Application of Monoferrocenylsumanenes Derived from Sonogashira Cross-Coupling or Click Chemistry Reactions in Highly Sensitive and Selective Cesium Cation Electrochemical Sensors

This paper reports the synthesis and characterization of novel monoferrocenylsumanenes obtained by means of the Sonogashira cross-coupling or click chemistry reaction as well as their application in cesium cation electrochemical sensors. A new synthetic protocol based on Sonogashira cross-coupling was developed for the synthesis of monoferrocenylsumanene or ethynylsumanene. The click chemistry reaction was introduced to the sumanene chemistry through the synthesis of 1,2,3-triazole containing monoferrocenylsumanene. The designed synthetic methods for the modification of sumanene at the aromatic position proved to be efficient and proceeded under mild conditions. The synthesized sumanene derivatives were characterized by detailed spectroscopic analyses of the synthesized sumanene derivatives. The supramolecular interactions between cesium cations and the synthesized monoferrocenylsumanenes were spectroscopically and electrochemically investigated. Furthermore, the design of the highly selective and sensitive cesium cation fluorescence and electrochemical sensors comprising the synthesized monoferrocenylsumanenes as receptor compounds was analyzed. The tested cesium cation electrochemical sensors showed excellent limit of detection values in the range of 6.0–9.0 nM. In addition, the interactions between the synthesized monoferrocenylsumanenes and cesium cations were highly selective, which was confirmed by emission spectroscopy, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), and cyclic voltammetry.

. 13 Figure S8. 13 Figure S25. Comparison of 1 H NMR spectra (500 MHz) of 1,4-diferrocenylbuta-1,3-diyne (12; brown spectrum), the product mixture from the first PTLC purification (green spectrum), and the pure 8 (from the second PTLC purification; blue spectrum). The inset of ESI-HRMS spectrum of the green spectrum sample is also presented. Labels of signals are also presented. The same color does not correspond to the same chemical shift. Selected insets of the spectra are presented. The signals of impurity 12 were clearly seen in the 1 H NMR spectrum of the sample obtained from the first PTLC purification process (eluent: 25% CH2Cl2/hexane) and the presence of residual 12 in that sample was also detected with highresolution mass.  Figure S26. The selected insets of the 1 H NMR spectra (500 MHz, CDCl3; 2-iodosumanene 2b -top and monoferrocenylsumanene 8 -bottom). The signals of the ferrocene protons are marked with the yellow frame. In a relation to the 1 H NMR spectrum of compound 2b, the spectrum of compound 8 showed: (a) signals of the ferrocene protons at 4.52-4.51 ppm and 4.26-4.25 ppm (multiplets, protons from the monosubstituted cyclopentadienyl (Cp) ring), and at 4.24 ppm (singlet, protons from the unsubstituted Cp ring), (b) signal of the sumanene aromatic proton from the substituted ring (brown) less separated from the signals of aromatic protons from the unsubstituted ring (violet), (c) downfield-shifted signals of the benzylic Hexo protons (light green) and benzylic Hendo protons (dark green). The linking of sumanene 1 with the 2-ferroceneethynyl moiety was also evidenced with the 13 Figure S27. 1 .. S59 Figure S43. Comparison between emission spectra (λex = 285 nm) of 7 in CHCl3 and CHCl3:CH3OH .. S60 Figure S44. Emission spectra (λex = 285 nm) of 7 in the presence of various amounts (equivalents = eq) of cesium cations.

Materials and methods
Chemical reagents and solvents for the synthesis were commercially purchased and purified according to the standard methods, if necessary. Thin layer chromatography (TLC) and preparative thin layer chromatography (PTLC) were performed using Merck Silica gel 60 F254 plates.
UV-vis measurements were performed with the PerkinElmer spectrometer Lambda 25, at room temperature in quartz cuvette of 1 cm length of optical window. For the UV-Vis measurements, the wavelengths for the absorption maxima λmax were reported in nm. Spectrofluorimetric analyzes were performed with a Hitachi F-4500 fluorescence spectrophotometer with the spectral resolution of 1 nm, the wavelengths for the emission maxima were reported in nm.
Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) experiments were performed in the three-electrode system with using a an Autolab potentiostat, model PGSTAT 12. The disc glassy carbon electrode (GC;  = 3 mm) was used as a working electrode, the Ag/AgCl/3 M KCl as a reference electrode and the platinum plate with an area of at least 1 cm 2 as a counter electrode. To minimize the electrical noise all experiments were carried out in Faraday cage. The electrochemical characteristic of the studied ferrocene derivatives (compound 7 and compound 8) was done in the dichloromethane (DCM) with addition of tetrabutylammonium hexafluorophosphate (TBAPF6) as a supporting electrolyte. The concentration of the studied ferrocene derivatives was 0.02 mM. Dry dichlorometane (DCM, Sigma-Aldrich), tetrabutylammonium hexafluorophosphate (TBAPF6, Sigma-Aldrich), tetrabutylammonium tetrafluoroborate (TBABF4, Sigma-Aldrich), cesium nitrate (CsNO3, Sigma-Aldrich), potassium nitrate (KNO3, Sigma-Aldrich), sodium nitrate (NaNO3, Sigma-Aldrich), barium nitrate (Ba(NO3)2, Sigma-Aldrich) and perfluorinated resin solution containing Nafion TM (nafion TM , Sigma-Aldrich) were used in electrochemical studies without additional purification.
The experimental data for LA-ICP-MS (Laser Ablation Inductively Coupled Plasma Mass Spectrometry) experiments were as follows. The Nd:YAG laser ablation system (LSX-213, CETAC, USA) was coupled to ICP-MS mass spectrometer (NexION 300D, Perkin Elmer, USA). The laser beam wavelength of λ = 213 nm, energy of 3mJ and diameter of 100 µm was used to ablate the surface layers of the analysed samples. During a multi-line ablation (n=5, 20Hz) with the constant scan rate of 100 µm/s transient signals were registered for 23 Na, 39 K, 57 Fe, 133 Cs and 137 Ba. The operating conditions of the used ICP-MS system are given in Table S1. For each isotope raw signals were individually background corrected for Ar flow before the start of ablation. Spikes were defined as a single data point exceeding the intensities of the neighbouring data for more than 2 times. They were replaced with the average value calculated based on the intensities of two neighbouring signals. All recalculations were done with the use of a custom written formula in Excel® (Microsoft Corp.).

S9
Note 2: The formation of side products, namely di-(ferrocenylethynyl)sumanenes (ca. 15 wt% of the mass of the crude mixture), was observed when 2-iodosumanene sample that was prepared from sumanene (1) using the different method (employing gold(III) chloride and Niodosuccinimide) 4 , was used in the Sonogashira cross-coupling reaction with ethynylferrocene (11). Those side products could be removed from the crude product using gel permeation chromatography (GPC; CHCl3), however, yield of 8 in that synthesis was lower (ca. 65%). 2-Ethynylsumanene (6) was synthesized in two steps.
Trimethyl(sumanenylethynyl)silane (9; 16.0 mg, 0.044 mmol, 1 eq) was placed in the reaction flask. The content of the flask was evacuated and purged with argon. Dry CH2Cl2 (3 mL) and MeOH (3 mL) were added, followed be the addition of dry potassium carbonate (K2CO3; 30.0 mg, 0.22 mmol, 5 eq). The reaction mixture was stirred for 24 hours at 27°C under argon atmosphere. Distilled water (10 mL) was added, and the crude product was extracted with CH2Cl2 (3x20 mL). Organic layers were combined, washed with water, and brine. After drying with MgSO4 followed by filtration, volatiles were distilled off on a rotary evaporator. The product was purified using a PTLC (SiO2; 25% CH2Cl2/cyclohexane) to provide the target compound 6 as a white solid (

Preparation of the cesium cations electrochemical sensor
The glassy carbon surface was first polished at the microcloth polishing pad with a slurry of alumina (1.0 m diameter, Buehler). Then, the GC surface was rinsed with distilled water to remove the Al2O3 residues. In the next step to produce more carboxyl groups at GC surface the electrode was oxidized in 0.                  Figure S25. Comparison of 1 H NMR spectra (500 MHz) of 1,4-diferrocenylbuta-1,3-diyne (12; brown spectrum), the product mixture from the first PTLC purification (green spectrum), and the pure 8 (from the second PTLC purification; blue spectrum). The inset of ESI-HRMS spectrum of the green spectrum sample is also presented. Labels of signals are also presented. The same color does not correspond to the same chemical shift. Selected insets of the spectra are presented. The signals of impurity 12 were clearly seen in the 1 H NMR spectrum of the sample obtained from the first PTLC purification process (eluent: 25% CH2Cl2/hexane) and the presence of residual 12 in that sample was also detected with highresolution mass. The second PTLC purification (eluent: 50% THF/hexane) yielded pure 8.

Explanation of the specific 1 H NMR profiles of compounds 2a, 2b and 6-8
The 1D and 2D NMR spectra were employed in order to explain the specific 1 H NMR profiles of compounds 2a, 2b and 6-8 (for the corresponding spectra, see Supporting Information, Section 2). Interestingly, in a relation to the 1 H NMR profile of sumanene 1, a signal corresponding to the H3 proton was separated from a set of signals of aromatic protons. Further, signals of the benzylic H1-, H4-and H7 protons were separated in the range of both the Hendo and Hexo protons (for the numbering system of the examined compounds, see Table  S2). The most significant changes in the spectra were observed in the rage of signals corresponding to the benzylic Hendo protons. For instance, in the spectrum of compound 7, three well separated doublets were observed. The signal assignments were based on the diagnostic 1 H-13 C HSQC and 1 H-13 C HMBC correlations (Table S2). Briefly, the applied methodology involved: (a) an identification of the C3 carbon atom signal from the H3C3 HSQC correlation (confirmed from the H3C3 HMBC correlations in compounds 2a and 2b); (b) a localization of both the C4 carbon atom signal from the H3C4 HMBC correlation and the H4endo proton signal from the H4endoC4 HSQC and H4endoC4 HMBC correlations (signals of H4endo proton and the H7endo proton in the spectra of compounds 7 and 8 were successfully differentiated); (c) a confirmation of the H4endo proton assignment by the H4endoC3 HMBC correlation in all the examined compounds, accompanied by the H4endoC3a and H3C3a HMBC correlations in compounds 2a and 2b; (e) an identification of the H1endo proton signal from two sets of the HMBC correlations: H1endoC1a and H3C1a (compounds 2b and 6-8), as well as H1endoC2 (compounds 2a, 2b and 6-8) and H3C2  (compounds 2a, 2b and 6). The H1exoC1 and H1exoC1a HMBC correlations in compounds 2a, 2b and 7 were also detected. The 1 H-13 C HMBC correlations within substituents of compounds 6-8 are also listed in Table S2.
Our analysis revealed the most significant changes in the chemical shifts of signals corresponding to the H1endo proton in the examined compounds, taking the chemical shifts of the Hendo protons in sumanene 1 as the reference ( Table S2). The downfield shift of these signals in the spectrum of compound 2a (δ(H1endo) = 3.50 ppm) was attributed to the deshielding effect of the highly electronegative bromine atom. On the contrary, the upfield shift of the H1endo proton signals in the spectrum of compound 2b (δ(H1endo) = 3.36 ppm) was assumed to result from two opposite effects, i.e., a lower electronegativity of the iodine atom (than that of the bromine atom) and the rigid structure of sumanene. Our hypothesis was supported by the literature data where the same tendency was reported for 1-chloro-9Hfluorene 6 and 1-iodo-9H-fluorene 7 when the 1 H NMR chemical shifts of signals corresponding to their benzylic protons were compared with those of 9H-fluorene. On the other hand, the 1 H NMR chemical shift of a signal corresponding to the benzylic protons in 1-benzyl-2bromobenzene or 1-benzyl-2-iodobenzene was reported to be essentially constant. 8 Therefore, based on the literature data on the bromine or iodine substituent 1 H NMR chemical shift effect in halocyklohexanes 9 , we believe that the steric H1endo····I shielding might be one of the factors determining the observed H1endo chemical upfield shift in compound 2b.
Further, the downfield shift of the H1endo proton signals in the spectra of compounds 6-8 were observed in a relation to the chemical shift of the Hendo protons in sumanene 1. Our structural optimizations of compounds 6 and 8 suggested a location of the H1endo proton in the deshielding area around alkyne group as a consequence of the bowl shape of the sumanene core (Figure 4). Thus, the anisotropic deshielding effect of the alkyne group might be considered one of the factors responsible for chemical shifts of the H1endo proton in compounds 6 and 8 ((H1endo): 3.58 ppm (6) vs 3.50 ppm (1), and 3.60 ppm (8) vs 3.50 ppm (1)). On the other hand, the strong downfield shift of signals corresponding to the protons H1endo, H1exo and H3 the observed in the spectrum of compound 7 ((H1endo), 3.66 ppm (7) vs 3.50 ppm (1); (H1exo), 4.95 ppm (7) vs 4.70 ppm (1), and (H3), 7.79 ppm (7) vs 7.18 ppm (1)), might be ascribed to a significant electron-withdrawing inductive effect of the 1H-1,2,3-triazole ring. 10 The abovementioned electron-withdrawing inductive effect of the 1H-1,2,3-triazole ring could also be considered responsible for the observed differences in the 1 H NMR pattern of signals corresponding to the aromatic protons of H5, H6, H8 and H9 spectrum of compounds 7 and 6 (see, Figure S19, and Figure S17 vs Figure S11). Based on the corresponding 1 H- 13 Cipso HMBC correlations in the spectrum of compound 7 (Figure 2, Figure S17 and Table  S2), the following assignments were found: H5 (7.18) and C5 (123.8); H6 (7.20) and C6 (124.5); H8 (7.20) and C8 (123.7); H9 (7.18) and C9 (123.8). The diagnostic 1 H-13 C HMBC correlations of H1endoC9, H4endoC5, H7endoC6 and H7endoC8 were crucial. Interestingly, the signal corresponding to the C6 carbon atom ( 124.1 ppm) featured a significant downfield shift in a relation to the chemical shift of signals corresponding to the carbon atoms of C5, C8 and C9 ( 123.73-123.79 ppm). Again, this phenomenon was attributed to the electron-withdrawing inductive effect of the 1H-1,2,3-triazole ring decreasing an electron density around the C6 carbon nuclei. Indeed, literature data showed a decreasing effect of the electron-withdrawing substituents on an electron density around the C6 carbon nuclei of the 2-nitro-, 2-formyl-or 2-acylsumanenes. 4  Table S2. The NMR signal assignments and diagnostic 2D NMR correlations in compounds 2a, 2b, 6-8. a,b

Calculations
Otherwise noted, all structure optimizations, self-consistent field (SCF) energies, and thermal energy correction calculations using density functional theory (DFT) were performed using Gaussian 16 suite of programs (revision C.01) 11 at ωB97X-D 12 level of theory in gas phase with Def2-SVP 13 as a basis set. The DFT optimized structures of 2-ethynylsumanene (6), monoferrocenylsumanenes 7 and 8 at ωB97X-D/Def2-SVP level of theory, are presented in Figure S32-S36. Figure S37 presents the differences in bowl depth values for those derivatives. The introduction of the substituents to the sumanene (1) molecule slightly affected the observed bowl depth. The bowl depth for newly synthesized 6, 7 and 8 was defined as the distance between the plane formed by the six-membered ring in the center of the sumanene skeleton and the rim carbon 14,15 , see Figure S37a. The bowl depth values were taken as the values for the carbon atom at the substituted aromatic ring of sumanene 15 (marked with a black arrow in Figure S37c). This way, the bowl depth for compounds 6, 7 and 8 equaled to 1.18, 1.16 and 1.19 Å, respectively. Those values are slightly higher than the bowl depth for the unmodified sumanene (1.15 Å, estimated from the DFT optimized structure; Figure S37b) 14,15 . It suggested that introducing acetylene or 1,2,3-triazole moieties to the aromatic sumanene ring might cause a slight distortion of bowl structure. 15 Optimized Cartesian coordinates of sumanene

Spectrofluorimetric analyzes of the interactions between monoferrocenylsumanene 7 or 8 and cesium cations
To provide the solubility of 7 and 8 and cesium salt, the measurements were carried out in methanol-chloroform mixture (1:1 v/v). Appropriate volumes of 110 −4 M CsCl solution were mixed with 110 −4 M solution of monoferrocenylsumanene (7 or 8) to reach given sumanene-to-metal cation molar ratio. The concentration of monoferrocenylsumanene in each sample was 210 −5 M. The excitation wavelength was 285 nm.
Complex stoichiometries were estimated with the Job's plot method (continuous variation method). [16][17][18][19] The apparent binding constants (Kapp) were estimated with the Benesi-Hildebrand method 20,21 , using the following equation: where I0 and I are the fluorescence intensities of sumanene-ferrocene conjugate in the absence and presence of cesium cations, respectively, a is a constant, and C(Cs + ) is the concentration of cesium cations in solution. The association constant was determined as a ratio of intercept-to-slope of 1/(I − I0) vs. 1/C(Cs + ) linear plot. The above-discussed spectra and data are presented below.

Electrochemical characterization of monoferrocenylsumanenes 7 and 8
Cyclic voltammetry (CV) experiments were performed to electrochemically characterize the synthesized monoferrocenylsumanenes 7 and 8. The voltammograms were recorded in DCM at different scan rates, ranging from 0.002 to 1 V·s −1 . The typical cycling voltammograms of 7 and 8 are presented in Figure S47. The voltammograms of both compounds are characterized by one pair of current signals (anodic and cathodic) corresponding to the Fe 2+/3+ redox couple in the ferrocene unit. [22][23][24] In the case of fast, reversible and one-electron process ,the peak potential separation (Ep = Epa -Epc) should equal to 0.059 V (298 K) and the peak current ratio (Ipa/Ipc) should equal to 1. 25 Considering the electrochemical parameters of the studied ferrocene derivatives 7 and 8 (refer to Table S3), it can be concluded that the conjugation of ferrocene unit with sumanene through acetylene or 1,2,3-triazole does not significantly affect the electrode process. The dependencies of anodic peak heights in the function of the square root of the scan rate are shown in the insets in Figure S47. In the whole studied scan rate range, the relationship of Ipa versus (v) 0.5 was linear, what indicates clear diffusion character of the electrode process. From the slope of the plot Ipa = f(v) 0.5 the diffusion coefficients of the studied ferrocene derivatives were calculated. The obtained values (Table S3) are very similar to the value for unmodified ferrocene. The ln(Ipa) = f(Epa-Ef) dependencies were plotted to get the information about the influence of the type of linker on the electron-transfer rate constant (k0). The value of the electron-transfer rate constant was determined from the slope of the curve, according to the formula: where: Ipa is a current intensity of the anodic peak, n − number of electron exchange during electrode process, F is Faraday constant, A − electrode surface area, C0 * concentration of the electroactive species, Epa − potential of the anodic peak, Ef − formal potential, R − gas constant, T − temperature and  is transition coefficient. Similarly, as in the case of the diffusion coefficient values, no significant differences were found compared to the standard (unmodified ferrocene).
The results of selectivity studies with cyclic voltammetry are presented in Figure S46.  Table S3. The NMR signal assignments Electrochemical parameters (Ipa: current intensity of the anodic peak, Epa: potential of the anodic peak, Ipc: current intensity of the cathodic peak, Epc: potential of the cathodic peak, Ef: formal potential, D: diffusion coefficient, k0: electrontransfer rate constant) of monoferrocenylsumanenes 7 and 8 estimated from CV voltammograms recorded at scan rate equal 0.

Additional data on LA-ICP-MS
Elemental distribution of Fe, Cs, Ba, Na and K baed on LA-ICP-MS analyzes at the surface of the samples labelled as compound 7 and compound 8 are presented in Table S4. Table S4. Elemental distribution of Fe, Cs, Ba, Na and K at the surface of the samples labelled as compound 7 and compound 8.

Fe
Cs Ba Na K Compound 7 Compound 8