Time-resolved Anisotropy Measurements Research Articles

Energy transfer at heterogeneous protein-protein interfaces to investigate the molecular behaviour in the crowding environment

Investigation of the behaviour of proteins in crowded environments is crucial for understanding the role of proteins in biological environments. In this study, the behaviour of bovine serum albumin (BSA) in crowded (highly concentrated) environments was investigated using time-resolved fluorescence spectroscopy as a model system. By using energy transfer as a molecular ruler, the crowding effect was clearly observed in the time resolved spectra. In addition, by using both time resolved anisotropy measurement and Raman spectroscopy, more detail insights from conformational and dynamic points of view were described. Consequently, it was revealed that in the highly concentrated solution, most of the BSA molecules are in the fast-reversible oligomeric state and the association at the “hard” and “soft” interfaces between protein surfaces occurred in a highly crowded environment with the aid of a charge-charge and short-range attractive interface. From both the conformational and dynamic aspects, the detail spectroscopic understanding of the behaviour of BSA in the crowding environment was obtained.

Apolipoprotein C-III Nanodiscs Studied by Site-Specific Tryptophan Fluorescence.

Apolipoprotein C-III (ApoC-III) is found on high-density lipoproteins (HDLs) and remodels 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles into HDL-like particles known as nanodiscs. Using single-Trp-containing ApoC-III mutants, we have studied local side chain environments and interactions in nanodiscs at positions W42, W54, and W65. Using transmission electron microscopy and circular dichroism spectroscopy, nanodiscs were characterized at the ultrastructural and secondary conformational levels, respectively. Nearly identical particles (15 ± 2 nm) were produced from all proteins containing approximately 25 ± 4 proteins per particle with an average helicity of 45-51% per protein. Distinct residue-specific fluorescence properties were observed with W54 residing in the most hydrophobic environment followed by W42 and W65. Interestingly, time-resolved anisotropy measurements revealed that Trp side chain mobility is uncorrelated to the polarity of its surroundings. W54 is the most mobile compared to W65 and W42, which are more immobile in a nanodisc-bound state. On the basis of Trp spectral comparisons of ApoC-III in micellar and vesicle environments, ApoC-III binding within nanodiscs more closely resembles a bilayer-bound state. Despite the nanodiscs being structurally similar, we found marked differences during nanodisc formation by the Trp variants as a function of temperature, with W42 behaving the most like the wild-type protein. Our data suggest that despite the modest mutations of Trp to Phe at two of the three native sites, the interfacial location of W42 is important for lipid binding and nanodisc assembly, which may be biologically meaningful as of the three Trp residues, only W42 is invariant among mammals.

Hydrodynamic Radii of Ranibizumab, Aflibercept and Bevacizumab Measured by Time-Resolved Phosphorescence Anisotropy.

PurposeTo measure the hydrodynamic radii of intravitreal anti-VEGF drugs ranibizumab, aflibercept and bevacizumab with μs time-resolved phosphorescence anisotropy.MethodsRuthenium-based dye Ru(bpy)2(mcbpy − O − Su − ester)(PF6)2, whose lifetime of several hundred nanoseconds is comparable to the rotational correlation time of these drugs in buffer, was used as a label. The hydrodynamic radii were calculated from the rotational correlation times of the Ru(bpy)2(mcbpy − O − Su − ester)(PF6)2-labelled drugs obtained with time-resolved phosphorescence anisotropy measurements in buffer/glycerol solutions of varying viscosity.ResultsThe measured radii of 2.76±0.04 nm for ranibizumab, 3.70±0.03 nm for aflibercept and 4.58±0.01 nm for bevacizumab agree with calculations based on molecular weight and other experimental measurements.ConclusionsTime-resolved phosphorescence anisotropy is a relatively simple and straightforward method that allows experimental measurement of the hydrodynamic radius of individual proteins, and is superior to theoretical calculations which cannot give the required accuracy for a particular protein.Electronic supplementary materialThe online version of this article (doi:10.1007/s11095-016-1940-2) contains supplementary material, which is available to authorized users.

A Comparative Study of the Influence of Sugars Sucrose, Trehalose, and Maltose on the Hydration and Diffusion of DMPC Lipid Bilayer at Complete Hydration: Investigation of Structural and Spectroscopic Aspect of Lipid-Sugar Interaction.

It is well-known that sugars protect membrane structures against fusion and leakage. Here, we have investigated the interaction between different sugars (sucrose, trehalose, and maltose) and phospholipid membrane of 1,2-dimyristoyl-sn-glycero-3-phoshpocholine (DMPC) using dynamic light scattering (DLS), transmission electron microscopy (TEM), and other various spectroscopic techniques. DLS measurement reveals that the addition of sugar molecule results a significant increase of the average diameter of DMPC membrane. We have also noticed that in the presence of different sugars the rotational relaxation and solvation time of coumarin 480 (C480) and coumarin 153 (C153) surrounding DMPC membrane increases, suggesting a marked reduction of the hydration behavior at the surface of phospholipid membrane. In addition, we have also investigated the effect of sugar molecules on the lateral mobility of phospholipids. Interestingly, the relative increase in rotational, solvation and lateral diffusion is more prominent for C480 than that of C153 because of their different location in lipid bilayer. It is because of preferential location of comparatively hydrophilic probe C480 in the interfacial region of the lipid bilayer. Sugars intercalate with the phospholipid headgroup through hydrogen bonding and replace smaller sized water molecules from the membrane surface. Therefore, overall, we have monitored a comparative analysis regarding the interaction of different sugar molecules (sucrose, trehalose, and maltose) with the DMPC membrane through DLS, TEM, solvation dynamics, time-resolved anisotropy, and fluorescence correlation spectroscopy (FCS) measurements to explore the structural and spectroscopic aspect of lipid-sugar interaction.

Tryptophan probes reveal residue-specific phospholipid interactions of apolipoprotein C-III

Apolipoproteins are essential human proteins for lipid metabolism. Together with phospholipids, they constitute lipoproteins, nm to μm sized particles responsible for transporting cholesterol and triglycerides throughout the body. To investigate specific protein–lipid interactions, we produced and characterized three single-Trp containing apolipoprotein C-III (ApoCIII) variants (W42 (W54F/W65F), W54 (W42F/W65F), W65 (W42F/W54F)). Upon binding to phospholipid vesicles, wild-type ApoCIII adopts an α-helical conformation (50% helicity) as determined by circular dichroism spectroscopy with an approximate apparent partition constant of 3×104M−1. Steady-state and time-resolved fluorescence measurements reveal distinct residue-specific behaviors with W54 experiencing the most hydrophobic environment followed by W42 and W65. Interestingly, time-resolved anisotropy measurements show a converse trend for relative Trp mobility with position 54 being the least immobile. To determine the relative insertion depths of W42, W54, and W65 in the bilayer, fluorescence quenching experiments were performed using three different brominated lipids. W65 had a clear preference for residing near the headgroup while W54 and W42 sample the range of depths ~8–11Å from the bilayer center. On average, W54 is slightly more embedded than W42. Based on Trp spectral differences between ApoCIII binding to phospholipid vesicles and sodium dodecyl sulfate micelles, we suggest that ApoCIII adopts an alternate helical conformation on the bilayer which could have functional implications.

Lateral Intermolecular Self-Exchange Reactions for Hole and Energy Transport on Mesoporous Metal Oxide Thin Films.

Intermolecular self-exchange energy and electron-transfer reactions occur without a loss of free energy. This behavior can be exploited for energy-transport applications when molecules that undergo self-exchange transfer reactions are immobilized on a solid support. This Article focuses upon lateral self-exchange reactions and the relevant interfacial chemistry that occurs on the mesoporous nanocrystalline (anatase) TiO2 thin films that are commonly used in dye-sensitized solar cells. It has been known for some time that all of the dye molecules (termed sensitizers) within such thin films can be reversibly oxidized and reduced by lateral self-exchange electron transfer provided that the sensitizer surface coverage exceeds a percolation threshold. Under conditions where excited-state electron injection into TiO2 is unfavored, lateral intermolecular energy-transfer reactions are also known to occur. The self-exchange rate constants have been quantified by electrochemical, absorption, and/or time-resolved anisotropy techniques and understood within the framework of Marcus theory. Such analysis reveals that the reorganization energy and the electronic coupling are sensitive to the identity of the molecular compound. Time-resolved anisotropy measurements have shown that lateral charge and energy-transfer reactions across the TiO2 surface occur in kinetic competition with charge recombination and excited-state relaxation, respectively. The extent to which lateral self-exchange reactions might be exploited for solar energy conversion applications is discussed, as are critical fundamental issues that remain unresolved.

Interaction of ANS with human serum albumin under confinement: Important insights and relevance

Human serum albumin (HSA) has been extensively studied over the years not only as a model protein but also as an important small molecule carrier with its ability to bind a variety of ligands. This study focuses on the modulation in the conformational disposition of HSA within the confinement of water pools of AOT reverse micelles, and its interactions with 1-anilinonapthelenesulfonate (ANS), the latter serving as a drug moiety. Circular dichroism studies show that while on one hand the incorporation of the protein in the reverse micelles leads to significant distortion in its secondary structure, however, at the same time, addition of ANS leads to a marked increase in helicity of HSA. A combination of FRET studies, time resolved anisotropy measurements and global analyses of temperature dependent spectra reveal little or no significant interaction between HSA and ANS inside the AOT water pools, this being expected, based on the observed distortion of the protein secondary structure on reverse micelle entrapment (the latter resulting in disruption of the binding pockets available to ANS). Taken together our data show possible insights into how HSA releases its bound species (when interacting with membranes or charged confined spaces) and thereby remains a viable drug carrier.

Polarization-modulation setup for ultrafast infrared anisotropy experiments to study liquid dynamics.

An infrared pump-probe setup using rapid polarization modulation has been developed to perform time-resolved vibrational anisotropy measurements. A photo-elastic modulator is used as a rapidly switchable half-wave plate, enabling the measurement of transient absorptions for parallel and perpendicular polarizations of the pump and probe pulses on a shot-to-shot basis. In this way, infrared intensity fluctuations are nearly completely canceled, significantly enhancing the accuracy of the transient-anisotropy measurement. The method is tested on the OD-stretch vibration of HDO in H2O, for which the signal-to-noise ratio is found to be 4 times better than with conventional methods.

Interligand electron transfer in heteroleptic ruthenium(II) complexes occurs on multiple time scales.

The time-dependent localization of the metal-to-ligand charge transfer (MLCT) excited states of ruthenium(II) complexes containing 2,2'-bipyridine (bpy) and 1,10-phenanthroline (phen) ligands was studied by femtosecond transient absorption spectroscopy. Time-resolved anisotropy measurements indicate that the excited state hops randomly among the three ligands of each complex by subpicosecond interligand electron transfer (ILET). Although the bpy- and phen-localized (3)MLCT states have similar energies and steady-state emission spectra, pronounced differences in their excited-state absorption spectra make it possible to observe changes in excited state populations using magic angle transient absorption measurements. Analysis of the magic angle signals shows that the excited electron is equally likely to be found on any of the three ligands approximately 1 ps after excitation, but this statistical distribution subsequently evolves to a Boltzmann distribution with a time constant of approximately 10 ps. The apparent contradiction between ultrafast ILET revealed by time-dependent anisotropy measurements and the slower ILET seen in magic angle measurements on the tens of picoseconds time scale is explained by a model in which the underlying rates depend dynamically on excess vibrational energy. The insight that ILET can occur over multiple time scales reconciles contradictory literature observations and may lead to improved photosensitizer performance.

Dynamics of the KcsA Selectivity Filter Probed using Intrinsic Tyrosine Fluorescence

KcsA is activated by intracellular protons through a large conformational change in the inner helical-bundle gate. The selectivity filter and surrounding structures play a crucial role in ion conduction as an inactivation gate. To monitor the conformational dynamics of the selectivity filter under various experimental conditions we engineered a KcsA construct devoid of tryptophan and tyrosine residues with the exception of residue Y78, locatd in the middle of the selectivity filter. Fluorescence lifetimes indicate that the microenvironment polarity of Y78-KcsA decreases upon gating, irrespective of the type of ion (K+, Rb+, NH4+, Cs+) present in the selectivity filter. Further, time-resolved anisotropy measurements indicate that the dynamics associated with the segmental mobility of the selectivity filter is significantly restricted, and this gating-driven motional restriction is more pronounced in High K+ and Rb+ conditions. Red Edge Excitation Shift (REES) measurements of Y78-KcsA show slow water relaxation dynamics under conditions that stabilize conductive and non-conductive filter conformations, suggesting the presence of bound/restricted water molecules in both filter conformations. Interestingly, opening the lower gate appears to further restrict the water molecules in the vicinity of the selectivity filter. Overall, our results show that the conformational dynamics associated with the selectivity filters of collapsed (Low K+) and open-inactivated KcsA are significantly different despite showing essentially identical conformations as derived from crystal structures. This highlights the importance of hydration dynamics in gating of K+ channels.

K(+) preference at the NaK channel entrance revealed by fluorescence lifetime and anisotropy analysis of site-specifically incorporated (7-hydroxycoumarin-4-yl)ethylglycine.

The fluorescent unnatural amino acid, (7-hydroxycoumarin-4-yl)ethylglycine (HC), was site-specifically incorporated at the Phe69 site, close to the entrance of the selectivity filter of the NaK channel. Decreased fluorescence lifetime and elevated time-resolved anisotropy of NaK-F69HC in buffers with high K(+)/Na(+) molar ratios indicated the K(+) preference at the entrance of the NaK channel, consistent with previous crystal structure results of the NaK channel.

A Comparison of Photophysical Characteristics of rHDL Encapsulated Anti-Cancer Drug Valrubicin and Free Valrubicin

Drug delivery via nanotechnology is one of the rapidly developing fields in cancer therapeutics. Targeted drug delivery has the advantage of having minimal interaction with healthy tissue, thereby reducing the toxicity of the drug to the rest of the body. rHDL nanoparticles are an efficient method of drug delivery for highly lipophilic anti-cancer drugs. Scavenger receptors class B type I (SR-BI), which are highly expressed on cancer cells interact with rHDL nanoparticles for effective drug delivery to the cancer cell and tumor. Valrubicin is an anti-cancer drug, with intrinsic fluorescence. In this experiment, we compared the photophysical properties of free valrubicin and rHDL valrubicin via steady state and time resolved fluorescence measurements. The steady-state anisotropy of rHDL valrubicin is higher as compared to free valrubicin, suggesting its encapsulation in rHDL nanoparticles. A longer rotational correlation time was observed for rHDL valrubicin in time resolved anisotropy measurements compared to free valrubicin, further supporting steady state anisotropy data.. We also studied the cellular internalization of free valrubicin and rHDL valrubicin using confocal microscopy. This could help track the movement of rHDL nanoparticles within the cancer cells.

Stimuli-sensitive breathing of Cucurbit[7]uril cavity: monitoring through the environment responsive fluorescence of 1'-hydroxy-2'-acetonaphthone (HAN).

In this work, we have focused on the supramolecular interactions of a water-soluble host Cucurbit[7]uril (CB[7]) with an excited state intramolecular proton transfer (ESIPT) probe 1'-hydroxy-2'-acetonaphthone (HAN) through steady-state and time-resolved fluorescence measurements. In water HAN is almost nonfluorescent in nature with a very low fluorescence quantum yield (Φ = 0.009). With gradual addition of CB[7] absorption maximum of HAN is red-shifted (292 cm(-1)) and hence confirming the formation of an inclusion complex in the ground state between HAN and CB[7]. Due to this complexation CB[7] offers a hydrophobic microenvironment to the HAN which is completely different from that of homogeneous water. Upon encapsulation into the nanocavity of CB[7], HAN exhibits a 20-fold increase in fluorescence intensity along with a 36 nm (1618 cm(-1)) hypsochromic shift in emission maxima. This hypsochromic shift is an indication about the modulation of excited state photophysical behavior of HAN due to the formation of HAN-CB[7] inclusion complex. Moreover, huge partition coefficient of HAN from water to CB[7] along with a∼ 12-fold increase in fluorescence lifetime confirm the favorable interaction between HAN and CB[7]. We have also observed the stimuli-sensitive (temperature and cationic stimuli) breathing of CB[7] cavity i.e., in the presence of different additives the portals of CB[7] open up to release HAN in water and take up the additives. Time-resolved anisotropy measurements further indicate about the probable location of HAN inside the CB[7]. The observation of a 1.7 ns component in the presence of CB[7] signifies the highly restricted rotational motion of HAN inside the cavity of CB[7] corroborates our finding.

Supramolecular interaction between a hydrophilic coumarin dye and macrocyclic hosts: spectroscopic and calorimetric study.

The photophysics of a hydrophilic molecule, 7-(diethylamino)-coumarin-3-carboxylic acid (7-DCCA), was studied in the presence of two macrocycles, (2-hydroxypropyl)-γ-cyclodextrin and cucurbit[7]uril. We have used steady-state absorption, fluorescence, and time-resolved fluorescence emission spectroscopy; Fourier transform infrared (FTIR) spectroscopy; (1)H NMR spectroscopy; and isothermal titration calorimetry (ITC) to confirm the supramolecular host-guest complex formation. The spectral properties of 7-DCCA were modulated in the presence of both macrocycles. It was assigned that 7-DCCA forms a 1:2 complex with (2-hydroxypropyl)-γ-cyclodextrin and cucurbit[7]uril. The large modulation of the emission properties of 7-DCCA in the presence of the macrocycles indicates the formation of supramolecular complexes. A significant shift in the bond vibration frequencies in the FTIR studies showed encapsulation of the dyes in the hydrophobic cavity of the macrocycles. This is further substantiated by the (1)H NMR studies, in which the upfield and downfield shifts of the protons were observed in both the aliphatic and aromatic region in the presence of macrocycles. The time-resolved anisotropy measurements further reinforce the conception of host-guest supramolecular complex formation because, in both cases, the rotational relaxation time increases significantly compared to that in water. A deeper understanding between the differences in interaction of an anionic molecule with cucurbit[7]uril and (2-hydroxypropyl)-γ-cyclodextrin will be achieved through this work. From the ITC measurement, we have formulated the forces due to complex formation.

Restricted State Selection in Fluorescent Protein Förster Resonance Energy Transfer

The measurement of donor lifetime modification by Förster resonance energy transfer (FRET) is a widely used tool for detecting protein-protein interactions and protein conformation change. Such measurements can be compromised by the presence of a significant noninteracting fraction of molecules. Combining time-resolved intensity and anisotropy measurements gives access to both molecular distance and orientation. Fluorescent proteins frequently used to detect energy transfer in biological systems often exhibit decay characteristics indicative of more than one excited state. However, little attention has thus far been given to the specific modes of energy transfer, in particular, which states are predominantly coupled. Here, we use a previously characterized dimerization system to study energy transfer between EGFP and mCherry. Optically excited EGFP and mCherry both exhibit biexponential decays, and FRET should therefore involve dipole-dipole transfer between these four states. Analysis of the sensitized fluorescence anisotropy and intensity decays indicates that FRET transfer is predominantly from the shorter lived EGFP emitting state (2.43 ns) to the longer lived (ca. 2.77 ns) minority component (ca. 16%) of the optically excited mCherry emission. This high degree of state selection between these two widely used FRET pairs highlights the fundamental differences that can arise between direct optical excitation of an isotropic molecular population and dipole-dipole coupling in a far from isotropic interaction geometry and has consequences regarding the accurate interpretation of fluorescent protein FRET data.

Photophysics of Delocalized Excitons in Carbazole Dendrimers

The photophysical properties in solution of three generations of carbazole-based dendrons and dendrimers with fluorenyl surface groups were studied using steady-state, time-resolved femtosecond transient absorption and anisotropy, and coherent two-dimensional ultraviolet spectroscopy. It was found that increasing the generation caused a switch in the nature of the emissive state between the first-generation compounds and the second- and third-generation dendrimers. Time-resolved anisotropy measurements revealed low initial anisotropies that decreased with increasing dendrimer generation consistent with increasing intradendrimer interchromophore coupling. Two-dimensional UV spectroscopy showed that the signal from the second- and third-generation dendrimers is the product of multiple chromophores interacting. The maximum number of interacting chromophores is reached by the second generation.

Homoconjugation in diporphyrins: excitonic behaviors in singly and doubly linked Zn(ii)porphyrin dimers

We have comparatively investigated excitonic features between singly and doubly alkyl bridged Zn(II)porphyrin dimers (SLZn and DLZn, respectively) with Zn(II)tetraphenylporphyrin (ZnTPP) as a reference by using various time-resolved anisotropy measurements. Time-resolved fluorescence anisotropy decay of the B-state of ZnTPP in toluene showed ultrafast dephasing times of 83 and 185 fs with an initial anisotropy value (r0) of ∼0.7 being consistent with the theoretically proposed one. On the other hand, the Q-state of ZnTPP exhibited a depolarization time of 120 fs with a smaller r0 of 0.25 than that of the B-state because of probing photo-induced absorption in transient absorption anisotropy measurements. Spectroscopic features in absorption, fluorescence, and excitation anisotropy spectra of SLZn are similar to those of ZnTPP indicating a relatively weak exciton coupling in the Q-state. As a result, the observed depolarization time with a time-constant of 1.4 ps can be explained by a Forster-type incoherent energy transfer process between two constituent subunits. When compared to ZnTPP and SLZn however, DLZn exhibited very different optical characteristics such as more red-shifted absorption and emission spectra, three times larger fluorescence excitation anisotropy values, and ultrafast sign inversion in transient absorption anisotropy. According to these results, it can be inferred that the overall π-electron densities of the electronic excited states in DLZn are fully delocalized in a whole dimer and that DLZn can be regarded as a coherently coupled single quantum system not as separate individual chromophores. Frontier π-molecular orbital structures as well as electron density difference maps between occupied and unoccupied molecular orbitals involved in the electron promotions for underlying excited states clearly represent weakly and strongly exciton-coupled natures of SLZn and DLZn, respectively. Accordingly, the extended conjugation of π-electrons over the whole DLZn molecule demonstrates an example for homoconjugation between two aromatic porphyrins.

Unusual Optical Resolution of All Four Tryptophan Residues in MPT63 Protein by Phosphorescence Spectroscopy: Assignment and Significance

MPT63, a secreted protein of unknown function that is specific to Mycobacterium tuberculosis and a potential drug target, contains four Tryptophan (Trp/W) residues located at positions 26, 48, 82, and 129 in the amino acid sequence. All of the four Trp residues have been optically resolved by simple inexpensive phosphorescence spectroscopy at 77 K. The protein architecture provides a delicate micro-environment and location of Trp residues giving rise to four different (0,0) bands in the phosphorescence spectra. Calculation of intra Trp energy transfer (ET) efficiency, accessible surface area (ASA) of Trp residues, and environment of Trp in the wild-type (WT) and the mutant W26F [where, Trp 26 is replaced by phenyl alanine (Phe/F)] reveal: E(T1) (W82) > E(T1) (W48) > E(T1) (W129) > E(T1) (W26), where E(T1) is the lowest (π-π*) triplet state energy of Trp. The (0,0) band observed at 421.6 nm assigned for Trp 26 is found to be the longest wavelength (0,0) band so far reported in the literature. Fluorescence in WT and W26F is dominated by buried or partially exposed Trp residues indicated by time-resolved spectra. Circular Dichroism (CD) studies and the time-resolved anisotropy measurement confirm the unaltered secondary and tertiary structure of the mutant compared to that of the WT. Excitation energy dependent phosphorescence spectra suggest that the intensity of the different (0,0) bands could be tuned and Tyrosine (Tyr/Y) residue is silent in emission. Optical resolution of all the Trp residues will help understand the role of each Trp residue in the folding/unfolding mechanism and in the interaction with other systems.

Fluorescence Study of Energy Transfer in PMMA Polymers with Pendant Oligo‐Phenylene‐Ethynylenes

A spectroscopic characterization of polymers containing rigid π-conjugated oligo(phenyleneethynylene) chromophores as well as oligo(phenyleneethynylene) and methyl methacrylate is presented. The polymers exhibit molar masses of up to 15,000 g mol(-1) and a degree of polymerization between 22 and 80. Emission measurements of the monomeric and polymeric species show that radiative as well as nonradiative rates are influenced by the degree of polymerization due to intramolecular interactions of chromophores pendant to the polymer backbone. Time-resolved emission anisotropy measurements suggest that energy migrates within the polymers. Steady-state emission anisotropy measurements also point to energy migration. Additionally, two oligo(phenyleneethynylene)s with different sizes of the conjugated system are copolymerized in order to enable energy trapping due to energy transfer. The shortened energy-donor fluorescence lifetime within the donor-acceptor copolymers suggest energy transfer. Depending on the degree of polymerization, dispersion of the donor fluorescence lifetime is observed.

Structural Spectroscopy and Dynamics of Inter- and Intramolecular H-Bonding Interactions of Topotecan, a Potent Anticancer Drug, in Organic Solvents and in Aqueous Solution

We report on the role of H-bonding interactions on the UV-visible absorption and emission (steady-state and time-resolved) spectroscopy of topotecan (TPT) in solution. In aprotic solvents, a very fast (less than 10 ps) excited-state intramolecular proton-transfer reaction occurs in the absorbing enol (E) form to give a zwitterion (Z) form, emitting with a large Stokes shift. In protic solvents like methanol, the time constant of Z* formation is longer (32 ps) due to the participation of solvent molecules in the proton-transfer reaction. In aqueous solution at near-neutral pH (6.24), a ground-state equilibrium is established between E, cation (C), and Z forms. Direct excitation of E leads to Z* through two channels: a very fast one (less than 10 ps) involving an intramolecular proton-tranfer and a slower one (680 ps) with the C* intermediate formation and reaction. A fast (42 ps) deprotonation of E* to give the excited anion (A*) also competes with the photoformation of Z* at the S(1) state. At pH =12.15, the A structures are the principal emitting species (τ(A) ~ 0.41 ns), showing the largest Stokes shift. In aqueous solutions, we cannot exclude the existence of an equilibrium between the lactone and carboxylate forms of TPT, whose spectroscopic (absorption and emission spectra) and dynamical behaviors should not be very different. Time-resolved emission anisotropy measurements in solvents of different viscosities suggest that the rotational relaxation time (φ) of TPT is mainly governed by the viscosity of the medium, increasing from 104 ps (in tetrahydrofuran, THF) to 156 ps (in water) and 338 ps (in dimethyl sulfoxide, DMSO). These results give spectroscopic and dynamical information on the structures, stability, and dynamics (picosecond to nanosecond time scale) of TPT in solution. They provide insights on the role of the intermolecular H-bonding surrounding medium on the ground- and excited-state structure and reaction of TPT. The finding should contribute to a better understanding of the relationship between the structures of the drug and its surroundings.