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. 2019 Mar 4;4(3):4785–4792. doi: 10.1021/acsomega.8b02357

Unique Photophysical Behavior of Coumarin-Based Viscosity Probes during Molecular Self-Assembly

Silvio Panettieri †,‡, Julian R Silverman †,‡, Riccardo Nifosí §, Giovanni Signore ∥, Ranieri Bizzarri §,⊥,*, George John †,‡,*
PMCID: PMC6648471  PMID: 31459662

Abstract

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Intermolecular interactions impact self-assembly phenomena having a variety of bio/chemical, physical, and mechanical consequences. Nevertheless, the underlying mechanisms leading to a controlled stereo- and chemo-specific aggregation at the molecular level often remain elusive because of the intrinsically dynamic nature of these processes. Herein, we describe two 3-styryl coumarin molecular rotors capable of probing subtle intermolecular interactions controlling the self-assembly of a small-molecule organogelator. Complementing the characterization of the gel via circular dichroism and atomic force microscopy, thorough spectroscopic investigations on these sensors were carried out to prove their high chemical and spatial affinity toward the 3D supramolecular network. The results were further supported by molecular dynamics simulations to reveal further critical insights into the gelator’s dynamic self-assembly mechanism. These sensors could potentially serve as templates to study a variety of soft-supramolecular architectures and the ways in which they assemble.

Introduction

Proposing an innovative design paradigm for smart functional materials in the field of soft matter requires fundamental understanding of both material and functional properties. A class of fluorescent probes known as molecular rotors exhibit predictable variations in their spectral properties in response to changes in their local environment.1−3 Such probes can sense and photophysically respond to the friction generated by intermolecular forces, thereby acting as tools to measure viscosity (η) at the nanoscale.2,4−6 As molecules self-assemble in solution, there are a myriad of factors which can control fibril and fiber growth, their directional and hierarchical architectures, and the resultant dynamic properties of these structures.7,8 To better understand both the static nature of the structuring gellant and dynamic nature of a gel’s bulk liquid, several different techniques have been developed to study both viscosity and flow.9,10 Leveraging the photophysical properties of small molecules, which can be demonstrated not to interfere in the dynamical processes allows researchers to study the gellant’s structure and gel’s potential functions.11,12 These techniques are of great interest to study biological systems in situ as the complex environments of both cytoplasm and thickened extracellular fluids can govern the reactivity and biochemistry of natural processes.9,13 Beyond this, understanding and learning to control the phase transitions and rheology of oil and gas mixtures and consumer products can help serve to impact the efficiency and economics of transferring and storing liquid, solid, and hybrid-phase solutions.14,15

Molecular organogels are soft viscoelastic solids comprised of self-organized unit structures, which can form fibers and trap solvent molecules within their assemblies.16,17 In particular, a clear description of the nature of molecular assembly and nanoscale morphology requires fundamental understanding of the molecular interactions leading to gelation.18 Although these organogel systems display an infinite bulk viscosity, most demonstrate liquid-like properties, being primarily composed of solvents.19,20 Nevertheless, various efforts have been made to understand their assembly process,21,22 the real-time probing of the self-assembly process through change in intermolecular interactions within the 3D network is rarely explored. In this context, fluorescent molecular rotors could potentially serve as an experimental platform to investigate the role of changing intermolecular forces in the 3D-network formation throughout gelation. Inspired by some of the previous reports on the use of stimuli-responsive systems toward probing the interactions in soft supramolecular assemblies,23−29 we scrutinized the dynamic and complex process underlying the gel formation of a low-molecular-weight gelator by leveraging the dependence of these fluorescent probes upon molecular motion restriction. By synthesizing glucose and acid group-containing derivatives tailored to distinctively complement an amphiphilic small-molecule glucoside ester gelator, we may be able to study fibril formation and gel setting thus leveraging complementary and programmed noncovalent interactions. Herein, we describe the experimental investigation of molecular self-assembly supported substantially through computational studies to explore the potential of these as-prepared functional smart materials as tools for characterizing soft-supramolecular assemblies. To better understand their structure-property relationship, we present here a combined aspect of spectroscopy and microscopy, correlated with computational evidence.

Results

The viscosity-dependence of the novel coumarin-based molecular rotor C1 was assessed in toluene–Polyethylene glycol (PEG) methyl ether methacrylate mixtures (Figure 1, Supporting Information Figure S7). C1’s fluorescence intensity displays a linear double-logarithmic dependence on bulk viscosity with a slope of 0.2 (Supporting Information Figure S8). This supports the rotor-like photophysical behavior of C1, even though a traditional molecular rotor strictly follows Forster–Hoffman behavior,28 that is, displaying a slope of around 0.5. This seems to suggest that C1 possesses a more complex photophysical mechanism than traditional rotors, which will be the object of the future study. The restriction of intramolecular motion in increasingly viscous media helps C1 to exhibit emission from the local excited state and modulation of the overall quantum yield via twisted intramolecular charge-transfer transition state, similar to other stilbene-based rotors.1,30,31

Figure 1.

Figure 1

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(a) Molecular rotor (C1) chemical structure of the molecular rotor C1 (b) and fluorescence spectra at increasing viscosity (viscosity values in cSt: 0.7, 53, 100, 280, and 440).

C1 was tested in low concentrations in toluene gelled with the raspberry ketone glucoside caprylate gelator (R8, Figure 2).19 This compound exemplifies a vast family of amphiphilic organogelators bearing a highly polar sugar core and at least one long partially hydrophobic alkyl chain.32,33R8 possesses a minimum gelation concentration of 0.46 wt % in toluene, and a temperature of gelation at 1.0 wt % (w/v) of 45–47 °C. Optically clear gels can be formed through thermoreversible gelation (Figure 2b).19,34 Oscillatory dynamic rheology demonstrates the thixotropic (mechano-reversible) behavior of the toluene gels (Supporting Information Figure S9) common to a variety of polymeric and molecular gels.35,36 In nonpolar solvents, X-ray diffraction highlights the β′ conformation of the fatty acid tails on the exterior of the 3D-network indicating that the gelator packs in a fashion similar to natural triglycerides in a 3D-epitaxial arrangement.19 UV–vis circular dichroism (CD) of the unassembled gelator in ethanol and the 1.0 wt % (w/v) toluene sol above the temperature of gelation (45–47 °C) (Figure 2c, red and green line, respectively) demonstrated primarily a small positive band corresponding to the ketone absorption at 270 nm. Around 4 min into gelation, the system exhibited a substantial negative excitonic CD signal arising from the alkyl phenol moiety at 290 nm (blue line, Figure 2c).37

Figure 2.

Figure 2

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(a) R8 gelator system: chemical structure of the gelator R8 and (b) 1% toluene solution of R8 before gelation (left vial) and after (right vial). (c) CD spectra of R8 toluene solutions in different conditions: R8 sol in ethanol at RT (red line), 1% R8 sol in toluene (green line) and 1% R8 gel in toluene (blue line).

These results suggest that the supramolecular gelator architecture is itself chiral in nature. This observation matches well with atomic force microscopy (AFM) images of a drop-cast 1.0 wt % (w/v) toluene gel, clearly showing right-handed nanoscopic helices about 6 nm thick and more than 200 nm-long (Figure 3a,b). Molecular and structural investigation of the fibrils via coarse-grained molecular dynamics (MD) simulations suggests that the nonpolar alkyl tail and phenolic head reside on the outside of the fibril, whereas the hydrophilic glucose moiety faces inwards, as expected in toluene (Figure 3c–e, and Supporting Information Figure S11). Large-scale MD simulations suggested that the helical architecture results from the intertwining of two tubular structures each of which consists of four-molecule discs stacked longitudinally (Figure 3c–e). Here, we propose that the gelator’s extended hydrophobic tails serve to reduce the surface tension in the solvent, thereby allowing the shielded glucose moieties to assemble and drive the sol–gel transition. A computational model obtained by fixing the diameter at the one observed in the AFM images (∼6 nm) shows the same pitch as in the gelator fibrils (∼3.5 nm) (Figure 3b).

Figure 3.

Figure 3

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(a) AFM image of a drop-cast 1% R8 toluene gel showing right-handed helices (scale bar: 40 nm). (b) Comparison between a gel double-helix obtained via AFM (left) and the MD-simulated double-helix (right) (scale bar: 3.5 nm). Views of the R8 quadruplet stacks: (c) top view of the R8 quadruplet from a minimized MD snapshot. The 4 molecules are not on the same plane. (d) Lateral view of the glucose rings only, showing the interlayer network of H-bonds. (e) Lateral view of 11 stacks from a minimized MD snapshot.

In monitoring the fluorescence of R8 toluene gels doped with the C1 probe, they exhibited a minor increase in fluorescence upon gelation (Supporting Information Figure S12). A similar response was observed by Geiger et al. with merocyanine-based dyes,20 while Raeburn et al. reported on the general and hardly rationalizable spectroscopic behavior of common fluorophores (ThT, DCVJ, and so forth.) to changes in viscosity.38 We can infer that C1 does not effectively intercalate within the gelator fibrils and remains mostly in highly fluid regions of the gel network. To dynamically probe the gelator architecture, tailored C1 analogues were designed to have a specific affinity to portions of the gelator assemblies (Figure 4a,b). Glucose and octanoic acid moieties, both of which comprise components of the gelator structure, were introduced into C1 via azide–alkyne Huisgen cycloaddition to give rise to Glu-C1 and Oct-C1 (Supporting Information 3 and Supporting Information Figure S7), following the idea that these moieties may interact preferentially with either the hydrophilic head and hydrophobic tails of R8 fibers, respectively. Glu-C1 and Oct-C1 both displayed a fluorescence behavior quite similar to the precursor C1 (Figure 4a,b and Supporting Information Figure S8), which suggests that the original photophysical properties remain unaffected with functionalization. Coarse grain and all-atom MD simulations supported this molecular design. Indeed, they confirmed that such chemical modifications cause the coumarin portions to reside in two spatially distinct regions of the gelator fibril: either exposed in the solvent (Glu-C1) or within the assembly (Oct-C1) (Figure 5a,b and Supporting Information Figure S15). Figure 5b shows that the coumarin portion of Oct-C1 resides among the sugar core causing a partial perturbation of the H-bond network that stabilizes successive sugar layers (Supporting Information Figure S16). At the same time, the probe forms H-bonds with adjacent hydroxyl groups through its lactone carbonyl and cyano group (Figure 5b, black dotted lines).

Figure 4.

Figure 4

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Molecular rotors Glu-C1 and Oct-C1. Chemical structure and fluorescence spectra at increasing viscosity of the two derivative Glu-C1 (a) and Oct-C1 (b) (viscosity values in cSt: 0.7, 53, 100, 200, 280, and 440).

Figure 5.

Figure 5

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Probe-assisted model for the mechanistic assembly of R8 in toluene. (a) Top view of Glu-C1 inserted within a gel fibril from a minimized all-atom MD snapshot. (b) Top view of Z-s-cis Oct-C1 inserted within a gel fibril from a minimized all-atom MD snapshot. (c) Fluorescence emission spectra of Glu-C1 acquired throughout the gelation process of 1% R8 in toluene (curve 1: 0 min; curve 2: 3 min; curve 3: 6 min). (d) Fluorescence emission spectra of Oct-C1 acquired throughout the gelation process of 1% R8 in toluene (curve 1: 0 min; curve 2: 3 min; curve 3: 6 min).

Solutions of R8 in toluene (1.0 wt % w/v) were gelled with either Glu-C1 or Oct-C1 (200 nM). Confocal fluorescence microscopy performed on doped gels shows the uptake of the fluorescent probes into grown microscopic fibers (Supporting Information Figure S14). Fluorescence spectra were monitored over the course of gelation starting from the hot sol at 100 °C to the gel at 25 °C. Figure 5c,d shows the marked difference between the fluorescence emission spectra of Glu-C1 and Oct-C1, respectively. The emission spectra of Glu-C1 highlight a monotonic increase in intensity, which is readily attributed to a progressive rigidification of the nearby environment of the rotor.10 Conversely, Oct-C1 shows an increase in fluorescence intensity, accompanied with a red-shift, subsequently followed by a diminution of the luminescence that is blue-shifted; this pattern suggests a multistep stiffening process.

According to the computational model in Figure 5a and Supporting Information Figure S11, the interaction of the glucose ring in Glu-C1 with the gelator’s sugar moieties causes its coumarin portion to project from the fibrils, and be exposed to the toluene phase. Such a peculiar spatial arrangement along the R8 fibrils allows Glu-C1 to respond to the change in molecular fluidity occurring at the hydrophobic interface of the fibrils. Figure 6a–f shows time-transient AFM images of drop-cast 1.0 wt % R8 gels obtained over the course of gelation (0–6 min). Starting from an apparently unassembled system (Figure 6a), protofibrils begin to grow in tight contact with each other forming oriented chiral bundles (Figure 6b–e). The attractive longitudinal association among fibrils captured by Glu-C1 and their epitaxial growth take place simultaneously, instead of constituting two distinct steps in the gelation process. After about 4 min, the increase in fluorescence emission plateaus, suggesting little response of the probe toward the helix formation observed after that time (Figure 6e,f). All normalized fluorescence intensities are greater than the limit of the calibration (Supporting Information Figure S8), indicating that the calculated local viscosity at the fibril–fibril interface reaches very high values; this effect arguably owes to the high level of interdigitation of R8 hydrophobic portions.

Figure 6.

Figure 6

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Time-transient AFM imaging mode of drop-cast samples and gel-induced atropoisomeric stabilization. (a–f) The epitaxial growth of the gelator fibers over gelation (6 min). Tubular structures visible after 30 s (scale bar: 25 nm) (b). between 4 and 6 min (e and f, respectively), the elongated fibers exhibits a torsion around their longitudinal axis that results in the formation of right-handed double helices (f).

The multiphase fluorescence readout of Oct-C1 is able to capture the actual liquid-to-gel transition occurring between 4 and 6 min (Figure 5d and Figure 6e,f). At time zero, when the system merely consists of nascent protofibrils (Figure 6a), Oct-C1 displays a broad band seemingly resulting from two overlapping bands. Between 0.5 and 3 min (Figure 5d, curve 1–2), the red-shifted band becomes more and more intense at the expense of the blue-shifted. The extrapolated values of local viscosity at time 0 and 3 min (Figure 5d, curve 1 and 2) suggest a liquid-like nature of the system (2.3 cSt and 4.1 cSt, respectively), in spite of the extremely high rigidity and stability of the sugar core acting as the protofibril’s central pillar (Supporting Information Figure S13): this observation matches well with computation, whereby Oct-C1’s environmental interactions are less effective from a photophysical standpoint owing to both a partial alteration of the fibril’s hydrophilic core upon intercalation and the labile H-bonds established with the surroundings (Figure 5b and Supporting Information Figure S13).

After minute 3 (Figure 5d, curve 2–3), the red-shifted band (556 nm) gradually decreases in intensity along with the blue-shifted band peaking at 528 nm that instead becomes preponderant. We attributed this phenomenon to two co-existing conformers of Oct-C1 by recording fluorescence emission spectra at different temperatures in pure toluene in the absence of the R8 gelator. Supporting Information Figure S17 shows that at RT, the red-shifted band is the main one. However, at higher temperature, the blue-shifted band surpasses the red-shifted one in intensity, clearly suggesting that there exists an energy barrier between the two putative species that can be overcome thermally. Interestingly, this conversion is completely reversible upon cooling.

On the basis of these observations, we hypothesized that Oct-C1 consists of two rotamers (i.e., rotational isomers) arising from the partially restricted rotation around the coumarin–stilbene single bond (Figure 7a).39,40 Time-dependent density-functional theory (TD-DFT) calculations (using CAM-B3LYP/6-31+G*, polarizable continuum model = toluene) confirmed that in toluene the ground state energy of the s-cis isomer is 25.3 kJ/mol lower than the s-trans one (Supporting Information Table S1). Moreover, similar to what was observed in both the gelation experiments (Figure 6) and the temperature-transient measurements (Supporting Information Figure S17), the TD-DFT calculation also confirms a Δλem ≈ 30 nm between the two isomers with the s-cis conformer’s emission being red-shifted (Supporting Information Table S1). Reverse-phase high-performance liquid chromatography–mass spectrometry (RP-HPLC–MS) analysis supports this evidence, showing that Oct-C1 exists as two chemically distinguishable isomers having the same mass ([M–H]− = 576.3m/z), but clearly distinct retention times (Supporting Information Figure S18). Interestingly, the fibrillar architecture greatly affects this equilibrium in a highly dynamic fashion: as shown in Figure 5d, at the very initial phase, (curve 1) the two rotamers co-exist in a ∼1:1 ratio; this ratio slightly differs from what was observed in pure toluene at high temperature (∼1:1.3, s-cis/s-trans) in virtue of the presence of nascent R8 protofibrils in solution. As the system cools down, the s-cis isomer becomes more and more abundant, which is in line with the temperature-transient experiment (Supporting Information Figure S17). Concurrently, as Oct-C1 molecules become incorporated into elongating fibrils and the H-bond network of the latter becomes more compact, the probe experiences steric hindrance (i.e., higher local viscosity). This effect, in turn, induces an increased fluorescence emission (Figure 5d, curves 1–2) because of the decrease in nonradiative decay. In the second phase (Figure 5d, curves 2–3), although the system is approaching room temperature, the emission from the least thermodynamically stable s-trans isomer unexpectedly rises and stabilizes indefinitely, indicating that the isomeric equilibrium is significantly shifted toward this form (final s-cis/s-trans ratio ≈ 1:1.3) (Figure 7). In addition to this, the spectroscopic transition observed took place simultaneously with the formation of chiral structures evident via AFM (Figures 3a and 6a–f) and CD (Figure 2c). To prove that Oct-C1 senses a rearrangement of the H-bond network within the fibril’s sugar core, which is arguably at the base of the fiber’s morphological change, we show via all-atom simulations that the H-bond interactions between the gelators’ glucose hydroxyl groups and the coumarin portion are affected during the structural transition. We found that the isomeric equilibrium results highly perturbed as a consequence of the stabilization of the s-trans isomer (Supporting Information Figure S19), which takes place through a three-H-bond network between the lactone carbonyl and the cyano group, and two adjacent hydroxyl groups of two spatially close glucose rings (Figure 7b, black dotted lines, and Supporting Information Figure S16). We can infer that the interactions stabilizing the s-trans isomer are established only in the fully formed fibers upon fine intermolecular rearrangements in the local structure at the end of gelation, which, in turn, drives the fiber maturation giving rise to double helices (Figure 6).

Figure 7.

Figure 7

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(a) Equilibrium between the Z-s-cis and Z-s-trans rotational isomers of Oct-C1. While the s-cis isomer is favored in nonpolar solvents at RT (such as toluene), this equilibrium is significantly shifted toward the s-trans isomer in the presence of the R8 gel network (R = octano-8-yl). (b) Top view of Z-s-trans Oct-C1 inserted within a gel fibril from a minimized all-atom MD snapshot. The s-trans isomer form a three-H-bond network (black dotted lines within blue circles) with the surrounding glucose rings which in turn, stabilizes this conformation.

Discussion

Understanding of the dynamic nature of molecular self-assembly process has been carried out through careful design of molecular fluorescent probes. The high selectivity of these fluorescent probes toward dynamic and predictable structural changes is justified by their abilities to investigate complex systems in situ without interfering with the overall self-assembly process. In this work, we show that the real-time changes in molecular self-assembly in a 3D network could be probed by introducing ad hoc fluorescent rotors at submicromolar concentrations, which intimately blend into the gelator’s nanostructure. The ability of the viscoelastic media to modulate the fluorescence signal from the probes in terms of both intensity and emission maxima provided significant insights into the gelator’s self-assembly mechanism, which was further corroborated with computational simulations. More specifically, to the viscosity-sensitive core of the coumarin/stilbene molecular rotor C1, we appended moieties that imparted high chemical affinity between the stacked gelator molecules and consequently underwent preferential spatial localization within individual fibrils. MD simulation helped to determine these intermolecular interactions, which ultimately plays a crucial role in unravelling the complexity behind R8’s fiber formation. The two completely distinct moieties, introduced into the probe C1 (i.e., glucose and octanoic acid), acted as molecular anchors allowing the viscosity-sensing portion to be securely localized either right outside of the fibrils’ hydrophobic layer or fully embedded within the hydrophilic fibril core, respectively. Interestingly for Oct-C1, MD simulations also show that the free carboxylic acid at the end of the aliphatic chain clearly interacts with the gelator’s ketone group via the H-bond. This further increases the level of interdigitation between the two molecules and may help shed light on the subtle changes in fibrillar growth in gel systems, as well as their systematic and often reversible responses to external stimuli including radiation, shear, temperature, and more.

Because of the instantaneous fluorescent response upon environmental changes, we were able to utilize these probes to monitor the growth of the nanofibrils in real-time. A particularly striking discovery was related to the observation of solid-like viscosity values at the interface of reversed fibrils where the hydrophobic tails reside. This finding yields important insights into the nature of the interfibrillar adhesive forces taking place within larger fibers. On the other hand, Oct-C1 revealed how 3-styryl-coumarin’s intrinsic s-cis/s-trans rotational equilibrium can serve as a tool to monitor subtle intermolecular interactions. In fact, we shed light on the intriguing multistep fibril formation process underlying a rearrangement of the H-bond network within the hydrophilic core that eventually leads to the generation of double helices, as also confirmed by MDs simulations. Furthermore, the intimate interaction between the gelator R8 and Oct-C1 is highlighted by the fact that during the gelation process the latter experiences a drastic and highly dynamic alteration of its isomeric equilibrium and corresponding fluorescence spectra.

Conclusions

In summary, by combining traditional material characterization techniques and computer simulations with these stimuli-responsive spectroscopic tools, we have demonstrated that it is possible to build an accurate model for the dynamic assembly of the organogelator under investigation. To do this, we first designed distinctive coumarin-based molecular probes, which are complementary to the assembled low molecular weight gelator’s dilute environment (1.0 wt % gelator in solution), while also possessing specific chemical moieties included for the specific interaction of the probe and the gelator structure. After demonstrating the activity and responsivity of the probe and gel system separately, we then tested the probes in the gel, and during gel formation to examine self-assembly to help understand the process of architectural growth and fiber formation. Fluorescent measurements served to characterize the probe’s photophysical response to changes in viscosity relating to the freedom or restriction of the probes intercalation into the gel’s network. AFM and stimulated emission depletion (STED) microscopy helped reveal the gel system’s architecture, dynamic growth, and localization of the probe within the gel fibers. To complement experimental work, computational investigations were conducted to reveal both the gelators potential molecular arrangements, and likely interactions and configurations between the different probes and the gel. This allowed us to highlight the distinctive interactions between the different probes and the assembled organogelator that are in good agreement with the experimental work. We believe that similar nano-sized tools can be designed to probe a vast array of molecular and polymer-based nanostructured materials, allowing for the future study in a broad range of applications.

Methods

Preparation and Characterization of Molecular Gels

Gels were prepared by adding the solid glassy glycosides (10.0–50.0 mg gelator) to toluene (1 mL). The mixture was then heated to disperse the gelator at 5° below the boiling point of the solvents, to produce a homogeneous sol. The sol was kept at this temperature for 5 min under constant agitation to fully disperse the gelator. The sol was then allowed to cool to room temperature to allow for self-assembly. No free flow of the 1% gels upon inversion was observed after 6 min. The samples were inverted to confirm gel formation after 1 h.

Spectroscopic Measurements

Samples (1500 μL) were used in a quartz cuvette (Thorlabs, Newton, NJ) with an optical path (l) of 1 cm. The temperature of the cell compartment was set at 25 °C, by a built-in Peltier cooler (Varian). Absorption data were recorded at 25 °C using 1 nm band pass, 1 nm resolution, and 200 nm/min scanning speed. Fluorescence intensity measurements were carried out at the absorption maximum of each compound, employing an excitation and emission band pass of 2.5 nm, 120 nm/min scan rate and 600 V PMT detector voltage. For every sample, the absorbance at the wavelength maximum was recorded and subsequently employed to normalize the corresponding fluorescence emission intensity.

Fluorescence emission calibration curves of the three probes were obtained in toluene–PEG methyl ether methacrylate mixtures at a final concentration of 400 nM.

The time-transient fluorescence emission measurements were obtained by adding the appropriate volume of a stock solution of the fluorophores (C1, Glu-C1, and Oct-C1) in hot R8 toluene solution (1% w/w) for a final concentration of 200 nM. The cuvette was kept sealed with a Teflon cap throughout the duration of the experiment to ensure no variations in concentration.

AFM Measurements

An R8 1% solution (w/v) was prepared by adding the solid gelator to 3 mL of toluene (1 mL). The mixture was then heated and stirred at 100 °C to produce a homogeneous sol. Sample solutions (5 μL) were drop-casted onto microscope cover glasses (Fisher Scientific, 12-542A, NY, NY) that were kept at 50 °C on a hot plate. The time points considered for AFM imaging were immediately after R8 completely dissolved (time 0 min) and after 0.5, 1, 1.5, 2, 4, and 6 min. After complete evaporation of the solvent (about 5 s), the samples were further air-dried in a dust-free environment for 12 h and then glued onto magnetic discs prior AFM imaging. The samples were scanned in the ScanAssyst mode.

STED Measurements

An amount of 10 μL of 1% R8 toluene gel doped with either Glu-C1 or Oct-C1 (100 nM) was deposited onto a microscope cover glass kept at 50 °C on a hot plate to ensure quick evaporation of the solvent. After further drying overnight at RT in a dust-free environment, the samples were imaged.

RP-HPLC–MS Analysis

Oct-C1 was analyzed with a gradient solvent system consisting of A (water with 0.1% formic acid) and B (acetonitrile with 0.1% formic acid). The flow rate was 250 μL/min and the gradient used was the following: hold at 35% B for 3 min, then 35–90% B in 23 min, then 90–100% B in 0.1 min, hold for 5.9 min and 100–35% B in 0.1 min, and finally hold for 7.9 min. The oven temperature was 40 °C.

Acknowledgments

The authors would like to thank Dr. Tai-De Li and Dr. Tong Wang in the Surface Science Facility and the Imaging Facility at the City University of New York’s Advanced Science Research Center for their help with the imaging experiments and Dr. Lorenzo Di Bari for the useful discussions.

Glossary

Nomenclature

AFM

atomic force microscopy

C1

coumarin-based molecular probe

CD

circular dichroism

Glu-C1

glucose derivative of coumarin-based molecular probe

Oct-C1

octanoic acid derivative of coumarin-based molecular probe

PCM

polarizable continuum model

R8

raspberry ketone glucoside octanoate

RP-HPLC–MS

reverse phase high performance liquid chromatography–mass spectrometry

RT

room temperature

TD-DFT

time dependent density functional theory

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02357.

  • Materials, analytical methods, synthetic protocols along with supplementary figures, parameters and details for molecular dynamics simulations and quantum mechanical calculations, and additional references (PDF)

Author Present Address

# CUNY Advanced Science Research Center, 85 St. Nicholas Terrace, New York, NY, 10031, USA.

Author Present Address

Center for Environmentally Beneficial Catalysis, the University of Kansas, 1501 Wakarusa Dr., Lawrence, KS, 66047, USA.

Author Contributions

S.P. and G.S. designed and synthesized the fluorescent probes. S.P. and J.R.S. designed and performed all spectroscopy and microscopy experiments. R.N. performed the computer simulations. S.P., J.R.S. and R.N. analyzed the data. S.P., J.R.S. and R.N. wrote the manuscript. All authors actively discussed, reviewed and edited the manuscript.

This research was made possible in part by Grants to G.J. from NIFA (National Institute of Food and Agriculture), US Department of Agriculture (GRANT11890945) and the PSC-CUNY Award 61778-00 49.

The authors declare no competing financial interest.

Supplementary Material

ao8b02357_si_001.pdf (6.2MB, pdf)

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