent accessible.29 A Stark shift of 2530 cm−1, corresponding to the bathochromic shift between the aqueous and proteinbound spectra, could be produced by a protein electric field component along the ground/excited state dipole moments of methenylTHF of ∼2 × 109 V/m, using ΔE = |Δμ⃗| |F⃗| cos θ, where θ = 0. This field strength is plausible for a protein binding site in which charged residues are located. This suggests that photolyase may have evolved such that the antenna binding pocket electric field was optimized for light-

Figure 9. Directions of the difference dipole moment of methyleneTHF in 8 M LiCl at pH 13 (green) and pH 6 (blue), projected onto the x−y plane. The calculated transition dipole moment is also shown (red).

suggesting that most of the polarizability change occurs along that direction.

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DISCUSSION Here we have shown that both 5,10-methenylTHF and 5,10metheleneTHF have substantial charge redistribution upon optical excitation. Interestingly, these large difference dipole moments appear to be due to the pendant PABA group and not solely due to the excited state properties of the pterin ring. We have investigated charge redistribution in the guanine analogue 6-methylisoxanthopterin (6MI, a fluorescent nucleobase analogue) by Stark spectroscopy previously.34 6MI is similar to the folates described here but lacks the methenyl/methylene and PABA groups. For 6MI, |Δμ⃗ 01| ∼ 2 D with ζ01 A ≈ 28° and a → → negligible TrΔα 01 Evidently the addition of the electron-rich PABA moiety to the pterin ring system, along with enhanced conjugation throughout the structure, leads to an almost threefold increase in charge redistribution for methenylTHF and methyleneTHF compared to 6MI and orients this difference dipole more parallel to the transition dipole. These results suggest that the addition of electron-rich groups to existing, or otherwise related, pteridine-based nucleobase analogues may be an additional point of modulation for the spectroscopic properties of these molecules. For example, a knowledge of Δμ⃗ affords an estimate of the two photon absorption (TPA) cross section, σ2P, through the relationship σ2P ∝ |Δμ⃗ f 0|2εf 0/υ̃f 0 where f 0 represents the final and ground states, respectively.35 To our knowledge, the TPA for 5,10-methenyl(methylene)THFs have not been reported. 8325



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harvesting efficiency by pushing the absorption band of the chromophore further toward the visible region of the spectrum. It is likely that folate-binding cryptochromes, photolyase analogues, have evolved on similar principles.8,44−46 Along these lines, it is true that the majority of tetrahydrofolates are utilized as one-carbon donors or acceptors in a variety of cellular processes.1,47,48 To this extent, the excited state electronic structure is of little importance. However, it has been shown by Boxer’s group that the specific Stark shifts of (vibrational) absorption bands may facilitate the description of electrostatic fields within protein binding sites.20−23 DFT calculations have shown that dihydrofolate reductase produces a significant electronic polarization of the folate substrate along the reaction pathway.19 Indeed, folate analogues,49,50 such as methotrexate, an antitumor drug, are often assayed by optical spectroscopic techniques. A thorough investigation of this molecule’s excited state electronic properties using Stark spectroscopy will improve our ability to detect it and determine its biological activity using in vivo optical methods.51 As such, detailed information on the Stark spectra of free folate molecules in nonperturbing (or minimally perturbing) solvents is paramount to the correct interpretation of protein field-induced spectral shifts. The data presented here may be used as a starting point for experimental studies that investigate the effect of electrostatics in many nonphotobiological folate proteins.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address ‡

G. K.: Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, 1004 Stellar-Chance Building, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104−6059, United States. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS R.F.P. and R.J.S. were supported in part by the NSF Grant C H E - 0 8 47 8 5 5 a n d b y NA S A E x o b i o l o g y G r a n t NNX13AH33G. G.K. acknowledges support from Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation Grant TGCHE130088. We wish to thank Dr. Madhavan Narayanan for help in estimating the TPA cross section of the folates.

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ABBREVIATIONS THF, tetrahydrofolate; FRET, Fö rster resonance energy transfer; FAD, flavin adenine dinucleotide; PL, DNA photolyase; FBA, fluorescent nucleobase analogue; EtOH, ethanol; (TD)DFT, (time dependent) density functional theory; 6MAP, 4-amino-6-methyl-8-(2-deoxy-b-D-ribofuranosyl)-7(8H)-pteridone; 6MI, 6-methylisoxantopterin; PABA, p-aminobenzoic acid

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CONCLUSIONS The excited state electronic structure of two biologically important folates, methenylTHF and methyleneTHF, were measured by Stark spectroscopy. Both molecules were found to have large difference dipole moments with the Δμ⃗ 01 of methenylTHF found to be 6.87 ± 0.12 D and 6.31 ± 0.01 D in ethanol and 8 M LiCl, respectively, while Δμ⃗ 01 of methyleneTHF was found to be 8.00 ± 0.12 D and 8.16 ± 0.12 D in 8 M LiCl at pH 13 and 6, respectively. The direction of charge redistribution upon excitation was found to lie primarily along the transition dipole moments of the molecules, with ζ01 A being 14 ± 3°, 16 ± 3°, 13 ± 5°, and 0.002 ± 0.004°, respectively for the experimental conditions described above. In all cases, the difference polarizabilities were found to be large,

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REFERENCES

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→ →

with TrΔ α 01 = 145 ± 12 and 82 ± 0.4 Å3 for methenylTHF in ethanol and 8 M LiCl, respectively, and 146 ± 14 Å3 and 105 ± 2 Å3 for methyleneTHF in 8 M LiCl at pH 13 and 6. The Stark analysis of these folates presents the groundwork for investigation of novel pteridine-derived fluorescent base analogues, folate-based nonlinear optical applications, and may be useful in the determination of electrostatic interactions of nonphotobiological enzymes which use these cofactors for catalysis.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information contains four figures, including MO maps from the TD-DFT calculations, examples of the finite-field calculations on the absorption line shape, and a Table containing values from the TD-DFT and FF methods, including transition energies, oscillator strengths, dipole moments, etc. This material is available free of charge via the Internet at http://pubs.acs.org. 8326



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Excited State Electronic Structures of 5,10-Methenyltetrahydrofolate

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