Water-Soluble Iridium(III) Complexes Containing Tetraethylene-Glycol-Derivatized Bipyridine Ligands for Electrogenerated Chemiluminescence Detection

Four cationic heteroleptic iridium(III) complexes containing a 2,2′-bipyridine (bpy) ligand with one or two tetraethylene glycol (TEG) groups attached in the 4 or 4,4′ positions were synthesized to create new water-soluble electrogenerated chemiluminescence (ECL) luminophores bearing a convenient point of attachment for the development of ECL-labels. The novel TEG-derivatized bipyridines were incorporated into [Ir(C∧N)2(R-bpy-R′)]Cl complexes, where C∧N = 2-phenylpyridine anion (ppy) or 2-phenylbenzo[d]thiazole anion (bt), through reaction with commercially available ([Ir(C∧N)2(μ-Cl)]2 dimers. The novel [Ir(C∧N)2(Me-bpy-TEG)]Cl and [Ir(C∧N)2(TEG-bpy-TEG)]Cl complexes in aqueous solution largely retained the redox potentials and emission spectra of the parent [Ir(C∧N)2(Me-bpy-Me)]PF6 (where Me-bpy-Me = 4,4′methyl-2,2′-bipyridine) luminophores in acetonitrile, and exhibited ECL intensities similar to those of [Ru(bpy)3]2+ and the analogous [Ir(C∧N)2(pt-TEG]Cl complexes (where pt-TEG = 1-(TEG)-4-(2-pyridyl)-1,2,3-triazole). These complexes can be readily adapted for bioconjugation and considering the spectral distributions of [Ir(ppy)2(Me-bpy-TEG)]+ and [Ir(ppy)2(pt-TEG)]+, show a viable strategy to create ECL-labels with different emission colors from the same commercial [Ir(ppy)2(μ-Cl)]2 precursor.

Numerous cyclometalated iridium(III) complexes have been synthesized and many have shown impressive annihilation and/or co-reactant ECL intensities in organic media (Bruce and Richter, 2002;Kapturkiewicz et al., 2004;Kim et al., 2005). For example, we recently re-examined a promising series of heteroleptic iridium(III) complexes containing an acetylacetonate anion (acac) ligand, with several exhibiting much greater ECL intensities than [Ru(bpy) 3 ] 2+ (with tri-n-propylamine (TPrA) co-reactant in acetonitrile solution), although the relative intensities were highly dependent on reaction conditions (Chen et al., 2017).
Nevertheless, very few of the iridium(III) complexes examined as ECL luminophores to date are soluble in the aqueous conditions in which most ECL assays are performed (Fernandez-Hernandez et al., 2016;Zhou et al., 2017). As previously reported, the solubility can be improved by incorporating polar functional groups such as sulfonates (Kiran et al., 2009;Jia et al., 2012) or saccharides (Li et al., 2011a,b) on one or more ligands of the complex. Li et al. (2011b), for example, reported intense ECL from a water-soluble bis-cyclometalated iridium(III) complex incorporating a bpy ligand appended with two sugar moieties. Similarly, we utilized bathophenanthroline-disulfonate (BPS) as an ancillary ligand to increase the solubility of the complexes in aqueous solution (Kiran et al., 2009;Zammit et al., 2011;Truong et al., 2014). In most cases, however, the dissolution of the complexes at relatively high concentrations often required the addition of some acetonitrile to the aqueous solution, and these approaches do not provide a convenient means to incorporate the luminophores into ECL labels. We recently examined the ECL of several water soluble [Ir(C ∧ N) 2 (pt)]Cl complexes (where C ∧ N = 2-phenylpyridine anion (ppy) or 2-(2,4-difluorophenyl)pyridine anion (df-ppy), and pt = 4-(2-pyridyl)-1,2,3-triazole) with either a tetraethylene glycol (TEG) or benzyl group attached to the triazole and/or methanesulfonate substituents on the ppy/df-ppy ligands (Doeven et al., 2015a;Kerr et al., 2015). Although the TEG and methanesulfonate groups improved the solubility of the complexes in water, the complexes with the pt-TEG ligand ( Figure 1A) gave greater co-reactant ECL intensities with TPrA and provide a convenient point of attachment of functional groups for bioconjugation Chen et al., 2019) for the development of iridium(III) complex ECL labels.
Herein, we prepare four novel [Ir(C ∧ N) 2 (N ∧ N)]Cl complexes ( Figure 1B), where N ∧ N is bpy with either one or two TEG groups attached in the 4 and 4 ′ positions (referred to hereafter as Me-bpy-TEG and TEG-bpy-TEG). Through the introduction of the TEG group(s) onto the commonly used bpy ligand, iridium(III) complexes previously studied in organic solvents can be examined in buffered aqueous solution. We incorporate the Me-bpy-TEG and TEG-bpy-TEG ligands into heteroleptic iridium(III) complexes with ppy or 2-phenylbenzo[d]thiazole anion (bt) ligands by reacting the bipyridine derivatives with commercially available ([Ir(C ∧ N) 2 (µ-Cl)] 2 dimers. We evaluate the influence of the TEG group(s) on the parent luminophore by comparing their spectroscopic and electrochemical properties with the corresponding [Ir(C ∧ N) 2 (Me-bpy-Me)] + complexes, and compare their co-reactant ECL intensities to the analogous water-soluble [Ir(C ∧ N) 2 (pt-TEG)] + complexes and [Ru(bpy) 3 ] 2+ .

General Method for Synthesis of Iridium Complexes
The chloro-bridged iridium dimer and almost two molar equivalents of ligand were added to a flask. A solvent mixture of dichloromethane and methanol (1:1 v/v) was added, and the mixture was sparged with N 2 for 20 min then sealed, shielded from light and heated at 50 • C for 20 h. Any solid that remained in the reaction mixture was removed by centrifuge and the supernatant was filtered through filter aid (Celite). The solvent was removed under reduced pressure, the residue was taken up in a minimum of dichloromethane and a precipitate was formed after addition of diethyl ether. The precipitate was isolated by centrifuge and washed with diethyl ether (× 3) then dried in vacuo.

Absorbance and Emission Spectra
Absorbance spectra were obtained using a Cary 300 Bio UV/Vis spectrophotometer (Agilent, USA). Conditions: Double beam mode; 2 nm SBW; 1 nm data interval; 600 nm/min scan rate; 0.1 s averaging time. Quartz cuvettes with a path length of 1 cm were used for all measurements. All room temperature photoluminescence spectra were collected using a Cary Eclipse spectrofluorometer. Conditions: 5 nm band pass; 1 nm data interval; 600 nm/min scan rate; 0.1 s averaging time; 250-395 nm excitation filter; 360-1,100 nm emission filter; PMT: 600 V; final spectrum is an average of 10 scans for [Ir(bt) 2 (Me-bpy-TEG)] + and [Ir(bt) 2 (TEG-bpy-TEG)] + , and 50 for [Ir(ppy) 2 (Me-bpy-TEG)] + and [Ir(ppy) 2 (TEG-bpy-TEG)] + (CAT mode). Quartz cuvettes with a path length of 1 cm were used for all measurements. Low temperature spectra were obtained using an OptistatDN Variable Temperature Liquid Nitrogen Cryostat (Oxford Instruments) with custom-made quartz sample holder, placed within the Eclipse sample chamber. Low temperature spectra were collected at 85 K to avoid damage to the spectroscopic cuvettes near 77 K. No difference was observed in the λ max at 77 K and 85 K for complexes such as [Ru(bpy) 3 ] 2+ under these conditions (Soulsby et al., 2018).
In both the low temperature and room temperature data, there is a wavelength dependence of the detector response. To account for this, a correction factor (established using a quartzhalogen tungsten lamp of standard spectral irradiance) was applied to both room temperature and low temperature emission spectra. All room temperature experiments were performed with deionized water or acetonitrile, and all low temperature experiments performed in an ethanol:methanol (4:1) glass.

Electrochemistry and ECL
An Autolab PGSTAT204 or PGSTAT128N potentiostat (Metrohm Autolab B.V., Netherlands) was used to perform cyclic voltammetry, squarewave voltammetry (0.005 V step, 0.02 V amplitude, 25 Hz), and chronoamperometry (CA). The system comprised of a flat-bottomed glass electrochemical cell with a Teflon custom-built lid designed for a three-electrode system. The electrodes were a glassy carbon working (CH instruments), Pt wire counter, and either a "leakless" Ag/AgCl reference (Innovative Instruments, FL, USA) or Ag wire pseudoreference. This configuration positioned the working electrode 2 mm from the bottom of the cell. Experiments were conducted with the electrochemical cell housed in a Faraday cage. All Experiments performed in acetonitrile were referenced to Fc +/0 in situ (at equimolar concentration to the analyte). The working electrode was polished on a felt pad with 0.05 µm alumina powder prior to use. A small blowtorch was used to polish the platinum electrode prior to use. All solutions were prepared in either deionized water with a 0.1 M phosphate buffer adjusted to pH 7.5, or dry acetonitrile with 0.1 M TBAPF 6 electrolyte, and were deaerated with nitrogen gas for 5 min. ECL spectra were collected using a QE65pro Ocean Optics CCD via optical fiber and collimating lens positioned below the base of the electrochemical cell. Each acquisition was triggered by the potentiostat in conjunction with a HR4000 Break-Out box. Relative ECL intensities were averages of two replicates using the integrated areas under the spectra.

Synthesis of Iridium(III) Complexes
To prepare the complexes shown in Figure 1B, the chlorobridged iridium(III) dimers ([Ir(C ∧ N) 2 (µ-Cl)] 2 , where C ∧ N = ppy or bt) were initially reacted with bpy derivatives furnished with either one or two TEG groups (Me-bpy-TEG (L 1 ) and TEGbpy-TEG (L 2 )). The bipyridine ligands were prepared using TEG mono-protected with a trityl group in excellent yield (91%) and converted to the corresponding tosyl ester (91%) to afford a suitable leaving group to react with hydroxyl methyl bipyridine derivatives (Scheme 1).
Trifluoroacetic acid was used to deprotect L 1 Trt followed by acid/base extraction to give L 1 , but L 2 proved difficult to isolate by this method, presumably due to the water solubility of the product. Therefore, L 2 Trt 2 was used directly to form iridium(III) dimers and subsequent removal trityl groups, in methanolic hydrochloric acid, gave [Ir(C ∧ N) 2 (TEG-bpy-TEG)]Cl in good to excellent yields. This strategy proved beneficial as the presence of the trityl group allowed the complex to be isolated via traditional silica gel chromatography, and then the desired water solubility could be introduced as the final step. Precipitation of complexes from dichloromethane occurred upon addition of diethyl ether allowing isolation by centrifugation. The [Ir(C ∧ N) 2 (Me-bpy-TEG)]Cl and [Ir(C ∧ N) 2 (TEG-bpy-TEG)]Cl complexes were sufficiently soluble for the preparation of aqueous stock solutions at 1 mM.

UV-Vis Absorption and Photoluminescence Spectra
UV-vis absorption spectra of the four novel iridium(III) complexes and [Ru(bpy) 3 ] 2+ were examined at 10 µM in water (e.g., Figure 2) and the peak maxima were compared to the [Ir(C ∧ N) 2 (Me-bpy-Me)] + analogs at the same concentration SCHEME 1 | Synthesis of trityl protected bipyridine ligands (L 1 Trt and L 2 Trt 2 ).
The photoluminescence spectra of the two [Ir(C ∧ N) 2 (TEGbpy-TEG)] + complexes (where C ∧ N is bt or ppy) were examined in 4:1 (v/v) ethanol:methanol at 85 K ( Figure 5). Lowtemperature spectra generally show greater detail of vibrational energy levels, and allow for a more accurate estimation of the energy gap (E 00 ) between the lowest vibrational levels of the ground and lowest excited state (Jones and Fox, 1994). The low temperature spectrum for [Ir(bt) 2 (TEG-bpy-TEG)] + is highly structured (Figure 5), even more than at room temperature (Figure 3), and was in close agreement with that of [Ir(bt) 2 (Me-bpy-Me)] + ( Table 2). The highest energy peak at 517 nm corresponds to an E 00 energy of 2.4 eV. The broad emission spectrum produced by [Ir(ppy) 2 (TEG-bpy-TEG)] + at low temperature is unusual for an iridium(III) complex, and the highest energy band at 471 nm is well over 100 nm blue-shifted FIGURE 5 | Normalized photoluminescence emission spectra obtained for [Ir(bt) 2 (TEG-bpy-TEG)] + (gray line), and [Ir(ppy) 2 (TEG-bpy-TEG)] + (orange line), at 5 µM in 4:1 (v/v) ethanol:methanol at low-temperature (85 K). Spectra were corrected for the change in instrument sensitivity across the wavelength range with a correction factor established using a light source with standard spectral irradiance.
from that of the room-temperature spectrum. The analogous hypsochromic shift of the closely related [Ir(ppy) 2 (Me-bpy-Me)] + complex has been ascribed to not only the rigidochromic phenomena typically observed with 3 MLCT emissions, but also contribution from higher energy transitions due to an unusually high barrier for relaxation to the 3 MLCT (bpy) (King and Watts, 1987;Wu et al., 2010;Yen et al., 2016;Connell et al., 2019). Although this complicates the approximation of E 00 for [Ir(ppy) 2 (TEG-bpy-TEG)] + , comparison with the interpretation of spectra of related complexes (Connell et al., 2019) enables an estimation at 2.4 eV.

Voltammetry
Cyclic voltammetry (CV) experiments were initially conducted using buffered aqueous solutions to mimic the analytical conditions for which they were designed (Figure 6, black lines). The oxidation of the four novel complexes appears irreversible in an aqueous environment. The shape of the cyclic voltammograms made assigning peak potentials difficult, so squarewave voltammetry (Figure 6, orange lines) was also conducted to inform the positions of the oxidation peaks ( Table 2).
The mechanism of co-reactant ECL depends on both the oxidation and reduction of the metal complex, so it is important that both are characterized. The reduction of the complexes, however, is obscured in voltammetric experiments due to the reduction of solvent, so these potentials were determined in acetonitrile and referenced to the ferrocenium/ferrocene couple (Figure 7). This internal electrochemical reference is more reliable that the reference electrode potential and provides a more accurate comparison to the previously reported potentials of related iridium complexes that were not sufficiently soluble in an aqueous buffer ( Table 2). The additional oxidation peak at ∼0.6 V (vs. Fc +/0 ) in the traces in Figure 7 arises from the chloride counter ion of these complexes. This peak could be removed by converting the compounds to their hexafluorophosphate salts, but this was deemed unnecessary.
The values obtained for the three [Ir(bt) 2 (N ∧ N)] + complexes were very similar (oxidation potentials within 30 mV and reduction potentials within 60 mV). Those obtained for the three  [Ir(ppy) 2 (N ∧ N)] + complexes were also consistent (oxidation potentials within 10 mV and reduction potentials within 70 mV). The similarity of these potentials indicates that the presence of TEG moieties on the bpy ligand has very little influence on the electrochemical properties of the complex.

Electrogenerated Chemiluminescence
The ECL intensities of the [Ir(C ∧ N) 2 (N ∧ N)] + complexes containing a Me-bpy-TEG or TEG-bpy-TEG ligand in buffered aqueous solution using TPrA as a co-reactant were compared those of the analogous complexes with a pt-TEG ligand. To remove the bias in sensitivity of typical photomultiplier tubes toward the hypsochromic emissions of these complexes, we used the integrated area of ECL spectra collected using a CCD spectrometer for these comparisons. Figure 8 shows the ECL intensities relative to that of the [Ru(bpy) 3 ] 2+ complex under the same conditions. The co-reactant ECL intensities of iridium(III) complexes relative to [Ru(bpy) 3 ] 2+ can be highly dependent on instrumental and chemical conditions (Chen et al., 2017). Using an applied potential pulse at [E ox p + 0.1 V] for 0.1 s, the co-reactant ECL intensities of most of the [Ir(C ∧ N) 2 (N ∧ N)] + complexes were greater than that of [Ru(bpy) 3 ] 2+ (Figure 8A), but when the pulse time was increased to 0.5 s ( Figure 8B) the intensities were below that of [Ru(bpy) 3 ] 2+ . Nevertheless, the trend in intensities between the [Ir(C ∧ N) 2 (N ∧ N)] + complexes was similar at the two pulse times. The intensities of the [Ir(C ∧ N) 2 (Me-bpy-TEG)] + complexes were between 1.2-and 2.2-fold those of the [Ir(C ∧ N) 2 (TEG-bpy-TEG)] + .
TPrA − e − → TPrA •+ (1) If the metal complex is also oxidized (reaction 3, where M = [Ru(bpy) 3 ] 2+ or [Ir(C ∧ N) 2 (N ∧ N)] + ), subsequent reaction with the α-aminoalkyl radical may generate ECL (reactions 4 and 5) if there is sufficiently energy to attain the excited state, which can be estimated using: , the co-reactant can also be oxidized via the 'catalytic' route shown in reaction 6.
M − e − → M + M + + TPrA • → M * + otherproducts (4) Based on the data presented above, reactions 1-8 are feasible for these iridium(III) complexes. An alternative pathway to the excited state, important for bead-based assays in which the majority of the metal complex luminophores cannot be electrochemically oxidized (Miao et al., 2002;Chen et al., 2019), involves reactions 1, 2, 7, and 9, where the reduced metal complex reacts with the aminium radical cation. This pathway requires not only Despite the similarity of their E • (M/M − ), the iridium(III) complexes exhibit higher excited state energies than [Ru(bpy) 3 ] 2+ . Considering the requirements noted above, the data presented in Tables 1, 2 suggest that reaction 9 will be slightly energy insufficient (∼0.1-0.2 V), limiting the application of these electrochemiluminophores to assays in which the metal complex can be oxidized. These calculations, however, involve considerable error, including those associated with estimating E • (TPrA), E • (TPrA • ) (Miao et al., 2002) and E 00 (M) (Jones and Fox, 1994;Connell et al., 2019), and the use of potentials measured in acetonitrile for a ECL reaction in aqueous solution. Further investigations are needed to confirm the ECL pathways for these novel luminophores. The ECL spectra of [Ir(C ∧ N) 2 (Me-bpy-TEG)] + and [Ir(C ∧ N) 2 (TEG-bpy-TEG)] + (Figure 9) were in good agreement with their photoluminescence emission spectra (Figure 3), taking into account the lower resolution of the CCD spectrometer used to collect the ECL spectra. The spectral distribution of [Ir(bt) 2 (pt-TEG)] + is similar to that of [Ir(bt) 2 (Me-bpy-TEG)] + and [Ir(bt) 2 (TEG-bpy-TEG)] + , but emission of [Ir(ppy) 2 (pt-TEG)] + is considerably blue-shifted from [Ir(ppy) 2 (Me-bpy-TEG)] + and [Ir(ppy) 2 (TEG-bpy-TEG)] + (Figure 9). A practical outcome of this shift is that much greater ECL intensities will be measured with [Ir(ppy) 2 (pt-TEG)] + than with [Ir(ppy) 2 (Me-bpy-TEG)] + or [Ir(ppy) 2 (TEG-bpy-TEG)] + when using photomultiplier tubes that are much more sensitive toward shorter wavelengths of light within the visible region. On the other hand, as both the Me-bpy-TEG and pt-TEG ligands can be readily adapted for bioconjugation, this shows a viable strategy to create two ECL-labels with distinctly different emission colors from the same commercial [Ir(ppy) 2 (µ-Cl)] 2 dimer that provide similar ECL intensities using a CCD spectrometer to distinguish their emissions. The novel [Ir(bt) 2 (TEG-bpy-TEG)] + and [Ir(ppy) 2 (TEG-bpy-TEG)] + complexes also exhibit distinct spectral distributions. As noted in previous studies (Doeven et al., 2012;Cao et al., 2020), the number of luminophores in multi-color ECL may be limited by the broad emission spectra of the metal complexes. The difference in their oxidation potentials (Table 1), however, may also enable "potential-resolved" ECL, which has recently been exploited to expand the scope of multi-color systems (Doeven et al., 2015b;Guo et al., 2018;Moghaddam et al., 2019;Bouffier and Sojic, 2020).

CONCLUSIONS
Four [Ir(C ∧ N) 2 (N ∧ N)]Cl complexes in which C ∧ N = ppy or bt, and N ∧ N = bpy with either one or two TEG groups attached in the 4 and 4 ′ positions, were successfully synthesized with acceptable yields for all reaction steps. Characterization of the complexes showed that the introduction of one or two the TEG groups to the bpy ligand of iridium(III) complexes is a useful strategy to enhance their solubility in aqueous solution while retaining the electrochemical and spectroscopic properties of the parent luminophore. The TEG groups also provide a convenient attachment point for the future development of ECL labels.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
PF and DH conceived and designed the study. BN synthesized the compounds under the guidance of DH and LH. LC and BN performed the spectroscopic and electrochemical characterizations under the guidance of ED and PF. All authors contributed to manuscript preparation and revision and have read and approved the submitted version.

FUNDING
This work was funded by the Australian Research Council (DP160103046). LH and DH thank the ARC Training Center for Lightweight Automotive Structures (IC160100032) and the ARC Research Hub for Future Fibers (IH140100018) funded by the Australian Government. LC was supported by a Deakin University International Postgraduate Scholarship.