Discovery of a size-record breaking green-emissive fluorophore: small, smaller, HINA

Astonishingly, 3-hydroxyisonicotinealdehyde (HINA) is despite its small size a green-emitting push–pull fluorophore in water (QY of 15%) and shows ratiometric emission response to biological relevant pH differences (pKa2 ∼ 7.1). Moreover, HINA is the first small-molecule fluorophore reported that possesses three distinctly emissive protonation states. This fluorophore can be used in combination with metal complexes for fluorescent-based cysteine detection in aqueous media, and is readily taken up by cells. The theoretical description of HINA's photophysics remains challenging, even when computing Franck–Condon profiles via coupled-cluster calculations, making HINA an interesting model for future method development.


Results
Systematic investigations by absorbance and emission spectroscopy were carried out to elucidate the photophysical behaviour of HINA ( Fig. 1). At 3.8 < pH < 7.0, HINA occurs in its neutral form and is blue emissive (l em,max ¼ 382 nm) with a respectable QY of 7% (Table 1). Upon addition of NaOH, a new absorbance centered at 385 nm arose while the band at 325 nm decreased (Fig. 1a). Simultaneously, the blue emission of neutral HINA vanished and an even stronger green emission (l em,max ¼ 525 nm) with a QY of 15% appeared; 33 the corresponding colour change can be seen by the naked eye ( Fig. 1b  and c). The pK a value for deprotonation of HINA was obtained by absorbance-based titration, pK a2 ¼ 7.05 AE 0.01, and agreed with the value from pH-meter recordings, see Fig. 1d and S10. † Coincidentally, this pK a value lies perfectly in the biologically relevant range. Interestingly, very similar pK a2 values ($7) were obtained by uorescence-based titrations regardless of the excitation wavelength, i.e. by exciting the neutral form of HINA, the anion or both at an isosbestic point. This indicates that deprotonation of the neutral form of HINA is not a signicant process during the lifetime of its excited state 34 despite its largely negative pK * a2 value estimate of À5.5, which was obtained by a Förster cycle analysis (see the ESI †). 35,36 Unlike many other hydroxarenes such as phenols and naphthols, 34,37 HINA does not function as a photoacid. HINA becomes N-protonated below pH 3.9, where it remains uorescent, albeit with a low QY of 0.9%. The emission band of cationic HINA is centred at 395 nm, and thus surprisingly bathochromically shied compared to neutral HINA (Fig. S1 † and Table 1). Conversely, the absorbance spectra showed the expected hypsochromic shi upon addition of acid. The emission lifetimes of the cationic, neutral and anionic forms of HINA identied them as uorophores (Table  1). Indeed, HINA is a fascinating emissive dye that occurs in three distinct emissive forms. We are unaware of any other small uorophore with this property.
Strongly solvent-dependent absorbance and emission spectra of HINA were observed. For instance, the characteristic $340 nm absorbance maxima of neutral HINA and a weak blue emission was found in anhydrous acetonitrile (ACN). In contrast, a longwavelength absorbance band and green emission appeared when water was added to ACN ( Fig. S13 †). Indeed, the emission of HINA is centred at $400 nm in all anhydrous organic solvents tested (DMSO, MeOH, ethylene glycol, Fig. S14-S18 †), but shis to the 450-600 nm range upon addition of water. Generally, the emission quantum yield of HINA increases upon deprotonation and is higher in polar protic than in polar aprotic solvents, e.g. reaching up to 24% in basied methanol (Table S8 †).
In aqueous environments, the aldehyde moiety of HINA equilibrates with its hydrate structure, as conrmed by UV/Vis 38 and NMR experiments (ESI, Fig. S42-S46 †). At rst, we wondered if one of the hydrated forms of HINA is the greenemitting species. However, uorescence in the 450 to 700 nm wavelength region was also detected when adding anhydrous base or anion-stabilising urea to a solution of HINA in organic solvents (see Fig. S16-S18 †), confuting hydrate-contributions. Unlike pyrenes (Scheme 1a) or other aggregation-induced emitting (AIE) systems, 3,24,39 HINA emission is not caused by aggregate formation (Fig. S27 and S28 †).
The photophysical properties of 3-hydroxypicolinealdehyde (HPA, an isomer of HINA), of 3-hydroxy-4-acetylpyridine (HAP, Scheme 1 Chemical structures, abbreviations, atom numbers (N), molecular weights (M w ) and the peak maximum of emission (l em, max ) of (a) representative green-emitting dyes, and of (b) herein investigated formyl/acetyl-hydroxy-pyridines. (c) (De)protonation equilibria of HINA in water.  Table 1. HPA showed spectroscopic features that were similar to that of HINA, but its absorbance and emission bands for both the neutral and anionic forms are hypsochromically shied. Moreover, HPA is a much weaker emitter than HINA. The ketone HAP is also poor emissive in its neutral form and as an anion. HAP requires a higher pH for deprotonation than HINA or HPA. Notably, HAP does not form hydrates in water as was conrmed by 1 H NMR experiments (Fig. S55 †), providing additional evidence that these are not the blue and green-emitting species. Like for HINA, absorbance and emission-based titrations of HPA and HAP yielded essentially overlaying pH-property plots (Fig. S6-S9 †). Again, effective deprotonation of the neutral form of the hydroxy-pyridines apparently does not occur during the lifetime of the excited state despite the strong acidity of the excited state ðpK * a2 Þ of the uorophores that was estimated by Förster's method (see ESI †).
7-Azaindole (7AI, 15 atoms) and SA (15 atoms) are to the best of our knowledge the smallest green-emitting uorophores that have been reported so far, but they require the use of an unphysiological basic pH of >13.5 and >8.4, respectively. 40-42 SA is structurally closely related to HINA (14 atoms) but shows despite its more electron-rich phenyl ring somewhat counterintuitively a hypochromic shied absorbance and emission spectra ( Fig. 1e and f). Compared to HINA, SA is an inferior uorophore because its neutral form is not uorescent and its anionic form is seven times weaker emissive (QY ¼ 2% at pH 10.0). Pyridoxal is both signicantly larger in size and much less emissive than the other formyl-hydroxy-pyridines.
The character of the emitting electronic states has been investigated by computing the Franck-Condon proles through coupled-cluster (CC) calculations with the def2-TZVPPD basis set, see Table S20. † For the anionic form of HINA, the predicted absorption and emissions bands, and the Stokes shi are in good agreement with the experimental ndings ( Fig. S72 and Tables S20-S22 †). The n / p* transition displayed an almost zero oscillator strength, whereas that of the p / p* transition is four orders of magnitude larger, resulting in computed RGB values (Table S22 †) that agreed well with the visually observed green emission. Also, the relative emission wavelength maxima trends between HINA, HPA and SA were correctly predicted (Table S20 †). Nevertheless, the theoretical description of HINA and its analogues is not simple, despite the small size of these uorophores. In fact, electronic states which are very close in energy were present and it was found that vibronic effects 23,24,43 are crucial for the description of the photophysical properties. 44 Very large discrepancies with the experiments were encountered when computing the emission transitions for the neutral forms of HINA and its analogues. Indeed, an excitedstate intramolecular proton-transfer process (ESIPT) 23,45-47 likely occurs for these substances (Fig. S73 †). An intramolecular ESIPT may also explain the large discrepancies seen between the expected pH-dependent deprotonation of the excited state based on calculated pK * a2 values, and the "ground-state like" protonation status observed in uorescence experiments. Thus, explicit theoretical treatment of the electron dynamics is required for computing the photophysical properties of (neutral) HINA, which is beyond the scope of this work. HINA and its analogues represent challenging test cases for advanced theoretical studies and method developments of the nuclear and electronic dynamics, because they combine fascinating photophysical properties with advantages for carrying out the computations (small number of atoms, electrons, and conformers).
The large observed Stokes shis, e.g. 6700, 4600 and 6900 cm À1 for protonated, neutral and deprotonated HINA characterize it as a push-pull dye, e.g. $5000 cm À1 for NBDs and $8400 cm À1 for recently reported benzoxazole-thiophenes 48 and stilbenes. 17 This raises the question if more compact but emissive push-pull uorophores are even conceivable because single aryl ring-based HINA, HPA and SA already feature some of the smallest but most powerful donor and acceptor moieties, i.e. -O À with a donor resonance const. R + of À2.04, and -CHO with an acceptor resonance const. R À of 0.70. 49 Note that similarly-sized cyanophenols (R À ¼ 0.49 for -CN) are not emissive.
Unlike most other push-pull uorophores, HINA shows a good emission quantum yield in water, which makes it attractive for applications. For instance, it may be used as ratiometric, uorescent pH indicator dye because its protonated, neutral and deprotonated forms display characteristic emission proles. Complementary to many other green emitting dyes that interact with hydrophobic species, HINA's high hydrophilicity diminishes its binding to b-cyclodextrin and cucurbit [7]uril as macrocyclic hosts, to serum albumin as a carrier protein, and to polymethacrylate-and polystyrene surfaces. HINA can also function as a uorescent indicator in supramolecular sensing assays: we observed that HINA coordinates to Pt-and Pd-complexes, resulting in a full uorescence switch off (see Fig. 2). From those complexes, the HINA ligand can be readily displaced by a stronger ligand, e.g. thiols, yielding in an emission switch-on response in aqueous and organic media. In fact, micromolar concentrations of L-cysteine (L-Cys) can be rapidly ($10 min) detected through a uorescence-based indicator-displacement assay in that way (Fig. 2b). Analogously, HINA can be displaced from a Pd-complex by pyridine (Fig. S65 †). We believe that HINA-capped metal-organic building blocks could be reacted on demand with stronger ligands to form metal-organic cages or -frameworks, [50][51][52] thereby providing an in situ uorescence response for monitoring reactions in real time. Noteworthy, HINA also forms covalent conjugates with L-Cys in aqueous media (Fig. S66-S68 †), via the formation of a 1,3-thiazolidine ring, which is reminiscent to other aldehyde moiety-containing cysteinereactive probes. [53][54][55] However, like for most reported CHObased probes, a large excess of L-Cys and long reaction times are needed, making it less attractive for the sensing applications than the HINA-based indicator displacement assay introduced above.
HINA, HPA and HAP readily permeate through biological membranes 19,56 as was conrmed for different cancerous and non-cancerous cell lines, see the ESI. † Preliminary results indicate that they localize in the perinuclear region of the cells (Fig. 3a) and the cell toxicity level of the dyes is low (Fig. S69 †). Interestingly, a rapid uorescence photoactivation of HINAtreated cells was observed within seconds during the uorescence imaging experiments ( Fig. 3b and Videos in the ESI †). A similar behaviour was seen in control experiments upon irradiation of HINA solutions with a strong light source, and in the presence of hydrogen peroxide (Fig. S71 †).

Conclusions
In conclusion, 3-hydroxyisonicotinealdehyde (HINA) has been identied as the smallest known green uorescent dye, and may be reaching the fundamental size-limit. Uniquely, HINA occurs in three different protonation states that are each distinctly uorescent. The QY of HINA in water is surprisingly high, despite being a push-pull chromophore. HINA's commercial availability, and its favourable photophysical properties (large Stokes shi, pH-dependent ratiometric emission properties that are switching in biorelevant pH 7 regime) will enable future applications, ranging from its use as a uorescent dye to its function as an indicator in supramolecular assays. The synthesis and investigation of additional hydroxyl-functional pyridine-aldehydes and -ketones will lead to the discovery of novel green-emissive labels with improved photophysical properties. These dyes will like HINA provide excellent test cases to evaluate theoretical predictions of emission spectra using highly advanced computational methods capable of considering both vibronic effects and excited-state intramolecular proton-transfer process (ESIPT).

Conflicts of interest
There are no conicts to declare.