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Molecular Insights on the Two Fluorescence Lifetimes Displayed by Warfarin from Fluorescence Anisotropy and Molecular Dynamics Studies Bjo¨rn C. G. Karlsson,† Annika M. Rosengren,† Per Ola Andersson,‡ and Ian A. Nicholls*,† Bioorganic and Biophysical Chemistry Laboratory, School of Pure and Applied Natural Sciences, UniVersity of Kalmar, SE-391 82 Kalmar, Sweden, and FOI CBRN Defence and Security, SE-901 82 Umeå, Sweden ReceiVed: December 19, 2008; ReVised Manuscript ReceiVed: April 8, 2009
A series of steady-state fluorescence anisotropy experiments has been performed to demonstrate the presence of a deprotonated open side chain form of warfarin in organic environments. We explain the observed emissionwavelength-dependent anisotropy of warfarin in ethanol, 2-propanol, and acetonitrile due to the coexistence of neutral isomers and deprotonated open side chain forms displaying different fluorescence decay kinetics. To investigate solvent-solute interactions in more detail, a series of molecular dynamics simulations was performed to study warfarin solvation and to predict the time scale of rotational diffusion displayed by this compound. Predictions obtained provide an explanation for the nonzero values in anisotropy observed for neutral isomers of warfarin associated with the short fluorescence lifetime (τ < 0.1 ns) and for an approximately zero anisotropy observed for the deprotonated open side chain form, which is associated with the longer fluorescence lifetime (τ ) 0.5-1.6 ns). Finally, we address the potential use of fluorescence anisotropy for an increased understanding of the structural diversity of warfarin in protein binding pockets. Introduction Warfarin is a highly potent anticoagulant commonly used to prevent, for example, strokes and heart attacks by inhibiting the active site of vitamin K dependent epoxide reductase (VKOR).1,2 Despite the frequent use of the drug and the drug’s narrow therapeutic window, the mechanisms of action underlying the inhibition process are not yet fully investigated. An important factor that would contribute to an increased understanding of drug function is the identification of the molecular structure of the biologically active form of warfarin when present at the site-of-action. When administered and once having reached the bloodstream, it has been reported that 99% is bound to the blood plasma protein human serum albumin (HSA), leaving only 1% available for interaction with other targets.3 Due to this distribution, it can be concluded that any factor affecting warfarin’s interaction with HSA4 will have severe effects on its drug action to VKOR. During the 1970s, detailed investigations by Valente et al.5-7 showed that warfarin exists as a dynamic equilibrium between isomeric forms, Chart 1. The distribution and types of isomers present were found to be dependent on solvent polarity and pH. Interestingly, although the cyclic hemiketal isomer was suggested to be the predominant structure in crystals and in nonpolar organic solvents, the form of warfarin when bound to HSA is the deprotonated open side chain form.8 Parallel investigations by others have, on the other hand, suggested that the cyclic hemiketal form is responsible for the interaction with the enzyme cytochrome P450 2C9 (CYP2C9)9 and the presence of an additional binding site on this enzyme, which preferably binds anionic species.10 Since this observed difference in the preferred active structure of warfarin is dependent on the structure and polarity of the * To whom correspondence should be addressed. Fax: +46 480 44 6262. Tel: +46 480 44 6280. † University of Kalmar. ‡ FOI CBRN Defence and Security.
CHART 1: Structures of Warfarin Reported in the Literature6,9
binding site to which the drug is directed, knowledge of the isomeric distribution of warfarin in different environments should provide insights on different protein binding sites (which provide different microenvironments for warfarin) and yield information on the underlying mechanisms favoring one isomeric structure of warfarin over another. We have recently characterized and assigned regions in fluorescence emission spectra generated for isomers of the anticoagulant warfarin in a series of organic solvents with differences in polarity, hydrogen bonding ability, and viscosity.11 The judicious selection of organic solvent or solvent mixtures allows us to vary the molecular environment of warfarin and even simulate the character of a binding site, for example, the hydrophobic interior of a target protein. Results from studies in ethanol, 2-propanol, and acetonitrile demonstrated the presence of a deprotonated open side chain form of warfarin with
10.1021/jp811242z CCC: $40.75 2009 American Chemical Society Published on Web 05/12/2009
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a higher quantum yield and a longer fluorescence lifetime as compared to the coexisting neutral forms. To our knowledge, fluorescence anisotropy studies of warfarin in organic solvents have not yet been reported. We hypothesized that the coexistence of different fluorescence lifetimes in polar organic solvents will have a profound impact on the observed anisotropy of warfarin. Through characterization of the fluorescence behavior of warfarin in organic solvents, it is envisaged that fluorescence anisotropy may be used as a tool for the study of warfarin-protein interactions. In this study, we use steady-state fluorescence anisotropy measurements in conjunction with a series of molecular dynamics simulations to clarify the presence of multiple fluorescence lifetimes in the emission anisotropy spectra of warfarin and their contribution to the steady-state anisotropy spectrum of warfarin in organic solvents. Experimental Section Chemicals. A racemic mixture of warfarin (3-(R-acetonylbenzyl)-4-hydroxycoumarin, min 98%) was obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Ethanol, EtOH (99.5%), was from Solveco Chemicals AB (Ta¨by, Sweden); acetonitrile, ACN (g99.8%), chloroform, CHCl3 (g99%), and 2-propanol, 2-PrOH (g99.9%), were all from Merck KGaA (Darmstadt, Germany). Acetic acid, AcOH (>98%), was from VWR (Stockholm, Sweden). All chemicals were used as received. Steady-State Anisotropy. Fluorescence steady-state anisotropy spectra were obtained on a SPEX FluoroMax-3 fluorimeter from Jobin-Yvon HORIBA using 14 × 10-6 M solutions of warfarin (absorption was kept below 0.2 to prevent inner filter effects and self-absorption of the emission). Quartz quvettes (1.0 cm, 3 mL of Suprasil from Helma) were used in all spectroscopic experiments. All emission anisotropy spectra were recorded between 315 and 500 nm (λexc ) 300 nm, 298 K) with the excitation and emission monochromator slit widths placed at 3 and 7 mm, respectively. In these analyses, four raw data files were collected, from which the anisotropy, eq 1, could be calculated. In this expression, G is the correction factor which compensates for instrument responses for horizontally and vertically polarized light (IHV/IVV). In the final presentation of data, solvent background emission was always removed from the spectra presented for warfarin.
r)
IVV - G × IVH IVV + 2G × IVH
(1)
Molecular Dynamics (MD) Simulations. Simulations of the neutral open side chain isomer (this structure of warfarin was chosen as a representative for all isomers) dissolved in either CHCl3, AcOH, 2-PrOH, EtOH, ACN, or water (TIP3P-WAT) were built and parametrized using the Amber9912 and GAFF13 force fields. For simulations of the dynamic behavior of warfarin in the solvents studied, solvent models of CHCl3 (as implemented in AMBER8), TIP3P-WAT,14 and ACN15 were implemented with parameters and structures as recommended from previous research on these solvent models. Single-molecule conformations of warfarin, AcOH, 2-PrOH, and EtOH were built and energy-minimized using the PRODRG-server16 program and final pdb coordinates were used as input to the AMBER suite of MD programs (v. 8.0, UCSF, San Francisco, U.S.A.).17 Prior to simulations, atomic partial charges were assigned using the AM1-BCC18 charge method in the ANTECHAMBER module, and all warfarin-solvent systems were built using XLEAP.
Mixtures were initially energy-minimized to remove adverse van der Waals contacts using 500 steepest-descent and 500 conjugate gradient steps. Second, systems were heated from 0 to 298 K at constant volume (NVT), allowing solvent molecules to relax around the restrained warfarin molecule (kr ) 500.0 kcal/mol × Å2) for 50 ps. After subsequent simulation at NVT for 100 ps, allowing the whole system to relax, equilibration was commenced in the isothermal and isobaric ensemble for 500 ps (NPT at 298 K and 1 bar) to ensure that the system evolved to achieve stable density and energy. In all steps of these simulations, periodic boundary conditions were employed together with an 8 Å nonbonded interaction cutoff for dealing with long-range electrostatics using the Particle Mesh Ewald (PME) summation method.19 The long-range van der Waal interactions in the system were treated using a continuum model correction to energy and pressure. All hydrogen atoms in the system were constrained using the SHAKE algorithm, allowing a time step of 0.002 ps. The temperature and pressure were held constant using Langevin dynamics with a collisional frequency of 1.0 ps-1 and isotropic position scaling associated with a pressure relaxation time of 2.0 ps. Finally, statistical data were extracted from equilibrated mixtures during a 3 ns production phase after additional equilibration for 500 ps at NPT. Final equilibrated box sizes and thermodynamic data are reported in Table S1 (Supporting Information). Production phase trajectories were typically analyzed using the PTRAJ module, saving data every 0.2 ps. The analysis tool hbond was utilized to follow all atoms and hydrogen bond interactions formed during simulations. Measurement of all interactions taking place in solution between specified hydrogen-bond-donating and -accepting atoms was undertaken. In this screening process, all interactions were extracted from the trajectory using a bond cutoff distance of 3.0 Å and an angle cutoff of 60°. Radial distribution functions (RDFs) were generated to quantify local densities of specified atom pairs at the optimal distance for interaction (ropt.). In these analyses, the radial distribution function, g(r) is defined as the ratio between the observed number density, Fij, of a specified solvent atom at a certain distance (r) from a solute atom (i) and the average bulk atom number density of the solvent, 〈Fj〉. Additionally, nij(r) is the number of atoms in a volume fragment, Vshell, which is dependent on the bin width used, δr in eq 2
g(r) )
Fij(r) nij(r) ) 2 〈Fj〉 〈Fj〉4πr δr
(2)
Rotational diffusion times, θ, for warfarin in the different solvents were obtained through the analysis of generated timecorrelation functions, C(τ), eq 3. From these analyses, vectors, r, on the warfarin molecule in the Cartesian coordinate systems were defined, and the positions of these vectors as a function of time were subsequently monitored during the production phase.
C(τ) ) 〈P2[r(t) · r(t + τ)]〉
(3)
In this expression P2 is a Legendre polynomial (P2(cos θ) ) (3 cos2 θ - 1)/2) and r is the time-dependent unit vector at position time t and time t + τ. Vectors analyzed for warfarin are defined in Figure S1 (Supporting Information). Brackets indicate a time and ensemble average for all conformations analyzed. Subsequent calculation of the average rotational diffusion time, 〈θ〉, was then performed according to eq 4
Fluorescence Lifetimes Displayed by Warfarin 〈θ〉
)
∫t)0t)∞ C(τ)dt
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(4)
Results and Discussion We have recently demonstrated that in polar organic solvents, warfarin displays two fluorescence lifetimes which are dependent on solvent character and show different contributions to the emission spectra with τ1 < 0.1 ns and τ2 ) 0.5-1.6 ns.11 Additionally, warfarin excitation in nonpolar organic solvents results in fluorescence associated with a fast decay of τ < 0.1 ns. Further elucidation made it possible to conclude that the short lifetime (τ1), observed in both nonpolar and polar organic solvents, originated from neutral isomeric forms of warfarin, W, whereas the longer lifetime (τ2), which only existed in polar environments, originated from the deprotonated open side chain form, (-)W. Fluorescence anisotropy experiments have frequently been used for the investigation of ligand-protein interactions.20 Here, fluorescence anisotropy for warfarin was investigated to elucidate the coexistence of two fluorescence lifetimes and their contribution to the observed anisotropy. Although the fluorescence behavior of warfarin in organic solvents has previously been carefully investigated, the influence of the mixed population of fluorescence lifetimes on anisotropy in organic solvents remains to be examined. The fluorescence anisotropy may provide a valuable tool for examining molecular environments, for example, upon protein binding of warfarin. Initially, excitation at 300 nm (absorbance maximum of the S0 f S1 electronic transition) in the nonpolar organic solvents chloroform (CHCl3) and acetic acid (AcOH), Figure 1, resulted in an emission-wavelength-independent anisotropy with values of r ≈ 0.15 for both solvents. For a small organic molecule, such as warfarin, this nonzero value in anisotropy may be explained by the existence of the short fluorescence lifetime, τ1, which is shorter or on the same order of magnitude as the time scale of rotational diffusion time, θ (θ ≈ τ). Excitation at 300 nm in the polar organic solvents studied, ethanol (EtOH), 2-propanol (2-PrOH), and acetonitrile (ACN), resulted in an emission-wavelength-dependent anisotropy, Figure 2. We suggest that this behavior is explained by the presence
Figure 2. Combined fluorescence emission (solid line) and anisotropy (dotted line) spectra for warfarin (14 µM) in 2-PrOH, EtOH, and ACN using λexc ) 300 nm.
of (-)W, associated with the longer fluorescence lifetime, τ2. Previously reported analyses of the structural contribution of warfarin to the overall emission spectrum at different emission wavelengths11 concluded that at shorter emission wavelengths, the contribution of the (-)W is less than the contribution of W. Since the overall anisotropy will be observed as an average of both decays, the contribution of τ2 increases as the emission wavelength increases, and finally, at long emission wavelengths, the only anisotropy observed will originate from (-)W. This behavior was found to be most evident for warfarin in 2-PrOH where τ1 < 0.1 ns and τ2 ) 0.53 ns and in ACN where τ1 ) 0.14 ns and τ2 ) 1.6 ns. In EtOH, similarity in the decay kinetics for the fluorescence lifetimes τ1 ) 0.2 ns and τ2 ) 0.45 ns resulted in what appeared to be an emission-wavelengthindependent anisotropy. Clear support for this observation was obtained using the Perrin equation, eq 5,20 which makes it possible to predict observed values in anisotropy
r ) r0
Figure 1. Combined fluorescence emission (solid line) and anisotropy (dotted line) spectra for warfarin (14 µM) in chloroform (CHCl3) or acetic acid (AcOH) using λexc ) 300 nm.
θ θ+τ
(5)
In order to compare calculated and experimentally observed values of anisotropy for warfarin in the series of organic solvents studied, a series of molecular dynamics (MD) simulations was performed. Results from these simulations showed that the obtained values for rotational diffusion times, θ, are typically of the same order of magnitude as the short fluorescence lifetime τ1. Notably, good correlation between the experimentally observed values in anisotropy and calculated values obtained from the Perrin eq 5 was obtained, Table 1 and Figures 1, 2, and 3. The higher value of the calculated compared to the experimentally observed anisotropy for warfarin in AcOH may have its origin in the simplified representation of the isomeric
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TABLE 1: Theoretical Predictions on the Time Scale of Rotational Diffusion Displayed by Warfarin as Studied by the Molecular Dynamics Method rotational diffusion θX solvents (ps)
θY (ps)
θZ (ps)
〈θ〉 (ps)a
water 34.7 23.3 32.6 30.2 ( 3.5 ACN 27.4 20.7 28.0 25.4 ( 2.3 EtOH 70.2 58.3 67.7 65.4 ( 3.6 2-PrOH 86.0 71.8 97.4 85.1 ( 7.4 AcOH 234.4 234.4 237.5 235.4 ( 1.0 CHCl3 60.8 67.1 73.7 67.2 ( 3.7
fluorescence lifetimesb anisotropyd τ1 (ns)
τ2 (ns)
0.05c 0.14 1.6 0.20 0.45 0.1 0.53 0.1 0.1
r1
r2
0.15 0.06 0.01 0.10 0.05 0.18 0.06 0.28 0.16
a Values presented as the mean ( standard error of the mean. Values used for predicting warfarin anisotropies as adapted from Karlsson et al.11 c Values adapted from Il’ichev et al.21 for estimations of warfarin fluorescence in PBS buffer. d Values calculated using the Perrin eq 5 from respective fluorescence lifetimes, estimating r0 ) 0.4. b
lecular hydrogen bonding between the -OH and side chain CdO functionalites of warfarin is less favored due to competition by the solvent. Interestingly, simulations in the less polar AcOH demonstrated that this solvent complexes strongly to warfarin through three solvent molecules, Table S2 (Supporting Information). This is in contrast to the situation found for the polar solvents water, EtOH, and 2-PrOH, where weaker hydrogen bonding to warfarin was observed. This observed strength of the hydrogen bonding between AcOH and warfarin may explain the higher value of θ observed in this solvent. Notably, the polar aprotic solvent ACN showed similar warfarin solvation behavior as nonpolar CHCl3, and in these solvents, intramolecular hydrogen bonding was highly favored, predicting a high stability for the cyclic hemiketals. This behavior is reminiscent of the isomeric status of warfarin as observed when bound to the active site of cytochrome P450 2C9 (CYP2C9).9 As the nature of warfarin solvation influences both the isomeric distribution, Chart 1, and the observed fluorescence behavior, judicious selection of the solvent allows for the mimicking of protein binding sites. Moreover, the structural interplay available to warfarin suggests its use as a probe for the study of protein binding sites. Conclusions
Figure 3. Time-correlation function (presented for the x-vector) shown for the first 300 ps for warfarin in the different solvents studied in a series of molecular dynamics simulations: water (s), ACN (- - -), EtOH ( · · · ), 2-PrOH (- · - · - · ), CHCl3 (- · · - · · - · · ) and AcOH (bold s).
distribution of warfarin simulated in this solvent system. Collectively, simulation data provided support for the hypothesis that a fluorescence lifetime shorter than the rotational diffusion time (τ < θ) will give rise to higher values of anisotropy. This behavior reflects that reported by Il’ichev et al.21 in studies of (-)W interacting with the protein HSA. They explained the high value in polarization of free unbound warfarin in PBS buffer (P ) 0.18) as being due to the short fluorescence lifetime, which they estimated from calculations to be ∼50 ps. Furthermore, predictions on the rotational diffusion time of warfarin in water (used as model for PBS buffer) performed by us resulted in a calculated anisotropy of r ) 0.15. Subsequent analysis of solute-solvent hydrogen bond interactions formed in the series of solvents by MD provided further insights concerning the origin of the rotational diffusion times calculated, Table 1. Radial distribution functions in conjunction with hydrogen bond analysis, Table S2 (Supporting Information), made it possible to calculate the average number of solvent molecules interacting with the studied functionalities of warfarin (phenolic -OH, coumarin ring CdO, and the side chain CdO) and the hydrogen bond occupational time (in percent of the total trajectory). Furthermore, the degree of intramolecular hydrogen bond formation, which is indicative of the stability of the cyclic hemiketal isomeric structure and/or hydrogen bond formation between the phenolic type -OH and the side chain CdO, was tracked and analyzed. In the polar solvents investigated, water, EtOH, and 2-PrOH, each demonstrated hydrogen bonding to all three of the functionalities of warfarin studied. In these cases, the intramo-
The emission-wavelength-dependent anisotropy observed for warfarin in organic environments is discussed in this paper, and we propose a molecular-level basis for this observation. A series of molecular dynamics simulations were performed to predict rotational diffusion times and to allow for a detailed study on the solvation of warfarin in the selected series of solvents. On the basis of the predictions obtained, a hypothesis was developed and used to explain the higher values of anisotropy observed for neutral isomers in contrast to the approximately zero anisotropy observed for the deprotonated open side chain form of warfarin. The observed solvent-dependent differences in the fluorescence anisotropic behavior of warfarin allow for the simulation for events taking place upon protein binding and potentially better insights into drug-protein recognition. Acknowledgment. The financial support of the Swedish Research Council (VR), the Knowledge Foundation (KKS), Carl-Trygger’s Foundation, and the University of Kalmar is most gratefully acknowledged. Supporting Information Available: Molecular dynamics simulation data. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Li, T.; Chang, C.; Jin, D.; Lin, P.; Khvorova, A.; Stafford, D. W. Nature 2004, 427, 541–544. (2) Rost, S.; Fregin, A.; Ivaskevicius, V.; Conzelmann, E.; Hortnagel, K.; Pelz, H.; Lappegard, K.; Seifried, E.; Scharrer, I.; Tuddenham, E. G. D.; Muller, C. R.; Strom, T. M.; Oldenburg, J. Nature 2004, 427, 537–541. (3) Yacobi, A.; Udall, J. A.; Levy, G. Clin. Pharmacol. Ther. 1976, 19, 552–558. (4) Ha, C.-E.; Petersen, C. E.; Park, D. S.; Harohalli, K.; Bhagavan, N. V. J. Biomed. Sci. 2000, 7, 114–121. (5) Valente, E. J.; Porter, W. R.; Trager, W. F. J. Med. Chem. 2002, 21, 231–234. (6) Valente, E. J.; Lingafelter, E. C.; Porter, W. R.; Trager, W. F. J. Med. Chem. 1977, 20, 1489–1493. (7) Valente, E. J.; Trager, W. F.; Jensen, L. H. Acta Crystallogr. 1975, 31, 954–960.
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J. Phys. Chem. B, Vol. 113, No. 22, 2009 7949 (16) Schu¨ttelkopf, A. W.; van Alteen, D. M. F. Acta Crystallogr. 2004, D, 1355–1363. (17) Case, D. A.; Cheatham, T. E.; Darden, T.; Gohlke, H.; Luo, R.; Merz, K. M.; Onufriev, A.; Simmerling, C.; Wang, B.; Woods, R. J. J. Comput. Chem. 2005, 26, 1668–1688. (18) Jakalian, A.; Jack, D. B.; Bayly, C. I. J. Comput. Chem. 2002, 23, 1623–1641. (19) Essmann, U.; Perera, L.; Berkowitz, M. L. J. Chem. Phys. 1995, 103, 8577–8593. (20) Lakowicz, Y. V. Principles of Fluorescence Spectroscopy, 2nd ed.; Plenum Press: New York, London, 1999; p 698. (21) Il’ichev, Y. V.; Perry, J. L.; Simon, J. D. J. Phys. Chem. B 2002, 106, 452–459.
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