Green-synthesized single-benzene fluorophores exhibiting room-temperature phosphorescence and solid-state fluorescence for biological and optical applications

Abstract

Single-benzene fluorophores (SBFs) offer minimalistic access to very small, bright, and electronically tunable emitters via push–pull effects. Herein, we report a green, neat S_NAr reaction of tetrafluoroterephthalonitrile (TFTPN) with five aliphatic amines that affords five SBFs in 70–99% yields without chromatography. These dyes absorb at 400–476 nm and emit in the range of 520–568 nm, achieving photoluminescence quantum yields of 51–68% in solution, up to 84% in polystyrene films, and up to 56% in crystalline form. X-ray crystallography and TD-DFT calculations confirmed their near-planar donor–acceptor geometries and large S₁–T₁ gaps, promoting efficient and fast fluorescence, alongside a polymer-induced exciplex delayed emissive component. Some SBFs crystallize as needle-like crystals that guide light with relatively low optical loss (∼0.11 dB mm⁻¹) and can be formulated into tunable hybrid room-temperature-phosphorescent materials in inert matrices for time-gated luminescence applications. Live-cell imaging using Arabidopsis thaliana roots demonstrates efficient tissue penetration and distinct staining patterns, highlighting their potential as minimal biolabels. This atom-economical multifunctional platform based on strongly emissive SBFs offers a sustainable blueprint for next-generation luminescent materials in photonic, security, and biological applications.

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Introduction

Single-benzene fluorophores (SBFs) have recently emerged as an intriguing class of minimalistic organic dyes that defy the conventional paradigm of organic emitters being extensively π-conjugated systems by achieving intense photoluminescence using a solitary aromatic core.^1–3 Unlike traditional fluorophores, the SBFs employ a donor–acceptor architecture on a benzene ring that induces efficient intramolecular charge-transfer (ICT) processes.^4–6 This design not only imparts remarkably high fluorescence quantum yields and large Stokes shifts, but also endows these dyes with prominent emission in both solution and solid state.^7–9 These unanticipated unusual photophysical properties of SBFs and their small size compared to the more common emitters have spurred significant interest in applications ranging from chemical sensing and bioimaging to optoelectronic devices.^7,10–13

A particularly powerful synthetic platform for SBFs is the tetrafluoroterephthalonitrile scaffold (TFTPN, 1). Its inherent electron-deficiency makes the benzene core highly amenable to nucleophilic aromatic substitution (S_NAr) reactions and facilitates the installation of diverse electron-donating groups. Previous studies^6–8 have demonstrated that such modifications can transform an initially non-emissive precursor into a bright fluorophore by the push–pull mechanism. As a notable example, amino-terephthalonitrile derivatives, when judiciously substituted, can deliver tunable emissions ranging from blue to red, and thus across most of the visible spectrum, with large Stokes shifts, while being devoid of and resistant to aggregation-caused quenching.¹⁴

Here we report a series of highly photoluminescent single-benzene fluorophores synthesized via a green, neat S_NAr reaction of TFTPN (1) with five different amines (2a–e) using the amine as the reaction medium to yield SBF products (3a–e) (Fig. 1). By varying the amine substituents, we demonstrate the effect of subtle changes in electronic donation and steric factors on the intramolecular charge-transfer dynamics and the overall photophysical performance. These minimalist fluorophores not only expand the SBF library but they also have significantly high photoluminescence quantum yields of up to 84%. They have potential for live cell imaging applications, and show promising waveguiding properties in the crystalline state. They are also phosphorescent at room temperature in both organic and inorganic matrices, providing valuable insights into the structure–property relationships essential for designing next-generation fluorescent probes and materials.

Fig. 1 Synthesis and substrate scope of TFTPN-derived SBFs 3a–e from tetrafluorophthalonitrile (TFTPN, 1) and the corresponding amines 2a–e.

Results & discussion

Synthesis

The synthesis of the SBFs was accomplished via a straightforward nucleophilic aromatic substitution (S_NAr) of TFTPN (1) with the desired amine using the amine as the reaction medium. Neat mixtures of TFTPN and the aliphatic amines (2a–e) added in excess were heated under reflux for 30 minutes, affording the products 3a–e in yields of 70–99% (Fig. 1). The procedure does not require any additional reagents or solvents, and the crude reaction mixtures were purified simply by filtration or recrystallization from ethanol to obtain pure fluorophores without the need for column chromatography (detailed synthetic protocols are provided in the SI). When considered in the context of green chemistry, this improved protocol aligns closely with several of the 12 principles of green chemistry.¹⁵ Namely, eliminating organic solvents reduces hazardous waste, and the potential for environmental pollution and health concerns associated with the use of high-boiling polar aprotic media can be circumvented.¹⁶ The reaction proceeds via an S_NAr mechanism that maximizes atom economy, incorporating nearly all atoms of the starting materials into the final product,¹⁷ with fluoride ions as the sole stoichiometric byproduct. The process energy efficiency due to short-term heating of only the neat reactants instead of large solvent volumes, and the straightforward purification by recrystallisation from a green solvent (ethanol) minimizes resource consumption.¹⁸ The simple aromatic framework may also suggest favorable end-of-life degradation pathways.¹⁹ Although our focus here was on compact aliphatic donors, we note that TFTPN is equally amenable to reaction with bulkier, aryl-based nucleophiles under more intense synthetic conditions. Literature precedents have demonstrated^20,21 that nucleophilic aromatic substitution with carbazole- or diphenylamine-derived anions (often generated using NaH or KOtBu) affords derivatives such as 4CzTPN for OLED TADF emitters and 4CzIPN as benchmark organic photocatalysts.²²

Photophysical & structural characterization

All investigated SBF derivatives (3a–e) exhibit intense photoluminescence emission both in solution and in the solid state. To elucidate the nature of this emission, we employed steady-state absorption and emission spectroscopy and time-correlated single-photon counting (TCSPC) to determine spectral features and emission lifetimes. These experimental investigations were integrated with time-dependent density functional theory (TD-DFT) calculations, which provided complementary insight into the vertical excitation energies and nature of the electronic transitions and fluorescence rates. To assess the extent of charge-transfer characteristics, experimental and theoretical photophysical studies were performed in solvents of varying polarity, including toluene, dichloromethane (DCM), and dimethyl sulfoxide (DMSO). Furthermore, photophysical experiments targeted the SBFs in the solid state, both as organic crystals and when embedded in polystyrene (PS) thin films. A comprehensive summary of the respective photophysical results is presented in Table 1, with representative spectra and fluorescence spectra shown in Fig. 2.

Table 1 Summary of the steady-state absorption and emission maxima as well as mean molar extinction coefficients, fluorescence quantum yields and rates of molecules 3a–e as collected in three different solvents, namely toluene, dichloromethane (DCM), and dimethyl sulfoxide (DMSO), as well as in the solid state as organic crystals and incorporated in polystyrene (PS) thin films. The simulated absorption and fluorescence band maxima and fluorescence rates are given in parantheses. The theoretical values labeled with asterisks were shifted by 0.63 eV for comparison

Molecule	State/solvent	λ max ^Abs/nm	ε/L mol^−1 cm^−1	λ max ^Emis/nm	QY^Emis/%	τ ^Emis/ns	τ ^delayed/ns
3a	Toluene	410 (393)	4061	529 (532*)	53.0	14.6 (11)	—
	DCM	411 (405)	2450	542 (564*)	64.6	18.2 (16)	—
	DMSO	414 (410)	4514	562 (575*)	53.7	17.0 (13)	—
	PS Film	417	—	533	83.3	16.0	171
	Crystals	—	—	556	37.8	13.7	—
3b	Toluene	404 (393)	4326	528 (518*)	63.0	14.9 (26)	—
	DCM	405 (403)	4160	541 (530*)	54.0	18.7 (14)	—
	DMSO	404 (407)	3357	555 (519*)	25.8	7.1 (18)	—
	PS Film	408	—	530	72.3	17.6	184
	Crystals	—	—	524	46.8	15.0	—
3c	Toluene	445 (423)	3210	540 (558*)	57.4	13.7 (6)	—
	DCM	448 (445)	4144	554 (594*)	54.9	17.2 (6)	—
	DMSO	436 (453)	4582	568 (608*)	67.8	18.6 (7)	—
	PS Film	476	—	554	68.6	17.7	219
	Crystals	—	—	562	22.7	5.8	—
3d	Toluene	414 (398)	4494	537 (531*)	51.0	14.8 (7)	—
	DCM	415 (411)	5276	548 (560*)	56.1	18.1 (8)	—
	DMSO	418 (416)	4134	565 (571*)	23.1	17.7 (8)	—
	PS Film	416	—	537	77.6	17.1	173
	Crystals	—	—	530	48.3	10.9	—
3e	Toluene	404 (391)	3508	525 (526*)	64.2	15.3 (8)	—
	DCM	400 (401)	5245	534 (549*)	66.9	17.9 (7)	—
	DMSO	404 (405)	4368	553 (556*)	56.2	16.7 (9)	—
	PS film	420	—	526	83.9	19.0	235
	Crystals	—	—	526	56.4	9.6	—

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Fig. 2 Photophysical characterization of molecules 3a–e. (a) Absorption spectra in different solvents, (b) fluorescence emission spectra in different solvents, (c) visible fluorescence color plotted onto the CIE1931 color space, (d) fluorescence lifetime decay curves in different solvents, (e) micrographs of crystals of 3a–e recorded by using fluorescence (blue excitation, ∼450 nm), dark field and fluorescence lifetime microscopy (355 nm excitation). All scale bars in panel (e) are 200 µm.

Ground-state absorption properties. The five investigated SBFs exhibit in all studied environments (solutions, PS film and as crystals) absorption maxima in the range of 400 to 476 nm, with compounds 3e and 3c defining the lower and upper boundaries, respectively (see Table 1). A slight solvatochromic effect is observed across the series, with absorption bands shifting bathochromically with increasing solvent polarity (toluene < DCM < DMSO), indicating the charge-transfer nature of the respective underlying transition. Moreover, the extinction coefficients are moderate but within the typical range for dyes featuring strong charge-transfer characteristics. This conclusion is supported by TD-DFT simulations, predicting for all five molecules a bright S₀-to-S₁ excitation, where charge density is shifted from the donor amine groups, through the central benzene ring, towards the electron-deficient cyano substituents (see the charge density differences in Fig. 3a).

Fig. 3 Theoretical and crystallographic investigations of SBFs 3a–e. (a) Charge density differences for the vertical S₀ → S₁ excitation as obtained for the S₀ equilibrium geometries. (b) Overlay of the minimum geometries of 3a–e in the S₁ (light-green, transparent) and T₁ (solid, element colors) state. All theoretical results are obtained at the (TD-)CAMB3LYP/def2TZVP(-F) level of theory, accounting for the solvent effects of toluene by means of an implicit solvent model. (c) Crystal packing of 3b and (d) crystal packing of 3c as well as visualization of key intermolecular interactions that govern the structural integrity and morphological features of the compounds. The BFDH (Bravais–Friedel–Donnay–Harker) morphology model was employed to illustrate the external crystal habit and highlight the dominant crystal faces.

Emission properties. The SBF derivatives 3a–e emit in the green to orange region of the visible spectrum, with emission maxima ranging from approximately 524 to 568 nm. Correlation analysis with the dielectric constants of the respective solvents indicates a strong positive correlation for the emission maxima with the solvent polarity (Fig. S5), as supported by TD-DFT simulations (see Table 1). Experimentally observed solvatochromic shifts, i.e., 0.05 eV between toluene and dichloromethane, and 0.12 eV between toluene and DMSO, are reproduced by TD-DFT, which predicts corresponding average bathochromic shifts of 0.11 eV and 0.13 eV, respectively. This behavior can be rationalized by stabilization of the highly polar charge-transfer excited state in solvents of increasing polarity, resulting in reduced emission energy and red-shifted fluorescence, i.e., supports the intramolecular charge transfer nature of the S₁ state. In polystyrene (PS) thin films, they exhibit remarkably high photoluminescence quantum yields of up to ∼84%, while in solution in air their quantum yields typically range from 51 to 68%, depending on the substituent.

Notably, all compounds also display bright solid-state emission in crystalline form, which is unique since many organic fluorophores suffer from aggregation-induced quenching.^23,24 In particular, derivative 3b stands out with strong visible brightness and a measured solid-state fluorescence quantum yield of around 47%, suggesting that aggregation-induced quenching, is effectively suppressed in this system. This highlights the suitability of the minimalist push–pull design for maintaining high emission efficiencies in both dispersed and condensed phases. While 3e shows advantageous features such as apparent aggregation-induced quenching resistance and higher quantum yield, we chose 3b for a detailed follow-up study because it pairs excellent photophysical performance with a distinctive crystal habit. In particular, 3b reproducibly forms centimeter-long, waveguiding needle crystals (Fig. S10), which are practical and technically suitable candidates for in-depth solid-state testing (micro-fading, waveguiding, and room-temperature phosphorescence).

Time-correlated single photon counting (TCSPC) measurements revealed photoluminescence lifetimes in the range between 6 and 19 ns for all SBFs in both solution and crystalline form. In each case, only rapid fluorescence decay component was observed (see Fig. 2d). This indicates that the compounds exhibit conventional fluorescence, rather than thermally activated delayed fluorescence (TADF), which typically involves longer-lived emissive states.

The absence of TADF can be directly traced to the molecular architecture of the studied SBFs. In contrast to known structurally similar TADF emitters such as 4CzTPN and 4CzIPN, which feature orthogonal donor–acceptor arrangements²⁵ and bulky amine substituents that promote spatial separation of the frontier orbitals, the SBFs described here possess more compact amine groups that are only modestly twisted relative to the central aromatic core. This configuration was confirmed by both experimental single-crystal X-ray diffraction analysis and (TD-)DFT structure optimizations (for further evidence for matrix-induced excited-state processes, see below).

The crystal structures of the derivatives were determined (for details see the SI, Fig. S8). As shown in Fig. 3c and d, compounds 3b and 3c crystallize in the triclinic space group P1̄ with half a molecule in the asymmetric unit. Analysis of the crystal packing of 3b reveals a face-to-face arrangement of molecules, similar to that observed in 3c. In 3b, the alkyl groups are positioned in a slip-stacked with a slight offset. However, due to the steric requirements of the two ethyl groups attached to the nitrogen atom, π–π interactions are nearly absent, explaining the ineffective photoluminescence quenching in the solid state. The crystal packing is primarily stabilized by halogen–halogen interactions (F⋯F contacts). Additionally, the cyano groups of adjacent molecules are aligned directly above each another, forming close contacts that contribute further to the stabilization of the crystal lattice. Crystal structures on all other derivatives 3a, 3d, and 3e are reported in the SI.

Torsional analyses based on (TD-)DFT optimized geometries reveal average dihedral angles of approximately 34° between the donor (amine) and acceptor (benzene) units across the S₀, S₁, and T₁ states for compounds 3a and 3c–e. Compound 3b presents a minor exception, with dihedral angles of 40°, 84°, and 37° in the S₀, S₁, and T₁ minima, respectively, due to the increased flexibility introduced by its ethyl substituents.

Unlike the orthogonally oriented donor units in TADF emitters like 4CzTPN or 4CzIPN,²⁰ this smaller twist leads to stronger electronic coupling between donor and acceptor moieties. As a consequence, the singlet–triplet energy gaps are significantly larger, thereby disfavoring reverse intersystem crossing (rISC). This is further corroborated by the minimal structural differences between the (TD-)DFT-optimized S₁ and T₁ geometries, as illustrated in the form of structural overlays in Fig. 3b, indicating minor structural excited-state relaxation between the T₁ and S₁ minimum geometries. Correspondingly, the computed singlet–triplet energy gaps remain comparatively large: on average, ∼1.25 eV between the T₁ minimum and vertical S₁ state, and ∼1.0 eV between the S₁ minimum and vertical T₁ state. Compound 3b displays a reduced gap of ∼0.5 eV with respect to the S₁ minimum geometry, consistent with its greater degree of relaxation (torsion angle of 84° vs. 37° in S₁vs. T₁ minimum). Nevertheless, in all cases, the S₁–T₁ energy separation is too large to enable efficient rISC, thereby precluding TADF. Consequently, the studied SBFs behave as conventional fluorescent dyes, with prompt emission originating exclusively from S₁ → S₀ transitions.

Interestingly, when the compounds were incorporated into polystyrene (PS) thin films, an additional delayed emission component was detected in the range of 171–235 ns. This result is consistent with TCSPC experiments of the SBFs in poly(methyl methacrylate) (PMMA) thin films with a delayed emission component and associated decay times of 200–400 ns (for details see the SI, Fig. S4). This longer-lived signal, which accompanies the prompt fluorescence decay, is tentatively assigned to the formation of an exciplex between the SBF molecule and the surrounding polymer matrix. Further evidence for matrix-induced excited-state processes is discussed in the subsequent section on room-temperature phosphorescence, where embedding the fluorophores in alternative hosts such as boric acid or phenyl benzoate leads to even longer-lived emission components on the order of seconds.

Summarizing the influence of the amine moiety on the photophysical properties of the resulting SBFs it can be stated that, across 3a–e the donors are all aliphatic secondary amines, and therefore the intrinsic donor strength is similar; the trends observed are governed by (i) sterics/conformation (amine–aryl dihedral angle) and packing and (ii) subtle electronic differences (e.g., reduced basicity of morpholine). TD-DFT shows that 3a and 3c–e adopt relatively small donor–acceptor twists (∼34°) across S₀/S₁/T₁, whereas 3b is exceptional: it relaxes to 84° in S₁ (40° in S₀; 37° in T₁), consistent with increased conformational freedom of the N,N-diethyl groups. This larger twist reduces the HOMO–LUMO overlap in S₁, changing oscillator strength/decay and increasing sensitivity to medium polarity (e.g., the PLQY drop for 3b in DMSO). The same calculations give large S₁–T₁ gaps (∼1.0–1.25 eV; ∼0.5 eV for 3b at the S₁ minimum), rationalizing prompt fluorescence and excluding TADF across the series. On the electronic side, ring-embedded donors (pyrrolidine/piperidine) vs. dialkylamino vs. morpholine manifest in the spectra as expected for push–pull systems. The relatively weaker morpholine donor in 3e (due to the electronegative O) gives the most blue-shifted absorption (≈404 nm) and emission (≈525–553 nm), while 3c (pyrrolidine) shows the most red-shifted absorption (≈445–448 nm) and emission (≈540–568 nm).

Photostability. Aimed at evaluating the photostability of the SBFs, microfading testing (MFT) was applied to molecule 3b. MFT is a well-established technique in conservation science and archaeometry, used to simulate accelerated photoaging by exposing a small, localized area (typically ∼200 µm) to very intense illumination under controlled conditions.^26–30

Dye 3b was subjected to focused, very high-intensity broad-band visible light (9.0–9.1 Mlux, ∼90 000 fold compared to ambient illumination) to induce accelerated photobleaching, and its resistance to color change was compared to that of the conventional fluorophore and commonly used reference dye Rhodamine B,³¹ a chromophore with a large extended π-electron system. The results demonstrate that 3b is approximately 4.8 ± 0.6 times more photostable than Rhodamine B (for details see the SI). Based on these data, a conservative estimate for the time until a barely noticeable color change (ΔE = 5, for further explanation see the SI, Fig. S9) occurs under ambient lighting conditions (100 lux) was calculated to be 24 644 ± 2153 hours, equal to about 2.81 ± 0.25 years of continuous ambient illumination. This result illustrates the durability of molecule 3b and its resistance to photobleaching, making it suitable for potential applications in materials and biological sciences such as photoluminescent staining dyes or other emissive materials.

Exemplary applications

To showcase the prospects brought about the SBFs described here beyond their fundamental photophysical properties, we aimed to explore their potential in application-relevant contexts. Specifically, we focused on the opportunities that these structurally exceedingly simple fluorophores offer across the materials and life sciences, such as optical waveguiding, room-temperature phosphorescence, and photoluminescent staining for biological tissue imaging, as some of the most active directions being pursued in research.

Optical waveguiding. Molecular organic crystals and, more recently, their hybrids with other materials, have been extensively explored as next-generation waveguiding materials.^32–36 The unique combination of high photoluminescence, structural tunability, and intrinsic molecular order makes them promising candidates for compact, flexible, and highly efficient optical components. The prospective applications range from optoelectronic circuitry and on-chip communication to novel display technologies. Notably, recent studies have demonstrated organic crystals that exhibit elastic or plastic deformation during light transport,^37,38 some even under very low temperature conditions.^36,39–41 Recent patents also underscore the additional potential of waveguiding organic crystals, especially those with high refractive indices, for enabling ultra-thin augmented and virtual reality (AR/VR) displays.^42–44

Given their bright solid-state emission, the SBFs presented in this work were thus evaluated for their optical waveguiding performance. As described above, all compounds studied exhibit intense photoluminescence in the crystalline state. Fluorescence microscopy (Fig. 2e) revealed that when they are exposed to light, the crystals exhibit directional emission, indicative of active (that is, transmission of fluorescence) waveguiding behavior (Fig. 4). The emission of 3b is particularly strong and has a high photoluminescence quantum yield. This compound is also particularly well-suited for this application as it naturally crystallizes as a long, slender needle-like habit, an ideal shape for optical waveguiding by internal total scattering. Slow evaporation of a dichloromethane/methanol (3 : 1) solution afforded single crystals of 3b measuring up to 4 cm in length. These optically uniform crystals enabled space-resolved micro-photoluminescence (µPL) measurements at room temperature to evaluate their waveguiding performance (for details, see the SI).

Fig. 4 Exemplary applications of the SBFs 3a–e. (a) Waveguiding of a single crystal of 3b, scale bar 1 cm, (b) room-temperature phosphorescence (RTP) spectra at different concentrations of 3b in boric acid, (c) RTP decay curves at different concentrations of 3b in boric acid, (d) micrographs of RTP of an 10⁻²% solid solution of 3b in boric acid, excited with an 365 nm UV LED, scale bar 1 cm, (e) fluorescence and bright field micrographs of SBFs 3a–e for live cell imaging staining 14-day-old seedlings of A. thaliana (Columbia wildtype), scale bar 50 µm.

Although the crystals of 3b were mechanically brittle and showed no indication of having elastic or plastic deformation ability, they demonstrated excellent waveguiding characteristics. Upon excitation with a focused 405 nm high-power LED, their emission was efficiently transduced along the longitudinal crystal axis. The optical loss factor was determined to be 0.10871 ± 0.02502 dB mm⁻¹, and this value lies at the lower end of the typical range reported for organic crystals (0.09–57 dB mm⁻¹),^{37,38,45–49} positioning compound 3b among some of the more efficient organic crystalline waveguiding materials known. These findings underscore the potential of SBFs such as 3b not only as small-molecule emissive materials, but also as functional optical elements in integrated photonic devices.

Room-temperature-phosphorescence. Besides waveguiding, certain formulations of the SBFs also show room-temperature phosphorescence (RTP). RTP refers to the emission of light from the triplet excited state of a molecule that persists even after the excitation source has been turned off, observable under ambient conditions without the need for cryogenic cooling.⁵⁰ Unlike fluorescence, which typically decays within nanoseconds, RTP lifetimes can range from milliseconds to seconds, enabling unique applications that rely on delayed emission.

Recent advances in RTP materials have opened new possibilities in areas such as anti-counterfeiting,⁵¹ dynamic security labeling, and time-gated imaging.^52,53 In particular, time-resolved afterglow materials can be precisely encoded into microstructured films for robust authentication and rewritable information storage.⁵⁴ Beyond this, RTP materials are also being explored for their roles in sensors, lasing media, and organic light-emitting diodes (OLEDs), where long-lived emissive states and delayed readout functionalities provide a distinct technological edge.⁵⁵

Various mechanisms have been proposed to rationalize the activation of RTP in organic materials, including intermolecular aggregation, crystallization-induced effects, and matrix-induced rigidification.^56–59 Here, we focused on compound 3b, which showed promising solid-state emission and was selected for detailed RTP investigations.

We discovered that embedding 3b into either an organic (phenyl benzoate) or inorganic (boric acid) matrix and excitation with UV radiation leads to visible RTP (see the SI, Video S1). Upon cessation of the excitation source, the doped samples exhibit a delayed green/teal afterglow that lasts for several seconds and is clearly visible to the naked eye under dark conditions (Fig. 4d). Similar matrix-activated phosphorescence has been reported in the literature for other small organic molecules in phenyl benzoate and boric acid hosts, where the matrix effectively suppresses non-radiative deactivation and stabilizes the triplet state.^60–62

To systematically investigate this behavior, we embedded 3b into boric acid at various concentrations. 3b was thoroughly ground with freshly dried boric acid, heated at 120 °C for 4 minutes to ensure homogeneity, and then cooled and re-ground. A series of doped samples with defined weight ratios (ranging from ∼10% to 10⁻³% w/w) were then prepared by serial reheating with adding boric acid. These solid solutions were characterized using both steady-state and time-resolved photoluminescence spectroscopy at room temperature and 77 K (for details, see the SI, Fig. S3).

Steady-state emission spectra revealed a clear concentration-dependent bathochromic shift: at high loading (10%), the emission maximum was observed at 533 nm, while at low loading (10⁻³%), the emission blue-shifted to 512 nm. The shift followed a logarithmic trend with concentration (Fig. 4b and c).

We note that at low 3b loading in boric acid the RTP maximum (512 nm) is slightly blue-shifted relative to the solution fluorescence in toluene (528 nm). This reflects distinct emitting states in different microenvironments: a higher-energy, more locally excited state of isolated 3b rigidified in a non-polar inorganic cage versus a strongly CT S₁ stabilized in a polarizable solvent. With increasing loading, π–π contacts generate excimer/J-aggregate triplets that emit at lower energy (≈533 nm) with shorter lifetimes (see below), confirming the monomer-to-aggregate evolution.

The RTP lifetime measurements showed two regimes: at low concentrations, the phosphorescence lifetime was ∼400–450 ms, and therefore mechanistically consistent with isolated monomeric species of the emitter that was firmly confined within the boric acid network. At higher concentrations, the lifetime dropped to ∼100–270 ms, attributed to the formation of π–π-stacked aggregates that introduce additional non-radiative decay pathways (Fig. 4c).

These observations can be explained by a dynamic equilibrium between two emissive species in the matrix. In the monomeric regime (low concentration), each 3b molecule is isolated and rigidified by the hydrogen-bonded boric acid cage. This suppresses vibrational relaxation and stabilizes the triplet excited state, resulting in high-energy emission with long lifetimes. In the excimer/J-aggregate regime (high concentration), closer packing promotes π–π stacking, forming dimers or oligomers with new, lower-energy triplet states. However, these aggregates exhibit faster non-radiative decay due to exciton migration and internal conversion, resulting in shorter lifetimes.

Live cell imaging. Given their favorable emissive properties, we also aimed to explore the usefulness of the SBFs as live cell imaging dyes. Live cell imaging is a powerful technique in biological research that enables visualization of dynamic cellular processes in real time. Unlike fixed-tissue imaging, it allows for non-destructive observation of living systems, offering critical insights into cell behavior, organelle dynamics, and developmental processes.^63,64 Recent studies have demonstrated its potential in plant sciences, for instance, in monitoring the dynamic behavior of storage organelles in developing cereal seeds⁶⁵ or for identification of tubular structures derived from protein storage vacuoles during barley germination.⁶⁶

To explore the applicability of the SBFs in this context, we investigated their performance as fluorescent stains in live cell imaging experiments using the model organism Arabidopsis thaliana (Columbia wildtype). Roots from 14-day-old seedlings of A. thaliana were incubated with each dye for 10 minutes and then mounted in tap water without fixation. Imaging was performed using confocal laser scanning microscopy with excitation at 405 nm and detection in the 539–713 nm range.

The results, summarized in Fig. 4e show that the dyes are readily taken up by the root tissue, with the exception of compound 3e, which displayed negligible uptake. Compounds 3a–c exhibited the most defined and specific staining patterns. Subcellular localization appears to involve the plasma membrane and/or cell wall, though precise identification requires further investigation. Notably, dye 3c revealed punctate fluorescent structures along the cell periphery (indicated by arrows in Fig. 4e), suggesting potential interaction with specific subcellular compartments. Follow-up studies involving colocalization with known markers will be carried out in the future to verify these observations.

These initial findings highlight the promising potential of the SBFs described here as minimally structured, bright, and biocompatible fluorophores for use in biological fluorescence microscopy. Their effective uptake and distinct staining patterns make them attractive candidates for future live cell imaging studies in plant and potentially other biological systems.

Conclusions

In summary, we have developed a green, neat S_NAr synthesis of five single-benzene fluorophores (3a–e) in excellent yields (70–99%) using the amine as the reaction medium without any chromatographic purification. These minimalist push–pull dyes absorb between 400 and 476 nm and emit tunable light from 524 to 568 nm, achieving high photoluminescence quantum yields in solution (51–68%), in polymer films (up to 84%), and in crystalline form (up to 56%). X-ray crystallography and TD-DFT calculations confirmed only slightly twisted donor–acceptor geometries that enforce large S₁–T₁ gaps, promoting efficient prompt fluorescence alongside a polymer-induced exciplex delayed component. Notably, 3b shows high photostability, exhibits low optical loss (0.11 dB mm⁻¹) in optical waveguiding, and can be formulated into a tunable hybrid RTP for material applications. Finally, live-cell imaging in Arabidopsis roots demonstrates these SBFs’ ability to penetrate tissues and produce distinct staining patterns, underscoring their promise as minimal, versatile biolabels. This atom-economical strategy lays the groundwork for next-generation minimalist luminescent materials with broad materials science and biological applications.

Materials & methods

All the methods and materials used are described in detail in the SI. These include detailed synthesis procedures, characterization techniques and computational details, and further supporting figures and data. For all discussed SBFs 3a–e extensive material characterization including ¹H-, ¹³C-NMR, HR-MS, FT-IR and melting point analysis is also reported in the (Fig. S1–S22) SI. The single-crystal X-ray diffraction data reported in this study have been deposited at the Cambridge Crystallographic Data Centre (CCDC), under deposition numbers 2431737, 2431017, 2430752, 2431319 and 2431255.

Author contributions

StS conventionalized, planned, executed, wrote the study, and provided funding, JCZ executed part of the synthesis and part of the photophysical investigations, MK executed and analyzed the room-temperature phosphorescence experiments, DPK carried out the XRD experiments and analyzed the results, PC carried out the NMR and HR-MS analysis of the samples, PN provided feedback on data analysis, partially supervised the experiments, edited the manuscript and provided funding and access to experimental facilities, TP was responsible for the MFT setup, CM conventionalized and executed the theoretical investigations of the molecules and wrote the study, and VI carried out the biological microscopy testing and analysis of the molecules.

Conflicts of interest

There are no conflicts to declare.

Data availability

All used methods and materials are described in detail in the supplementary information (SI). Supplementary information: a detailed synthesis procedure, characterization techniques and computational details, and further supporting figures and data. For all discussed SBFs 3a–e extensive material characterization inducing ¹H-, ¹³C-NMR, HR-MS, FT-IR and melting point analysis are reported. See DOI: https://doi.org/10.1039/d5tc03134f.

CCDC 2431737, 2431017, 2430752, 2431319 and 2431255 contain the supplementary crystallographic data for this paper.^67a–e

Acknowledgements

This research was partially performed using the Core Technology Platform resources at New York University Abu Dhabi. We thank Dr Rachid Rezgui for his help with the FLIM and epifluorescence microscopy experiments and Prof. Christoph Herm for providing the MFT setup. Live-Cell-Microscopy was performed at the Core Facility Cell Imaging and Ultrastructure Research, University of Vienna – member of the Vienna Life-Science Instruments (VLSI). We thank Asem Bock for the expert help with the in vitro work. All high-performance computations were carried out at the Erlangen National High Performance Computing Center (NHR@FAU). AI-based tools were used to refine clarity, sentence structure, grammar and spelling of this manuscript.

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67. (a) CCDC 2431737: Experimental Crystal Structure Determination, 2025 DOI:10.5517/ccdc.csd.cc2mmf4v; (b) CCDC 2431017: Experimental Crystal Structure Determination, 2025 DOI:10.5517/ccdc.csd.cc2mlnxt; (c) CCDC 2430752: Experimental Crystal Structure Determination, 2025 DOI:10.5517/ccdc.csd.cc2mldc0; (d) CCDC 2431319: Experimental Crystal Structure Determination, 2025 DOI:10.5517/ccdc.csd.cc2mlznw; (e) CCDC 2431255: Experimental Crystal Structure Determination, 2025 DOI:10.5517/ccdc.csd.cc2mlxlr.

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