Synthesis of versatile fluorescent isoquinolinium salts and their applications

Abstract

Isoquinolinium salts are well-known N-heterocyclic cationic compounds that have been applied in various research fields, such as materials chemistry and pharmaceutical applications. In particular, isoquinolinium salts with polyaromatic structures have been widely investigated due to their intrinsic photophysical properties, including fluorescence emission maxima, fluorescence lifetime, and quantum yield. Notably, fluorescent isoquinolinium salts have been synthesized via various synthetic strategies, such as alkylation of isoquinoline, oxidation of dihydropyridines, rearrangement reactions, and transition-metal-catalyzed C–H activation. In this review, we summarize the synthetic methodologies for diverse fluorescent isoquinolinium salts and their applications in pharmaceutical applications, materials chemistry, theranostics, DNA binding, fluorescent sensing, and bioimaging.

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Ye Ri Han

Dr Ye Ri Han received her PhD degree in Chemistry from Yonsei University in 2018. She is currently an Assistant Professor in the Department of Chemistry at Duksung Women's University. Her research interests include transition-metal-catalyzed C–H activation, materials chemistry, medicinal chemistry, and fluorescence-based bioimaging.

Sang Bong Lee

Sang Bong Lee is currently Executive Director and Principal Researcher at SimVista Inc., a company-affiliated research institute. He previously held research and senior research positions at national institutes and biotechnology companies, with expertise in drug development, microrobotics, vaccine R&D, and preclinical strategy.

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1. Introduction

Among various N-heterocycles, isoquinolinium salts represent privileged N-heterocyclic cationic scaffolds that have attracted considerable attention owing to their unique physicochemical properties.^1–3 In recent years, numerous N-heterocyclic small-molecule fluorophores such as pyridinium, quinolinium, imidazolium, and related scaffolds also have been developed for live-cell imaging and organelle-specific visualization, driven by their structural tunability and mitochondrial membrane targeting capability.^4,5 These compounds have also been actively explored in radiolabeling-based bioimaging studies through incorporation of isotopic atoms into their molecular frameworks.^6,7

Notably, isoquinolinium salts have been widely employed in materials chemistry and pharmaceutical applications.^8–12 Despite concerns about toxicity and the poor pharmacokinetic properties of their structure, they are effectively applied in neurology, antimicrobial therapy, local anesthesia, and cancer therapy.^13–17 Isoquinolinium cores are also found in natural alkaloid compounds such as coralyne, sanguinarine, and berberine.^18,19

In addition, isoquinolinium salts are used in the development of various fluorescent dyes due to their advantages such as photophysical activity, structural diversity, ease of detection, and high solubility in polar solvents.^3,20–22 With these intrinsic properties, fluorescent isoquinolinium dyes have been employed in a broad range of recent studies, including cell and in vivo bioimaging, photocatalysis, organic light-emitting diodes, fluorescent sensors, DNA binders and theranostics.^23–29 Notably, several isoquinolinium-based compounds exhibiting aggregation-induced emission (AIE) characteristics have been effectively utilized in photodynamic therapy and in vivo bioimaging.^30–33 Bioimaging and theranostics, in particular, are closely interconnected fields that have garnered substantial attention in contemporary biomedical research due to their effectiveness in the rapid diagnosis and treatment of diseases.^34–36 These studies are expected to enable more accurate diagnosis, effective treatment, and real-time disease monitoring. To advance fluorescence-related research, fluorescent isoquinolinium salts offering high-resolution and sensitive imaging without photobleaching and toxicity are still under development. To further promote their application, future research may focus on integrating isoquinolinium-based fluorophores into real-time in vivo imaging platforms and designing target-specific probes for precise diagnostics. Herein, the recent reports on diverse fluorescent isoquinolinium salts are classified and summarized according to various synthetic methods and their applications including bioimaging, DNA binding, and theranostics (Fig. 1). The synthetic methodologies and biological applications of fluorescent isoquinolinium salts are discussed in detail. Notably, the first application of isoquinolinium salts as fluorescent probes was reported in 1949 by Huang Hsinmin's research group, who developed a novel type of cyanine dye following cyclization.³⁷ Despite the long-standing interest and historical development of fluorescent isoquinolinium salts, comprehensive review articles on this topic remain limited. This review is expected to provide valuable insights for the development of novel and advanced fluorescent isoquinolinium salts.

Fig. 1 Fluorescent isoquinolinium salts and their representative applications in bioimaging and theranostics. (Adapted with permission from ref. 31, 35 and 38. Copyright 2020, 2019, and 2021, respectively. © The Royal Society of Chemistry. Adapted with permission from ref. 23. © 2023, American Chemical Society. Adapted with permission from ref. 18 and 39. Copyright 2024 and 2022, respectively. © Wiley-VCH.)

2. Various synthetic methods of fluorescent isoquinolinium salts

Isoquinolinium salts are relatively easy to synthesize, and structural modifications allow for the design of fluorescent compounds with diverse properties. As shown in Fig. 2, the synthetic methods can be broadly classified into five major categories, including transition-metal-catalyzed N-annulation and metal-free N-annulation using ethynyl benzaldehyde. Isoquinolinium salts can also be obtained as final products through sequential alkylation, protonation, rearrangement, and oxidation of isoquinoline. In particular, among these various synthetic approaches, transition-metal-catalyzed N-annulation has been the most extensively studied due to its facile functionalization and simple reaction steps. Each synthetic method, along with representative examples, is described in detail.

Fig. 2 Synthetic strategies for fluorescent isoquinolinium salts.

2.1. Transition-metal-catalyzed C–H activation and N-annulation

We summarized fluorescent isoquinolinium salts synthesized via transition-metal-catalyzed C–H activation and N-annulation, as shown in Fig. 3. These reactions have emerged as powerful synthetic strategies for the efficient construction of target compounds, offering several advantages. They play a crucial role in forming new bonds and enable the installation of key functional groups or the construction of core frameworks. This approach not only reduces the number of synthetic steps but also provides high reactivity and minimizes by-product formation, thereby improving overall synthetic efficiency.

Fig. 3 Examples of isoquinolinium salt synthesis via Rh(III)-catalyzed C–H activation and N-annulation to obtain: (A) polycyclic purinium-based cationic fluorescent compounds (adapted with permission from ref. 22. © 2024, American Chemical Society); (B) isocoumarin-conjugated isoquinolinium salts (adapted with permission from ref. 1. © 2023, American Chemical Society); (C) AIE fluorophores (adapted with permission from ref. 33. © 2019, Wiley-VCH); (D) isocoumarin and isoquinolinium conjugated compounds (adapted from ref. 40 with permission from the Royal Society of Chemistry. Published under a Creative Commons Attribution (CC BY) licence); (E) imidazo[5,1-a]isoquinolinium tetrafluoroborate derivatives (adapted with permission from ref. 21. © 2024, Elsevier B.V.); (F) pyrrolo[2,1-a]isoquinolinium salts (adapted with permission from ref. 41. © 2023, Wiley-VCH); (G) phenaleno-isoquinolinium salts (adapted with permission from ref. 24. © 2021, The Royal Society of Chemistry); (H) N-heterocyclic quaternary ammonium salts (adapted with permission from ref. 27. © 2020, The Royal Society of Chemistry); and (I) berberine derivatives (adapted from ref. 28 with permission from the original authors. Published under a Creative Commons Attribution (CC BY 4.0) license. © 2023 The authors).

In 2022, Yang et al. designed and synthesized polycyclic purinium-based cationic fluorescent compounds via Rh(III)-catalyzed C–H activation with various functionalized C6-aryl purine nucleosides and an internal alkyne in the presence of [Cp*RhCl₂]₂, Zn(OTf)₂, and Cu(OAc)₂ in DCE (Fig. 3A).²² Interestingly, purinium salts bearing diverse counter anions were obtained using different silver additives, zinc salts, or sodium salts in each reaction. These counter anions play an important role in regulating the physicochemical properties of the fluorescent isoquinolinium salts.

In 2023, Arsenov et al. reported a tandem C–H N-annulation reaction for the synthesis of isocoumarin-conjugated isoquinolinium salts (Fig. 3B).¹ Both isocoumarin and isoquinolinium salts are well-known fluorescent scaffolds, and the study discussed their electron push–pull interactions. For the tandem synthesis, functionalized benzaldehydes, aminobenzoic acids, and internal alkynes were subjected to Rh(III)-catalyzed C–H activation in the presence of 3 equivalents of Cu(OAc)₂ and AgBF₄. After the reaction, saturated KPF₆ solution was added to obtain the final isoquinolinium salt with a PF₆⁻ counterion. Furthermore, different reactivity was observed when only 0.5 equivalent of oxidant was used; under these conditions, only the isocoumarin product was obtained.

Tang's research group designed isoquinolinium salts with aggregation-induced emission (AIE) properties by using a tetraphenylethylene derivative—a classical AIE fluorophore—as the starting material (Fig. 3C).³³ The reaction, carried out using [Cp*RhCl₂]₂ and AgBF₄, provided high yields ranging from 65% to 80%.

In 2020, Tang's research group reported another AIEgen, synthesized via Suzuki coupling followed by Rh(III)-catalyzed N-annulation, in which isocoumarin and isoquinolinium salt moieties were conjugated in the final structure (Fig. 3D).^32,40 This compound exhibits a donor–acceptor fluorescent structure effective for visualizing pathogen types, as the isoquinolinium salt moiety functions as a strong electron acceptor.

Li's research group designed and synthesized imidazo[5,1-a]isoquinolinium tetrafluoroborate derivatives via a [4 + 2] annulation reaction using a Rh(III)/Cu(II) catalytic system, achieving a high yield of 98% (Fig. 3E).²¹ In this reaction, a work-up process was performed using NaBF₄ for anion exchange, and through a similar procedure, various cation–anion complexes including OTf⁻, SbF₆⁻, Cl⁻, and PF₆⁻ were obtained in high yields of over 90%.

In 2023, Zelenkov's group designed and developed pyrrolo[2,1-a]isoquinolinium salts using 3H-pyrrole derivatives and internal alkynes in the presence of [Cp*RhCl₂]₂ as a catalyst, Cu(OAc)₂ as an oxidant, and AgBF₄ as an anion source in DCE (Fig. 3F).⁴¹ The reaction was generally carried out at 60 °C, although some compounds were obtained in high yields even at room temperature. These reaction conditions demonstrate the high reactivity of the pyrrolo[2,1-a]isoquinolinium salt synthesis approach.

Phenaleno-isoquinolinium salts were developed and reported by Han et al. in 2021, and derivative compounds were also reported in 2023 as theranostic agents. In both papers, fluorescent isoquinolinium salts were prepared using the facile Rh(III)-catalyzed C–H activation reaction. Interestingly, a pyrene moiety was employed as the starting material in an N-annulation reaction with an internal alkyne in the presence of a Rh(III)/Cu(II) complex, affording phenaleno-isoquinolinium salts in high yields of 60–86% (Fig. 3G).²⁴ These compounds exhibited high absolute quantum yields of 63–88%, except for those functionalized with a dimethylamino group.

An N-annulation reaction under Rh(III) catalysis with 1-phenyl-3,4-dihydroisoquinoline derivatives and functionalized 1,2-diphenylethyne led to the formation of fluorescent N-heterocyclic quaternary ammonium salts in yields exceeding 83% (Fig. 3H).²⁷ After the reaction, the desired product was obtained as a cation–trifluoroacetate ion pair, which functioned as a photosensitizer in subsequent application studies.

Another Rh(III)-catalyzed N-annulation was performed for the synthesis of berberine derivatives, using benzaldehyde and silylalkynyl-substituted compounds (Fig. 3I).²⁸ After the reaction, silylated compounds were treated with trifluoroacetic acid (TFA) to obtain the desired product. In the N-annulation reaction, the substrate scope for both benzaldehydes and internal alkynes afforded fluorescent isoquinolinium salts as 11- and 13-substituted berberine derivatives, which were evaluated for their DNA binding affinity in comparison to a reference compound.

As described above, various N-annulation reactions utilizing Rh(III) catalysts have been developed for the preparation of fluorescent isoquinolinium salts. However, metal-free N-annulation reactions have also been reported, particularly involving ethynyl benzaldehydes and primary amines, offering high selectivity and sensitivity (Fig. 4A).²⁶ This transformation is identified as a cascade reaction, wherein isoquinolinium salts are formed via a 6-endo-dig cyclization following imine formation.

Fig. 4 Isoquinolinium salts synthesized via metal-free approaches, including: (A) catalyst-free N-annulation (adapted with permission from ref. 26. © 2024, Elsevier B.V.); (B) iodocyclization (adapted with permission from ref. 42. © 2023, Wiley-VCH); and (C) silver-mediated cyclization (adapted with permission from ref. 39. © 2022, Wiley-VCH).

In 2023, Kawakubo et al. developed an alternative synthetic approach involving iodocyclization using iodine as a reagent, which led to the formation of pyrido[1′,2′;2,3]imidazo[5,1-a]isoquinolinium salts (Fig. 4B).⁴² In this study, the isoquinolinium salt intermediates were commonly functionalized with an iodo group, enabling a subsequent palladium-catalyzed Suzuki coupling to yield the desired products. Thus, various boronic acids were utilized in the reaction to diversify the final cationic imidazolium moiety.

Cyclization of pyridino-alkynes to afford N-heterocyclic cation derivatives in the presence of silver triflate was reported by Mule et al. (Fig. 4C).³⁹ The fundamental concept of this research is the design of novel AIEgens based on anion–π⁺ interactions. Following a facile silver-mediated 6-endo-dig cyclization, various isoquinolinium triflate derivatives were obtained in moderate to high yields ranging from 68% to 94%.

2.2. Alkylation and protonation of isoquinoline

Isoquinolinium salts can also be obtained via simple protonation or alkylation, as reported by several research groups. In 2020, Tervola et al. designed and synthesized the target compounds via protonation, expecting fluorescence enhancement (Fig. 5A).⁴³ In this study, six different organic acids were used in the protonation step to compare the effects based on their differing pK_a values. Notably, TFA was the strongest acid used in this study, and it induced a significant change in the absorption spectrum, identified as a bathochromic shift. In addition, an increase in fluorescence intensity was observed due to the sharing of the nitrogen lone pair with hydrogen upon protonation of the isoquinoline nitrogen atom, resulting in a π to π* transition from the S₁ excited state.

Fig. 5 Isoquinolinium salts synthesized via protonation (A) and (F) or N-alkylation (B)–(E) of isoquinoline-based precursors. ((A) Adapted from ref. 43. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence; (B) adapted with permission from ref. 44. © 2019 Georg Thieme Verlag KG; (C) adapted with permission from ref. 45. © 2017 Georg Thieme Verlag KG; (D) adapted with permission from ref. 46. © 1985 American Chemical Society; (E) adapted with permission from ref. 37. © Royal Society of Chemistry; (F) adapted with permission from ref. 47. © 2018 American Chemical Society).

A simple protonation reaction was also reported by Miura's group, carried out after Rh(III)-catalyzed annulative coupling to form naphtho[1,8-bc]pyran, yielding isoquinolinium salts (Fig. 5B).⁴⁴ Interestingly, the naphtho[1,8-bc]pyran and isoquinoline moieties are connected via a biaryl linkage, forming a polycyclic core structure. Thus, strong fluorescent spectra were observed in the study.

In 2017, Zhu et al. synthesized a series of imidazolyl isoquinolinium salts after N-alkylation of the corresponding isoquinoline intermediate (Fig. 5C).⁴⁵ Isoquinoline derivatives were first prepared by double C–H activation in the presence of Pd(OAc)₂ and Cu(OAc)₂. Subsequently, alkyl halides such as MeI, BuI, and BnBr were reacted with imidazolyl isoquinoline to afford the desired products in good yields (62–88%).

The Mariano group also reported a similar N-alkylation reaction of dihydroisoquinolines using methyl iodide or ethyl iodoacetate to afford various N-substituted isoquinolinium salts (Fig. 5D).⁴⁶ Subsequently, through an ion-exchange process, N-alkyldihydroisoquinolinium perchlorate was obtained as the final product. These compounds were further subjected to photocyclization, leading to the formation of spirocyclic products, and their physical properties were compared based on structural differences.

Fluorescent isoquinolinium salt derivatives were also designed and synthesized via N-alkylation of l′-R–l-R′-indolo(3′:2′-3:4)isoquinolines by Huang-Hsinmin et al. (Fig. 5E).³⁷ Three types of cation–anion combination salts—hydrochloride, picrate, and methiodide—were obtained, and the intrinsic color of each compound was investigated.

Isoquinolinium salts were also efficiently produced through alkynylation, as reported by Toriumi et al. (Fig. 5F).⁴⁷ This study represented the first development of N-alkynylpyridinium structures and other heterocyclic ynammonium compounds. Among the various N-alkynylpyridinium compounds examined, isoquinolinium salts were readily prepared from isoquinoline and terminal alkynes. In addition, an intramolecular cyclization strategy employing alkynyl-λ³-iodanes as alkynyl transfer reagents was used to obtain a broader range of fluorescent isoquinolinium salts. This one-pot reaction demonstrated high synthetic efficiency.

2.3. Other synthesis methods

We have described various synthetic strategies for the preparation of isoquinolinium salts. However, additional synthetic approaches—such as rearrangement and oxidation—have also been reported. In 2008, Tian's group designed and synthesized 3,4-dihydroisoquinolinium derivatives from acridizinium ions and primary alkylamines (Fig. 6A).⁴⁸ This reaction proceeds via an initial nucleophilic ring-opening, followed by an intramolecular aza-Diels–Alder reaction. Subsequent nucleophilic aromatic substitution at the fluorine atom afforded the desired products in low to moderate yields (15–81%). A representative fluorescent isoquinolinium salt was investigated by measuring its absorption and emission spectra, and its photophysical properties were evaluated in various solvents. Only a marginal solvatochromism was observed in the study.

Fig. 6 Strategies for the synthesis of isoquinolinium salts. (A) Rearrangement. Adapted with permission from ref. 48. © 2008 American Chemical Society. (B) Oxidative strategy. Adapted with permission from ref. 49. © 2022 Wiley-VCH GmbH.

In another study, simple oxidation following substitution of 1H-benz[de]isoquinolines afforded 2-aryl-5,8-di-tert-butyl-1H-benz[de]isoquinolinium tetrafluoroborates as the final products (Fig. 6B).⁴⁹ These compounds exhibited strong fluorescence and were isolated as yellow to orange-colored crystals. Photophysical properties, including both absorption and fluorescence maxima, were measured in various solvents. Moreover, solid-state fluorescence maxima were also analyzed and compared. It was found that, in the solid state, the fluorescence data strongly depended on molecular stacking interactions, whereas the electronic properties of the substituents showed poor correlation.

3. Analysis of physical properties

3.1. Full-color tunable fluorescent spectra

Full-color tunable fluorescent probes are widely utilized in various research fields owing to their numerous advantages. In particular, they are easily tuned to suit experimental conditions or research purposes. Such full-color tunable fluorescent spectra have also been observed in isoquinolinium salts, and the colorimetry of each is identified by its emission wavelength range, as shown in Table 1. Among several studies, these results were confirmed in five representative structures, as described below.

Table 1 Wavelength ranges corresponding to perceived visible colors

Wavelength	Fluorescent color
400–420 nm	Violet
420–440 nm	Indigo
440–490 nm	Blue
490–570 nm	Green
570–585 nm	Yellow
585–620 nm	Orange
620–780 nm	Red

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Treatment of ethynylbenzaldehyde with primary amines led to the formation of isoquinolinium salts, displaying fluorescence in the 410–535 nm range, covering the violet to green region (Fig. 7A).²⁶ These emission wavelengths correspond to structural variations, including functional group effects and degrees of conjugation. In the study, EBA-1a, functionalized with an electron-donating methoxy group and treated with butylamine, exhibited emission at 535 nm. In contrast, EBA-1d, bearing an electron-withdrawing bromo group, showed emission at 495 nm, which can be compared with EBA-1b containing only a proton substituent. These results clearly demonstrate that electron density influences the HOMO–LUMO band gap, resulting in distinct fluorescence emission colors. The effect of polyaromatic conjugation was also examined by comparing molecular structures and their corresponding emission spectra, which showed similar trends.

Fig. 7 Structure-dependent emission properties of isoquinolinium salts with full-color tunability; (A) EBA derivatives (410–535 nm). Adapted with permission from ref. 26. © 2024 Elsevier B.V. (B) Imidazo[5,1-a]isoquinolinium salts (433–633 nm). Adapted with permission from ref. 21. © 2024 Elsevier B.V. (C) Polycyclic purinium salts (436–587 nm). Adapted with permission from ref. 22. © 2024 American Chemical Society. (D) Benzoquinolizinium salts (460–683 nm). Adapted with permission from ref. 50. © 2017 American Chemical Society. (E) Isoquinolinium salts (362–400 nm). Adapted with permission from ref. 47. © 2018 American Chemical Society.

Studies on imidazo[5,1-a]isoquinolinium salts reported by Li's research group showed full-color emission in the 433–633 nm wavelength range, corresponding to the colorimetric range from violet to red (Fig. 7B).²¹ In particular, emission wavelengths were carefully compared to assess the influence of functional groups at different substitution positions. Interestingly, no clear trend was observed with respect to changes in the R¹ substituent on the imidazole moiety. However, the R² substituent exhibited a significant influence on the fluorescence properties. A blue shift in emission was observed with increasing electron-withdrawing character at the R² position. In contrast, red-shifted emission was observed upon introduction of electron-donating groups at the same site. These findings clearly indicate that the R² position in the imidazo[5,1-a]isoquinolinium scaffold plays a key role in modulating fluorescence emission.

In another study on the development of polycyclic purinium salts, the photoluminescence spectra of these compounds were examined, revealing intense fluorescence emission peaks ranging from 436 to 587 nm, corresponding to blue to orange colors (Fig. 7C).²² By varying the substituents, fluorescence emission could be effectively tuned, highlighting the potential of these compounds in materials chemistry and pharmaceutical applications. Further investigations into the photophysical properties and bioimaging applications of these compounds are currently being conducted by Yang et al.

The design and synthesis of benzoquinolizinium salts via double N-annulation yielded fluorescent emission spectra ranging from 460 to 683 nm, covering the blue to near-infrared (NIR) region (Fig. 7D).⁵⁰ These full-color tunable fluorescent dyes were efficiently synthesized under Rh(III)-catalyzed conditions, allowing for incorporation of various functional groups into their structures. Functionalization at the R² position of the benzoquinolizinium salt with electron-donating groups led to a pronounced red shift, as shown in Fig. 7D, indicating that electron density is strongly correlated with the emission maximum.

Although the fluorescence emission range was relatively narrow (362, 398, and 400 nm), due to the limited number of fluorescent isoquinolinium salts developed in this study, it serves as a representative example demonstrating structure-dependent fluorescence behavior (Fig. 7E).⁴⁷ A notable feature of these compounds, synthesized via cyclization of N-alkylpyridinium salts, is the bathochromic shift in fluorescence wavelength resulting from extended conjugation. In subsequent reactions, polyaromatic N-heterocyclic cations were developed to broaden the fluorescence emission range from violet to yellow, corresponding to 357–577 nm.

3.2. Aggregation induced emission (AIE)

The concept of aggregation-induced emission (AIE) is the opposite of aggregation-caused quenching (ACQ), which typically involves self-quenching due to intermolecular interactions at high fluorophore concentrations. AIE-active fluorescent probes exhibit strong fluorescence intensity, excellent biocompatibility, and outstanding optical stability, primarily due to their large Stokes shifts. As a result, AIE materials have been applied in diverse fields such as bioimaging, photodynamic therapy, and chemical sensing, and are considered promising candidates for next-generation light-emitting systems. AIEgens hold great potential in bioimaging and photodynamic therapy; however, it is important to recognize and systematically investigate their limitations, such as potential biotoxicity, variability in targeting selectivity, and limited light penetration in deep tissues. In this context, several studies have reported AIE-active fluorescent isoquinolinium salts.

The first example of an AIE-active compound is the study on isoquinolinium triflate derivatives synthesized via silver-mediated 6-endo-dig cyclization. Interestingly, all of these fluorescent probes exhibited a prominent Stokes shift of over 100 nm in solution.³⁹ Due to this property, investigations of fluorescence emission in both solutions and solids have shown that the quantum yield in the solid state is much higher than in solution. To further examine AIE characteristics, representative compound 2f was analyzed in mixed solvents of hexane and dichloromethane (DCM) (Fig. 8A), comparing fluorescence intensity in solution and solid states. The hexane fraction in DCM was gradually increased from 0% to 100%, and the corresponding fluorescence intensities were recorded and tabulated. At hexane fractions below 85%, only weak fluorescence was observed. However, at 88% hexane, the fluorescence of 2f significantly increased, reaching maximum intensity at 95% hexane. Thus, the DCM–hexane mixed solvent system proved effective for evaluating the AIE properties of these derivatives. Another equally important parameter that needs due consideration is the requirement of low bio- and immunotoxicity of the developed AIEgens, especially when aiming for in vivo imaging applications. While the photophysical properties are promising, future studies must evaluate the biosafety profile to ensure compatibility for biological and clinical use.

Fig. 8 AIE properties of isoquinolinium salts. (A) Compound 2f shows fluorescence enhancement at 95% hexane (Adapted with permission from ref. 39. © 2022 Wiley-VCH); (B) TPE-IQ-O exhibits yellow emission at 539 nm in 99% hexane and selectively targets mitochondria (Adapted with permission from ref. 33. © 2019 Wiley-VCH); (C) IQ-Cm displays red emission in aqueous media with lysosomal specificity (Adapted from ref. 40 with permission from the Royal Society of Chemistry. Published under a Creative Commons Attribution (CC BY) licence).

Tang's research group has reported numerous studies in this field. In particular, several studies have focused on fluorescent isoquinolinium salts as AIE-active fluorophores. In 2019, Tang and his colleagues designed and synthesized tetraphenylethylene-isoquinolinium (TPE-IQ) derivatives.³³ These compounds have typically been obtained through cyclization of the existing AIE scaffold, TPE. Additionally, cyano and triphenylamino groups were introduced into the core structure to generate structurally diverse fluorescent probes. These functional groups are well known to facilitate AIE behavior. Unlike the previous study, a hexane–THF mixed solvent system was used to investigate the AIE behavior of TPE-IQ-O (Fig. 8B). Fluorescence intensity of TPE-IQ-O gradually increased with increasing hexane content, reaching a maximum at 99 vol% hexane. The maximum fluorescence emission was observed at 540 nm, and the AIE-active fluorophore appeared bright yellow-green in the 99 vol% hexane–THF solution. For the application of these compounds, Gu et al. further explored TPE-IQ-based photosensitizers for effective photodynamic antiviral therapy by leveraging their aggregation-induced emission (AIE) properties.⁵¹

In 2020, the group further developed a hybrid system combining isoquinolinium salt (IQ) and coumarin (Cm) as a novel fluorescent dye.⁴⁰ Structurally, the molecule can be divided into three components: an isoquinolinium core, a coumarin derivative, and a phenyl linker. The isoquinolinium part is a key position to express AIE due to its highly twisted molecular structure. This core structure well represents the extended and twisted structure of the linked donor and acceptor of IQ and Cm, which can clearly explain twisted intramolecular charge transfer (TICT) properties. Notably, the study demonstrated a pronounced solvatochromic effect when the solvent was changed from dioxane to water (Fig. 8C). The fluorescence color shifted from blue to red, indicating a red shift induced by solvent polarity. The fluorescence intensity at 643 nm gradually increased with increasing water content in the DMSO–water mixture. Conversely, as the 643 nm emission increased, the fluorescence intensity at 501 nm gradually decreased. This IQ-Cm fluorescent dye was subsequently applied in various fields, and further studies were reported in 2021 in Biomaterials.³² While low tissue penetration limits the broader application of PDT using AIEgens, this limitation is effectively addressed in the present study by targeting areas with high light transmittance, such as the eye.

4. Applications

Various applications of fluorescent isoquinolinium salts have been widely explored owing to their advantageous photophysical properties. These fluorescent dyes exhibit strong emission due to their high photostability and quantum yield. Applications such as cell imaging, in vivo imaging, DNA binding, and theranostics are closely related to biological studies. In particular, numerous bioimaging studies employing fluorescent isoquinolinium salts have been reported. Additionally, applications based on the photophysical properties of these dyes have also been explored in fluorescent sensors and exciplex systems.^52–54 Interestingly, some studies have utilized fluorescent isoquinolinium salts as intermediates for synthesizing isoquinolines.^45,55–57 These studies are categorized in the following sections, with representative examples described in detail.

4.1. Cell imaging

In 2022, Mule et al. reported live-cell imaging studies using fluorescent isoquinolinium dyes (Fig. 9A).³⁹ They hypothesized that cancer cells preferentially absorb cationic compounds due to their relatively more hyperpolarized mitochondrial membrane potential compared to normal cells. In the study, among various fluorescent probes, 2m and 2u were selected for cell imaging due to their tunable photophysical properties. The cytotoxicity of these fluorescent dyes was first evaluated to assess their biocompatibility. Using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assays with live BHK-21 cells, appropriate staining concentrations of 2m and 2u were determined. Intracellular localization was subsequently examined by comparing the fluorescent probes with commercial organelle trackers—MitoTracker Red, ER Tracker Red, and LysoTracker Red—in BHK-21 cells. Notably, these fluorescent isoquinolinium salts showed high correlation with MitoTracker Red for cell imaging. Treatment of BHK-21 cells with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a well-known mitochondrial oxidative phosphorylation uncoupler, reduced the staining efficiency of the fluorescent isoquinolinium salts. This result confirmed that the cationic probes are sensitive to and dependent on mitochondrial membrane potential (MMP) in live-cell imaging.

Fig. 9 Cellular imaging and photophysical properties of the compounds. (A) Structure of 2m and 2u which were utilized staining BHK-21 cells for CLSM images (Adapted with permission from ref. 39. © 2022 Wiley-VCH). (B) Unlike 4a, styryl-substituted derivatives 4h displays enhanced intracellular fluorescence and ER specificity. (Adapted with permission from ref. 42. Copyright 2023, Wiley-VCH.) (C) Pyridoimidazoisoquinolinium salts (2c) exhibit selective mitochondrial accumulation with cytoplasmic localization. (Adapted from ref. 58. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.)

Additionally, several studies have investigated cell imaging using human cervical cancer (HeLa) cells. In 2023, cell staining assay using pyridoimidazoisoquinolinium salts was reported by Yasuike's research group (Fig. 9B).⁴² In this study, HeLa cells were used to evaluate the bioimaging capabilities of the fluorescent probes, excluding compound 4a, which exhibited no detectable fluorescence quantum yield. After staining, the cells were observed using confocal laser scanning microscopy (CLSM). Due to structural differences among the dyes, they exhibited distinct fluorescence intensities correlating with their quantum yields and localized in specific subcellular regions such as the cytoplasm or nucleus. Notably, compound 4h exhibited co-localization with a commercially available ER-Tracker Green, indicating its ability to stain the endoplasmic reticulum (ER). Although compound 4h possesses a cationic character, it exhibits specific localization to the ER, which is likely attributable to the structural influence of the 5-styryl substituent. The emission maximum of the fluorescent dye was observed at 454 nm, corresponding to blue fluorescence in imaging experiments. In addition, compound 4h accumulated in the endoplasmic reticulum of rat basophilic leukemia (RBL-2H3) cells, without affecting cell viability as determined by MTT assays. These findings suggest that the fluorescent dye selectively stains specific cell types or organelles.

Yasuike's research group reported another application in cellular imaging utilizing pyridoimidazoisoquinolinium salts (Fig. 9C).⁵⁸ This study also involved cellular imaging of HeLa cells and fluorescence analysis using CLSM. Some compounds exhibited structural characteristics that favored localization in the cytoplasm rather than the nucleus. MTT assays confirmed that none of the isoquinolinium salts affected cell viability during bioimaging. Among the fluorophores developed, compound 2c exhibited the strongest fluorescence intensity and efficiently stained HeLa cells. In subcellular localization studies of 2c, co-staining with MitoTracker Green showed clear overlap in fluorescence, indicating mitochondrial localization. Interestingly, these results demonstrate that 2c functions effectively as a mitochondrial tracker, likely due to the cationic nature of the isoquinolinium salt structure.

In 2025, Wong's research group investigated cellular imaging studies of fluorescent isoquinolinium salts in HeLa cells using fluorophores synthesized from ortho-ethinylbenzaldehyde (EBA).²⁶ This study focused on the turn-on fluorescence properties arising from the facile cyclization of EBA and primary amines to yield fluorescent probes. Thus, the ethynyl group must be positioned ortho to the aldehyde in EBA to enable intramolecular cyclization with cellular primary amines. EBA derivatives with differently positioned ethynyl groups were also synthesized for comparison; these compounds did not exhibit fluorescence in cell imaging. Additionally, structural differences among EBA derivatives resulted in varying degrees of cytotoxicity, and suitable candidates were selected accordingly. Mitochondrial tracking ability, cell permeability, and fluorescence quantum yield were all influenced by the structural features of the isoquinolinium salts. In particular, quantum yield was a key criterion; fluorophores with low quantum yield were excluded from further cell imaging studies. Consequently, EBA-1a–1e and EBA-4 were identified as efficient bioimaging agents for labeling primary amines in live cells, without the need for additional mito-targeting functionalization.

4.2. In vivo bioimaging

In vivo imaging studies using fluorescent probes are an important field of research as they enable real-time monitoring of various physiological and biochemical processes. Among the various in vivo bioimaging approaches, sentinel lymph node (SLN) imaging has recently been reported by several research groups. In particular, SLN imaging is a significant area of research, as it enables monitoring of cancer metastasis through the detection of lymphatic spread. A variety of SLN imaging agents—including radiolabelled fluorophores, nanoparticles, and small-molecule fluorescent dyes—have been developed and extensively studied. Novel fluorescent isoquinolinium salts were also investigated by Han et al. for SLN bioimaging. The first study, reported in 2019, involved the synthesis of benzoquinolizinium salts via a double N-annulation reaction between primary allylamines and internal alkynes, followed by application in SLN bioimaging (Fig. 10A).³⁵ In this study, the MF1 fluorescent probe was selected based on its photophysical properties, particularly its emission maximum at 683 nm, corresponding to the far-red region. Notably, this fluorescent dye was used to detect inflammatory lesions in a carrageenan-induced mouse model. A strong fluorescent signal from MF1 was detected in the inflamed paw, in contrast to the control paw injected with PBS. However, 24 hours post-injection, the fluorescence intensity in both locations equalized, indicating that the probe had been successfully released and cleared. These results suggest that this fluorophore can be utilized for in vivo tracking of acute inflammation. Further studies were conducted to evaluate the physiological response of this fluorescent dye to anti-inflammatory treatments in inflammation imaging. The well-known anti-inflammatory drugs dexamethasone and sulfasalazine were used in this study. Upon administration of these drugs to inflammation-induced mice, subsequent intravenous injection of MF1 showed significantly reduced uptake in the inflamed paw. Therefore, this fluorescent isoquinolinium salt serves as a useful tool for imaging and evaluating inflammation in vivo.

Fig. 10 In vivo imaging applications of isoquinolinium-based fluorophores. (A) MF1 accumulates in inflamed tissue and enables fluorescence imaging of acute inflammation in a mouse paw edema model. Adapted with permission from ref. 35. © The Royal Society of Chemistry. (B) MF37–40 selectively highlight sentinel lymph nodes within 15 minutes post-injection with strong in vivo fluorescence. Adapted with permission from ref. 24. Copyright 2021, The Royal Society of Chemistry.

In 2021, new phenalenoisoquinolinium salts were designed, synthesized, and applied as SLN bioimaging agents (Fig. 10B).²⁴ With the exception of one probe exhibiting low fluorescence intensity, these compounds were intravenously injected into live mice as lymphatic imaging agents, enabling effective SLN visualization within 1 minute. Subsequently, ex vivo fluorescence imaging of the organs confirmed that these MF fluorophores are suitable for accurate lymphatic imaging and hold promise for clinical staging applications.

4.3. Theranostics

Recently, numerous research groups have published studies advancing in vivo imaging combined with therapeutic functions, collectively referred to as theranostics. MF33, a phenalenoisoquinolinium salt, has been applied in theranostic studies as an anticancer agent beyond its use in in vivo bioimaging (Fig. 11A).²³ Based on in vitro and in vivo biocompatibility data, MF33 was evaluated for its antitumor activity in HCT-116 tumor-bearing mice. After daily intraperitoneal injection of MF33 into mice at the indicated doses for up to 18 days, effective antitumor activity was observed in a dose-dependent manner. In contrast, control mice treated only with vehicle exhibited rapid tumor growth. Moreover, no significant weight loss was observed in either group during the anticancer treatment. These results suggest that this probe holds strong potential as both an anticancer and theranostic agent for colorectal cancer treatment.

Fig. 11 Application of isoquinolinium salts as theranostic agents. (A) MF33 shows in vivo anticancer efficacy and sentinel lymph node imaging in mouse models. Reproduced with permission from ref. 23. © 2023 American Chemical Society. (B) LIQ-6 enables photodynamic cancer therapy and antibacterial activity via mitochondrial targeting. Reproduced with permission from ref. 31. © 2020 The Royal Society of Chemistry.

In several studies, fluorescent isoquinolinium salts have been applied in photodynamic therapy (PDT). In PDT studies, fluorescent dyes act as photosensitizers that are activated by specific wavelengths of light. After activation, these dyes generate reactive oxygen species (ROS) to destroy targeted cells, especially useful for treating certain cancers or bacterial pathogens.

In 2020, Feng's research group developed and reported theranostic agents that are applicable to both photodynamic anticancer and antibacterial therapies (Fig. 11B).³¹ The core structure of the photosensitizer was derived from N-annulation of 1-phenyl-3,4-dihydroisoquinoline derivatives. Notably, the LIQ-6 compound, modified with a strong electron-donating group, exhibited markedly enhanced phototoxicity. This fluorescent dye was first applied to induce apoptosis in cancer cells under white light irradiation. In addition, the probe showed broad efficacy against various cancer cell lines, including HeLa, breast cancer, hepatoma, and melanoma cells. LIQ-6 also enabled imaging of these cancer cells, allowing real-time monitoring of the apoptosis process. In addition to its anticancer effect, the probe was also employed as an antibacterial agent owing to its efficient ROS generation capability. LIQ-6 binds to bacteria and eliminates them via a photodynamic mechanism. This therapy was effectively applied in vivo to promote wound healing. As a result, even in wounds infected with S. aureus, treatment with LIQ-6 combined with light irradiation exhibited the strongest therapeutic effect compared to control groups. Thus, this fluorescent isoquinolinium salt is considered a versatile fluorophore for efficient photodynamic theranostic applications. Additionally, isoquinolinium structures with systematically tuned donor–acceptor characteristics may further enhance PDT efficacy.

To date, Tang's research group has reported various PDT studies, several of which are based on isoquinolinium salt core structures.⁵⁹ In one such study, TPE-IQ-based AIEgens were employed as PDT agents capable of generating ROS upon light irradiation (Fig. 12A).³³ The phototoxicity of the developed fluorescent dyes was evaluated using MTT assays under white light irradiation. Depending on their structural features, the probes exhibited varying photodynamic therapeutic effects in HeLa cells, attributed to their targeting of different organelles such as lysosomes or mitochondria. In this study, mitochondrial-targeting probes showed superior PDT efficacy, with TPE-IQ-O identified as the most potent PDT agent. These findings strongly correlate with the cell imaging results obtained using the TPE-IQ-based AIEgens. Under identical staining conditions, the bioimaging properties of these fluorophores were investigated using CLSM. Interestingly, TPE-IQ-O effectively stained the cells and exhibited strong co-localization with MitoTracker Red CMXRos, suggesting its high specificity for mitochondria. In contrast, TPE-IQ-CN and TPE-IQ-TPA were not detected in the cytoplasm, likely due to delayed cellular uptake associated with lysosomal targeting. This delayed cellular uptake is attributed to their higher zeta potentials and smaller hydrodynamic diameters, which collectively increase the energy barrier for direct membrane permeation.

Fig. 12 Theranostic applications of isoquinolinium-based AIEgens in cell and microbial systems. (A) TPE-IQ derivatives enable organelle-targeted photodynamic cancer therapy via mitochondria or lysosome localization and light-induced ROS generation. Adapted with permission from ref. 33. © 2019 Wiley-VCH. (B) IQ-Cm selectively images and eradicates S. aureus through fluorescence-guided PDT in a bacterial keratitis model. Adapted with permission from ref. 32. © 2017 Elsevier Ltd.

Tang's research group also developed new AIEgens for the treatment of bacterial keratitis (BK) using PDT (Fig. 12B).³² The IQ-Cm structural fluorescent probe was utilized as an effective theranostic agent. This fluorescent probe selectively illuminated S. aureus over normal cells and exhibited superior cytocompatibility toward human corneal epithelial cells (HCECs) compared to the commercial PDT agent Rose Bengal (RB). Interestingly, the combination of dark antibacterial activity and light-induced ROS generation by IQ-Cm resulted in significant apoptosis of S. aureus in vitro. For in vivo evaluation, BK rabbit models were established via S. aureus infection, and IQ-Cm was administered via intrastromal injection. As a result, strong antibacterial activity was observed upon light irradiation following intrastromal injection of the probe in the BK rabbit model. These results suggest the potential of this agent as an effective antibacterial photosensitizer that integrates photodynamic therapeutic properties with antibacterial activity to combat bacterial drug resistance.

In 2022, Guo et al. published a review on AIE-based photosensitizers for mitochondria-targeted PDT.³⁰ In this research, the authors highlighted the importance of AIEgens and their ROS-generating capability in therapeutic applications. The mechanism of ROS generation was explained in detail. Additionally, mitochondria-targeting moieties such as phosphonium and pyridinium salts were introduced as functional groups. Examples of fluorophores functionalized with these groups were presented in the research. Notably, several isoquinolinium dyes possessing AIE characteristics were also introduced as promising candidates for mitochondria-targeted PDT.

In recent studies, fluorescent isoquinolinium salts have been explored in PDT research based on AIE frameworks. These molecules show key features such as high singlet oxygen generation efficiency, excellent biocompatibility, and selective targeting via mitochondrial membrane potential or bacterial membrane interactions. Interestingly, despite structural similarities, each study targets a different biological system. These works demonstrate the broad therapeutic potential of isoquinolinium-based AIE photosensitizers in fungal infections, cancer, and bacterial resistance, providing a versatile and selective platform for next-generation PDT.^60–62

4.4. DNA binding

A DNA intercalating agent is a compound that inserts between the base pairs of the DNA helix, thereby altering its structure. Such compounds play crucial roles in biological and medical research, with potential applications in cancer therapy, gene delivery, diagnostics, and drug transport systems.⁶³ Several studies have reported the application of fluorescent isoquinolinium salts in DNA binding.

In 2009, Rubis et al. reported a study on a papaverine-derived ligand that binds to G-quadruplex DNA (Fig. 13A).²⁹ This study focused on telomerase, an enzyme closely linked to carcinogenesis and considered a key biomarker for distinguishing normal from cancerous cells. Motivated by this, the authors designed and synthesized a new papaverine oxidation compound and evaluated its effects on telomerase activity in breast cancer cells. Interestingly, the telomerase activity varied depending on the concentration of ligand 1, as shown in Fig. 13A. At concentrations below 0.1 μM, ligand 1 increased telomerase activity, whereas higher concentrations led to inhibition of polymerase function. The proposed mechanism involves π–π stacking with the G-quartet, leading to stabilization of the quadruplex and reduced telomerase accessibility. These findings suggest that isoquinolinium-based ligands act as selective telomerase inhibitors via G4-DNA interaction, with ligand 1 showing concentration-dependent selectivity. This property may be advantageous for developing more effective anticancer agents.

Fig. 13 Isoquinolinium-based systems for DNA-targeted anticancer and imaging applications. (A) Ligand 1 targets G-quadruplex DNA and induces telomerase inhibition. Adapted from ref. 29. © 2008 Springer Science and Business Media, LLC. (B) PMSAILs compact DNA and alter fluorescence intensity by binding to DNA–EB complexes, leading to the formation of DNA–surfactant assemblies. Adapted from ref. 64. © 2018 Published by Elsevier B.V.

Research on DNA interaction using paramagnetic surface-active ionic liquids (PMSAILs) was published by Kumar's group in 2018 (Fig. 13B).⁶⁴ PMSAILs bearing pyridine and isoquinoline-type headgroups were synthesized and evaluated. Notably, PMSAILs with an isoquinolinium head group [C12iQ] showed a pronounced ability to precipitate DNA at specific concentrations. DNA binding ability was assessed via fluorescence displacement assays using ethidium bromide (EB) as the intercalating dye. Addition of PMSAIL solution to the DNA-EB complex showed a gradual decrease in fluorescence intensity due to the formation of a DNA-surfactant complex that displaces the EB. The interaction occurs through electrostatic attraction between the cationic isoquinolinium head and the DNA phosphate backbone, displacing intercalated EB and decreasing fluorescence intensity. Additional evidence from zeta potential and electrophoresis confirmed strong DNA condensation, suggesting that isoquinolinium-type PMSAILs are promising for nucleic acid delivery applications.

In 2023, Ihmels's research group designed and synthesized a series of berberine derivatives and investigated their DNA-binding properties.²⁸ Notably, it was observed that upon addition of DNA to the berberine derivative solution, the initial absorption spectrum gradually decreased, accompanied by a red shift. The study also showed that DNA binding efficiency was influenced by the structural characteristics of each probe. Methoxy groups promoted dipole–dipole interactions with G-quadruplex DNA, whereas with duplex DNA, molecular size played a more significant role in intercalation. Corresponding results were also observed using circular dichroism (CD) spectroscopy. These compounds offer potential for selective DNA structure detection, particularly through fluorescence analysis combining intensity and lifetime measurements.

In 2024, Solomonov's group studied the binding interactions of ionic liquids with bovine serum albumin (BSA). Various cationic fluorophores, including isoquinolinium salts, were investigated as components of the ionic liquids.⁶⁵ In particular, these cations have different strengths of binding affinity for BSA, with isoquinolinium showing significantly increased binding to BSA due to the hydrophobic nature of the isoquinolinium core. Isoquinolinium particularly interacts with the aromatic pockets of the protein, as confirmed by circular dichroism and fluorescence spectroscopy. The fluorescence intensity of BSA effectively reflects its binding affinity with ionic liquids, supporting the potential of isoquinolinium-based ionic liquids in protein modulation and drug delivery applications.

Ihmels and co-workers reported the DNA-binding properties of 9-aryl-substituted isoquinolinium derivatives based on a berberine scaffold.¹⁸ The binding affinity toward G-quadruplex DNA was investigated using photometric titration and fluorescence spectroscopy. Upon DNA addition, a red shift in absorption and a pronounced fluorescence light-up effect were observed, along with significantly extended fluorescence lifetimes for methoxy-substituted derivatives. Notably, the compounds interact with G4 DNA via terminal π–π stacking on the guanine tetrads, while restricted intramolecular rotation within the DNA binding site contributes to enhanced fluorescence. Thus, these methoxy-functionalized berberine-type isoquinolinium fluorophores represent promising ligands for the selective detection of various DNA structures via fluorescence analysis.

In 2012, Juskowiak and co-workers investigated three papaverine-derived isoquinolinium compounds, ligands I–III, for their selective binding to various DNA architectures using equilibrium dialysis (Fig. 14A).⁶⁶ Targeting G-quadruplexes using small ligands can contribute to the development of new anti-cancer agents and the exploration of their biological roles. Among the tested forms (ssDNA, dsDNA, triplex, quadruplex), ligands II showed markedly higher binding affinity toward G-quadruplex DNA, particularly the c-myc oncogene and human telomeric sequences. Ligand I displayed moderate interaction with different DNA structures such as G-quadruplexes and triplexes, whereas ligand III exhibited negligible affinity for all DNA types. The high binding affinity is attributed to the planar, π-conjugated aromatic frameworks that promote π–π stacking interactions with guanine quartets.

Fig. 14 Isoquinolinium-based ligands for DNA binding and selective anticancer activity. (A) Papaverine-derived ligands I–III exhibit differential nucleic acid binding, with ligands I and II showing high selectivity for G-quadruplex DNA over single- and double-stranded forms. Adapted with permission from ref. 66. © 2011 Springer Science and Business Media, LLC. (B) Cadein1 induces apoptosis via p38 signaling and selectively targets p53-deficient cancer cells. Adapted with permission from ref. 16. © 2010 The American Society for Biochemistry and Molecular Biology, Inc. This is an Open Access article under the CC BY license.

In 2010, Jang et al. investigated Cadein1, a novel isoquinolinium salt derived from a protoberberine scaffold, as a selective inducer of apoptosis in p53-deficient cancer cells (Fig. 14B).¹⁶Cadein1 exhibited potent cytotoxicity in MMR (DNA mismatch repair)-proficient, p53-deficient carcinoma cell lines, but not in normal cells. Notably, it induces G2/M arrest and caspase-dependent apoptosis via p38 MAPK activation. MMR-deficient cells were resistant to Cadein1, highlighting the importance of functional mismatch repair in its activity. These findings support Cadein1 as a promising lead compound for targeting cancers with defective p53 but intact MMR systems.

4.5. Fluorescent sensors

Recently, Wong's research group applied fluorescent probes for paper-based detection of amine groups (Fig. 15A).²⁶ This sensing method is advantageous for amine detection due to its simplicity, low cost, and portability. In this study, ortho-ethynylbenzaldehyde was utilized, which readily forms fluorescent isoquinolinium salts through cyclization. Accordingly, the EBA compounds were loaded onto paper strips and used to detect primary amines under various conditions. Different concentrations of primary amines were evaluated based on changes in fluorescence intensity. Interestingly, this approach was extended to detect food spoilage, as biogenic amines are released from decomposing food. The EBA-loaded paper effectively detected spoiled shrimp, which exhibited enhanced green fluorescence intensity. These results indicate that EBA compounds are effective in detecting primary amines under various conditions via facile N-annulation.

Fig. 15 Isoquinolinium-based fluorescent probes for selective molecular sensing. (A) EBA-derived fluorophores detect primary amines via fluorescence turn-on in various formats. Reproduced with permission from ref. 26. © 2024 Elsevier B.V. (B) pH-Responsive isoquinolinium dyes show OFF–ON fluorescence toward O-nucleophiles. Reproduced with permission from ref. 67. © 2024 Wiley-VCH GmbH. (C) Zn(II)–boronic acid complexes selectively recognize L-DOPA through fluorescence quenching. Adapted with permission from ref. 38. © the Royal Society of Chemistry.

In 2024, Schmidt's research group reported isoquinolinium–quinolinium–substituted acetylenes capable of detecting hydroxides on glass surfaces with high sensitivity (Fig. 15B).⁶⁷ In this study, five different glass types were examined and compared based on their thermal expansion coefficients (ECs) and metal oxide compositions. The elements of the glass have a significant effect on the absorption intensity observed at approximately 500 nm. These results suggest that the fluorescent isoquinolinium–quinolinium–substituted acetylenes are highly sensitive dicationic molecules that act as switchable dyes, detecting minute traces of hydroxide from water or other O-nucleophiles, with adduct formation reversible by protons.

Bazany-Rodríguez et al. designed and synthesized novel fluorescent Zn(II)-terpyridine complexes incorporating a cationic N-isoquinolinium nucleus for the detection of biological catecholamines (Fig. 15C).³⁸ Catecholamines such as L-DOPA and dopamine serve as important chemical markers for various human diseases. Among various neurotransmitters tested, the fluorescent dye showed strong affinity and high selectivity for L-DOPA. Upon binding of neurotransmitters to the fluorescent compounds, significant changes in photophysical properties such as fluorescence intensity and emission maxima were observed. Due to the pronounced changes in fluorescence, visual detection of L-DOPA using these fluorophores represents an accurate and sensitive analytical approach with high practical applicability.

Tian's research group developed a sensitive and selective method for detecting fluoride anions using fluorescent isoquinolinium dyes.^27,68 The initial development was reported in 2020 in the Russian Journal of General Chemistry, with more detailed application studies published later that year in Dyes and Pigments (Fig. 16). These fluorescent dyes were synthesized using similar approaches to that of Wong's research, which involved a cyclization reaction with 2-ethynylbenzaldehyde in the presence of AgNO₃. The fluorescence emission spectra of each molecule were influenced by their substituents. Due to the strong affinity between silicon and fluoride ions, these fluorophores were efficiently investigated for fluoride detection. As the fluoride ion concentration changed, both the fluorescence intensity and emission wavelength gradually changed. Interestingly, successful paper-based detection of fluoride was also demonstrated, offering a portable and practical approach for onsite fluoride sensing.

Fig. 16 BIQS, an isoquinolinium-based fluorescent probe, was synthesized via Ag(I)-mediated cyclization and applied for intracellular fluoride detection through fluorescence quenching in HeLa cells. Adapted with permission from ref. 27. © 2020, The Royal Society of Chemistry.

5. Conclusions

This review summarizes recent advances in the development of versatile fluorescent isoquinolinium salts. These fluorescent dyes have been readily synthesized through various methods, including simple protonation, alkylation, transition-metal-catalyzed N-annulation, and oxidation. During their synthesis, structurally diverse probes were obtained and investigated to evaluate the influence of electronic effects on fluorescence emission maxima. Fluorescent isoquinolinium dyes exhibiting notable photophysical properties such as aggregation-induced emission (AIE), which are highly applicable in biological studies, were also described. In particular, recent research has highlighted their effectiveness in a range of biological applications, including cell imaging, in vivo sentinel lymph node (SLN) imaging, theranostics, and DNA binding. We expect that this review will inspire the development of new fluorescent isoquinolinium salts in various application studies. In particular, greater structural diversity may be achieved by integrating photocatalytic or electrochemical annulation strategies—approaches that have recently emerged as powerful tools in heterocyclic compound synthesis—into the construction of isoquinolinium-based fluorophores. These strategies may enable more sustainable and structurally diverse access to functionalized isoquinolinium frameworks. Such strategies may also enable the development of novel photocatalytic transformations using isoquinolinium salts as alternatives to acridinium or quinolinium salts, which are commonly employed as photocatalysts. Furthermore, the rational design of isoquinolinium-based fluorescent materials with enhanced biocompatibility and reduced cytotoxicity is expected to expand their utility in interdisciplinary research, extending beyond bioimaging and theranostics into broader biomedical and materials science domains.

Author contributions

Y. R. Han and S. B. Lee supervised the project and wrote the manuscript.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was financially supported by the NRF-2021R1C1C1009541, 2022R1FA1063012.

Notes and references

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