Switchable fl uorescence by click reaction of a novel azidocarbazole dye †

Department of Chemistry, Karlsruhe Inst 76131 Karlsruhe, Germany. E-mail: stefan.b Department of Chemistry, University of Hels Helsinki, Finland. E-mail: martin.nieger@h Institute of Toxicology and Genetic, Karlsruh Hermann-von-Helmholtz-Platz 1, 76344 Egg ute.schepers@kit.edu Cynora GmbH, Hermann-von-Helmholtz-P Germany † Electronic supplementary information (E computation method and X-ray crystallogr 2 and 3 (CIF). CCDC 936576 and 936577. F or other electronic format see DOI: 10.103 ‡ Both authors contributed equally. Cite this: RSC Adv., 2014, 4, 11528


Introduction
Carbazole moieties are a structural key motif in both dyes and luminophores.Pigments like Violet 23 or Hidrotin Blue 2R are used e.g. for the staining of clothing and strongly benet from carbazole units in terms of photostability and high absorbance. 1,28][9] However, because of the p-p* and n-p*-transitions occurring upon excitation of the carbazole core, stokes shis for carbazole-substituted compounds are usually small.This hampers the discrimination between excitation and emission when using them as dyes in uorescence microscopy and biological applications.
To resolve this issue, we attempted to design a new uorescent label, combining the benecial properties of carbazole such as high photostability and extinction, with the large stokes shi of a charge-transfer (CT) system.CT systems typically consist of a donor-moiety, which may be excited, and a matching acceptor-unit, to which the electron density is shied.
Typically, this leads to broad, ill-structured spectra as well as large stokes shis.In our case, the carbazole unit acts as a donor-unit, while aryl-substituted 1,2,3-triazoles are used as acceptors (Schemes 1-3).
Here, we report the synthesis and the photophysical properties of new carbazole dyes.Single-crystal X-ray diffraction revealed the molecular structure, while DFT-and TD-DFT calculations provided valuable insights into the photophysical mechanism leading to light-emission and a large stokes shi.The dyes themselves are formed by Click-functionalization of a non-uorescent precursor, N-(4-azidophenyl)-carbazole 1, which is non-uorescent due to vibronic quenching by the free azido-group.This enables an easy discrimination of labelled material and traces of remaining, non-reacted precursor, e.g. in uorescence microscopy.

Synthesis of precursor 1 and the fluorescent dyes
The synthesis of the precursor 1 is summarized in Scheme 1. Aer an Ullmann-type coupling of a straight carbazole with 4-bromo-nitrobenzene, the resulting N-(4-nitrophenyl)-carbazole is reduced with hydrazine, diazotized with HNO 2 and converted to N-(4-azidophenyl)-carbazole 1 by reaction with sodium azide.Further functionalization of non-uorescent N-(4-azidophenyl)carbazole 1 with terminal alkynes in a Cu(I)-catalysed alkyneazide cycloaddition (CuAAC) (Scheme 2) as well as with cyclooctynes (Scheme 3) yielded the uorescent carbazole dyes. 10,11or the CuAAC click reaction, we chose copper(I) iodide in N,N-diisopropylethylamine (DIPEA) as the catalyst and several alkyne moieties such as the commercially available phenylacetylene as well as 2-methyl-3-butyn-2-ol, yielding the uorescent dyes 2 and 3, respectively.High conversion was achieved for both examples (92% yield each) within 4 hours.Furthermore, azide 1 was coupled to a polycationic peptoid containing an alkyne side chain on solid phase in a submonomer approach under the same conditions (see ESI †). 12,13This peptoid is a heptameric compound with six aminohexyl-and one propargylside chain and was used to investigate the inuence of charged moieties on the carbazol uorescence. 14,15n contrast to the CuAAC reaction, the reaction between azides and cyclooctynes does not require any catalysts at room temperature due to the strained ring structure of cyclic alkyne.This makes the strain-promoted azide-alkyne cycloaddition (SPAAC) 16 suitable for click reactions in the presence of cells or whole organisms.All reactions depicted in Scheme 3 were carried out by simply dissolving the reactants (azide 1 and one equivalent of cyclooctynes 5a-8a) in dichloromethane (DCM) and stirring overnight to yield dyes 5-8 in good to very high yields (74-98%).

Single-crystal X-ray diffraction
We were able to characterize molecules 2 and 3 by single-crystal X-ray diffraction (Fig. 1 and ESI †). 17 The most striking observation for both structures was the twisting of all three p-systems, which breaks the conjugation between the three ring systems into electronically independent localized moieties.The smallest angle between the triazole ring and the phenyl ring is 29.9 for component 2 and 38.7 for component 3. Between the carbazole moiety and the phenyl ring, an angle of 49.4 for component 2 and 66.2 for component 3 has been found.The molecular structures of 2 and 3 have also been used as starting structures of DFT-calculations and to interpret the photophysical properties (see below).

Absorption spectroscopy
It is tempting, yet illegitimate to interpret a twist of the p-systems in solution from the results found in single-crystals without further evidence.We thus recorded UV-Vis spectra in various solvents.In summary, absorption spectra of both the unreacted precursor 1 and the uorescent compounds 2 to 8 were quite similar.Carbazole moieties are very strong chromophores, which dominate the UV-Vis spectra; Peaks varied only in a 4 nm range (Fig. 2, Table 1 and ESI †).Molar absorption coefficients were quite high and depended on the solvents (Table 1 and ESI †).Substitutions with aromatic rings and modications of side groups did not lead to a bathochromic shi or strong changes in the absorption spectra (Fig. 2 and Table 1).This suggested that the twisting of the conjugation found in solid state by X-ray diffraction also occurred in solution.Subtle variations manifested themselves in the differences in height of the shoulders in the normalized absorption spectra.Due to the broken conjugation, the UV-Vis spectra may be  described as a superposition of the expected spectra of the isolated chromophores (phenyl, triazol, carbazole) with additional bands due to a charge-transfer transition from the carbazole to the aryl-triazole (300-350 nm).

Emission spectroscopy
To evaluate the strongest transition between absorption and emission 3D spectra of compound 7 in ethyl acetate and cyclohexane have been recorded.The result for cyclohexane is given in Fig. 3, while the corresponding plot for ethyl acetate is given in the ESI.† For both solvents, 3D-spectra were similar and in agreement with the 2D-measurements (uorescence over wavelength, see Fig. 2) over a range of excitation wavelengths.The strongest transition was found to occur at an excitation wavelength of 292 AE 1 nm.Excitation with lower energies (345 nm) did not lead to detectable luminescence.We thus dene the strong transition at 292 AE 1 nm as the preferred excitation wavelength and refer the stokes shis to this transition.

Solvent-and polarity-influences on the emission
Emission spectra of the novel carbazole dyes were highly dependent on the solvent, with varying emission maxima between 341 nm and 492 nm (Table 2, Fig. 4).However, differences between compounds 2-4 and 5-7 were noted probably due to structural variations.While compounds 2-4 have been reacted with terminal alkynes, compounds 5-8 have been reacted with cyclooctynes and contain bicyclic (5-7) and polycyclic (8) moieties.Stokes shis were large, ranging from 49 nm to 200 nm and strongly solvent dependent (see Tables 1 and 2).The Lippert-Mataga-Plot, which was plotted for substance 7, also served to rationalize this (see Fig. 4).A linear correlation of the solvent orientation polarisability and the obtained stokes shi was found (slope of the line and standard deviation in ESI †).The orientation polarisability of the   environment interacted with the dipole moment of the chromophore.This led to a shi of the emission maxima and consequently to the large stokes shis.
For methanol (MeOH), we noticed signicant deviations from the tted line in the Lippert-Mataga plot.Interestingly, no such effects were found in absorption spectra (Fig. 5).We recorded emission spectra of compound 7 in different mixtures of MeOH and DCM as solvents.In non-protic solvents such as DCM, two small band maxima were found, while an additional, broad band appeared when protic methanol was mixed with the non-protic solvent.When increasing the amount of protic solvent, the broad band became more pronounced.
This behaviour is indicative for the occurrence of different emissive states.The blue-shied, structured transition could be attributed to carbazole-centered uorescence, which was apparently not inuenced by the surrounding solvents.The second, red-shied transition was stabilized by protic solvents and was thus more pronounced when moving from pure DCM to pure MeOH.Under aqueous conditions, the carbazole-based emission was fully suppressed in favour of the CT-type band as shown for the water soluble compound 4. The fact that increasing polarity and the presence of protic solvents seem to cause a very large stokes shis makes these dyes interesting candidates for biological applications.

pH-influences on the emission for compound 4
Compound 4 carried six amino groups and was therefore expected to be inuenced by the pH value of the medium due to the introduction of positive charges in close proximity to the emissive chromophore.The absorption and emission spectra have been measured in phosphate buffered saline (PBS) under acidic, neutral and basic conditions (spectra see Fig. 6 and    ESI †).The emission spectra (Fig. 6) were pH sensitive: The band was shied towards lower energies, when moving to more acidic solvents.This was accompanied by decreased quantum efficiency.Similar to the inuences of protic solvents on compound 7, the pH-value did not seem to affect the absorption spectra of compound 4. We thus presume that the positive charge of the ammonium groups inuenced the molecules in excited state, which is typical for CT emission.This decreased the intensity and shied the emission to longer wavelength (normalized spectra see ESI †).

Determination of the quantum efficiency and emission decay time
Also measurements of the photoluminescence quantum yield (PLQY) 19 were done in different solvents as well as in the solid state (Table 1).The PLQY values were quite high, despite the large stokes shi.They mostly varied between 30% and 72%.An exception was compound 4 due to the high interactions with the protonated amino side chains, as stated above (ESI †).Using the time-correlated single photon counting (TCSPC) technique, the emission decay time of components 2 and 3 was measured in degased DCM at room temperature.Both compounds featured a monoexponential decay with s ¼ 3 and 4 ns, respectively.These lifetimes indicated a uorescence emission without an involvement of triplet state (T 1 ).We can exclude delayed uorescence as the cause for the broad spectra and large stokes shis in the solid state.

DFT calculations
Density functional theory (DFT) geometry calculations for compounds 2, 3 and 7 were performed on B3-LYP/def2-TZVP level 20,21 using D3 dispersion corrections. 22Comparison of the calculated and measured bond lengths and angles (see ESI †) showed a good agreement for the applied functional.Representative frontier orbitals (HOMO and LUMO) for compound 3 are depicted in Fig. 7 (see ESI † for compounds 2 and 7).For all calculated compounds, the highest occupied molecular orbital (HOMO) is mainly localized at the carbazole moiety, whereas the lowest unoccupied molecular orbital (LUMO) is localized on the triazole and bridging phenyl ring.As seen for the ground state HOMO-LUMO energies in Table 3, HOMO energies remain basically unchanged (around À5.7 eV) with varying substituents at the triazole, whereas the LUMO energies are affected.Hence, it appears that major differences in the observed emission maxima of compounds 2, 3 and 7 may result from variations in the LUMO energies.
On the basis of these results, the type of electronic transition that led to the lowest excited states may be classied as being largely based on intramolecular charge-transfer character.This assumption was further supplied by gas phase time-dependent density functional theory (TD-DFT) calculations at the same level, which shows 99% participation of HOMO / LUMO for the S 0 to S 1 vertical singlet excitation.However, it is known that exact DFT calculations of charge-transfer (CT) excitations are very difficult.Since all excitations show a distinct CT character, the excited states and transition energies are not described quantitatively by simple application of TD-DFT.As found for compound 3 (Table 3), the energies of HOMO and LUMO are just slightly affected by varying solvent polarity (conductor-like screening model, COSMO). 23

Twisted internal charge transfer mechanism
All results indicates that a twisted internal charge transfer (TICT) as described by Wang et al. 24 is responsible for the emission of our new dyes.We believe that the strong polarity dependence of the stokes shi originates from solvent dependent slow reorientations of the molecule dipole in the excited states.This is in good agreement with all photophysical measurements, the TCSPC measurements, X-ray results and DFT calculations.The reason for this is the polarity-dependent twist of the three connected p-systems of the carbazole, phenyl and triazole moieties.

Biological application
To verify that the specic conditions in biological media lead to a twisting conformation that allows for efficient light detection, we tested compounds 2 and 7 for their applicability in cell culture.Eventually, 10 4 human cervix carcinoma (HeLa) cells were treated with 30 mM of 2 and 7, respectively.Fluorescence confocal microscopy showed that in cells both compounds revealed a strong uorescence with a bathochromic shi of the measured emission maxima towards 450-550 nm, when excited with a UV laser at 351 nm.Both compounds 2 and 7 showed a strong accumulation within the endosomal-lysosomal system (Fig. 8), which suggests an endocytotic uptake.A xyl-scan resulted in maximum uorescence emission at about 481 nm for 2 and 543 nm for 7.While the bathochromic shi of compound 2 is less pronounced, the shi in 7 is in accordance with the uorescence measurements in protic solvents with increasing polarity (Fig. 5).
As the pH of the endocytic pathway is decreasing from the plasmamembrane towards the lysosomal compartment (from 7.4 to 4.4), the red-shied transition of compound 7 is shied towards lower energies when moving to more acidic (and thus, more protic) compartments.This is even more pronounced in cells than in solution.Although the excitation wavelength is not optimal, the strong uorescence emission of the compound promises an application in biological systems.

Conclusions
In this study, we present a new class of uorescent carbazole dyes.We demonstrate that the non-uorescent N-(4-azidophenyl)-carbazole moiety becomes uorescent by click reaction with an alkyne or cyclooctyne.This specic property allows uorescent control in samples aer click reaction, for example in biological samples or material science.The spectral properties show that absorption spectra are not changed aer click reaction with various substrates.The uorescence of the resulting dyes results from a twisted internal charge transfer mechanism, which has been veried by detailed photophysical experiments, single-crystal X-ray diffraction and DFT-calculations.The leads to a high solvent dependency accompanied by large stokes shis from 49-200 nm.Most samples have a high quantum yield between 30 and 72%.The feasibility of the new dyes has been demonstrated in a rst series of confocal microscopy experiments.

Fig. 5
Fig. 5 Absorption (top) and emission (bottom) spectra of compound 7 in solvent mixtures of MeOH and DCM.Excitation wavelength l ex ¼ 290 nm.

Fig. 6
Fig. 6 Fluorescence spectra of compound 4 in different pH, the intensities are recalculated to the lamp intensities and detection sensitivity.Excitation wavelength l ex ¼ 290 nm.

Table 1
Spectroscopic data for the compounds at room temperature a Solid, b DCM, c DMF, d H 2 O, e MeOH, f DMSO, g Cyclohexane, h Diethyl ether, i Ethyl acetate, j DMF.

Table 2
Solvent dependency for compound 7 in numbers a a DCM ¼ dichloromethane, DMF ¼ dimethylformamide; calculation of stokes shis see ESI.Df numbers for methanol and ethyl acetate, as well as formula according to ref.18.