Anion Binding Studies on Receptors Derived from the Indolo[2,3-a]carbazole Scaffold Having Different Binding Cavity Sizes

2.1. Synthesis of Receptors 1–3

The synthesis of the studied receptors was carried out as depicted in Scheme 1. The access to the indolo[2,3-a]carbazole scaffold was accomplished using a one-pot procedure involving a double Fischer indolisation between 4 and the corresponding phenylhydrazine in AcOH/TFA mixtures. The reaction with o-hydrazinobenzoic acid, 5, led to the formation of the dicarboxylic acid 6, which was treated with BH₃·SMe₂ in THF to obtain the diol 1.

Using a similar synthetic route, the reaction of 1,2-cyclohexanedione (4) with o-bromo-phenylhydrazine enabled the isolation of the bis-brominated derivative 8. The protection of the indolocarbazole NH groups with TMSCl, followed by the lithiation of the heteroaromatic system and subsequent reaction with DMF produced the dialdehyde 9. The expansion of the binding cavity was achieved via a Horner-Wadsworth-Emmons reaction on 9, which stereospecifically led to the α,β-unsaturated diester 10. Finally, hydrolysis of ester groups led to the dicarboxylic acid 11 and reaction of the latter with butylamine or p-nitroaniline in the presence of 1,1′-carbonyldiimidazole (CDI) in DMF produced receptors 2 and 3, respectively.

2.2. Binding Studies

Initially, binding studies on receptor 1 were performed by ¹H-NMR in CD₃CN (Figure 2). When 1,10-di(hydroxymethyl)-indolo[2,3-a]carbazole, 1, was titrated with a series of anions, namely acetate, benzoate, dihydrogenphosphate, hydrogenpyrophosphate, chloride and bromide, the expected downfield shift was detected for the NH protons which formed hydrogen bonds with the anionic guests.

Unfortunately, the OH protons rapidly exchanged and disappeared after the addition of the first aliquots of anions. Nevertheless, the peak ascribed to the CH₂ protons still exhibited a subtle shift induced by the complexed anion. The detected evolution of the NMR peaks clearly evidenced a preferential binding towards oxyanions.

The fused polyheteroaromatic structure of indolocarbazole allows that it can be simultaneously used as binding unit and as signaling unit. The UV-vis spectrum of 1 showed five bands at λ = 261 nm (ε = 43,000 cm⁻¹·M⁻¹), λ = 269 nm (ε = 45,000 cm⁻¹·M⁻¹), λ = 285 nm (ε = 19,500 cm⁻¹·M⁻¹), λ = 324 nm (ε = 21,500 cm⁻¹·M⁻¹), and λ = 359 nm (ε = 4300 cm⁻¹·M⁻¹) assigned to the π-π* transitions of the indolocarbazole system (Figure 3). Accordingly, titration experiments carried out by absorption spectroscopy showed a bathochromic shift of all the bands in the spectrum upon anion complexation. Additionally, four isosbestic points could be detected indicating the establishment of a well-defined equilibrium between host and guest [27].

Job plot analysis of the titrated anions confirmed a 1:1 stoichiometry for most of them (see Supplementary Information). However, a different evolution was detected for the hydrogen-pyrophosphate anion. This caused the saturation of the binding curve right after the addition of 0.5 equivalents, which could be related to the formation of a 2:1 (H:G) complex due to the larger size of this anion. This result was further confirmed by the Job plot, which displayed a minimum at a value of 0.66 for the molar fraction of the receptor. The previously mentioned preference towards oxyanions was corroborated by the binding constants determined from non-linear regression of the experimental binding curves. In this regard, the angular and tetrahedral geometries of acetate, benzoate, dihydrogenphosphate and hydrogenpyrophosphate anions enabled a better geometrical correspondence with the di(hydroxymethyl)indolocarbazole 1.

The weak sensitivity resulting from the UV-vis experiments, led us to the study of anion complexation by the more sensitive fluorescence spectroscopy (Figure 4). Concerning the emission spectrum (λ_exc = 300 nm) of diol 1, it showed a band at 387 nm accompanied by a shoulder at 371 nm, ascribed to the π-π* transitions of the indolocarbazole system. The titration experiments with AcO⁻ and H₂PO₄⁻ only caused a subtle decrease in the fluorescence spectrum along with a weak bathochromic shift. As far as the HP₂O₇³⁻ anion is concerned, a two-phase sigmoidal profile proved the higher 2:1 stoichiometry discussed above. Conversely, a more sensitive response was obtained for the benzoate anion which provoked the almost complete quenching of the indolocarbazole emission. Finally, a comparison of the diol 1 with the plain indolo[2,3-a]carbazole evidenced an increase in the stability of the complexes as a result of the attached hydroxymethyl groups (Table 1).

Motivated by the promising results obtained from a very simple functionalisation of the indolocarbazole skeleton, we decided to examine the possibility of expanding the size of the indolocarbazole binding cavity. Since most of the reported 1,10-disustituted indolocarbazole-based anion receptors describe a conformationally restricted cavity, the widening of the receptor represents an unexplored option. As it has been previously anticipated our approach consisted in the synthesis of (E)-double bonds appended to the polyheteroaromatic system. Subsequently amide functional groups were incorporated to the expanded π-conjugated system.

Preliminary studies with the N-butyl amide 2, denoted a red shift of the UV-vis spectrum as a result of the extended conjugation. Once again, the titration with the series of tested anions (AcO⁻, BzO⁻, H₂PO₄⁻, Cl⁻ and Br⁻) produced a weak bathochromic shift (Figure 5). It is worth highlighting that differently from the diol 1, the diamide 2 could bind halide anions such as chloride and bromide. Regarding the experiment with the HP₂O₇³⁻ anion, it described a complex titration isotherm denoting a dissimilar binding mode than the rest of the anions. Anyhow, the association constants determined for compound 2 manifested a lack of selectivity for any of the assayed anions (Table 1).

With the aim of investigating the effect of the environment on the anion binding ability of the expanded indolocarbazole receptors, the diamide 3 was analysed in a more competitive solvent such as DMSO. The ¹H-NMR titrations confirmed the expected downfield shift of the pyrrolic NHs taking part in the complexation of the anions (Figure 6). Although these peaks vanished during the initial part of the titration, they came up again as the titration progressed. Interestingly, the olefinic protons in receptor 3 also showed a downfield shift caused by the deshielding effect of hydrogen bonding the anionic guests. Nevertheless, the peaks ascribed to the amide NHs did not display any significant displacement, which could be interpreted in terms of the perhaps too long distance from the center of the binding cavity. In any case, it was the benzoate anion which formed more stable complexes compared to the rest of the series of anions. Therefore, the increase in the polarity of the environment produced a noticeable improvement in the binding selectivity which was evidenced even for the two analysed carboxylate anions, i.e., benzoate (K_assoc. = 1.02 × 10⁴ M⁻¹) and acetate (K_assoc. = 2.09 × 10³ M⁻¹).

Concerning the UV-vis experiments, the higher competitiveness of the environment, joined to the more diluted concentration of the samples, resulted in a negligible response for most of the studied anions. Nevertheless, those anions with a stronger basicity induced a colour change in the solution which, in the case of hydrogenpyrophosphate, was especially meaningful since the solution turned from yellow to dark red (Figure 7). Control experiments performed with a strong base such as TBAOH, confirmed that the detected colour change corresponded to a deprotonation process. Additionally, the presence of the p-nitrophenyl groups in 3, favoured the colorimetric response which had not been observed for other indolocarbazole receptors.

By virtue of very simple structural modifications in the binding cavity of the indolocarbazole a modulation of the selectivity and sensitivity of a multichannel response has been achieved.

3.1. General Information

Solvents were dried following the usual protocols. THF, Et₂O and toluene were distilled from sodium wire with benzophenone indicator; CH₃CN and CH₂Cl₂ were distilled from CaCl₂; EtOH and MeOH were distilled from magnesium and stored with molecular sieves. All anions were employed as their tetrabutylammonium salts. Unless stated otherwise, all reactions were carried out under nitrogen atmosphere. Column chromatography was run with silica gel 60 Å CC 70–200 μm as stationary phase and using HPLC grade solvents. Melting points were measured in a Reichert instrument and are not corrected. ¹H-NMR, ¹³C-NMR and NOESY experiments were recorded on a Bruker AV200, AV300, AV400 or AV600 instruments. Chemical shifts are referred to the residual peak of the solvent. In the experimental data “bp” stands for broad peak and “Cq” for quaternary carbon atom. Mass spectrometry was recorded on HPLC-MS TOF 6220 instrument. Absorption spectra were recorded on a Cary 5000 UV-vis-NIR spectrophotometer. Emission spectra were recorded on a Cary Eclipse spectrophotometer. Microanalyses were performed on a Carlo Erba 1108 instrument. All binding constants were calculated by a non-linear fitting procedure using the software SPECFIT/32 Global Analysis System.

3.2. Synthesis