Ultrafast dynamics of a molecular rotor in chemical and biological nano-cavities

Abstract

Molecular rotors have become indispensable tools in monitoring several important processes in chemistry and biology owing to their sensitivity towards viscosity. Despite their importance, less attention has been paid to understanding the excited state properties of molecular rotors. Recently, Maroncelli and coworkers unraveled the excited state photochemistry of a julolidine based molecular rotor, 9-(2-carboxy-2-cyano)vinyl julolidine (CCVJ), and claimed that CCVJ is not a simple rotor probe. Unlike other molecular rotors, photoisomerization is believed to be the main non-radiative decay pathway for this molecule. Inspired by their report, herein, we tried to understand how the excited state dynamics of CCVJ is affected inside the nano-cavities of cyclodextrins (CDs) and human serum albumin (HSA) protein using steady-state and femtosecond fluorescence up-conversion techniques. We observed a pronounced enhancement in fluorescence quantum yield when CCVJ is encapsulated in CDs (β- and γ-CD) and HSA. Femtosecond up-conversion studies reveal that the ultrafast dynamics of CCVJ are drastically retarded inside the nano-cavities of CDs and protein. All these results suggest that photoisomerization, which is believed to be the major non-radiative decay pathway of CCVJ, is severely restricted inside the abovementioned bio-mimetic and biological nano-cavities. The molecular images of orientations of CCVJ inside the nano-cavities of CDs and protein have been discussed by theoretical and molecular modeling studies. We believe the present results might be helpful in exploiting this molecule more in biological and viscosity sensing applications.

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Introduction

Molecular rotors, a special class of fluorescent molecules, are used to probe many important chemical and biological processes such as polymerization progress, peptide–protein interaction, assembly processes of tubulin and phase transitions of phospholipid bilayers.^1–6 They have also been employed to probe local viscosity in simple and complex biological media.^7–15 Among the various molecular rotors, 9-(dicyanovinyl)-julolidine (DCVJ) and 9-(2-carboxy-2-cyano)vinyl-julolidine (CCVJ) are the most commonly used molecular rotors. Different mechanisms have been proposed regarding the excited state process responsible for the environmental sensitivity of these probes. Computational and experimental studies suggested that the isomerization about the double bond (similar to the well-known behavior of styryl compounds¹⁶) is mainly accountable for the excited state non-radiative decay pathway of these types of molecules.¹⁷ The TICT mechanism, which involves the twisting of the aryl–alkenyl single bond, is also still widely accepted as non-radiative decay channels of these types of molecules.^13,18–20 In order to address this issue, very recently, Maroncelli and co-workers explored the excited state photochemistry of CCVJ.²¹ They reported that isomerization, rather than the commonly assumed TICT process, dictates the viscosity-sensing ability of CCVJ. In hydroxylic solvents, CCVJ exists primarily in the carboxylate form of the E isomer (Scheme 1).²¹ Apart from the abovementioned studies, limited research on this molecule is devoted to understanding its excited state dynamics. In the present study, we attempted to understand how the non-radiative decay pathways, including the photo-isomerization process, would be affected in confined environments. Moreover, to exploit this molecule in biological applications, it is necessary to have a proper knowledge about the environment-dependent fluorescence properties of CCVJ, which is lacking in the literature. In an endeavor to address the aforesaid points, we investigated the excited state fluorescence properties inside bio-mimicking nanocavities, i.e., cyclodextrins (CDs), as model supramolecular nano-cavities and protein nano-cavities, i.e., human serum albumin (HSA) as a model protein.

Scheme 1 Chemical structure of CCVJ (E isomer).

Herein, for the first time, we explored the ultrafast dynamics of CCVJ in an aqueous solution, CDs and HSA using the femtosecond fluorescence up-conversion technique. The excited state dynamics of CCVJ are found to be strongly dependent on the surrounding confinement effect of the medium. Importantly, the fluorescence quantum yield of CCVJ is enhanced many times in CDs and protein due to the retardation of non-radiative processes inside the nano-cavities of the hosts and HSA. Fluorescence lifetime of CCVJ becomes several times slower inside the nano-cavities of CDs and HSA, confirming the restriction of rotational motions of CCVJ inside the abovementioned constrained media. Finally, the possible orientations of CCVJ inside the nano-cavities of CDs and protein have been predicted by molecular modeling and theoretical studies.

Materials and methods

CCVJ (purity ≥ 97%, HPLC), CDs and HSA were purchased from Sigma Aldrich and used without further purification. All the experiments were conducted in 10 mM sodium phosphate buffer of pH 7. CD/HSA was gradually added to the solution containing CCVJ, and the solution was gently shaken after each addition until CD was completely solubilized.

Absorption measurements were performed on a Perkin-Elmer UV-visible spectrophotometer (Lambda-45) and steady-state fluorescence spectra were obtained with a Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon). Time-resolved fluorescence measurements were initially collected on a time correlated single photon counting (TCSPC) spectrometer (Horiba Jobin Yvon IBH, U.K.). The detailed description of the instrument is described elsewhere.²² Briefly, in the present study, a 440 nm diode laser (∼100 ps, 1 MHz repetition rate) was used as the excitation source and a MCP-PMT detector was used for collecting the fluorescence signal. The analysis of the lifetime was done by the IBH DAS6 analysis software.

Ultrafast fluorescence decay transients were measured using a femtosecond fluorescence up-conversion setup (FOG 100, CDP) with a description given elsewhere.²³ Briefly, the sample was excited at 420 nm using the second harmonic of a mode-locked Ti-sapphire laser (Mai-Tai, Spectra Physics). The fluorescence emitted from the sample was up-converted in another nonlinear crystal using a gate pulse (840 nm) of the fundamental beam. The sum frequency of the fluorescence and gate pulse was detected as a function of the time delay between excitation and gate pulses. The angle between the polarization of the pump and gate pulses was maintained at the magic angle to eliminate effects from rotational diffusion. The up-converted signal was dispersed in a monochromator and detected using photon counting electronics. A cross-correlation function obtained using the Raman scattering from ethanol provided a full-width at half-maximum (fwhm) of ∼350 fs. Estimated uncertainties in the up-conversion measurements are ∼15–20%.

Molecular docking was carried out by the standard protocol and the details are described elsewhere.²⁴ CD was taken as the receptor and the anion form of CCVJ, i.e., the E isomer was used as the ligand in the docking protocol. During docking, the receptor was kept rigid and the ligand was flexible. For all docking, we used Gasteiger charges for both the CDs and the ligand. The grid was generated on a whole receptor with grid points of 60 on each of the orthogonal directions with a spacing of 0.16 Å. The search was performed using a genetic algorithm. Finally, all the docked complexes were fully optimized by PM3 semi-empirical methods using Gaussian software.²⁵

Results and discussion

Steady-state results

CCVJ exhibits an absorption maximum at 430 nm in an aqueous buffer solution (Fig. 1). In the presence of CDs, the absorption profiles exhibit some changes depending upon the cavity sizes of the CDs. As shown in Fig. 1a, no change is observed in the absorption spectra in the presence of α-CD, suggesting no significant interaction between CCVJ and α-CD. Unlike the α-CD case, the absorption maximum of CCVJ undergoes a hypsochromic shift in the presence of β-CD (Fig. 1b), and this observation infers that there exists a considerable interaction between CCVJ and β-CD. In the presence of γ-CD, analogous results are observed indicating that similar types of interactions are present in both of these host molecules. The hypsochromic shift in absorption profiles in the presence of CDs (β- and γ-CD) is indicative towards the change in polarity of CCVJ surroundings when it interacts with β- and γ-CD. In summary, the absorption results infer that the ground state of CCVJ molecules interact with β- and γ-CD.

Fig. 1 Absorption spectra of CCVJ (10 μM) in the presence of (a) α-CD (0 to 10 mM), (b) β-CD (0 to 10 mM) and (c) γ-CD (0 to 10 mM). Arrows indicate the direction of increasing concentration of CD.

To gain insight into the excited state properties, emission profiles of CCVJ are monitored in the absence and presence of various cavity sized CDs. CCVJ exhibits weak fluorescence (Φ_f = 0.0016)¹¹ with an emission maximum situated at 500 nm in a neutral buffer solution (Fig. 2). It is pertinent to mention that CCVJ exists mainly in the carboxylate form (E isomer) in buffer or water. However, when E isomers are excited, they convert to non-fluorescent Z isomers. Hence, the observed fluorescence spectrum with a peak of ∼500 nm is originated from the S₁ state of the E isomers of CCVJ. In the presence of α-CD, a slight increase in fluorescence quantum yield is found (∼1.3 times) without showing any hypsochromic shift (Fig. 2a, Table 1). However, a significant alteration in emission profiles is observed in the presence of β-CD. Herein, a ∼10 fold enhancement in fluorescence quantum yield is noticed (Fig. 2b, Table 1) at the highest concentration of β-CD. Besides the increase in quantum yield, a hypsochromic shift from 500 nm to 475 nm is observed. The abovementioned findings infer a strong interaction between CCVJ and β-CD, whereas a weak interaction may prevail with α-CD. The observed hypsochromic peak shift suggests that E isomers are in a less polar environment in the presence of β-CD and it has been reported that the E isomer is sensitive to the polarity of the surrounding environment.²¹ This confirms that E isomers are displaced from the water environment, and subsequently, they are encapsulated inside the less polar nano-cavity of β-CD. The increase in fluorescence quantum yield can be ascribed to the inhibition of the photoisomerization process, which is believed to be the main non-radiative decay pathway of CCVJ.²¹ As there is no hypsochromic shift observed in the case of α-CD, the possibility of inclusion complex formation between α-CD and CCVJ can be ruled out. This is further justified considering the cavity size of CD (the internal cavity diameter of α-CD is 5.7 Å) and size of a CCVJ molecule (according to the geometry optimized structure, the width of a CCVJ molecule is ∼7 Å along the julolidine group and the length of a CCVJ molecule is ∼9.2 Å). However, partial inclusion complexes (incorporation of cyano, carboxy-vinyl group of CCVJ) as well as hydrogen bond interaction between the carboxyl group and –OH groups of α-CD cannot be ruled out. To confirm the latter possibility, we performed a control experiment with glucose. However, no significant enhancement in fluorescence intensity is observed (Fig. S1†), confirming that the hydrogen bond interaction between the carboxyl/cyano group of CCVJ and –OH groups is not responsible for the observed change in the presence of α-CD. Similar to β-CD, the fluorescence quantum yield of CCVJ increases continuously with gradual addition of γ-CD (Fig. 2c, Table 1), and at maximum γ-CD concentration, a ∼4.5 fold increase in fluorescence quantum yield along with a 10 nm hypsochromic shift is observed. Certainly, this increase as well as the shift is less compared to β-CD. The dissimilar extent of the rise in quantum yield in CDs is due to the different extent of rigidity felt by E isomers of CCVJ in various CDs. Therefore, the cavity size of the host has a major role in the observed changes in fluorescence quantum yield of CCVJ in different CDs. Among all three CDs, the increase in fluorescence quantum yield of CCVJ is more in the case of β-CD, and it is attributed to the more restricted environment inside the β-CD nano-cavity. Since the internal cavity diameter of β-CD is ∼7 Å and the depth of the cavity is ∼8 Å, a CCVJ molecule can perfectly fit inside the nano-cavity of β-CD, thereby, restricting its rotational motion along the vinyl bond as well as the twisting motion along the aryl–alkenyl single bond. On the other hand, the internal cavity diameter of γ-CD (∼9 Å) is large enough compared to the size of the E isomer. As a result, the incorporation of a CCVJ molecule into the γ-CD cavity cannot fully restrict the photo-isomerisation process, which is believed to be the main non-radiative decay pathway of the CCVJ molecule.²¹ The large hypsochromic shift inside the β-CD cavity is attributed to the less polar nano-cavity experienced by the E isomer of CCVJ compared to the γ-CD nano-cavity. To gain more insight into the inclusion complex formation, stoichiometry and association constants are calculated using the Benesi–Hildebrand (BH) equation.²⁶

where F₀, F and F₁ are the fluorescence intensities of the E isomer of CCVJ in the absence and presence of the host and in the inclusion complex, respectively. The double reciprocal plot of 1/ΔF vs. 1/[CD] yields a straight line (Fig. S2†), confirming 1 : 1 stoichiometry for both β- and γ-CD. The association constants for β- and γ-CD are calculated to be ∼1300 (±100) M⁻¹ and 450 (±20) M⁻¹, respectively. The three times higher binding constant for β-CD is in good agreement with our conjecture that CCVJ forms a more stable inclusion complex with β-CD than γ-CD.

Fig. 2 Emission spectra (λ_ex = 430 nm) of CCVJ (10 μM) in the presence of (a) α-CD (0 to 10 mM), (b) β-CD (0 to 10 mM) and (c) γ-CD (0 to 10 mM). Arrows indicate the direction of increasing concentration of CD.

Table 1 Lifetime, quantum yield, radiative and non-radiative decay rate constants of CCVJ in a buffer and in the presence of various host molecules

Sample	a1^a	τ1 (ps)	a2^b	τ2 (ps)	Φf^c	kr (ns)^−1	∑knr (ns)^−1
a Relative amplitude for τ1.b Relative amplitude for τ2.c ∼10% error in measurement.
CCVJ in buffer	1.00	2.87	—	—	0.0016	0.56	347.87
α-CD 15 mM	0.90	2.90	0.10	16.00	0.0024	0.15	62.35
β-CD 14 mM	0.35	2.90	0.65	35.00	0.02	0.57	28.00
γ-CD 15 mM	0.56	3.00	0.44	24.00	0.0087	0.36	41.30
HSA 100 μM	0.25	3.00	0.75	84.00	0.0895	1.07	10.84

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Time-resolved fluorescence study

Since the internal molecular rotations of CCVJ are expected to be severely restricted on forming an inclusion complex with CDs, it is expected that the fluorescence lifetime of CCVJ should be affected inside the nano-cavities of CDs. Therefore, we probed the inclusion complex formation by monitoring the fluorescence lifetime of CCVJ in the presence of CDs. In buffer solutions, CCVJ exhibits a fast decay comparable with the time-response of our TCSPC set-up, i.e., IRF. Hence, it cannot be resolved using the TCSPC set-up (Fig. S3†). Thus, we used the fluorescence up-conversion technique to unravel the ultrafast dynamics of CCVJ (Fig. 3). In buffer solutions, the emission decay of CCVJ is well represented as a mono-exponential function with a lifetime of ∼2.87 ps (Table 1). Notably, this is the first ever report regarding the excited state lifetime of CCVJ in a water environment. As the E isomer of CCVJ is only emissive in nature, the detected lifetime certainly reflects the excited state lifetime of the E isomer in the water environment. The short lifetime (∼2.87 ps) observed in water indicates that the non-radiative decay pathway, mainly via photoisomerization process,²¹ reduces its excited state lifetime. In the presence of α-CD, the decay fits well with two lifetime components of 2.90 (90%) and 16 ps (10%) (Fig. 3, Table 1). The fast lifetime component is attributed to the un-complexed CCVJ in a solution, whereas the longer lifetime component (16 ps) may appear as a result of interaction between CCVJ and α-CD. The smaller amplitude of long component indicates that minute populations of E isomers interact with the host. As the molecule cannot be accommodated inside the cavity of α-CD (confirmed from steady state results), the observed 16 ps lifetime may be attributed to some sort of partial inclusion complex formation, which slightly suppresses the photoisomerization process and is believed to be main non-radiative decay pathway for CCVJ.²¹ More evidences for the partial inclusion complex formation are provided by the docking and computational studies discussed later in the manuscript. In the presence of β- and γ-CD, the decay profiles of CCVJ are also bi-exponential in nature. In both cases, a long lifetime component (35 and 24 ps, for β- and γ-CD, respectively) along with a fast component (originated from the uncomplexed CCVJ molecules) is detected (Fig. 3, Table 1). The large increase in lifetime values can be explained in terms of restriction of photoisomerization process around the vinyl bond (which is believed to be main non-radiative decay pathway of CCVJ) in the inclusion complexes. As a result, the non-radiative decay pathways are significantly reduced. The longer lifetime of CCVJ in the case of β-CD is attributed to the formation of a more rigid complex with β-CD than γ-CD due to the perfect fitting of CCVJ inside the β-CD cavity, whereas γ-CD has a bigger cavity size, which may not hold the CCVJ molecule as tightly as the β-CD cavity.

Fig. 3 Femtosecond resolved fluorescence decays of CCVJ (∼100 μM) in the presence of α-CD (15 mM), β-CD (14 mM), γ-CD (15 mM) and HSA (100 μM). Excitation wavelength is 420 nm and collection wavelength is at emission peak maximum.

For the further understanding of radiative and non-radiative decay pathways, we calculated radiative (k_r) and non-radiative (k_nr) decay rate constants using the following equations:

The calculated decay rate constants (considering the lifetime component of bound CCVJ) are summarized in Table 1. Herein, it is pertinent to mention that the long lifetime components, which reflect the lifetime of bound CCVJ, are considered for calculating k_r and k_nr. The very low k_r and high k_nr values are in good agreement with the very low fluorescence quantum yield as well as the faster lifetime component of CCVJ in water. In the presence of α-CD, the k_r value increases ∼3.5 times; however, the k_nr value decreases almost 5.5 times. Considering the steady state and lifetime results, the increase in k_r and decrease in k_nr values may be attributed to the formation of a partial inclusion complex between CCVJ and α-CD. In the presence of β-CD, an almost 14 times reduction in k_nr confirms that the non-radiative internal rotations are drastically reduced inside the nano-cavity of the supramolecular host. For γ-CD, similar observations are found. However, in this case, the suppression of k_nr is less (∼8.75 times) when compared to β-CD. The lower k_nr value inside the β-CD nano-cavity can be attributed to the formation of a more rigid inclusion complex with the E isomer, hence the restriction of internal molecular rotations (includes photoisomerization process²¹) is increased. In summary, lifetime results and decay rate constants are in good agreement with steady state findings.

Binding interactions with HSA

It is not enough to only examine the dynamics of CCVJ inside bio-mimicking chemical nano-cavities, it is also necessary to understand the effect of confinement of biological medium on the excited state properties of CCVJ. Therefore, we investigated the environment-dependent fluorescence of CCVJ inside a model protein (HSA) in the context of specific biomolecular interactions. Absorption and emission spectra of CCVJ in the absence and presence of HSA are shown in Fig. 4. The absorption maximum of CCVJ undergoes a blue shift in the presence of HSA. In the emission spectra, a huge enhancement in quantum yield (∼55 times) accompanied by a hypsochromic shift is observed with gradual addition of HSA. The vast increase in fluorescence quantum yield confirms the strong binding affinity of CCVJ with the protein, whereas the blue shift in the emission peak is attributed to the reduced polarity experienced by E isomers of CCVJ inside the protein binding pocket. These results bear a close resemblance to the observations with CDs (β-CD and γ-CD). However, the huge increase in quantum yield and blue shift (Fig. 4) indicates the strong binding affinity of E isomers towards HSA. The binding constant (K_a) with HSA is calculated using eqn (1). The association constant (K_a) is found to be 1.0 × 10⁵ M⁻¹ (Fig. S4†). Certainly, this value is several times higher than the binding constant observed in the presence of β-CD, inferring that CCVJ binds more strongly to HSA compared to β-CD.

Fig. 4 Absorption (a) and emission (b) spectra of CCVJ (10 μM) in the presence of HSA (0 to 100 μM). Excitation wavelength is 430 nm. Arrows indicate the direction of increasing concentration of HSA.

To gain deeper insight into the underlying photo-physical behaviour of the rotor upon interaction with the protein, fluorescence lifetime measurements were performed for CCVJ in the presence of a protein environment. The fluorescence up-converted decay profile of CCVJ in the presence of HSA exhibits a comparably slow lifetime (∼84 ps) component (Fig. 3). This time constant is certainly higher than for the CCVJ:β-CD complex, wherein a ∼35 ps lifetime component is observed. Moreover, a 30 times decrease in the k_nr value compared to uncomplexed CCVJ is observed. Lower k_nr and longer lifetime values compared to CDs confirm that the inhibitions of non-radiative internal molecular rotations are more inside the protein nano-cavity. As the E to Z conversion is one of the major non-radiative decay pathways,²¹ we believe that this conversion process is restricted more inside the protein nano-cavity compared to cyclodextrin nano-cavities.

Molecular docking and computational study

To obtain the molecular image of the orientation of CCVJ in the inclusion complexes, we docked the ligand into CDs and simulated the structure using quantum chemical calculations. The optimized structures are shown in Fig. 5. As shown in Fig. 5, CCVJ is encapsulated inside the CDs (β- and γ-CD) in such a way that both the vinyl bond (having cyano and carboxylate groups) as well as aryl–alkenyl single bond stay inside the nano-cavities, thereby the twisting and/or conversion of E to Z isomers are believed to be reduced drastically inside the nano-cavity. While in the case of α-CD, CCVJ is not completely encapsulated in the nano-cavity, instead it resides at the hydroxyl portals of CD with cyano and carboxylate groups projected partially towards the cavity. This partial encapsulation of CCVJ results in the slight restriction of internal molecular rotation, which is believed to be responsible for the observed changes in steady state and time resolved results. The smaller cavity size of β-CD compared to γ-CD might be responsible for the observed dramatic changes in the presence of β-CD. In summary, the computational studies further support and confirm the experimental evidences.

Fig. 5 Geometry optimized structures of inclusion complexes of CCVJ with (a) α-CD, (b) β-CD and (c) γ-CD.

To locate the dye in a protein nano-cavity, CCVJ is docked with HSA. The negatively charged E isomer of CCVJ is taken as the ligand because it exists as an anion in solutions. Fig. 6 shows the CCVJ docked protein complex with minimum energy in the cluster analysis. The most stable or least energetic structure clearly depicts that CCVJ resides at the domain II (IIA) binding pocket of the protein. This binding site is well-known to have higher binding affinities for negatively charged ligands,^27–30 and therefore the location of CCVJ in this binding pocket is justified. The less polarity at this binding site^27–30 is believed to be responsible for observed blue shift in steady state emission spectrum. Moreover, the negatively charged E isomer of CCVJ is surrounded by positively charged basic amino acid residues, Lys286, Arg257, Lys289 and His242. These basic amino acids may be the main driving force for attracting CCVJ towards the binding pocket IIA of the protein. In particular, carboxy and cyano groups of CCVJ are involved in hydrogen bond interactions with two basic amino acids (Lys199 and Arg222). Therefore, the E isomer of CCVJ is believed to be stabilized by positively charged amino acids located at this binding site through both electrostatic and hydrogen bonding interactions (Fig. 6b).

Fig. 6 (a) Docked structure of CCVJ complex with HSA. (b) Interaction of CCVJ with amino acids at the binding pocket.

Conclusion

We investigated the excited state dynamics of CCVJ in the presence of molecular container (cyclodextrins (CDs)) and protein (human serum albumin (HSA)) environments. The marked enhancement in fluorescence quantum yield as well as hypsochromic shift indicates that CCVJ molecules are displaced from water and are encapsulated inside the less polar nano-cavities of CDs (β- and γ-CD) and HSA, whereas partial inclusion complex formation is observed for α-CD. Our femtosecond fluorescence up-conversion study reveals that the ultrafast dynamics of CCVJ are severely hindered inside the nano-cavities of CDs and protein due to the restriction imposed on free internal molecular rotation of CCVJ. As a result, the fluorescence lifetime significantly increases inside the abovementioned constrained media. Interestingly, in accordance with quantum yield and lifetime results, the non-radiative rate constants decrease several times inside the abovementioned nano-cavities. However, the extent of decrease depends on the cavity size. All the abovementioned observations lead us to conclude that the photo-isomerization process, which is believed to be major non-radiative decay pathway of CCVJ, is severely restricted inside the abovementioned bio-mimetic and biological nano-cavities. The possible orientations of CCVJ inside the nano-cavities of CDs and protein have been discussed by theoretical and molecular modeling studies.

Acknowledgements

Authors thank IISER-Pune for providing excellent experimental and computation facilities. Authors are thankful to anonymous reviewers for their valuable comments and suggestions.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra13298c

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