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Synlett 2024; 35(01): 3-20
DOI: 10.1055/a-2091-0916
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Functional Dyes

Functional α-Cyanostilbenes: Sensing to Imaging

Rahul Dahiwadkar
a   Department of Chemistry, Indian Institute of Technology Gandhinagar, Palaj- 382355, India
,
Masood A. Kaloo
b   Department of Chemistry, Government Degree College Doda, Doda City, Jammu and Kashmir-182202, India
,
Sriram Kanvah∗
a   Department of Chemistry, Indian Institute of Technology Gandhinagar, Palaj- 382355, India
› Author Affiliations

The authors acknowledge financial support from various funding agencies, Council of Scientific and Industrial Research (CSIR) (01(2487)/11/EMR-II), the Department of Science & Technology [SR/S1/PC-24/2010(G)], the Board of Research in Nuclear Sciences (BRNS) (37(2)/14/05/2016) and Science and Engineering Research Board, India (SERB; CRG/2018/004020), that helped us to achieve the research goals. R.D. acknowledges a research fellowship from IIT Gandhinagar, M.A.K. is highly thankful to DST [INSPIRE/04/2016/000098] for financial support.
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Abstract

In recent years, there has been considerable interest in cyanostilbenes due to their unique photophysical properties. The compounds emit light when aggregating, commonly called aggregation-induced emission (AIE). This remarkable feature makes cyanostilbenes ideal for various sensing applications, especially in aqueous environments. The detection of various analytes, such as metal ions and nitroaromatic compounds, has been accomplished using these compounds through various sensing mechanisms from chelation-enhanced fluorescence to fluorescence quenching. Furthermore, cyanostilbenes have shown great promise in biological imaging applications and have been employed for intracellular imaging, tracking, and targeting of sub-cellular organelles. The development and utilization of cyanostilbenes can significantly impact advanced sensing and imaging technologies in both analytical and biological fields. This potential stems from the unique properties of cyanostilbenes, such as their AIE characteristics, which sets them apart from other compounds and makes them highly useful for various applications. Further exploration and development of cyanostilbenes could lead to the creation of novel sensing and imaging technologies with wide-ranging applications in both academic and industrial settings.


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Key words

aggregation-induced emission - acrylonitriles - bioimaging - cyanostyrenes - chromophores

Biographical Sketches

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Sriram Kanvah obtained his PhD in chemistry from the Indian Institute of Technology Bombay, Mumbai. He was a post-doctoral fellow at the Georgia Institute of Technology in Atlanta and worked as a research-intellectual property analyst at General Electric Company in Bangalore. Thereafter, he joined the Indian Institute of Technology Gandhinagar, Department of Chemistry, in December 2009. His research interests lie in the synthesis and development of photoresponsive systems for organic electronics, biological imaging, and chemosensing applications. Apart from research, Sriram also has a keen interest in science promotion activities in schools and undergraduate colleges.

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Rahul Dahiwadkar completed his PhD (Chemistry) from Indian Institute of Technology Gandhinagar in 2023. He obtained his Master’s degree in chemistry from Savitribai Phule Pune University in 2017. His PhD research interests relate to the design and synthetic aspects of novel fluorescent donor-π-acceptor molecules such as α-cyanostilbenes and stilbene analogues for sensing and imaging applications along with the development of novel functional organogel materials.

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Masood Ayoub Kaloo earned his PhD from the Indian Institute of Science Education and Research Bhopal, in 2016. Currently, he serves as an Assistant Professor in the Department of Chemistry at Government Degree College Doda (HED, J&K, India). His research focuses on the design and development of molecular-recognition-based strategies for environmental and material applications. He has received numerous prestigious awards, including the DST INSPIRE-Faculty Award, SERB INDO-US Postdoctoral Fellowship, M.P. Young Scientist Fellowship Award, Scientific Events Award, INSA Visiting Scientist Award, and Young Scientist Award (IARDO). Additionally, he actively participates in various chemistry and environmental societies, and has presented at numerous national and international conferences.

Introduction

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Figure 1 Representative timelines summarizing the discovery and application of aggregation-induced emission properties of cyanostilbenes

Aggregation-induced emission (AIE) is a phenomenon in which a molecule’s fluorescence or light-emitting ability is enhanced upon aggregation, in contrast to its weak emission in the solution state. AIE, which is typically caused by structural changes in the molecular geometry and by changes in the electronic distribution upon aggregation, contributes to enhanced fluorescence quantum yield.[1] Molecules that exhibit AIE are generally characterized by the following structural attributes: (1) Extended conjugation that helps suppress fluorescence in the isolated state, enabling enhancement in the aggregated state; (2) Bulkier substituents that hinder intermolecular interactions and preventing aggregation in solution; (3) Rigid or semi-rigid structures that prevent rotation around bonds and intermolecular interactions in solution but allow for stacking in the aggregated state, and (4) Hydrophobicity, which helps to drive their self-association in the solid state. Many organic fluorophores have thus been designed based on these structural scaffolds. Examples include derivatives based on tetraphenylethenes,[2] triphenylethene,[3] distyrylanthracene,[4] distyrylbenzene,[5] [6] perylenes, pyrene,[7] and naphthalene,[8] and have been widely used in various fields, such as chemical sensing, bioimaging, and organic light-emitting diodes (OLEDs).

An important scaffold exhibiting such characteristic AIE emission is α-cyanostilbene.[5] [8] [9] Cyanostilbenes are π-conjugated molecules with two terminal phenyl or aromatic groups connected by a cyanovinyl π-bridge. The design therefore provides synthetic flexibility for incorporating different functional moieties, particularly on the aromatic ring, and they are consequently characterized by excellent electrical and photophysical properties. Their properties are attributed to their ‘elastic twist’ feature that allows significant torsional changes in response to intermolecular interactions. When in a dilute solution, the molecules twist due to internal repulsions, but they still assume a more conjugated, planar conformation during self-assembly. This planar structure leads to tight molecular packing and improved charge transport, and their self-assembly results in enhanced optical properties, namely aggregation-induced emission (AIE).[9,10] The aggregation-induced emission (AIE) behavior in cyanostilbenes is a direct result of their unique molecular structure, which is characterized by a rigid, planar conjugated backbone containing a cyano (-CN) group attached to the phenyl ring. In solution, the rotation of the cyano group around the C–C bond adjacent to the phenyl ring is relatively free, leading to a non-radiative decay pathway that decreases the fluorescence quantum yield. However, when the concentration of cyanostilbenes increases or when they are in a solid state, the intramolecular motion is restricted due to intermolecular interactions, such as van der Waals forces and π-π stacking, which stabilizes the molecule and enhances its fluorescence emission. The restriction of intramolecular motion leads to a decrease in non-radiative decay pathways, such as vibrational relaxation and energy transfer, and increases the radiative decay pathway, leading to a higher fluorescence quantum yield and AIE behavior. Additionally, the cyano group in cyanostilbenes contributes to the AIE behavior by promoting electron delocalization along the conjugated backbone of the molecule, which enhances the fluorescent emission. The rigid and planar structure of the cyanostilbenes also contributes to the AIE behavior by reducing the non-radiative decay pathways. Since the pioneering work of Park et al. in 2002,[11] the α-cyanostilbenes have attracted tremendous attention for the development of materials for various applications, from investigating the photophysical properties to biological applications (Figure [1] and Figure [2]). In this account, we consolidate various applications of cyanostilbenes specific to the sensing of several analytes, drugs, environmental pollutants, bioactive species such as ROS, RNS, RSS, and finally, bio-imaging. We also describe the synthetic strategies employed.

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Figure 2 Overview of the applications of α-cyanostilbenes

Choice and Design of Fluorophores for Sensing Applications

The choice of fluorophore is crucial for the design of a fluorescent probe. It is essential to choose a fluorophore that emits light at an appropriate wavelength for the intended application, with high quantum yield, photostability, and low background signal. Typically, to achieve strong fluorescence, one must control the probe’s intramolecular motion by incorporating rigid structural elements or by introducing bulky substituents. This design helps to reduce non-radiative transitions and emit strong fluorescence. Organic fluorophores offer a powerful tool for sensing and imaging applications due to their flexibility in design. For sensing applications, the design can be tailored to the specific analyte of interest by incorporating sensing elements or reactive groups in the molecules. Incorporating sensing elements or reactive groups into the design should also result in measurable photophysical changes such as changes in emission wavelength, intensity, or fluorescence lifetime. Based on these basic design principles, several researchers have developed a variety of organic fluorescent systems[12] in both solution and solid phases. Cyanostilbenes offer an excellent platform for sensing and imaging applications due to the enhanced emission observed in these compounds. The suppression of non-radiative emissive processes in cyanostilbenes is enabled by the aggregation of the fluorophores in aqueous media. In a crystalline or aggregated state, the molecule tends to become planar, and aggregation-induced planarization extends the effective conjugation length and increases the oscillator strength, thereby activating the radiative process.[11] Considering the general functional utility of the cyanostilbenes, several molecular scaffolds were designed for various sensing applications.


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Stimuli-Responsive Sensing

pH Sensing

pH sensing can be brought about by incorporating several functional units such as carboxylic acids, amines, imidazoles, phenols, and sulfonamides that undergo protonation or deprotonation as the pH changes.[13] Such strategies were employed for sensing pH using cyanostilbene as a scaffold. Multi-stimuli responsive luminogens 1–4, with carboxylic substituents, were synthesized and used to sense pH under aqueous environments and for vapor sensing of amines (Figure [3]). Different fluorescence intensity or wavelength changes were attributed to the conversion between protonated and deprotonated states.[14] Under strongly acidic conditions (pH ≤4.0), a broad UV absorption band and a bathochromic shift are observed. The shifts correspond to the aggregation of their protonated states in aqueous media. Compound 2 emits intensely in acidic conditions, but fluorescence was turned off at pH >4.0. At pH >4.0, molecule 2 becomes deprotonated to form the carboxylic salt, aiding solubility and activating the phenyl rings’ intramolecular motion, causing emission quenching. The variation in the emission response of the neutral state of 4 is due to the difference in the ionic intensity in the environment, which leads to decreased solubility of 4 and enhanced aggregation. At low pH (<4.0), compound 3 is green luminescent and emits around 500 nm. When the pH is increased (>4.0), green emission disappears, and a new blue emission peak emerges at about 450 nm. The blue and green fluorescence of 2 corresponds to its dissolution and aggregation.

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Figure 3 Molecular structures of α-cyanostilbenes 1–23

Molecules 5–7, with a dimethylamine and phenolic group, show excellent acidic and basic pH sensing. The addition of acid or base leads to a distinct color change. The 1H NMR evidence reveals the formation of a phenoxide ion (5) in the presence of a base and a protonated amine derivative in acid media, contributing to the emission changes.[15] The bent α-cyanostilbene 8, tethered with amino groups on two benzene rings,[16] shows stimuli-responsive emission for acid and base due to the presence of the amine groups. A switchable emission is observed upon adding trifluoroacetic acid (TFA) and base triethylamine (TEA) with conversion between protonated and deprotonated states accompanied by color changes.[16] TFA was also detected using a pyridyl acrylonitrile derivative 9, which, without any standard auxiliary groups such as alkyl chains, cholesterols, or amide units, formed stable organogels. The molecule also formed a two-component gel upon adding a second cyanostilbene molecule (10). Upon exposure to TFA vapors, the organogels underwent distinct color changes and lost their gelation ability (Figure [4]).[17]

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Figure 4 (A) Molecular structure of α-cyanostilbene 5 (B), (C), and (F) The plots indicate interaction with different surfactants, CTAB, SDS, and Triton, respectively. (D), (G), and (H) show SEM images in aqueous solution and with 15 × 10–4 M SDS and 2 × 10–4 M CTAB, respectively.[18] (I) Molecular structure of α-cyanostilbenes 9 and 10. (J) Absorption spectra of 3 upon addition of TFA (0–1.7 equiv) measured at a concentration of 5 μM in CHCl3, and (K) associated color change and product formation upon exposure to TFA vapor. (L) Two-component organogel morphology (Xerogel SEM) and (M) associated color and gel morphology changes upon exposure to TFA.[17]

Isomeric π-conjugated derivatives bearing dimethylamine units and a nitrile group, 11 and 12, showed good solvatochromic behavior. The presence of dimethylamine units allowed their use in sensitively detecting volatile acids, such as TFA, with a limit of detection (LOD) of 2.0 and 8.82 nM, respectively. The emission losses due to TFA were recovered upon adding triethylamine (TEA).[19] α-Cyanostilbenes 13 and 14 were used for sensing p-nitroaniline (PNA). These molecules exhibit AIE characteristics in both the solid state and solution. Due to the better charge separation and electron-rich properties of N,N-dimethylaniline, PNA was detected in tetrahydrofuran (THF). The results showed that the sensors displayed extraordinary sensitivity to PNA with fluorescence quenching upon the addition of PNA, with a LOD of 0.42 and 0.60 μmol L–1, respectively, for 13 and 14. The sensing behavior is attributed to photoinduced electron transfer (PET).[20] The molecules were also used to demonstrate the detection of p-nitroaniline in different environmental samples. The α-cyanostilbene containing a primary amine group 15 was utilized to detect CO2.[21] The molecule displayed a ‘turn-on’ emission response in the presence of CO2. The reaction of CO2 with the primary amine moiety forms carbamic acid, which leads to the formation of salt bridges due to electrostatic interaction between the carbamate and ammonium salts. Such forces induce self-assembly and increase the size of aggregates, yielding fluorescence enhancement. The molecule also exhibited reversible behavior upon purging N2 into the solution. Another fluorescent sensor for CO2 was developed based on a bis-cyanostilbene derivative (16)[22] and was utilized in two different ways: dispersed gel aggregates and solid-supported xerogels. In the former, the sensing of CO2 is driven by H-bonding interactions of the carbamate ionic liquid (CIL) anion, which is formed in situ by the reaction of CO2 with diethyl amine (DEA) with 16. This alters the aggregation, leading to emission quenching. In the latter case, the xerogel formed in a quartz vial also responds to the presence of CO2, with wavelength tuning from blue to green, apart from emission quenching.


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Metal Analyte Detection: Hg, Zn, Ag, Cu, Pd

Detecting metal analytes is vital for the health of living organisms to diagnose metal-related diseases such as those emanating from lead, mercury, and cadmium, for the assessment of treatment efficacy and essential metal nutritional status, and for environmental monitoring. Monitoring the levels of these metals can thus provide information on the extent of the metal ions present and guide appropriate environmental or medical interventions. To detect metal analytes, the approaches utilize internal charge transfer (ICT), excited-state intramolecular proton transfer (ESIPT) upon binding to a metal ion, fluorescence resonance energy transfer (FRET), chelation enhanced fluorescence (CHEF), and photoinduced electron transfer (PET). All the strategies involve the complexation or binding of metals, leading to changes in the fluorescence behavior.[23] One such design strategy involves the utilization of Schiff bases[24] and monitoring the changes in the emission response. A linear π-conjugated bis-Schiff base derivative 17, with an α-cyanostilbene scaffold,[25] was synthesized for sensing Hg2+. Distinct color and ‘turn-on’ emission changes were observed in the presence of Hg2+ in THF and a mixed solvent system (THF/water, 2:8: v/v) with high selectivity and sensitivity in the presence of other competitive ions, with a LOD of 2.4 × 10−7 M in THF/water. In the presence of Hg2+, 17 assembled into a separated, tilted arrangement, blocking non-radiative intramolecular rotation, suppressing C–N isomerization, and avoiding strong stacking. The Schiff base design was also utilized as a ‘turn-on’ sensor for Zn2+ ions.[26] The α-cyanostilbene 18 shows a redshift due to the intramolecular charge transfer phenomenon, and, upon complexation with Zn2+, the C=N isomerization is inhibited, giving a fluorescence enhancement via chelation-enhanced fluorescence (CHEF) effect.[26] The self-assembled cyanostilbene AIE platform 19 is also utilized for sensing Hg2+ ions in aqueous solutions.[27] The monomer 19, which contains hydrophilic methoxy polyethylene glycol and hydrophobic components, forms spherical micelle-like self-assembled structures with a critical micelle concentration of 2.63 × 10–5. Upon self-assembly formation, rhodamine B is encapsulated within the micelle, and exposure to Hg2+ ions is a ring-opening reaction resulting in a strong fluorescence due to the FRET. A thiourea-functionalized π-conjugated cyanostilbene 20 with excellent AIE characteristics[28] was utilized for selective recognition of Cu2+ and Hg2+ analytes even in the presence of a range of interfering metal ions in water. The selectivity was attributed to the strong affinity of Hg2+ ions to sulfur, and the complexation drives the desulfurization reaction. Detection of Cu2+ was observed through complexation with the addition of EDTA. A vinyloxy-substituted cyanostilbene 21 was also utilized as a ‘turn-off’ probe to detect Hg2+ with a detection limit of 37 nM. The initial yellowish fluorescence shows a sevenfold drop in the fluorescence intensity with concomitant color changes (colorless) upon adding Hg2+. Mechanistic investigations reveal the reaction of Hg2+ with the vinyl group via oxymercuration reaction yielding the corresponding phenol.[29] Precious metals such as palladium were also detected by adopting synthetic strategies incorporating a reactive site. Instead of a methoxyvinyl, a propargyl group 22 is used where the palladium-catalyzed depropargylation leads to a loss of fluorescence accompanied by visual changes.[30] Metal ions such as silver were also detected using the cyanostilbenes. A gel is formed with a sharp fluorescence increase by adding a silver salt to the non-fluorescent solution of 23 bearing a pyridine functionality, Upon the addition of TBAF, the fluorescent gel switches back to its non-fluorescent state. The formation of gel is due to metal coordination with pyridine N between two units of cyanostilbene.[31]


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Surfactants

Surfactants are widely used in industrial and household products, such as detergents, cosmetics, and agricultural and oilfield applications. Monitoring the levels of surfactants in environmental samples can provide information on the extent of contamination and guide interventions to reduce the impact of surfactants on the environment.[32] Compound 5, which contains dimethylaniline and a hydroxyl group, exhibits AIE behavior and has been used to study the effect of added surfactant on aggregation-induced emission (Figure [4]). At lower concentrations of the surfactant, AIE emission is preserved, while at higher concentrations, the loss of AIE emission is observed, which is correlated with micellization. Additionally, distinct colors are observed with different surfactants (CTAB, SDS, and Triton X-100), and the emission properties are utilized to calculate the critical micelle concentration.[18]


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Trace Water

The phenomenon of AIE is characterized by enhanced emission in water. In most cases, a gradual emission shift is observed upon adding water. This characteristic is used for detecting trace amounts of water. This is particularly useful in identifying water in solvents typically used for moisture-sensitive organic reactions. Carbazole-containing cyanostilbenes with D-π-A-π-D compounds showing excellent fluorescence were utilized for trace water detection. Compound 24 has an aniline group, while compound 25 with N,N-dimethylaniline group is present (Figure [5]). With an increase in water percentage, a loss of fluorescence is noted along with concomitant emission shifts with up to 77% emission intensity losses due to solvent polarity and H-bonding effects.[33] A D-π-A α-cyanostilbene (26) skeleton with NO2 as an acceptor moiety and methoxy group as a donor moiety was synthesized to detect trace water (Figure [6]). Unlike the other compounds, the compound showed strong fluorescence quenching upon adding water due to the presence of the nitro group, which promotes intersystem crossing. While it shows reasonable solvatochromic behavior with a change in solvent polarity from non-polar toluene to dichloromethane, the quenching ability upon the addition of water is utilized to detect trace amounts of water present in THF with a 71 ppm water fraction in THF and dioxane.[34]

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Figure 5 Molecular structures of α-cyanostilbenes 24–43
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Figure 6 (A) Molecular structure of α-cyanostilbene 26. (B) Normalized fluorescence spectra of 26 with varied water content. (C) (i) Photographs of test papers before and after absorbing a certain amount of 1. Fluorescence photographs of test papers after soaking in (ii) THF and (iii) dioxane solutions with different amounts of water.[34]

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Detections of Explosives

Nitroaromatic compounds are explosive due to their high energy content and the ease with which they release that energy. The presence of the nitro (-NO2) group in these compounds makes them highly reactive, and the release of energy occurs when the compound undergoes rapid oxidation or combustion and therefore requires careful attention to safety and detection procedures. The detection of explosives using fluorophores typically involves the design of a fluorophore that can selectively bind to the explosive molecule. When the explosive molecule binds to the fluorophore, the fluorescence of the fluorophore is quenched, providing a signal indicating that the explosive is present. This can also be achieved by utilizing some functional groups that can induce emission changes by protonation.

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Figure 7 Synthetic scheme for α-cyanostilbene 28

Picric Acid

The cyanostilbene units with imidazole substitution (27 and 28; Figure [7]) containing α-cyanostilbene units were utilized to detect explosives such as picric acid (PA).[35] The designed and synthesized molecules possess a D–π–A structure, with -CN and imidazole units acting as electron-withdrawing units, while diphenylamino acts as an electron-donating group. The D–π–A design gives ICT characteristics to the molecule with wavelength shifts from 494 nm in toluene to 548 nm in DMF. Due to intermolecular interactions and restriction of intramolecular rotation in the aggregated state, the molecules exhibit AIE behavior. The possibility of Coulombic interactions of electron-rich imidazole moieties with electron-deficient PA was utilized to detect picric acid. Apart from this interaction, PA may bind with the imidazole ring’s basic N atom. This interaction propensity led to its use for ‘turn-off’ sensing of picric acid and 2,4-dinitrophenyl hydrazine in a THF–water mixture with a detection limit below 10−6 mol L−1. Our group has reported the synthesis of AIE-active cyanostilbenes 29, 30, and 31 with a benzimidazole moiety.[36] When PA is added, the compounds exhibit excellent AIE emission in aqueous conditions and show quenched emission with color demarcation with –11,–50, and –85 fold decrease in the emission intensity for 29, 30, and 31, respectively. The compounds also show excellent selectivity for PA at ppm detection levels compared to other nitroaromatic and phenolic substrates. The H-bonding interaction of PA with the fluorophores was proposed as the primary mechanism for its sensing. Trifluoromethyl-substituted cyanostilbenes were also used for sensing picric acid (PA). Allen and co-workers synthesized three isomeric (o-, m-, p-) cyanostilbenes[37] 32, 33, and 34 that exhibit strong fluorescence in dioxane–water binary mixtures. The molecules showed excellent sensitivity to picric acid in water. Density functional theory calculations indicate the involvement of photoinduced electron transfer in its detection.

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Figure 8 (A) Molecular structure of α-cyanostilbene 40. (B) Emission spectra of 40 in the presence and absence of PA (50 μM). (C) Color demarcation in solution and gel disruption in the gel state. (D) Chromatographic strip test for PA detection with varied concentrations (0–20 μM).[38] (E) Molecular structure of α-cyanostilbene 43. (F) Emission spectra of 43 in the presence of various nitroaromatics. (G) Emission spectra of 43 with increasing concentration of PA. (H) Chromatographic strip tests with varied concentrations of PA.[39]
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Figure 9 Molecular structures of α-cyanostilbenes 44–64
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Figure 10 Synthetic strategy for α-cyanostilbene 44

Similarly, trifluoromethyl-substituted cyanostilbenes 35–40, containing dialkylamine substitution with remarkable AIE properties, were also used to sense picric acid (Figure [8]). Due to the alkyl chain and trifluoromethyl groups, the compounds formed stable and thermo-reversible organogels.[38] A stable gel was formed when the molecules had two trifluoromethyl (CF3) groups and showed gelation loss upon adding the picric acid. Sensing of picric acid was also observed in the solution state. Amine functionalized α-cyanostilbenes 41 and 42 were utilized as selective chemosensors for PA through ion-pair formation between the analyte and the compounds.[40] The sensitivity of 41 is greater than that of 42 due to reduced steric hindrance as observed from their LOD of 2.85 × 10–7 and 1.96 × 10–7, respectively. After addition of the picric acid, the molecules show inhibited intramolecular charge transfer caused by the effective protonation of amino groups, leading to fluorescence quenching. A difluoroboron derivative 43, containing α-cyanostilbene and tetraphenylethylene units, was also utilized for sensing PA.[39] Compound 43 showed strong fluorescence in dilute solutions (Φ = 19.3% in THF) and solid state (Φ = 49.3%), and displayed highly selective sensing of picric acid (PA) in THF and aqueous media, with limits of detection of 497 and 355 nM, respectively (Figure [8]). Test strips prepared using dip-coating also achieved the detection (50 μM PA). The sensing mechanism is based on the synergy of fluorescence resonance energy transfer (FRET) and photoinduced energy transfer (PET), which results in emission quenching in the presence of PA.

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Figure 11 (A) Molecular Structure of α-cyanostilbene 45. (B) Images of 45 in 1,4-dioxane/water binary mixture under 365 nm UV illumination; (C) The normalized fluorescence spectra of 45; (D) Kinetic behavior of 45 with N2H4 at different times (0–60 min). (E) Photographs of 45 after the addition of different concentrations of hydrazine.[42]

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Detection of Biologically Relevant Analytes

While α-cyanostilbenes have traditionally been investigated for their unique aggregation-induced enhanced emission properties, several research groups have also reported using these probes for biological investigations by incorporating heterocyclic scaffolds or introducing other cationic groups (Figure [9]).

The fluorescent probe 44 (Figure [10]) showed selective detection of Al3+ ions among 16 metals in an aqueous solution (pH 5 to 7.4) by ratiometric red emission at 600 nm and a concomitant decrease in the emission at 535 nm upon excitation.[41] The Al3+ binding with the probe triggered self-assembly with the dipeptide receptor yielding an aggregation-induced emission recognition output. The peptide receptor, having a glycine-aspartate residue substituted on the aromatic ring, helps in the coordination bond formation with Al3+, resulting in emission changes with a detection limit of 145 nM. The designed and developed probe efficiently penetrated living cells and showed good biocompatibility for detecting Al3+ in live cells. An AIE-active fluorescent probe, 45, was developed for sensing hydrazine (N2H4) in live cells. The probe has diethylamino coumarin conjugated with benzothiazole. This architecture enhances the fluorescence and provides strong ICT. In water, 45 displays strong AIE behavior with emission at 650 nm, solid solvatochromism in various solvents, and high selectivity for N2H4, with a LOD of 0.101 μM, even in the presence of various related analytes. The 2-benzothiazole acetonitrile moiety is also a reactive site for N2H4. After reacting with N2H4, the structure of 45 is destroyed, and both the ICT and AIE effects disappear, resulting in distinct changes in emission.[42] Notably, 45 is biocompatible and can detect trace amounts of N2H4 in living cells (HeLa cells) and Zebrafish (Figure [11]). An effective α-cyanostilbene with a thiophene substitution 46 has been used to sense hypochlorite. The design takes advantage of the potential reaction of the sulfur with the hypochlorite, and the sulfone that is formed results in an emission quenching. The emission quenching is associated with a blueshifted emission from 508 to 435 nm.[43]


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Amyloid Fibrils

Molecule 47, with a cationic pyridinium group, was utilized as a probe for detecting fibrillar α-synuclein and amyloid fibrils. A strong redshifted (605 nm) fluorescence is noted with a significant Stokes shift (145 nm) when bound with the protein aggregates (Figure [12]). The bathochromic shift is attributed to changes in local polarity due to electrostatic solute-solvent interactions. The molecule also showed higher binding affinity (K b = 5.5 ± 0.7 μM) to fibrillar α-synuclein than thioflavin T (K b = 9.9 ± 1.5 μM). Notably, the molecule can be used to detect amorphous aggregates and oligomeric species formed early during aggregation.[44] The AIE fluorogens were also utilized for detecting and imaging amyloid fibrils.[45] Four AIE-active fluorescent probes 48–51, with a piperidine and dimethylamino group as electron donors and binding groups for Aβ, were used to detect in vitro Aβ fibrils and perform super-resolution imaging of ex-vivo Aβ deposits in mouse brain. The molecules show more than 90% colocalization with a known amyloid binder: NIR dye (8c). The fluorophores exhibit superior sensitivity to amyloid fibrils of Hen Egg White lysozyme (HEWL) in vitro but not native HEWL, with a detection limit of 63.71 nM.[45] The imaging of mouse brain slices reveals radiant nanofibrils with detailed information on the structure, which would help elucidate the physiological mechanisms of protein conformational disorders.


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Heparin

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Figure 12 (A) Molecular structure of α-cyanostilbenes 47 and ThT. (B) The fluorescence emission spectra of both ThT and (C) 47 (0–50 μM) in the presence of monomeric or fibrillar α-synuclein (20 μM). (D) and (E) Binding affinity and competitive binding of 47 and ThT to α-synuclein fibrils, and (F) Monitoring and visualizing the elongation of α-synuclein fibrils using ex-situ fluorometry and TIRF microscopy from each time point. Scale bar represents 5 μm.[44]
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Figure 13 Synthetic scheme for 61, 64, and 20

Heparin is an essential molecule with a wide range of functions in the body, having critical roles in blood clotting, anti-inflammatory properties, anticancer properties, and cardiovascular diseases. To design synthetic binders for anionic polysaccharides such as heparin, the binder typically contains positive charges that aid in electrostatic binding. Following this strategy, Banerjee et al. employed dicationic cyanostilbene derivatives 52, 53, and 54 with alkyl groups on pyridyl ring with a range of carbon chain lengths (n = 2,4,6), for sensitive and ratiometric detection of heparin-induced enhanced emission.[46] The derivatives exhibit weak cyan-blue monomeric emissions in solutions around 450 nm but display significant enhancement and redshifted (90 nm) emissions around 540 nm in the presence of heparin. The enhancement is also associated with longer lifetimes and high quantum yields.[46] Heparin was also detected by amphiphilic and dicationic cyanostilbene derivatives 55–57. Alkyl groups characterize the molecules with carbon chain lengths of 8, 10, and 12. The hydrophobic alkyl groups enable self-assembly, and positive charges in the form of pyridinium cation help detect anionic polysaccharides such as heparin with a detection limit of 20 nM.[47] The detection is achieved even in the presence of competing biological species in medically relevant concentrations, including 50% serum and 60% human plasma.[46] [47]


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Albumin Detection

The cationic benzimidazole–Michler’s ketone derivative 58 with an emission at 650 nm showed a significant increase (66-fold) in fluorescence intensity and quantum yield in the presence of serum albumins (BSA and HSA), accompanied by distinct color changes. The binding phenomenon is driven by electrostatic, hydrophobic, and H-bonding interactions. Competitive binding studies showed that the probe binds selectively to albumin proteins over other substances commonly found in serum. Cellular imaging studies reveal localization in the cytoplasm, and serum depletion studies show a reduction in fluorescence intensity, indicating the probe’s binding affinity to albumins.[48]


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Sensing of Sulfite

The coumarin pyridinium scaffold 59 and 60, with a nitrile group on the double bond, was utilized by our group to detect sulfite through a Michael addition reaction.[49] The molecules are characterized by strong donor moieties, N,N-diethylamine, or Julolidine groups, with a pyridinium acceptor. With an emission at 646 nm for 59 and 673 nm for 60, the fluorophores showed emission quenching upon the addition of sulfite, with the formation of new absorption and emission bands, with a limit of detection of 1.47 μM and 2.8 μM, respectively. Upon treating the dye-incubated cells with two different concentrations of sulfite SO3 2− (0.3 mM and 30 mM), a decrease in emission in the 630–720 nm range followed by an increase of emission in the 430–510 nm range is observed, indicating the exogenous detection of sulfite. The sulfite was also generated endogenously, and similar observations were noted with the decrease in emission intensity.[49]


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Detection of H2S

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Figure 14 Molecular structures of α-cyanostilbenes 65–67 and synthetic scheme for 66
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Figure 15 (A) Molecular structure of α-cyanostilbene 65; (B) Absorption and emission spectra of 63 with and without GHB; (C) Chromatographic strip paper test for GHB detection.[53] (D) Molecular Structure of α-cyanostilbene 66; (E) Emission spectra of 66 with vitamin B1 and competitive analytes; (F) Representation of vitamin B1 detection via complex formation.[54]
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Figure 16 General representation of α-cyanostilbenes employed for imaging applications and structures of various cyanostilbenes 68–89

A derivative of cyanostilbene 61 with dual reactive sites for H2S, namely the double bond of cyanostilbene and maleic anhydride, susceptible to H2S reaction, has been developed (Figure [13]). Compound 61 exhibits AIE with enhanced fluorescence intensity at λem = 530 nm with an increased water fraction (70% water/ethanol). With the addition of H2S, a further enhancement of fluorescence intensity is noted with naked yellow fluorescence. Encapsulation of 61 with α-cyclodextrin disaggregates the AIE behavior, and the supramolecular assembly also shows enhanced fluorescence intensity upon H2S addition.[50] Tian et al. utilized Julolidine bearing cyanostilbene scaffold and introduced a hydrophilic viologen group 62 to achieve dual mode sensitivity to viscosity with photoswitchable behavior.[51] The presence of the viologen group has an affinity to form inclusion complexes with cyclodextrin, allowing tunable behavior. A linear relationship of fluorescence intensity increment with increasing viscosity is observed for 62 in ethylene glycol/glycerol mixtures as well as in an aqueous solution of dextran. The dual-mode tunable viscosity sensitivity is well distinguished when the inclusion complex is subjected to environmental viscosity changes. Fluorescent α-cyanostilbenes 63 and 64, owing to the presence of NO2 (63) or azide (64) functionality, were utilized for H2S detection in aqueous medium as well as in HeLa cells. Upon adding H2S, 63 (λem = 616 nm) shows a gradual decrease in fluorescence intensity, with the formation of a new band at λem = 525 nm, accompanied by a colorimetric and ratiometric response with a detection limit of 6.87 μM. In contrast, molecule 64, with N3 as the functional group (λem = 570 nm, weaker emission), shows a gradual 8-fold increase in fluorescence intensity with a –35 nm emission shift and a detection limit of 0.52 μM. The compounds could also be used to detect H2S in HeLa cells when cells were pre-treated with NaHS.[52]


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Biomolecular Sensing

γ-Hydroxy butyric acid (GHB) is a sedative that can be added to alcoholic drinks to impair a victim, and detecting such drugs is achieved by utilizing a hydroxyl-substituted cyanostilbene 65 that interacts with GHB via H-bonding interaction supported charge transfer (Figure [14]). The molecule yields a significant redshifted emission of +110 nm and a +35 nm bathochromic absorption shift upon adding GHB (Figure [15]).[53] Several other molecules were designed and synthesized for analyte detection using different design strategies. In one such example, a ‘turn-on’ fluorescent macrocycle with cyanostilbene was designed for sensitively detecting guest molecules.[54] The crown-ether macrocycle 66, exhibiting the AIE, effect showed strong selectivity for Vitamin B1 with a detection limit of 7.8 × 10−7 M. Upon Vitamin B1 inclusion, a dramatic increase in fluorescence was noted (Figure [15]). The enhancement is attributed to the strong binding of the macrocycle due to H-bonding and π-π stacking. Such a macrocycle was also used to detect the amino acid lysine. In this case, cage 67 is slightly smaller than 66. The Z-cyanostilbene cage shows weak fluorescence, but adding lysine results in a substantial turn-on fluorescence enhancement with a detection limit of 1.34 × 10–7 M. The sensing selectivity is attributed to the spatial matching of lysine within the cage and the subsequent intermolecular interactions involving H-bonding and π-π interactions.[55]


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Imaging of Sub-Cellular Organelles

The imaging of cellular organelles allows investigations of various structures, functions, and interactions of different organelles within living cells. This information is crucial for understanding the mechanisms of various cellular processes and pathways, as well as for the development of new therapies for diseases. Various strategies can target the sub-cellular organelles by targeting specific proteins or molecules on their surface. For example, mitochondria are often targeted using probes that selectively bind to their membrane, such as pyridinium or triphenylphosphonium moieties. Similarly, lysosomes can be targeted by incorporating pH-sensitive groups such as morpholine and the presence of hydrophobic groups to help target the lipid droplets (Figure [16]).

The structures of various cyanostilbene molecules that are utilized for sub-cellular imaging are shown in Figure [16]. A cyanostilbene derivative with a pyridinium group was synthesized to target fluorophores to specific organelles (Figure [17]). The fluorophore is characterized by a strong triphenylamine donor and pyridinium as an acceptor, with a sulfonate or bromobenzyl linker. The zwitterionic compounds 68 and 69, with propyl and butyl sulfonate linkers, were utilized for imaging the endoplasmic reticulum (ER) due to their electrostatic interactions with the helical domain of the phosphocholine cytidylyltransferase (CCT) enzyme. Meanwhile, compound 70, with a bromobenzyl linker, showed specificity to the mitochondria.[56] Similar molecular scaffolds (71) with propyl sulfonate and methyl groups (72) were synthesized (Figure [16]) for in vitro and ex vivo bioimaging. The compounds show near-IR emission, a significant Stokes shift (>180 nm), and a high fluorescence quantum yield (12.8–13.7%) with an excellent two-photon absorption cross-section. The molecules are also characterized by an octyloxy chain on one of the aromatic terminals. Upon incubation in cells, 70 showed staining in the membrane, while 71 showed staining within the mitochondria with a good Pearson correlation coefficient. Furthermore, deep-tissue penetration of about 100 μM was also demonstrated with 72 in live rat skeletal muscle tissues upon continuous irradiation with a 900 nm NIR pulsed laser.[57]

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Figure 17 Representative synthetic scheme to access cationic α-cyanostilbenes 68, 70 and 76

A julolidine-substituted α-cyanostilbene with a pyridinium moiety (73) was synthesized for dual imaging of mitochondria and the nucleolus (Figure [18]). The molecule, with donor-acceptor substituents emitting at ca. 610 nm, showed improved water solubility and localization in mitochondria, with a Pearson correlation coefficient of 0.71. Compound 73 also showed substantial, ca. 155-fold, fluorescence intensity enhancement with glycerol, attributed to the restriction of intramolecular rotations. The compound showed enhanced fluorescent intensity due to increased cellular viscosity induced by the addition of Nystatin, Staurosporine, and Lipopolysaccharide. Additionally, the nucleolar staining of 73 was confirmed by rRNA digestion with the help of enzyme RNase A in HeLa cells, which disrupts the RNA binding, signaling potential localization in the nucleolus.[58] Other cyanostilbene derivatives with pyridinium moieties include 47 and 74. Compound 47 is used for dual-color imaging of the nucleolus and mitochondria with organelle-specific emission. The molecule bears α-cyanostilbene as the AIE skeleton, a dimethylamino donor, and a pyridinium moiety that functions as both an acceptor and a mitochondrial targeting unit, exhibiting excellent colocalization with Mito-Tracker Green in HeLa cells. In addition, distinct fluorescence is observed in the nucleolus, attributed to the interaction with nucleic acids.[59] Compound 74 bears an α-cyanostilbene with a pyridinium cation as the mitochondrial targeting group and a diethylamino donor. The fluorophore is highly sensitive to changes in viscosity due to the restriction of intramolecular motion, resulting in a ca. 117-fold increase in fluorescence intensity at 625 nm, and shows sensitivity to endogenous cellular viscosity with Nystatin and Monensin agents.[60] The authors also demonstrate a monitored decrease in viscosity with temperature from 37 to 45 °C through fluorescence. Apart from the pyridinium cation, a triphenylphosphonium pendant attached to the cyanostilbene scaffold was also utilized to target mitochondria. A long hydrocarbon chain compound 75 enables self-assemblies as nanoparticles or nanorods in water, yielding a solid ‘turn-on’ emission. The molecule accumulates within the mitochondria and shows greater uptake in cancer cells than in normal cells. The authors also demonstrate the utility of the nanoparticles for drug delivery in a xenograft mouse model.[61]

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Figure 18 (A) Molecular structure of α-cyanostilbene 73. (B) Emission spectra of 73 in glycerol/water mixtures. (C) The effect of viscosity on the mitochondrial fluorescence signal of 73 with cells with and without nystatin treatment. (D) Nucleolus imaging. (a–c) Cells stained with Hoechst and 73, in which the nucleolus is noted. (d–f) The same dye channels but the cells were treated with RNase A. (E) Effect of apoptosis and inflammation on mitochondrial viscosity. (a–c) The effect of staurosporine treatment and (d–f) the effect of LPS treatment (10 μg mL−1) on mitochondrial viscosity. Scale: 10 μm.[58]

An α-cyanostilbene 76 with dihydroxanthene (DHX) derivative with a pyridinium cationic moiety and a benzyl chloride group was utilized for selective mitochondrial localization (Figure [19]). The molecule emitting at 755 nm shows 67-fold enhanced emission intensity with glycerol. The improved viscosity sensing ability of molecule 76 is further employed to detect viscosity in cells and mice.[62] Inducing the viscosity by adding Nystatin and LPS yields a 10 times higher enhanced fluorescence intensity. While cationic derivatives are known and show possible localization within mitochondria, neutral pyridine derivatives show localization within the lipid droplets. Neutral pyridyl derivatives (77 and 78), with coumarin and julolidine donor groups, shows selective staining to lipid droplets with excellent Pearson correlation coefficient of 0.97 for 77 and 0.96 for 78 when colocalized with commercial tracker Oil Red O. A double rotor structure 79, with cyanostilbene and benzothiazole, was used for lipid droplet imaging[63] and also to investigate the viscosity of lipid droplets during erastin induced ferroptosis. The rotor structure enables a ca. 17-fold fluorescence intensity enhancement by adding glycerol. The molecule could also be used to sense changes in the cellular viscosity induced with Nystanin and Monensin, with similar intensity enhancement. Triphenylamine-substituted α-cyanostilbenes 80, 81, and 82 were synthesized with pyridyl acetonitrile acceptor moieties for pH sensing and cellular imaging. The molecules exhibit a significant Stokes shift with λem at 556, 560, and 567 nm for 80, 81, and 82, respectively, and bright fluorescence in the solid state. The enhanced emission intensity in acetonitrile/water binary mixtures is attributed to the aggregation, and this aggregation emission behavior is utilized for pH sensing (pH 2.0–7.0). Distinct color changes were noted with a change in pH from red to green in solution, and the emission response is attributed to the twisted intramolecular charge-transfer phenomenon.[64] The cellular imaging reveals no specific organelle staining. Recently in 2023, we have developed trifluoromethyl substituted α-cyanostilbene derivatives 83–86 with naphthalene and julolidine scaffolds for specific staining of lipid droplets.[65] The probes were further used to quantify the number and size of the LDs upon oleic acid stimulation (Figure [20]). Cyanostilbene scaffolds 87–89, substituted at one end with tetraphenylethylene and the other end with triphenylamine unit were synthesized and examined in cellular internalization studies.[66] The molecules showed excellent AIE properties. The cellular imaging studies revealed good cellular uptake and the imaging studies reveal predominant localization in the cytoplasm.

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Figure 19 (A) Molecular structure of α-cyanostilbene 76. (B) Effect of nitrile group on viscosity enhancement (emission spectra in water/glycerol mixture). (C) Colocalization experiment with 76 and Mito-Tracker Green. (D) Inflammation treatment of mice with LPS and recognition of LPS-mediated inflammation. (E) Effect of starvation (media devoid of FBS) on fluorescence intensity of 76 in cells. (F) Recognition of cellular viscosity induced during inflammation (LPS treatment) and Nystatin.[62]
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Figure 20 (A) Molecular structure of α-cyanostilbene 84. (B) CLSM images of cos-7 cells stained with 84 without (–) and with (+) oleic acid pre-treatment for 4 h. (C) and (D) Analysis of number and size of lipid droplets without (–) and with (+) oleic acid pre-treatment for 4 h.[65]

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Conclusions and Future Prospects

This account highlights the versatility and utility of cyanostilbene-based fluorescent probes for various sensing and imaging applications. Their AIE properties enable sensitive and selective detection of analytes, while their biocompatibility and targeting abilities make them promising candidates for sub-cellular imaging applications. However, cyanostilbenes still have low Φ f and Stokes shift, and, hence, there is a need to develop better designs for greater sensitivity, photostability, brightness, and signal-to-noise ratio. Despite their many known applications, there is still scope for improvement, especially in their use as gelators for the development of low molecular weight or supramolecular gels for strong biomedical applications, such as drug delivery, tissue engineering, and wound healing. Integrating the robust fluorescent moieties with biomedical gel strips could enable real-time tracking of drug action or cancer. Further, tuning the architecture of cyanostilbenes can yield near-IR emitting materials for multiplexed imaging, super-resolution microscopy, and in vivo and in vitro imaging with probes that have greater penetration depth through biological tissues and minimal toxicity. As the research field continues to expand and newer molecules are developed, cyanostilbenes can be further tuned to enable, for example, (1) signal amplification for analyte detection, (2) wearable and flexible sensors that are photochromic for applications such as medical diagnosis or environmental monitoring, (3) multifunctional sensors by incorporating suitable analyte detecting functional groups, and (4) real-time monitoring of the quality and safety of food, water and air. It is essential to take the lessons from the studies and integrate the fluorescence probes with other technologies, such as microfluidics and lab-on-a-chip devices. Overall, the prospects for novel fluorescence probes with a multifunctional cyanostilbene scaffold are promising.


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Conflict of Interest

The authors declare no conflict of interest.

Acknowledgment

S.K. thanks all his students who were instrumental in the research progress.


Corresponding Author

Sriram Kanvah
Department of Chemistry, Indian Institute of Technology Gandhinagar
Palaj- 382355
India   

Publication History

Received: 10 March 2023

Accepted after revision: 10 May 2023

Accepted Manuscript online:
10 May 2023

Article published online:
11 July 2023

© 2023. Thieme. All rights reserved

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Figure 1 Representative timelines summarizing the discovery and application of aggregation-induced emission properties of cyanostilbenes
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Figure 2 Overview of the applications of α-cyanostilbenes
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Figure 3 Molecular structures of α-cyanostilbenes 1–23
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Figure 4 (A) Molecular structure of α-cyanostilbene 5 (B), (C), and (F) The plots indicate interaction with different surfactants, CTAB, SDS, and Triton, respectively. (D), (G), and (H) show SEM images in aqueous solution and with 15 × 10–4 M SDS and 2 × 10–4 M CTAB, respectively.[18] (I) Molecular structure of α-cyanostilbenes 9 and 10. (J) Absorption spectra of 3 upon addition of TFA (0–1.7 equiv) measured at a concentration of 5 μM in CHCl3, and (K) associated color change and product formation upon exposure to TFA vapor. (L) Two-component organogel morphology (Xerogel SEM) and (M) associated color and gel morphology changes upon exposure to TFA.[17]
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Figure 5 Molecular structures of α-cyanostilbenes 24–43
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Figure 6 (A) Molecular structure of α-cyanostilbene 26. (B) Normalized fluorescence spectra of 26 with varied water content. (C) (i) Photographs of test papers before and after absorbing a certain amount of 1. Fluorescence photographs of test papers after soaking in (ii) THF and (iii) dioxane solutions with different amounts of water.[34]
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Figure 7 Synthetic scheme for α-cyanostilbene 28
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Figure 8 (A) Molecular structure of α-cyanostilbene 40. (B) Emission spectra of 40 in the presence and absence of PA (50 μM). (C) Color demarcation in solution and gel disruption in the gel state. (D) Chromatographic strip test for PA detection with varied concentrations (0–20 μM).[38] (E) Molecular structure of α-cyanostilbene 43. (F) Emission spectra of 43 in the presence of various nitroaromatics. (G) Emission spectra of 43 with increasing concentration of PA. (H) Chromatographic strip tests with varied concentrations of PA.[39]
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Figure 9 Molecular structures of α-cyanostilbenes 44–64
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Figure 10 Synthetic strategy for α-cyanostilbene 44
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Figure 11 (A) Molecular Structure of α-cyanostilbene 45. (B) Images of 45 in 1,4-dioxane/water binary mixture under 365 nm UV illumination; (C) The normalized fluorescence spectra of 45; (D) Kinetic behavior of 45 with N2H4 at different times (0–60 min). (E) Photographs of 45 after the addition of different concentrations of hydrazine.[42]
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Figure 12 (A) Molecular structure of α-cyanostilbenes 47 and ThT. (B) The fluorescence emission spectra of both ThT and (C) 47 (0–50 μM) in the presence of monomeric or fibrillar α-synuclein (20 μM). (D) and (E) Binding affinity and competitive binding of 47 and ThT to α-synuclein fibrils, and (F) Monitoring and visualizing the elongation of α-synuclein fibrils using ex-situ fluorometry and TIRF microscopy from each time point. Scale bar represents 5 μm.[44]
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Figure 13 Synthetic scheme for 61, 64, and 20
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Figure 14 Molecular structures of α-cyanostilbenes 65–67 and synthetic scheme for 66
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Figure 15 (A) Molecular structure of α-cyanostilbene 65; (B) Absorption and emission spectra of 63 with and without GHB; (C) Chromatographic strip paper test for GHB detection.[53] (D) Molecular Structure of α-cyanostilbene 66; (E) Emission spectra of 66 with vitamin B1 and competitive analytes; (F) Representation of vitamin B1 detection via complex formation.[54]
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Figure 16 General representation of α-cyanostilbenes employed for imaging applications and structures of various cyanostilbenes 68–89
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Figure 17 Representative synthetic scheme to access cationic α-cyanostilbenes 68, 70 and 76
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Figure 18 (A) Molecular structure of α-cyanostilbene 73. (B) Emission spectra of 73 in glycerol/water mixtures. (C) The effect of viscosity on the mitochondrial fluorescence signal of 73 with cells with and without nystatin treatment. (D) Nucleolus imaging. (a–c) Cells stained with Hoechst and 73, in which the nucleolus is noted. (d–f) The same dye channels but the cells were treated with RNase A. (E) Effect of apoptosis and inflammation on mitochondrial viscosity. (a–c) The effect of staurosporine treatment and (d–f) the effect of LPS treatment (10 μg mL−1) on mitochondrial viscosity. Scale: 10 μm.[58]
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Figure 19 (A) Molecular structure of α-cyanostilbene 76. (B) Effect of nitrile group on viscosity enhancement (emission spectra in water/glycerol mixture). (C) Colocalization experiment with 76 and Mito-Tracker Green. (D) Inflammation treatment of mice with LPS and recognition of LPS-mediated inflammation. (E) Effect of starvation (media devoid of FBS) on fluorescence intensity of 76 in cells. (F) Recognition of cellular viscosity induced during inflammation (LPS treatment) and Nystatin.[62]
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Figure 20 (A) Molecular structure of α-cyanostilbene 84. (B) CLSM images of cos-7 cells stained with 84 without (–) and with (+) oleic acid pre-treatment for 4 h. (C) and (D) Analysis of number and size of lipid droplets without (–) and with (+) oleic acid pre-treatment for 4 h.[65]