pubs.acs.org/JACS Article Fast Photoswitchable Molecular Prosthetics Control Neuronal Activity in the Cochlea Aida Garrido-Charles,△ Antoine Huet,△ Carlo Matera,△ Anupriya Thirumalai, Jordi Hernando, Amadeu Llebaria, Tobias Moser,* and Pau Gorostiza* Cite This: J. Am. Chem. Soc. 2022, 144, 9229−9239 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. ABSTRACT: Artificial control of neuronal activity enables the study of neural circuits and restoration of neural functions. Direct, rapid, and sustained photocontrol of intact neurons could overcome the limitations Downloaded via 158.109.42.72 on September 29, 2022 at 07:34:01 (UTC). of established electrical stimulation such as poor selectivity. We have developed fast photoswitchable ligands of glutamate receptors (GluRs) to enable neuronal control in the auditory system. The new photoswitchable ligands induced photocurrents in untransfected neurons upon covalently tethering to endogenous GluRs and activating them reversibly with visible light pulses of a few milliseconds. As a proof of concept of these molecular prostheses, we applied them to the ultrafast synapses of auditory neurons of the cochlea that encode sound and provide auditory input to the brain. This drug-based method afforded the optical stimulation of auditory neurons of adult gerbils at hundreds of hertz without genetic manipulation that would be required for their optogenetic control. This indicates that the new photoswitchable ligands are also applicable to the spatiotemporal control of fast spiking interneurons in the brain. ■ INTRODUCTION Ultrafast signaling is a feature of several important neural CIs. Moreover, electrical SGN stimulation results in super- natural synchronization of neural activity and hence CIs circuits such as those in the auditory pathway, the brainstem, employ high rates to generate pseudostochasticity of SGN the cerebellum, and the cerebrum.1−3 Such signaling builds on firing. Recently, optogenetics has been proposed for improved specialized synapses for synchronous neurotransmission as well bionic SGN stimulation, as light can be better confined in as on suitable neural membrane properties for speedy action space. Rapidly gating channelrhodopsins enable optogeneti- potential generation and propagation. The resulting neural cally evoked firing with near-physiological temporal fidelity. firing features high rates and submillisecond precision. For an Yet, genetic manipulation for expression of channelrhodopsins example, synaptic sound encoding builds on ultrafast presents a risk to consider. Avoiding the need for gene therapy, photopharmacology could help reduce the complexity of glutamatergic transmission at specialized ribbon synapses optical SGN stimulation. Reversibility of chemical photo- formed by inner hair cells (IHCs) achieving firing at rates of switches9 makes them interesting candidates for controlling several hundreds of hertz with submillisecond precision in neural excitation via endogenous neuronal receptors, such as spiral ganglion neurons (SGNs).4,5 Utmost precision of the ionotropic glutamate receptors (GluRs) of the postsynaptic neural time code of the incoming sound forms the basis of SGN boutons in the cochlea. sound localization in dedicated neural circuits of the brainstem Photoswitchable tethered ligands (PTLs) are covalently that feature powerful calyceal synapses and neurons with attached to their receptor and seem particularly attractive for extremely short membrane time constants owing to their this purpose. If conjugated precisely, they can provide finer specialized set of ion channels.1,2 photocontrol than freely diffusible photochromic ligands. PTLs Dissecting the function of such time-critical neural circuitries produce higher local concentrations10 and cannot diffuse away, requires ultrafast control of neuronal activity. Likewise, functional restoration, for example, following degeneration of the sensory receptor cells, needs bionic approaches that Received: November 22, 2021 reinstate the physiological information processing as closely as Published: May 18, 2022 possible. Restoration of hearing to the deaf currently employs electrical stimulation of SGNs by cochlear implants (CIs).6−8 Due to wide spread of current from each electrode contact, encoding of sound frequency information is heavily limited in © 2022 The Authors. Published by American Chemical Society https://doi.org/10.1021/jacs.1c12314 9229 J. Am. Chem. Soc. 2022, 144, 9229−9239 Journal of the American Chemical Society pubs.acs.org/JACS Article Figure 1. Fast-relaxing targeted covalent photoswitch of endogenous GluRs. (A) Schematic representation of the click chemistry strategy adopted for the preparation of TCP ligands. Modular parts are the ligand (red), the photoswitch (blue), and the reactive anchoring group (green). (B) Molecular design showing “head” (1) and “tail” (2) precursors of TCPfast (3) that are freshly coupled via a copper(I)-catalyzed azide−alkyne cycloaddition prior to incubation in neuronal tissue. (C) Chemical structure and photoisomerization of TCPfast between the cis (blue light, λ = 460 nm) and trans (dark, fast relaxation) conformations. which yields a sharper separation in biological activity between regulatory proteins) for extended periods of time, offering the two isomeric states.11,12 Genetic manipulation can be untapped potential for functional restoration.6 avoided with affinity-labeling conjugation of PTLs to target Here, we developed a fast-relaxing glutamate TCP (TCPfast, nucleophilic residues in the native protein (e.g., lysine,13 Figure 1) that fulfills the abovementioned requirements. We histidine14). These targeted covalent photoswitches (TCPs) show the ability of TCPfast to produce photocurrents in naive can be applied to intact neurons and readily provide hippocampal neurons likely via tethering to endogenous GluRs photocontrol for hours.13 However, they display some and reversibly modulating their activity with visible light pulses limitations. The standard bistable photoswitches can be as short as a few milliseconds. As an original proof of concept, isomerized in milliseconds using dual-color light flashes we demonstrate the application of this molecular tool to the (UV−visible), but these wavelengths penetrate poorly in tissue ultrafast first synapses of the auditory system that mediate cochlear sound encoding. This drug-based method affords and are problematic in vivo. In addition, their slow-relaxing ultrafast rate stimulation of SGNs in naive adult gerbils, photochromism (lifetime ∼80 min for TCPs) limits to ∼80% matching the performance of optogenetic photostimulation the fraction of trans isomer that can be achieved with light15−19 that requires gene therapy. In general, these photoswitches and thus the reversibility of effects. To address these provide a fundamental resource of broad interest to shortcomings, photoswitches have been modified to isomerize spatiotemporally control endogenous receptors in intact with longer wavelengths and rapidly relax in the dark to 100% neuronal circuits with ultrafast signaling. ■ trans,22,26 but these compounds were designed to target genetically modified receptors instead of the native proteins,13 RESULTS AND DISCUSSION which has so far hindered their use in one-wavelength Design and Synthesis of a Fast-Switching TCP of applications to ultrafast synapses and in vivo preparations. Ionotropic GluRs. The molecular design of TCPfast was based Thus, methods for direct, rapid, and sustained photocontrol of on the recently reported TCPs13 and is shown in Figure 1A. activity in intact neurons constitute an unmet need, both to TCPs have a modular structure obtained by combining an study neuronal circuits for basic research purposes and to affinity “head” (1), which bears both the bioactive ligand explore new phototherapies. Interestingly, when sensory cells (glutamate moiety) and the modified photoisomerizable unit or neurons are damaged or absent, fast and sustained (azobenzene), and a reactive “tail” (2) bearing the protein neurotransmitter release is impaired, but the postsynaptic anchoring group (NHS ester). NHS ester-activated linkers are neurons and receptors can retain their full capacities short-lived groups that promptly react with primary amines (activation kinetics, localization, and complex formation with (e.g., lysine residues) under neutral or slightly alkaline 9230 https://doi.org/10.1021/jacs.1c12314 J. Am. Chem. Soc. 2022, 144, 9229−9239 Journal of the American Chemical Society pubs.acs.org/JACS Article Scheme 1. Chemical Synthesis of the “Head” Module (1) Bearing the Glutamate Moiety, the Photoswitchable Unit, and the Terminal Azide conditions (pH 7.2−9). To avert self-reactivity, TCPs are advanced intermediate 9, which was finally converted into the readily generated prior to attachment to the target protein via a desired compound 1 via removal of the tert-butoxycarbonyl copper(I)-catalyzed azide−alkyne cycloaddition reaction protecting group under acidic conditions (see also the (CuAAC, also known as “click chemistry”) (Figure 1B). Supporting Information for further details). This photoswitch Previous TCPs featured azobenzene moieties that were showed an absorption maximum at about 460 nm (blue light) photoisomerized using two different illumination wavelengths in aqueous solution at neutral pH (Figure S8), as previously (380 and 500 nm) and characterized by slow thermal cis-to- reported.22 Moreover, no variation of the absorption spectrum trans relaxation.13 Azobenzenes displaying faster relaxation could be detected by steady-state UV−vis spectroscopy, kinetics and single, longer wavelength switching can be suggesting that the cis isomer rapidly (<1 s) relaxes back to obtained with minimal variation of their chemical structure trans when the light is turned off. We used transient absorption by generating an electron “push−pull” system. It consists of spectroscopy to confirm photoswitching in aqueous phosphate including connected electron donating groups on one aromatic buffer saline (PBS) solution, to measure the absorption ring of the azo unit and electron withdrawing groups on the spectrum and optimal wavelength of isomerization (450−460 other, which lower the energy barrier of the thermal cis-to- nm), the relaxation lifetime in the dark (∼8 ms), and to trans isomerization,20,21 increasing the photoisomerization demonstrate the high fatigue resistance of the compound rate. This also results in a red-shifting of the azobenzene (Figures S9−S11). The head (1) was coupled via CuAAC with absorption spectrum, which is useful to reduce light scattering a commercial tail (compound 2) providing a fast-relaxing and in tissue and phototoxicity, which is lower for longer red-shifted ligand (3) with a similar length to the successful wavelengths in applications in vivo. Thus, we designed a TCP9 ligand previously described13 (Figure 1C). To reach a TCPfast head in which one of the two amide groups at the para satisfactory head-tail click coupling rate in minutes at room positions of the azobenzene was replaced by a tertiary amine as temperature, we replaced the conventional sodium ascorbate the electron-donating group.22 To avoid perturbing the ligand by ascorbic acid (see the Supporting Information for details). region, we chose to introduce this modification on the We hypothesized that the buffering effect of the ascorbic acid opposite side of the azobenzene core (head compound 1). (to the tertiary amine in compound 1) could create more Compound 1 was prepared via a five-step synthesis starting proper pH conditions to promote the catalytic cycle of the from commercially available materials (Scheme 1). Azoben- reaction23 as well as favor the formation of active copper(I) zene 5 was obtained by reduction of its nitro precursor 4 with species from the copper(I) oxide catalyst.24 Because NHS- sodium sulfide nonahydrate. Pyroglutamate derivative 6, based ligands are constitutively short-lived, we confirmed the prepared as previously described,15 was coupled to compound formation of the desired TCPfast by liquid chromatography− 5 using HOBt/EDC activation to give the intermediate 7 and mass spectrometry (LC−MS) of the click reaction crudes and then converted via mesylation into the corresponding azide verified their ability to conjugate primary amine-containing derivative 8. Hydrolysis of the pyroglutamate moiety with biomolecules by reacting them with pure lysine as a mock concomitant saponification of the ethyl ester provided the protein residue (see the Supporting Information for details). 9231 https://doi.org/10.1021/jacs.1c12314 J. Am. Chem. Soc. 2022, 144, 9229−9239 Journal of the American Chemical Society pubs.acs.org/JACS Article Figure 2. TCPfast conjugation produces photocurrents in untransfected hippocampal neurons in the presence of glutamate via endogenous non- NMDA GluRs. (A) Current recording in whole cell voltage clamp mode in dissociated hippocampal neurons after incubation in TCPfast (100 μM for 2 min at pH9). Inward current induced by bath perfusion of 300 μM glutamate (red bar) and 473 nm light (blue bars, 1 s). Time gap between traces corresponds to immediate subsequent recordings (<2s). Inset: photocurrent elicited by 100 ms light pulse. Exponential fit indicated in black correspond to Toff. (B) Increasing TCPfast concentration during incubation (2 min, pH9, 25−100 μM) leads to a higher fraction of cells displaying photoresponses. (C) Photocurrent amplitude increases relative to concentration of TCPfast incubated. Note: from the total number of cells responding to perfusion of 300 μM glutamate, 56% had light response. Per concentration, 47, 62% of cells incubated at 25 μM had light response (n = 21); 20% of cells incubated at 50 μM had a light response (n = 10); 75% of cells incubated at 75 μM had a light response (n = 8); 90% of cells incubated at 100 μM had a light response (n = 11). Means comparison by the Bonferroni test: 25 μM vs 50 μM (ns. p-value = 0.149); 25 μM vs 75 μM (ns; p-value = 0.198); 25 μM vs 100 μM (*, p-value = 0.0154); 50 μM vs 75 μM (*, p-value = 0.018); 50 μM vs 100 μM (**p-value = 2.6 × 10−4); and 75 μM vs 100 μM (ns, p-value = 0.3758). (D) Current recordings in whole cell voltage clamp mode in rat hippocampal neurons maintained 15 days in culture and incubated with TCPfast (100 μM for 2 min at pH 9). Example traces of photocurrents elicited by irradiation at λex = 473 nm (1 s, blue bars) in the presence of 300 μM glutamate (red bar). Photocurrents are reversibly blocked by perfusion of 100 μM DNQX (orange bar) and not affected by 100 μM AP5 (green bar). After washout and reperfusion of 300 μM glutamate photocurrents are recovered, indicating covalent conjugation. Note that the absence of sharp responses may be indicative of GluR desensitization as a result of the high glutamate concentration. (E) Quantification of the effect of DNQX (100 μM, n = 7, p-value = 0.008) and AP5 (100 μM, n = 3, p-value = 0.56) on the photocurrent amplitude obtained from hippocampal neurons incubated with TCPfast (25-75-100 μM for 2 min at pH9). Control and wash-out measurements were obtained after bath solution and glutamate perfusion. White dots indicate mean ± SE. Note: p-values obtained after performing Friedman test: non-parametric, data from any distribution; small samples; and related samples. Characterization of TCPfast in Cultured Neurons. We receptors, followed by wash-out of physiological solution, pH made use of the several GluRs subunits expressed by 7.4) previously shown to be favorable for TCP conjugation.13 hippocampal neurons25 to evaluate, on dissociated neuron 473 nm illumination did not elicit photocurrent measured by cultures, the ability of TCPfast to photocontrol GluR activity. whole-cell patch-clamp recordings directly after incubation TCPfast was conjugated to GluRs by using the same incubation with TCPfast. However, in the additional presence of glutamate conditions (i.e., 2 min at 25−100 μM, pH 9 to favor (300 μM), 473 nm illumination elicited 2.5−220 pA deprotonation and reactivity of nucleophilic residues in the photocurrents (Figure 2A). As intended by the chemical 9232 https://doi.org/10.1021/jacs.1c12314 J. Am. Chem. Soc. 2022, 144, 9229−9239 Journal of the American Chemical Society pubs.acs.org/JACS Article Figure 3. Fast photoswitchable control of SGN activity mediated by TCPfast. (A) Mongolian gerbil cochlea was exposed, through the middle ear, by performing a retro-auricular approach. (B) Silver ball electrode was placed in the RW niche (dashed line) to record CAP in response to sound or to light stimulation. The light stimulation was delivered to the cochlea via an optical fiber coupled to a 470 nm-laser. (C) Experiment time course description: TCPfast application (2.5−12.5 μM, 10 min) is preceded and followed by artificial perilymph application (AP) and acoustically CAP measurements. After the second AP application, the optical fiber is inserted in the RW and the optically evoked potentials (E−J) are recorded. (D) Representative acoustically (black, left, aCAP) and optically evoked CAP (blue, right, oCAP) following 2.5 μM application of TCPfast. aCAPs (black, 200 averages, 8 kHz toneburst, 30 dB SPL, repetition rate = 20 Hz) are similar before and after TCPfast application. oCAP (blue, 80 μs, 30 mW, repetition rate = 10 Hz) is abolished following application of competitive antagonist DNQX (1 mM). Stimuli are represented at the top. (E1,G1) Representative oCAP in response to various radiant fluxes (80 μs at 10 Hz, E1), pulse durations (27 mW at 10 Hz, F1), and repetition rates (80 μs at 27 mW, G1). In (E1,G1), the blue line indicates the light stimuli and in F1 the blue arrow indicates the beginning of the light pulse. A color scale is used to represent the variable. (E2,G2). Quantification (n = 6 cochleae) of the oCAP amplitude (N1−P1, black axis) and oCAP latency (N1, gray axis) as a function of the radiant flux (E2), the pulse duration (F2), and the repetition rate (G2). In F2 and G2, oCAP amplitudes were expressed as relative to the highest amplitude recorded for the given measure. design, the cis isomer of TCPfast, induced by blue light Next, we showed that the number of photosensitized illumination, evoked inward photocurrents. thus supporting hippocampal neurons (Figure 2B) and the amplitude of the the notion that TCPfast enabled fast-one-wavelength photo- photocurrent evoked by blue light (Figure 2C) increased with modulation of GluRs (Figure 2A inset). Next, we tested the TCPfast concentration during the incubation (25, 50, 75, and effect of light in absence of TCPfast incubation (Figure S16). 100 μM). At 25 μM of TCPfast, 47.62% of neurons (n = 21) The currents measured at the onset of 300 μM glutamate had a measurable photocurrent (on average: 25 ± 12 pA), perfusion were of similar amplitude in both cases (793 ± 131 while 100 μM raised this to 90% of the neurons (n = 11) with pA with TCPfast and 1161 ± 445 pA without TCPfast), a photocurrent of 93 ± 25 pA. In contrast, the relaxation discarding any non-specific effect of light stimulation. Direct lifetime in the dark of TCPfast-conjugated neurons was perfusion of TCPfast in the dark indicates that trans-TCPfast can independent of TCPfast concentration and amounted to 220 activate GluRs as a free ligand, an effect that is enhanced by ± 48 ms (Figures 2A inset and S18). As intended by our light stimulation (Figure S17). chemical design, the achieved relaxation lifetime was faster 9233 https://doi.org/10.1021/jacs.1c12314 J. Am. Chem. Soc. 2022, 144, 9229−9239 Journal of the American Chemical Society pubs.acs.org/JACS Article than shown for TCPs (∼80 min)13 and comparable to one- employed a lower TCPfast dose (2.5 μM, <0.05% of organic wavelength PTLs (MAG460: 710 ms,22 MAG2P: 150 ms and solvent), we observed stable acoustically and optically evoked MAGA2P: 265 ms26). For a subset of neurons, we also found cochlear potentials in 60% of the incubated cochleae (Figures that the photocurrent increased in amplitude with the radiant 3D−G, S22A−C). The other 40% of the cochleae were similar flux (Figure S19) and could be evoked by light pulse as short to previously reported using the highest concentration with a as 3 ms (Figure S20). loss of mass potentially (Figure S22D−F), which indicates a Finally, we showed that the photoresponses can be reversibly narrow therapeutic window. blocked by the AMPA receptor antagonist DNQX (100 μM) For the positive cochleae, oCAPs were similar in shape and but not by the NMDA receptor antagonist AP5 (100 μM) amplitude to aCAP evoked by a 40 dB SPL toneburst but (Figure 2D,E). This demonstrates that TCPfast photocurrents consistently showed a shorter latency (∼1.5 ms, Figure 3D) are receptor-specific and that the photoswitchable ligands are indicating a direct optical SGN excitation bypassing the outer indeed covalently tethered to GluRs (as the TCPfast-mediated and middle ear transmission and inner ear mechanotransduc- photoresponses persist even after washout and competition tion of sound in acoustic hearing. oCAPs could be abolished by with an antagonist). DNQX (1 mM) application at the RW, supporting a response In Vivo Photocontrol of Neural Activity in Gerbil’s mediated by AMPA receptors (Figure 3D, light blue trace). Cochlea. The potential of TCPfast to photosensitize SGNs in We further characterized the TCPfast-mediated optical vivo was tested in the Mongolian gerbil cochlea using the same response by measuring oCAP amplitude and latency as a TCPfast batch produced under the same click reaction function of radiant flux (n = 6) using 80 μs light pulses at a conditions as for the in vitro characterization demonstrated repetition rate of 10 Hz (Figure 3E1,2). oCAP threshold above. The goal was to administer TCPfast to SGNs, where it amounted to 12.5 ± 0.62 mW (1 ± 0.05 μJ). From there, could covalently tether to the AMPA receptors in the oCAP amplitude increased linearly with radiant flux up to 20.5 postsynaptic density. ± 4.09 μV at 30 mW and latency decreased from 1.62 ± 0.05 To do so, we surgically exposed the cochlea by performing a to 1.51 ± 0.05 ms. Next, we measured the effect of the pulse middle ear surgery (posterior tympanotomy) and applied duration on the oCAP (Figure 3F1,2, 30 mW, repetition rate = TCPfast to the niche of the round window (RW, a membrane- 10 Hz): the biggest oCAPs were recorded in response to 80 covered bony window of the cochlea, Figure 3A,B) for passive and 100 μs light pulses. From there, oCAPs decreased in diffusion into the cochlea. In order to evaluate the potential amplitude for shorter and longer durations. In 50% of the toxicity induced by TCPfast, we recorded cochlear mass cases, sizable oCAPs (i.e., > 1 μV) were measured in light potentials evoked by sound, before, during, and after TCPfast pulses as short as 20 μs, corresponding to 0.6 μJ. In response to diffusion. These mass potentials reflect the status of three light pulses shorter than 100 μs, oCAPs were characterized by cochlear cell types: outer hair cells, inner hair cells, and SGNs a single negative wave occurring at ∼1.54 ms. In contrast, (for details, see Supporting Information, Figure 3C, Figure oCAPs evoked by a longer pulse had multiple negative peaks, S21A1‑4). In this experiment, the acoustically evoked potentials which might reflect the firing of multiple synchronous action reflecting the outer hair cells (i.e., the cochlear microphonic, potential across SGNs in response to the longer stimuli. Figure S21A3,B), IHCs (i.e., the summating potential, Figure Finally, we measured the effect of the repetition rate on oCAPs S21A4,C), and SGNs (i.e., the compound action potential, using 80 μs light pulses (Figure 3G1,2, 30 mW). The oCAPs CAP, Figure S21A4,D) remained largely unchanged following were stable in amplitude up to 60 Hz and decreased a 10 min application of 12.5 μM TCPfast (0.25% of organic exponentially at a higher repetition rate until not being solvent) and wash-out (n = 4, Friedman’s test). This indicates observable for rates exceeding 1 kHz (the best performer that TCPfast incubation did not interfere with cochlear cochlea is illustrated in Figure S23). The reported in vivo data physiology. support the concept of fast control of the AMPA receptor with We then tested for a TCPfast-mediated photopharmaco- TCPfast in the SGNs. logical SGN stimulation by inserting an optical fiber coupled to a blue laser (λ = 473 nm) into the RW. Transient optically evoked CAPs (oCAPs) were recorded from 12 out of 12 ■ CONCLUSIONS In this work, we developed a chemical-biological method, cochleae treated with TCPfast. oCAPs were similar in shape to called TCPfast, which allows fast photoswitching of neuronal acoustically evoked CAP and had a maximal amplitude of activity in native neurons. TCPfast is a photoswitchable ligand 40.88 ± 12.58 μV (at maximum radiant flux: 30 mW), which is that is chemically attached to endogenous receptors at the equivalent to the amplitude of a 50 dB SPL tone burst (Figure postsynaptic side.13 The molecular design of TCPfast optimized S21E). oCAPs vanished within minutes of optical stimulation, the properties of the bistable photoisomerizable group of which was accompanied by a disappearance of the acoustic former TCPs,13 that is, thermal relaxation lifetime and potentials reflecting the IHC and SGNs activation. The absorption wavelength, without altering the ligand and the number of synapses per IHC (i.e., the juxtaposition of pre- reactive group. TCPfast functions as a molecular prosthesis that [CtBP2] and postsynaptic [Homer 1] markers) was similar bypasses the neurotransmitter-encoded signal by a photonic between treated and non-treated cochleae, at all frequencies, signal. Photosensitization of cochlear SGNs by locally arguing against glutamate excitotoxicity induced by TCPfast administered TCPfast enabled temporally precise light-evoked mediated optical stimulation (Figure S21F-G and Supporting SGN firing up to a rate of approximately 1 kHz, thus matching Method S2 for details). We can only speculate about a putative the fastest optogenetic SGN stimulation (≤1 kHz27). Hence, functional impairment of IHCs, SGNs, and their synapses, TCPfast-mediated photopharmacology might serve as an which might involve mechanisms such as phototoxic damage, interesting alternative to the optogenetic approach6,28 for the for example, via generation of free radicals and desensitization development of an optical CI. and/or endocytosis of AMPA receptors. Future studies will be TCPfast elicited depolarizing photocurrents that are activated needed to elucidate such mechanisms. Interestingly, when we by the cis isomer (“cis-on”), which is generally preferred to 9234 https://doi.org/10.1021/jacs.1c12314 J. Am. Chem. Soc. 2022, 144, 9229−9239 Journal of the American Chemical Society pubs.acs.org/JACS Article avoid activation in the dark. Such behavior has been reported case, the effect of light in the presence of the endogenous for similar photoswitchable ligands10,13 and has been attributed neurotransmitter glutamate can also be envisaged as a way of to compound lengths and conjugation sites in the protein that potentiating physiological rather than generating totally allow ligand binding to the active site only in a “bent” (cis) exogenous signals, which is often preferable. conformation.29 Molecular docking studies (Figures S25 and Considering the unique properties of TCPfast to modulate S26) and the cis-on agonist behavior of the freely diffusible native GluR activity, TCPfast was tested in vivo at the synapse compound in neurons (Figure S17) agree with this view. between IHC and SGNs of the cochlea, where excitatory TCPfast photoresponses are reversed in the absence of responses are mainly mediated by AMPA receptors.37,38 illumination with a thermal relaxation lifetime ranging between TCPfast-mediated control of the SGNs was obtained in minutes 8 ms for the TCPfast head in the cuvette (Figure S9) and 220 by diffusing (and washing out) the compound into the cochlea. ms in hippocampal neurons in culture (Figure S18). While Interestingly, neural responses were observed in response to slower lifetimes have been observed upon photoswitch light pulses with energies between 0.5 and 1 μJ, which is the conjugation and interaction with the target protein,26 we can lower range of the optogenetic approaches applied to the adult currently only speculate on how such a thermal relaxation rate gerbil cochlea (CatCh: 1−2 μJ,;39 f-Chrimson: ∼5 μJ).40 measured in vitro yields the ultrafast photostimulation of Additionally, maximal oCAP responses were obtained in SGNs in vivo eliciting SGN CAPs with interstimulus intervals response to light pulse as short as 80 μs, which is substantially ranging from a few milliseconds to submillisecond. Of note, shorter than reported for optogenetic tools in the coch- postsynaptic boutons of SGNs likely contain hundreds of lea.27,39,41,42 Finally, TCPfast enabled neural responses up to a AMPARs such that each light stimulus might hit a sufficient stimulation rate of 1.5 kHz. Although this observation should number of AMPARs with the TCPfast being in the trans state. be confirmed by single SGN recording, it is interesting to note Moreover, CAPs represent population responses and so light that this performance is similar to what was reported using an pulses might be recruiting variable subsets of SGNs. Finally, optically evoked auditory brainstem response, for SGNs AMPA receptor desensitization might contribute to the expressing the fastest opsins to date (Chronos:;27 vf- transient response properties. Future work could consider to Chrimson:42), while presenting a lower activation threshold further speedup thermal relaxation potentially by replacing the (TCPfast ∼ 1 μJ, Chronos-ES/TS and vf-Chrimson-ES/TS ∼ 6 current azobenzene unit in TCPfast with faster photoswitches μJ). such as (hydroxy-substituted) phenylazopyrimidine (milli- TCPfast provides similar or better performance than current second to nanosecond range30−32). However, such accelerated optogenetic approaches in terms of speed and light require- back-switching might in turn result in a lower cis conversion ments, while avoiding the gene therapy required to optogeneti- percentage in the photostationary state and, therefore, would cally modify SGNs. Yet, long-term availability, efficacy, and require higher light intensity. In addition, further increasing the safety of TCPfast or more advanced photoswitches will need to wavelength for photoswitching33 might improve the overall be established. Moreover, neural degeneration often starts with utility of the method due to better tissue penetration and lower the loss of postsynaptic structures,43,44 which may limit the phototoxicity risk. usefulness of TCPfast photopharmacology for optical CIs. We demonstrated, in dissociated hippocampal neurons, Future studies should focus on designing safe compound- specific photomodulation of AMPA/kainate receptors by targeting channels expressed at the surface of the surviving TCPfast. Such specificity is due to the affinity-labeling SGN somas, while showing similar target specificity, fast mechanism of PTLs.10,13,29 In this experimental setting, TCPfast kinetics, and longer wavelength. This way, photopharmacology photomodulation required the presence of glutamate in the might establish a toolkit for photosensitization tailored to the bath solution for all the tested concentrations (Figure 2C), but specific neural status of the cochlea or other structures. In in the range of 0.03−3 mM free glutamate, a weak correlation addition, a drug-based approach would appeal to patients and with the photocurrent amplitude was observed. One the pharmaceutical industry and facilitate regulatory processes. explanation could be that conjugated TCPfast acts as a trans- Photopharmacology has previously demonstrated its potential on antagonist. However, because tethered glutamate has never to photosensitize other sensory systems such as the been reported to act as an antagonist in GluRs and freely retina.13,45−47 To the best of our knowledge, this is the first diffusible TCPfast acts as an agonist, we argue that the proof-of-concept photopharmacology study in the auditory requirement of glutamate is due to incomplete conjugation of system and opens up an avenue for auditory research and TCPfast to a heterogeneous population of endogenous GluRs. clinical applications such as in optical CIs designed to This concept may be translated into a distinctive subunit fundamentally improve hearing restoration. ■ occupancy resulting in differential agonist affinity and efficacy, thus reducing the channel open probability (gating).12,34,35 Enhancing the reactivity of the electrophilic group could EXPERIMENTAL SECTION improve the photoswitch performance. Another possible Click Reaction. To a 1.5 mL glass vial containing azide 1 (“head”, mechanism would be the presence of native GluR hetero- 1.00 mg, 1 equiv) and copper(I) oxide (0.82 mg, 3 equiv) in tetramers36 with subunits displaying different TCP conjugation tetrahydrofuran (47 μL) and equipped with a magnetic stir bar was and photoswitching properties.12 We cannot rule out the added a solution of ascorbic acid (1.34 mg, 4 equiv) in water (94 μL) possibility that TCPfast behaves as a positive allosteric and the resulting mixture was vortexed for 1 min. Then, a solution of alkyne 2 (“tail”, 0.47 mg, 1.1 equiv) in tetrahydrofuran (47 μL) was modulator or as a partial agonist, allowing only partial receptor added and the resulting mixture was stirred at room temperature for activation by partial closure of the ligand binding domain. We 45 min. The so obtained final mixture was taken up in argue against this possibility because cis-TCPfast photocurrent dimethylsulfoxide (193 μL), vortexed, centrifuged for 1 min to in the presence of the orthosteric antagonist DNQX is blocked, separate the insoluble copper(I) oxide particles, and finally divided and in the presence of glutamate is not reduced, but enhanced, into aliquots of the final compound stock solution (Figure 1B,C and indicating that it is not competing with glutamate.10,35 In any Scheme S2). 9235 https://doi.org/10.1021/jacs.1c12314 J. Am. Chem. Soc. 2022, 144, 9229−9239 Journal of the American Chemical Society pubs.acs.org/JACS Article Alternatively, the click reaction can be performed in an Eppendorf Animal Preparation for Cochlear Potential Recordings and tube and stirred with a suitable mixer. Cochlear Pharmacology. All experiments were done in compliance We observed that the catalytic performance of the copper(I) oxide with the German national animal care guidelines and were approved may vary significantly from batch to batch, therefore the actual by the board for animal welfare of the University Medical Center reaction time should be adjusted to obtain at least a 95% conversion Göttingen and the animal welfare office of the state of Lower Saxony of the starting material and a (TCPfast):(hydrolyzed TCPfast) ratio (agreement 2014/1726 and 2019/3188). Experiments were per- greater than 3 (Figure S12). In vitro and in vivo biological formed on adult (>8 weeks old) Mongolian gerbils (Meriones characterizations were done using the same batch and results were unguicalatus) of both sexes. reproducible across experiments. Gerbils were anesthetized by isoflurane (5% for anesthesia Rat Hippocampal Neural Primary Culture. All experiments induction, 1−3% for maintenance, and frequent testing for the were done in compliance with the national animal care guidelines and absence of hind-limb withdrawal reflex), and analgesia was obtained were approved by the board for animal welfare of the University by subdermal injection of buprenorphine (0.1 mg/kg body weight) Medical Center Goettingen and the animal welfare office of the state and carprofen (5 mg/kg body weight). Body temperature was of Lower Saxony. maintained at 37 °C using a custom-designed heat plate. The cochlea Primary hippocampal cultures were prepared from newborn P0−P3 was exposed following a retroauricular approach and a bullostomy. pups Wistar rats. Brains were collected in a 10 cm Petri dish The recording electrode was placed against the bony edge next to the containing ice-cold dissection media (HBSS (Gibco) + 10 mM Hepes RW leaving enough space to access the RW for pharmacological (Gibco)). Hippocampi were separated from the brain, and meninges manipulation and optical stimulation. The pharmacological manipu- were removed. Hippocampi were digested with 2 mL prewarmed 37 lation was made by filling the RW niche with the solution of interest. °C 0.1% trypsin−EDTA (Gibco, Germany) for 20 min at 37 °C. The RW membrane was punctured to increase the fluid exchange Trypsin was removed, and the tissue was washed three times with between the cochlea and RW niche. Between each solution, the RW dissection medium. Dissection medium was replaced with 1 mL niche was emptied by capillarity. prewarmed complete DMEM (DMEM with 1× Glutamax, 10% FCS/ Acoustically Evoked Cochlear Potential. Acoustic cochlear FBS and 1% penicillin (100 U/mL)/streptomycin (100 μg/mL); all potentials were obtained in response to 8 kHz tone burst (pulse from Gibco). Tissue was triturated by gentle pipetting. The tissue duration = 8 ms, rise/fall time = 1 ms, repetition rate = 20 Hz, level: suspension was filtered through a 100 μm cell strainer (BD 20 to 80 dB SPL per 10 dB step, and 200 repetitions per level). The Biosciences). mass potentials were amplified using a custom-made physiological Cells were counted using the trypan blue exclusion method and amplifier and sampled at a rate of 50 kHz (NI PCI-6229, National cultured on 12 mm glass coverslips (Thermo Scientific) coated with Instrument). Stimulus generation and data acquisition were made poly-D-lysine (Sigma). Neurons were plated in 24-well plates at a using custom-written software (MATLAB, MathWorks) employing density of 50,000 hippocampal neurons per coverslip in 500 μL of NB National Instrument data acquisition cards in association with a + medium (Neurobasal with 2% B-27 supplement, 1% Glutamax and custom-built acoustic and laser-controller. 1% penicillin (100 U/mL)/streptomycin (100 μg/mL); all from The cochlear microphonic was extracted by averaging the band- Gibco) at 37 °C and 5% CO2. Half of the medium in each well was pass filtered (cut-off frequencies = 5.6 and 11.1 kHz) mass potential changed every 3−4 days. recorded using the RW electrode and its amplitude defined as the Electrophysiology Recording Conditions for Rat Hippo- RMS value. The CAP and summating potentials were obtained by campal Neurons. Before starting the recording, neurons between 10 averaging the low-pass filtered (cut-off frequency = 3.5 kHz) mass- and 18 days in vitro (div) were incubated with TCPfast at potential. The CAP amplitude was defined as the amplitude between concentrations: 25−50−75−100 μM (≤2% DMSO) for 2 min, in the first negative peak (N1) and the following positive peak (P1). The the absence of light and in pH 9 bath solution composed of (in mM): summating potential amplitude was defined as the difference between 100 NaCl, 1 MgCl2, 2.5 KCl, 2.5 CaCl2, 10 glucose, and 50 the plateau response (between 5 and 7 ms) and the baseline prior to sodiumcarbonate/sodiumbicarbonate, 310 mOsm/kg, pH 9 adjusted the stimulation onset. with NaOH. Optically Evoked Cochlear Potential. Optical cochlear Voltage and current-clamp recordings under whole-cell config- potentials were obtained in response to blue light pulse delivered uration were done using an Axopatch 200B amplifier, filtered at 5 by a 200 μm optical fiber coupled to a 473 nm laser (MLL-FN-473- kHz, and digitized with an Axon DigiData 1440A interface (Axon 100, 100 mW, diode pumped solid state [DPSS]; Changchun New Instruments). Acquisition software used was Clampex 10.5.2.6 (Axon Industry Optoelectronics). Irradiance was calibrated with a laser Instruments). power meter (LaserCheck; Coherent Inc). The CAP was obtained by Light stimulation was performed at saturating radiant flux (5−14 averaging the low-pass filtered (cut-off frequency = 3.5 kHz) mass mW) using diode-pumped solid-state lasers (λ = 473 nm) focused potential. into a 400 μm optic fiber. Light pulses were applied using a fast IHC Synapse Counting. Cochleae were fixated in 4% form- computer-controlled shutter (Uniblitz LS6ZM2, Vincent Associates, aldehyde for about 15 min and decalcified in 0.12 M EDTA solution Rochester, USA). Radiant flux was adjusted by placement of density for about 12 h (at 4 °C). Organs of Corti were then: (i) isolated in filters between the laser output and the optic fiber. PBS; (ii) incubated for an hour in Goat serum dilution buffer (GSDB, During recordings, neurons were maintained at room temperature comprised of: 16% normal goat serum, 450 mM NaCl, 0.3% Triton X- (r.t., 25−27 °C) in a continuous perfusion of bath solution and 100, 20 mM phosphate buffer, pH 7.4); (iii) treated for 2 h with the clamped at −70 mV. Bath solution was composed of (in mM): 140 primary antibodies: mouse anti-CtBP2 (BD Biosciences, 1:200), NaCl, 1 MgCl2, 2.5 KCl, 10 HEPES, 2.5 CaCl2, and 10−20 mM rabbit anti-myo6 (Proteus Biosciences, 1:200), and chicken anti- glucose to fix osmolarity to 310 mOsm/kg, pH 7.42 adjusted with Homer1 (SYsY, 1:200) for staining presynapse, IHCs, and NaOH. Borosilicate glass pipettes were pulled with a typical resistance postsynapse, respectively. After washing the primary antibodies with of 3−8 MΩ for neurons. Pipette solution contained (in mM): 129 PBS for 20 min, the secondary antibodies were incubated: Goat-anti- potassium gluconate, 10 HEPES, 10 KCl, 4 MgATP, and 0.3 Chicken 488 (Invitrogen, 1:500), Goat-anti-Rabbit 568 (Thermo Na3GTP. Osmolarity is adjusted at 289 mOsm/kg, and pH is adjusted Fisher, 1:500), and Goat-anti-Mouse 647 (Invitrogen, 1:500). After to 7.2 with KOH. another washing step with PBS, samples were mounted on a glass Drug Preparation for In Vivo Cochlea Infusion. Artificial slide in Mowiol (Carl Roth) mounting medium. Confocal images of perilymph solution consisted of the following (in mM): 137 NaCl; 5 the organ of Corti were first obtained using a 10× magnification KCl; 2 CaCl2; 1 MgCl2; 1 NaHCO3; and 11 glucose, pH 7.4 adjusted (LSM 510 microscope, Carl Zeiss, Jena) in order to fit a Greenwood with NaOH and osmolarity: 304 ± 4.3 mOsm/kg. Before each function48,49 to them and localize the tonotopic position of the IHCs. experiment, TCPfast was diluted in artificial perilymph to a final The synapses were visualized at different tonotopic regions (0.5, 1, 2, concentration ranging from 2.5 to 12.5 μM. 4, 8, 16, and 32 kHz) using a 100× magnification (Abberior 9236 https://doi.org/10.1021/jacs.1c12314 J. Am. Chem. Soc. 2022, 144, 9229−9239 Journal of the American Chemical Society pubs.acs.org/JACS Article Instruments Expert Line STED microscope) and counted using the Carlo Matera − Institute for Bioengineering of Catalonia spot function from Imaris software (version 7.6.5). (IBEC), Barcelona Institute for Science and Technology, Data Analysis and Statistics. Amplitude of photocurrents was 08028 Barcelona, Spain; Network Biomedical Research analyzed using IgorPro (Wavemetrics). Displayed whole-cell current Center in Bioengineering, Biomaterials, and Nanomedicine traces have been filtered using the infinite impulse response digital filter from IgorPro (low-pass filter with a cutoff of 50 Hz). The drift in (CIBER-BBN), 28029 Madrid, Spain; Department of current was corrected where appropriate with the IgorPro (Wave- Pharmaceutical Sciences, University of Milan, 20133 Milan, Metrics) software using a custom-made macro for drift correction. Italy; orcid.org/0000-0001-6939-3859 Statistics were done with OriginPro 8.5 (OriginLab) and Matlab Anupriya Thirumalai − Institute for Auditory Neuroscience (Mathworks). In vivo electrophysiological data were analyzed using and InnerEarLab and Auditory Circuit Lab, Institute for custom-made Matlab routines. Auditory Neuroscience and InnerEarLab, University Medical ■ * ASSOCIATED CONTENT sı Supporting Information Center Göttingen, 37075 Göttingen, Germany; Auditory Neuroscience and Optogenetics Group, German Primate Center, 37077 Göttingen, Germany The Supporting Information is available free of charge at Jordi Hernando − Departament de Química, Universitat https://pubs.acs.org/doi/10.1021/jacs.1c12314. Autònoma de Barcelona (UAB), Cerdanyola del Vallès General materials and methods; detailed description of 08193, Spain; orcid.org/0000-0002-1126-4138 the chemical synthesis and physicochemical character- Amadeu Llebaria − Consejo Superior de Investigaciones ization of TCPfast; biological in vitro measurements; Científicas (IQAC-CSIC), Institute of Advanced Chemistry of supporting in vivo method description and measure- Catalonia, 08034 Barcelona, Spain ments; and sequence alignment and molecular docking Complete contact information is available at: simulations (PDF) https://pubs.acs.org/10.1021/jacs.1c12314 ■ AUTHOR INFORMATION Corresponding Authors Author Contributions △ A.G.-C., A.H., and C.M. contributed equally to this work. Tobias Moser − Institute for Auditory Neuroscience and T.M. and P.G. contributed equally to this work. InnerEarLab, University Medical Center Göttingen, 37075 Notes Göttingen, Germany; Auditory Neuroscience and The authors declare the following competing financial Optogenetics Group, German Primate Center, 37077 interest(s): Prof. Dr. Tobias Moser is a co-founder and CEO Göttingen, Germany; Cluster of Excellence “Multiscale of OptoGenTech company. The other authors declare no Bioimaging: from Molecular Machines to Networks of conflict of interests. ■ Excitable Cells” (MBExC), University of Göttingen, 37075 Göttingen, Germany; Email: tmoser@gwdg.de ACKNOWLEDGMENTS Pau Gorostiza − Institute for Bioengineering of Catalonia (IBEC), Barcelona Institute for Science and Technology, This research received funding from the European Union 08028 Barcelona, Spain; Network Biomedical Research Research and Innovation Programme Horizon 2020Human Center in Bioengineering, Biomaterials, and Nanomedicine Brain Project SG3 (945539), DEEPER (ICT-36-2020− (CIBER-BBN), 28029 Madrid, Spain; Catalan Institution 101016787), Agency for Management of University and for Research and Advanced Studies (ICREA), 08010 Research Grants/Generalitat de Catalunya (CERCA Pro- Barcelona, Spain; orcid.org/0000-0002-7268-5577; gramme; 2017-SGR-1442 and 2017-SGR-1604 projects), Email: pau@icrea.cat Fonds Européen de Développement É conomique et Régional (FEDER) funds, Ministerio de Ciencia e Innovación, Agencia Authors Estatal de Investigación and ERDF-FEDER European Fund Aida Garrido-Charles − Institute for Bioengineering of (projects CTQ2017-89222-R and PID2020-120499RB-I00), Catalonia (IBEC), Barcelona Institute for Science and and Fundaluce and “la Caixa” foundations (ID 100010434, Technology, 08028 Barcelona, Spain; Network Biomedical agreement LCF/PR/HR19/52160010). The project Clúster Research Center in Bioengineering, Biomaterials, and Emergent del Cervell Humà (CECH, 001-P-001682) is Nanomedicine (CIBER-BBN), 28029 Madrid, Spain; cofinanced by the European Union Regional Development Institute for Auditory Neuroscience and InnerEarLab, Fund within the framework of the ERDF Operational Program University Medical Center Göttingen, 37075 Göttingen, of Catalonia 2014−2020 with a grant of 50% of total eligible Germany; Auditory Neuroscience and Optogenetics Group, cost. A.G.-C. was supported by the fellowship BES-2014- German Primate Center, 37077 Göttingen, Germany; Cluster 068169 and Alexander von Humboldt Foundation. J.H. was of Excellence “Multiscale Bioimaging: from Molecular supported by Agencia Estatal de Investigación (PID2019- Machines to Networks of Excitable Cells” (MBExC), 106171RB-100/AEI/10.13039/501100011033) and General- University of Göttingen, 37075 Göttingen, Germany itat de Catalunya (2017 SGR00465). We thank chemical Antoine Huet − Institute for Auditory Neuroscience and facility PCB staff (Unai Elezcano, Sònia Varon) and IBEC InnerEarLab and Auditory Circuit Lab, Institute for Auditory infrastructure staff (Isabel Oliveira, David Izquierdo). We Neuroscience and InnerEarLab, University Medical Center thank Christiane Senger-Freitag, Sandra Gerke, and Sina Göttingen, 37075 Göttingen, Germany; Auditory Langer for technical support, Gerhard Hoch for his expert Neuroscience and Optogenetics Group, German Primate technical support, and Patricia Räke-Kügler for excellent Center, 37077 Göttingen, Germany; Cluster of Excellence administrative support. We thank Thomas Mager for providing “Multiscale Bioimaging: from Molecular Machines to the patch clamp setup. This work was funded by the European Networks of Excitable Cells” (MBExC), University of Research Council through the Advanced Grant “OptoHear” to Göttingen, 37075 Göttingen, Germany T.M. under the European Union’s Horizon 2020 Research and 9237 https://doi.org/10.1021/jacs.1c12314 J. Am. Chem. Soc. 2022, 144, 9229−9239 Journal of the American Chemical Society pubs.acs.org/JACS Article Innovation program (grant agreement no. 670759), the (16) Gorostiza, P.; Isacoff, E. Optical Switches and Triggers for the Fraunhofer and Max-Planck Cooperation Program (NeurOpto Manipulation of Ion Channels and Pores. Mol. BioSyst. 2007, 3, 686− grant) to T.M., was further supported by the German Research 704. Foundation through the Priority Program 1926 “Next (17) Gorostiza, P.; Isacoff, E. Y. Optical Switches for Remote and generation optogenetics” to A.H. and T.M., and the Leibniz Noninvasive Control of Cell Signaling. Science 2008, 322, 395−399. (18) Berlin, S.; Szobota, S.; Reiner, A.; Carroll, E. C.; Kienzler, M. Program (to T.M.) and the Deutsche Forschungsgemeinschaft A.; Guyon, A.; Xiao, T.; Trauner, D.; Isacoff, E. Y. A Family of (DFG, German Research Foundation) under Germany’s Photoswitchable NMDA Receptors. eLife 2016, 5, No. e12040. Excellence StrategyEXC 2067/1-390729940 to A.H. and (19) Kienzler, M. A.; Isacoff, E. Y. Precise Modulation of Neuronal T.M. In addition, this research was supported by the Activity with Synthetic Photoswitchable Ligands. Curr. Opin. Fakultätinternes Forschungförderungsprogramm from the Neurobiol. 2017, 45, 202−209. Universitätsmedizin Göttingen, Georg-August-Universität Göt- (20) Chi, L.; Sadovski, O.; Woolley, G. A. A Blue-Green Absorbing tingen to A.H., and Fondation Pour l’Audition (FPA RD-2020- Cross-Linker for Rapid Photoswitching of Peptide Helix Content. 10) to T.M. Molecular graphics and analyses were performed Bioconjugate Chem. 2006, 17, 670−676. with UCSF Chimera, developed by the Resource for (21) Bandara, H. M. 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JOURNAL OF THE AMERICAN CHEMICAL SOCIETY READ Restoring Vision to the Blind with Chemical Photoswitches. Chem. Rev. 2018, 118, 10748−10773. In Vivo Photopharmacology Enabled by Multifunctional (48) Greenwood, D. D. Critical Bandwidth and the Frequency Fibers Coordinates of the Basilar Membrane. J. Acoust. Soc. Am. 1961, 33, James A. Frank, Polina Anikeeva, et al. 1344−1356. OCTOBER 27, 2020 (49) Müller, M. The Cochlear Place-Frequency Map of the Adult ACS CHEMICAL NEUROSCIENCE and Developing Mongolian Gerbil. Hear. Res. 1996, 94, 148−156. READ Optical Control of Cannabinoid Receptor 2-Mediated Ca2+ Release Enabled by Synthesis of Photoswitchable Probes Roman C. Sarott, James A. Frank, et al. JANUARY 05, 2021 JOURNAL OF THE AMERICAN CHEMICAL SOCIETY READ Bistable Photoswitch Allows in Vivo Control of Hematopoiesis Lea Albert, Olalla Vázquez, et al. DECEMBER 22, 2021 ACS CENTRAL SCIENCE READ Get More Suggestions > 9239 https://doi.org/10.1021/jacs.1c12314 J. Am. Chem. 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