A ratiometric substrate for rapid evaluation of transfer hydrogenation efficiency in solution

Abstract

A cyclometalated iridium(III) complex bearing a self-immolative quinolinium moiety was developed as a ratiometric substrate for transfer hydrogenation studies. This photoluminescent probe allowed the rapid screening of a variety of Ir catalysts using a microplate reader, offering a convenient method to assess activity using a minimum amount of catalyst sample.

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Transition metal catalysts capable of promoting transfer hydrogenation (TH), which are reactions involving the exchange of H⁻ equivalents between hydride donors and acceptors, have numerous applications.^1–3 For example, they are used in the stereoselective reduction of C=O and C=N groups in chemical synthesis^4,5 and the conversion of dioxygen to hydrogen peroxide in small-molecule activation⁶ (Scheme 1A). Researchers have shown that TH catalysts can also be applied in living systems, including as redox modulators for anti-cancer therapy,^7,8 activity-based platforms for sensing endogenous formate,⁹ detoxification agents against harmful aldehydes,¹⁰ and cofactors in artificial metalloenzymes.^11,12 Although intracellular catalysts^13–15 are commonly derivatized with various ligand substituents or targeting groups,^11,16,17 such modifications risk negatively impacting their catalytic performance. Thus, having a fast and reliable method to evaluate TH catalysts could accelerate their discovery and development.

Scheme 1 Various applications of transfer hydrogenation (TH) catalysts (A) and off–on (B) and ratiometric (C) substrates used to study their activity.

Herein, we introduce a user-friendly photoluminescence-based method to screen for TH catalyst activity using a microplate reader. Our strategy relies on the use of a ratiometric substrate that allows in situ quantification of the reaction progress.^18,19 This approach enables simultaneous monitoring of multiple reactions using minimum amounts of sample, which is particularly attractive for studies of catalysts that are costly (e.g., those containing Ir,²⁰ Ru,²¹ or Os²²) or difficult to obtain on large scales.

The design of our ratiometric probe was inspired by a self-immolative umbelliferone-quinolinium substrate (UmQN) developed by Ward and coworkers (Scheme 1B).^11,12 It was shown that when this compound was reduced via TH catalysis, release of umbelliferone (Um) led to an emission turn-on. Although this off–on substrate was used to evaluate TH reactions in the periplasm of E. coli, the total amount of starting substrates inside the cells could not be quantified by fluorescence microscopy because they are non-emissive. To further complicate matters, small molecules such as substrates and molecular catalysts can diffuse in and out of cells so obtaining accurate yields in living systems is highly challenging. To overcome these limitations, we sought to develop a fluorophore-quinolinium construct that would exhibit different emission properties in the starting and product forms so that the reaction yields could be determined by monitoring the emission intensity ratio at two different wavelengths. Such ratiometric substrates may allow non-invasive studies of intracellular TH reactions in real time without having to lyse the cells and analyse their contents by methods such as high performance liquid chromatography. After considering a variety of fluorophore candidates, we selected to use a cyclometalated iridium(III) complex as the reporter due to its tunable emission, chemical stability, and ease of preparation (Scheme 1C).^23–28 In addition, its phosphorescence may be more easily distinguished from biological self-fluorescence.²⁹

The synthesis of our ratiometric iridium complex (RIC₅₇₅, where the subscript denotes its emission maximum) proceeded starting from 2,6-dimethylquinoline (Scheme S1†). Monobromination using N-bromosuccinimide (NBS) afforded compound 1 (68% yield), which was then subjected to nucleophilic attack by 2-bromo-4-hydroxypyridine to furnish 2 in 73% yield. Combining 2 in the presence of 2-(tributylstannyl)pyridine under Stille cross-coupling conditions gave the bipyridine species 3 (62% yield). Metalation of 3 was accomplished by treatment with [Ir(ppy)₂Cl]₂ (ppy = 2-phenylpyridine), anion exchange using AgOTf (OTf⁻ = triflate anion), and then alkylation using methyl triflate to afford RIC₅₇₅ in 38% yield (based on 3, Scheme 2A).

Scheme 2 Synthesis of RIC₅₇₅ (A) and RIC₅₂₅ (B) starting from their corresponding bipyridine ligands. The molecular structures of RIC₅₇₅ and RIC₅₂₅ are depicted with 50% thermal ellipsoids based on their respective X-ray crystallographic data (orange = iridium, blue = nitrogen, red = oxygen atoms). The two OTf⁻ anions in RIC₅₇₅ were omitted for clarity. In part C, the Ir catalysts used for TH studies are shown.

The cleaved cyclometalated iridium species RIC₅₂₅ (where the subscript denotes its emission maximum) was also prepared (Scheme S2†). The starting 2-bromo-4-hydroxypyridine was protected using 2-methoxyethoxymethyl (MEM) chloride to give 5, followed by Stille cross-coupling with 2-(tributylstannyl)pyridine to furnish bipyridine 6. Combining 6 with [Ir(ppy)₂Cl]₂ and then treatment with HCl gave the corresponding anionic iridium complex (RIC_525′). To obtain the neutral form, it was stirred in the presence of NaOH, followed by the addition of AgOTf to afford RIC₅₂₅ with a yield of 79% based on the amount of [Ir(ppy)₂Cl]₂ used in the previous step.

To obtain structural characterization, X-ray crystallography was used to analyse single crystals of RIC₅₇₅ and RIC₅₂₅. The structure of RIC₅₇₅ showed the expected octahedral iridium center with a quinolinium moiety attached to the bipyridine ligand (Fig. S62†). The presence of two triflate anions supports the dicationic nature of the complex. The structure of RIC₅₂₅ also reveals an octahedral Ir geometry but the absence of any counterions in the crystallographic unit cell indicates that this species is charge neutral overall (Fig. S63†). Comparison of the bond metrics in RIC₅₇₅vs.RIC₅₂₅ suggests that the former contains a 4-alkoxypyridyl donor whereas the latter contains a 4-pyridonate donor coordinated to iridium (Fig. 1A).^30–32 For example, in RIC₅₇₅, the C–C and C–N bond distances of ∼1.33–1.39 Å in the functionalised pyridine ring are typical of an aromatic structure (Fig. 1A).³³ Additionally, its C(3)–O(1) bond length of 1.35 Å is expected for a carbon–oxygen single bond (average = ∼1.37 Å). In contrast, the corresponding six-membered ring in RIC₅₂₅ exhibits contracted or elongated C–C/C–N bonds relative to those in RIC₅₇₅, suggesting that it is non-aromatic. The only exception is that the C(4)–C(5) bond distance in RIC₅₂₅ is similar to that in RIC₅₇₅ (∼1.37 Å), which may be due to the conjugation of its C4 and C5 p-orbitals with the π-system of the adjacent pyridine ring. Lastly, the C(3)–O(1) bond distance of 1.28 Å in RIC₅₂₅ is consistent with a C=O carbonyl group.³³

Fig. 1 Comparison of key bond distances (Å) between RIC₅₇₅ and RIC₅₂₅ (A) and their corresponding photophysical properties (B). The emission spectra were recorded in anhydrous MeOH at 20 °C under N₂ with excitation at 350 nm.

Due to the electronic differences in their ancillary ligands, RIC₅₇₅ and RIC₅₂₅ were expected to display different photophysical properties.²³ As shown in Fig. 1B, when RIC₅₇₅ was excited with 350 nm light in MeOH under N₂, a broad emission peak was observed with a maximum at 575 nm and shoulders at 490 and 520 nm. Our data indicate that the non-radiative decay rate (k_nr) was about 160-fold faster than the radiative decay rate (k_r), presumably due to the emission quenching effects of its quinolinium moiety.³⁴ For comparison, the iridium precursor 4 bearing an unmethylated quinoline ring exhibited a k_nr that is only 1.9-fold faster than k_r (Table S4†). When RIC₅₂₅ was irradiated with 350 nm light, its emission spectrum showed a peak maximum at 525 nm. This hypsochromic shift, relative to the λ_em of RIC₅₇₅, is consistent with the more electron-rich nature of the RIC₅₂₅ bipyridine ligand, which destabilizes the LUMO energy and consequently, increases the Ir complex's HOMO–LUMO gap. Our results also indicate that RIC₅₂₅ has a higher quantum yield (Φ_PL = 0.438 vs. 0.006) and longer luminescence lifetime (τ = 1.26 vs. 1.03 μs) than that of RIC₅₇₅ (Fig. 1B). A chromaticity analysis shows that the photoluminescence of RIC₅₇₅ appears yellow whereas that of RIC₅₂₅ appears green (Fig. S4†). Because RIC₅₇₅ and RIC₅₂₅ have poor solubility in water, we were unable to perform photophysical measurements in aqueous solutions such as biological buffers or cell culture media.

Prior to conducting reaction studies with RIC₅₇₅, we created a calibration curve using standard solutions containing different ratios of RIC₅₇₅/RIC₅₂₅ (Table S6†). Sodium bicarbonate was also added to the mixtures to ensure that RIC₅₂₅ remains in the neutral form. We found that measuring the emission intensities at 550 (I₅₅₀) and 600 (I₆₀₀) nm and plotting the I₅₅₀/I₆₀₀ ratio gave a linear correlation with the amount of RIC₅₂₅ present (Fig. S6†).¹⁹ Due to the greater than 70-fold difference in quantum yield between RIC₅₂₅ and RIC₅₇₅, the choice of 550 and 600 nm for reaction monitoring (rather than at their emission maxima of 525 and 575 nm, respectively) ensures that signals from the product do not overwhelm signals from the starting material for accurate quantification.

Next, we proceeded to study the TH activity of a series of previously reported [Cp*Ir(4-R-picolinamidate)Cl] complexes (where Cp* = pentamethylcyclopentadienyl anion, R = various functional groups; Scheme 2C).^11,35–37 To establish that RIC₅₇₅ is a viable substrate for TH,^11,12 a small-scale reaction was performed using a 1-dram vial (Fig. S61A†). When Ir4 (R = NMe₂), RIC₅₇₅, HCOONa, and NaHCO₃ were dissolved in 1 mL of MeOH and stirred at RT, the reaction mixture showed increasing luminescence over the course of 1 h with a change in color from yellow to green (Fig. S61B†), suggesting that RIC₅₂₅ had formed. The presence of the RIC₅₂₅ product was further confirmed by emission spectroscopy and mass spectrometry. Reduction of the quinolinium moiety was supported by showing that a model substrate, 1,2,6-trimethylquinolinium triflate, was converted to 1,2,3,4-tetrahydro-1,2,6-trimethylquinoline in the presence of Ir4, HCOONa, and NaHCO₃ (Fig. S59†). These results indicate that the conversion of RIC₅₇₅ to RIC₅₂₅ by the Ir catalysts and HCOONa occurs on a timescale that is suitable for quantification using a microplate reader.

Once we identified the optimal reaction conditions for TH, we carried out our catalyst screening experiments in 24-well microplates (Fig. 2A). In these investigations, each well was charged with an Ir catalyst (2.0 μM), HCOONa (0.7 mM), and NaHCO₃ (1.25 mM) in MeOH at 28 °C (Fig. 2A). The mixtures were injected with RIC₅₇₅ (0.2 mM) and then the microplate was inserted into a microplate reader for continuous emission monitoring at 550 and 600 nm. By converting the I₅₅₀/I₆₀₀ ratio to the %yield of RIC₅₂₅ (see Table S7† for example with Ir1), kinetic plots of the reactions were generated (Fig. 2B, left). The kinetic data showed that in all cases, an induction period of ∼10 min was observed, which may be due to catalyst activation (e.g., formation of the initial iridium-hydride species).³⁶ Based on the RIC₅₂₅ yields after 1 h, the catalyst activity trend follows the order: Ir4 (R = NMe₂) > Ir3 (R = pyrrolidinyl) > Ir2 (R = OMe) > Ir1 (R = H) > Ir5 (R = CF₃) > Ir6 (R = CN). The poor solubility of Ir7, which bears a nitro group on the pyridyl ring, precluded studies using this complex. The trend observed that electron-rich Ir catalysts are more active than their electron-poor counterparts is fully consistent with previous studies performed in aqueous media.^35,36 It has been demonstrated that hydride formation is typically rate-limiting in TH reactions by [Cp*Ir(4-R-picolinamidate)Cl] catalysts and that this step occurs faster with electron-rich than electron-poor catalysts.³⁶ Although a more in-depth analysis of the kinetic data was not pursued in this work, these results validate our ratiometric substrate-based approach for rapid screening of TH catalysts. Because each reaction in our studies requires only 20 nmol (<∼90 μg) of Ir catalyst, a significant amount of material could be saved compared to performing larger scale reactions in standard vials or flasks.

Fig. 2 (A) Scheme depicting our TH reaction studies using 24-well microplates. The photo shows a portion of the microplate after the reaction proceeded for 30 min (excitation = 325 nm light). (B) Kinetic plots of TH reactions using RIC₅₇₅ with catalysts Ir1–Ir6 (left) and Ir4/Ir8–Ir10 (right) acquired using a microplate reader.

To illustrate the utility of our method, we used our protocol to evaluate the performance of several new TH catalysts. When developing a catalyst for in cell or in vivo applications, it is often desirable to customize its biological properties, such as cellular uptake, interactions with biomolecules, subcellular localization, etc., without diminishing its catalytic activity.¹³ Because Ir4 was the most active in our series (Fig. 2B, left), we chose to derivatize this complex to determine whether the catalyst structure and activity could be tuned independently of each other. By adapting previously reported procedures (see ESI†), we successfully prepared three iridium variants featuring tert-butoxyphenyl (Ir8), triethylene glycol phenyl (Ir9), and 1,3-benzodioxole (Ir10) groups on the picolinamidate ligand (Scheme 2C). These substituents were chosen to represent groups with varying shapes and sizes. The new Ir catalysts were tested in TH reactions by combining them with HCOONa, NaHCO₃, RIC₅₇₅, and MeOH in 24-well microplates. Analysis of the photoluminescence data acquired by the microplate reader produced the kinetic traces shown in Fig. 2B (right). The results revealed that Ir8–Ir10 exhibit nearly identical kinetic profiles as that of their parent catalyst Ir4. This finding corroborates our previous observations that for [Cp*Ir(4-R-picolinamidate)Cl] complexes ligand modifications at the N-phenyl moiety do not impact their catalyst activity.³⁶

In summary, we have developed a self-immolative substrate for photoluminescence monitoring of TH catalysts using a microplate reader. Because the emission maximum of the cleaved product RIC₅₂₅ is blue-shifted relative to that of the starting RIC₅₇₅, ratiometric quantification of the reaction yields was possible by measuring the intensity changes at two different wavelengths. Application of our method to studies of several new [Cp*Ir(4-R-picolinamidate)Cl] variants revealed that functionalization of the N-phenyl group does not negatively impact their catalytic activity. Our photoluminescence-based method is user-friendly because it is rapid, requires minimal catalyst sample, and can be readily used by researchers with access to microplate readers, which are available in many synthetic and biochemistry laboratories.

There are numerous non-aqueous applications that may benefit from this new molecular tool, such as in high throughput automation for chemical synthesis or metallodrug discovery.^38,39 To enable TH reaction imaging in biological cells,⁴⁰ the RIC₅₇₅ probe could be rendered more water soluble by functionalizing it with sulfonate groups; however, how this change may affect its cell permeability remains to be investigated.⁴¹ Since different cellular compartments have different pH values, modifying the probe so that its emission properties are not sensitive to pH changes could broaden its biological range. Although in-cell applications were not achieved in this work, it provides proof-of-concept demonstration that cyclometalated Ir-quinolinium substrates are useful for ratiometric quantification of TH reactions in solution.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors thank the Welch Foundation (grant no. E-1894 to L. H. D.) and the National Institutes of Health (grant no. R01GM129276 to L. H. D.) for funding this work. We are grateful to Prof. Tom Teets for helpful suggestions and allowing us to use their fluorimeter.

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Footnote

† Electronic supplementary information (ESI) available: Experimental details, characterization data, photophysical studies, and reaction data. CCDC 2336124 and 2336125. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d4dt00891j

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