Monodirectional Photocycle Drives Proton Translocation

Photoisomerization of retinal is pivotal to ion translocation across the bacterial membrane and has served as an inspiration for the development of artificial molecular switches and machines. Light-driven synthetic systems in which a macrocyclic component transits along a nonsymmetric axle in a specific direction have been reported; however, unidirectional and repetitive translocation of protons has not been achieved. Herein, we describe a unique protonation-controlled isomerization behavior for hemi-indigo dyes bearing N-heterocycles, featuring intramolecular hydrogen bonds. Light-induced isomerization from the Z to E isomer is unlocked when protonated, while reverse E → Z photoisomerization occurs in the neutral state. As a consequence, associated protons are displaced in a preferred direction with respect to the photoswitchable scaffold. These results will prove to be critical in developing artificial systems in which concentration gradients can be effectively generated using (solar) light energy.


Introduction
2][3] Release of protons to the extracellular medium and reprotonation from the cytoplasm creates a concentration gradient, which serves as energy reservoir to produce ATP.Artificial systems thatto some extent imitate such function [4][5][6] may provide new perspectives on solar energy conversion, and could shed light on fundamental aspects of biological pumps.3][14][15][16][17][18] However, directional translocation of small substrates, including ions, has not been achieved to date, while it would be of particular interest in the context of developing artificial molecular pumps. 17,18It is worth to note that pH-and photoswitchable scaffolds have been used for intramolecular transport of small-molecule cargo, 19,20 albeit without any repetitive, autonomous operation.
We became interested in transforming hemi-indigo into a pH-sensitive molecular switch for several reasons.Earlier studies had shown that incorporation of a pyrrolic ring can energetically favor the otherwise metastable E isomer owing to formation of an intramolecular hydrogen bond. 21,22Furthermore, while hemi-indigo recently regained interest as potential photoswitch, 23 its pyridyl derivative was found to not undergo photoisomerization, 21,22 which is likely due to excited state intramolecular proton transfer (ESIPT) as has been demonstrated for indigo. 24Likewise, for the E isomer of some structurally related indole-containing hemithioindigo derivatives, which are stabilized by intramolecular hydrogen bonding, 25,26 E→Z photoisomerization was inhibited, which was also ascribed to ESIPT. 27Based on these findings, we hypothesized first that protonation of N-heterocyclic hemi-indigo would induce thermal isomerization over the double bond by disruption and (re-)formation of a stabilizing intramol- As the forward isomerization takes place in the protonated state and the backwards isomerization occurs after deprotonation, protons are displaced effectively (from bottom to top, with respect to hemi-indigo) under continuous illumination using a single wavelength of light.
Here, we demonstrate unique protonation-controlled isomerization behavior for N-heterocyclic hemi-indigos 1-6 (Fig. 1a).Almost quantitative conversion from Z to E isomer and vice versa is induced by addition of acid and base, respectively.This conversion is strongly accelerated by visible-light irradiation.More remarkably, the forward Z→E photoisomerization takes place only when protonated, while the reverse E→Z photoisomerization occurs in the neutral state as is shown for all six derivatives.Such behavior is to our best knowledge unprecedented for molecular photoswitches, as usually a dynamic photoequilibrium is established under continuous irradiation (using a wavelength at which both isomers absorb).Since in the presence of acid, the neutral and protonated species are in equilibrium, they are interchanged photochemically in a specific order under irradiation with a single wavelength of light as is depicted in Fig. 1b.We came to the realization that by maintaining a constant flux through the cycle, associated protons are being displaced in one direction (with respect to the hemi-indigo scaffold), where the system operates by an information ratchet mechanism. 30,31

Results and discussion
Energy Minimization by DFT First, DFT calculations on possible isomers of hemi-indigo pyridyl derivative 1 were carried out (Supplementary section 10).For the E and the Z isomers, in their neutral and protonated states, two possible rotational isomers were considered.The energetically favored structures are depicted in Fig. 2 alongside the relative energies.In the neutral form there is a substantial thermodynamic preference for the Z over the E isomer (G = 32.9kJ mol -1 ) whereas, in the protonated state, the E isomer is considerably lower in energy (G = -30.5 kJ mol -1 ).These large energy differences can be mainly ascribed to stabilization by intramolecular hydrogen bonding.That is, in (Z)-1 a hydrogen bond is formed between the pyridyl nitrogen and the N-

Synthesis and Crystallographic Analysis
Encouraged by the computational results, we synthesized hemi-indigo 1 and its derivatives 2-6 containing different N-heterocycles (Fig. 1a).The imidazole rings in compounds 5 and 6 were methylated to avoid tautomerization.These derivatives were accessed through a condensation reaction of commercially available indoxyl acetate with the respective aldehydes (Supplementary section 1). 32,23The desired products were obtained in good yields (65-82%) as the thermodynamically most stable Z isomers (Z/E ratio >99%).Importantly, the 1 H NMR spectra recorded in CDCl3 revealed downfield-shifted NH-signals for all compounds ( = 10.47-9.14 ppm) with respect to parent hemi-indigo containing a phenyl instead of the N-heterocyclic ring (7,  = 6.82 ppm, Supplementary section 2), which indicates involvement of the NH proton in hydrogen bonding with the nearest nitrogen atom of the N-heterocycles.
All products, except quinolyl derivative 4, were additionally characterized by single crystal X-ray crystallography (Supplementary section 3).The solid-state structures are (as expected) of the energetically most stable Z isomers.The structural similarity between these Z isomers is high, i.e., the torsional angles between 3-oxindole and N-heterocycle moieties are all below 3° and the N(H) … N bond distances are found between 2.72-2.86Å, which is within hydrogen bond range (Supplementary Table 6).The presence of a stabilizing intramolecular hydrogen bond is thus supported in all cases.Important to note is that for (Z)-1 this hydrogen bond distance (2.82 Å) is similar to the one in the DFT calculated structure (2.81 Å).

Acid-Induced Isomerization
The effect of incremental addition of acid (TFA) was monitored by 1 H NMR spectroscopy in CDCl3 for (Z)-1.It resulted in downfield shifts of all pyridyl proton signals, indicative of protonation (Fig. 3 and Supplementary section 4).When a solution containing excess TFA (32 equiv.) was then left to equilibrate, the 1 H NMR signals for the protonated species (Z)-1 .H + disappeared and a new set of signals emerged (Fig. 3 and Supplementary section 5).A NOEdiff. experiment revealed a through-space interaction between the olefinic proton (Hf) and the 3-oxindole NH-proton (He) (Supplementary Fig. 37), supporting formation of the E isomer.Furthermore, the NOESY spectrum of (E)-1 .H + indicated that the olefinic proton (Hf) is in close proximity to an N-heterocyclic proton (Hj), while no coupling was observed with the pyridinium proton (Hk) (Supplementary Fig. 38).Very likely, hydrogen bonding between the pyridinium and carbonyl group leads to a fixed orientation of the N-heterocycle as was predicted by DFT calculations.Gratifyingly, within a day, nearly quantitative isomerization to the E isomer was observed, i.e., a mixture enriched to 95% in (E)-1 .H + was obtained at thermal equilibrium.When the amount of TFA was lowered, however, the E/Z ratio decreased (Supplementary Figs.23-24), even while protonation of 1 was established.A possible explanation was sought in pyridinium-trifluoroacetate ion-pair formation (Supplementary section 4), which could compete with the stabilizing intramolecular hydrogen bond formation.This ionpair is proposed to be solvated in the presence of larger amounts of TFA, 33,34 explaining the difference in conversion to the E isomer.Further, increasing the amount of TFA accelerated isomerization (Supplementary section 5-6), hinting at an acid-catalyzed process.[37] The Z isomer could be regained by basification of the solution.When Et3N was added (48 equiv.) to an equilibrated sample of 1 in the presence of TFA (32 equiv.),most pyridyl proton signals now shifted back upfield (Hg, Hh, Hi, Fig. 4 and Supplementary Fig. 26), indicative of deprotonation.Over the course of several days, (E)-1 fully isomerized back to (Z)-1, hence completing the thermal acid/base-controlled switching cycle as outlined in Fig. 1b.
For hemi-indigos 2-6, the thermal isomerization behavior was comparable (Supplementary Table 7).The dimethylamino-and bromo-substituted pyridyl derivatives 2 and 3, as well as the quinolyl derivative 4, showed identically high conversion to the E isomer when protonated (>95%), while differences in isomerization rates among the different compounds were small.For imidazole-based derivatives 5 and 6, conversion to the E isomer was lower (85% and 88%, respectively), and while the former compound exhibited the slowest isomerization in the series, this process was the fastest for the latter.Where all compounds displayed quantitative isomerization back to their Z isomer after neutralization of the solution by Et3N addition, in this case large variations were observed in the E→Z isomerization rates (Supplementary Table 7).
It is worth noting that for structurally related hemi-thioindigo, Hammett analysis indicated that electron-donating para-substituents can lower the thermal isomerization barrier, most likely because of the increase in donor-acceptor character. 38A similar effect may play a role here and, to summarize, the choice of N-heterocyclic ring can thus be used to tune the thermal isomerization rates.(400 MHz) of (Z)-1 (5.7 mM in CDCl3) before and after addition of TFA (32 equiv.) to give (Z)-1 .H + , followed by flame-sealing of the NMR tube and thermal equilibration at rt for 3 days to afford (E)-1 .H + , opening of the NMR tube with a diamond glass file and addition of Et3N (48 equiv.) to give (E)-1, and flame-sealing of the NMR tube and thermal equilibration at rt for 9 days to reobtain (Z)-1.
Protonation and thermal E/Z isomerization were accompanied by distinct color changes and were additionally monitored using UV-vis spectroscopy (Fig. 4a-b and Supplementary section 7).All compounds absorbed in the visible region having absorption maxima between 478-508 nm.In the case of pyridyl derivative (Z)-1, addition of TFA was accompanied by a large bathochromic shift (max = 482 to 504 nm).When the solution was allowed to equilibrate, the absorption shifted to longer wavelength (max = 549 nm) and decreased in intensity.This redshifted absorption is in agreement with time-dependent DFT calculations for (Z)-1 .H + (max = 505 nm) and (E)-1 .H + (max = 554 nm) and thus, supportive of E→Z isomerization (Supplementary section 10).Subsequent addition of excess Et3N to basify the solution led to a hypsochromic shift (max = 489 nm) and over time, the original UV-vis spectrum was recovered, demonstrating reversibility of the protonation-induced isomerization.
For hemi-indigos 2-4, similar shifts were observed upon TFA addition, whereas the imidazole-based derivatives 5 and 6 mainly showed a decrease in absorptivity with only a minimal change in absorption maximum.Nevertheless, in all cases, Z→E isomerization was accompanied by a large bathochromic shift and clear isosbestic points were observed, while basification caused hypsochromic shifts.a solution of (Z)-1 (0.76 mM in degassed CHCl3, 1 mm quartz cuvette) before and after addition of excess TFA (4.3 × 10 2 equiv.),followed by equilibration at rt for 15 h, treatment with Et3N (1.6 equiv. with respect to TFA), and equilibration at rt for 24 h.Please note that direct addition of TFA and Et3N to the same solution of (Z)-1 also results in a slight decrease in molar absorptivity as is observed here after the full switching cycle has been completed (Supplementary Fig. S53).c, UV-vis spectra starting with (Z)-1 (0.076 mM in degassed CHCl3, 1 cm quartz cuvette) where irradiation with 365 nm, 385 nm, 455 nm, 465 nm, 525 nm, for 2-10 min did not cause any spectral changes, yet after addition of TFA (5.5 μL, 4.7 × 10 2 equiv.)and irradiation with 455 nm the absorption shifted bathochromically.

Monodirectional Photoisomerization Cycle
In line with reported data, 21,22 exposure of a solution of (Z)-1 to various wavelengths of light did not cause any UV-vis spectral changes (Supplementary section 8), which confirms that no isomerization takes place.Remarkably, after addition of TFA, irradiation at 455 nm afforded a bathochromically shifted absorbance profile similar to the one observed for the thermally generated (E)-1 .H + species (Fig. 4c).It is important to note that the irradiation studies were performed at lower temperature (≤ 0 °C) and higher dilution than the isomerization experiments described above to suppress the thermally activated process.The spectral changes thus reveal that photoisomerization can occur in the protonated state.Likewise, in the 1 H NMR spectrum, the same set of signals that was observed upon acid-induced thermal isomerization appeared after irradiation of a solution of Z isomer in presence of TFA, supporting that the E isomer can be generated photochemically upon protonation (Supplementary section 9).Interestingly, when a sample of (E)-1 .H + (generated using 455 nm light) was subsequently exposed to other wavelengths, including those at which the (Z)-1 .H + species does not absorb (>625 nm), no further spectral changes were noted.This suggests that backwards photoisomerization is inhibited, presumably through an ESIPT reaction, as was similarly reported for (Z)-1 21,22 and the E isomer of an indole-derived hemi-thioindigo. 27Nevertheless, after addition of excess Et3N, now giving the absorption spectrum characteristic for (E)-1 (Fig. 4c), full isomerization back to original (Z)-1 was achieved upon irradiation at 455 nm.Also here, thermal isomerization was negligible under the experimental conditions used.
These results confirm that Z→E isomerization is enabled in the protonated form, while the reverse isomerization process takes place in the neutral form.To our best knowledge, such a feature has not been observed in photoswitchable systems before.It is also fundamentally different from earlier reported examples of acid-gated photoresponsivity, 37,[39][40][41] which allowed both switching directions in one of the states.In our case, only a single (and opposite for neutral and protonated forms) direction of photoswitching is suppressed, affording quantitative conversion by default.It leads to a unique monodirectional cycle of interconversion between species under continuous illumination once a protonation equilibrium is established.

Conclusions
In summary, unprecedented photo-and thermal isomerization behavior was discovered for Nheterocyclic hemi-indigos.Disruption and (re-)formation of intramolecular hydrogen bonding interactions were found to be of major importance.It must be recognized that beside hydrazone, 28 these compounds represent the second family of switches that undergo pH-activated double bond isomerization.More striking though, is the observed protonation-controlled oneway photoisomerization, giving rise to an exceptional monodirectional interconversion cycle.
Unidirectional translocation of associated protons (with respect to the hemi-indigo scaffold) takes place during this cycle.Future efforts in our lab will focus on immobilization in porous materials and membranes of these N-heterocyclic hemi-indigos, in addition to time-resolved spectroscopic studies.Furthermore, we envision that the selective inhibition of either forwards or backwards photoisomerization paths by binding of a substrate 42 can be applied as a general approach for directional transport and pumping of that substrate.This is a tantalizing prospect, as it would give the ability to effectively generate concentration gradients in artificial systems using (solar) light as the energy source.

General procedure for the synthesis of hemi-indigo dyes 1-7
Based on previously reported literature procedures, 23,32 1H-indol-3-yl acetate (50 mg, 0.29 mmol) was suspended in 1.6 mL degassed 1.5 M aqueous NaOH solution (purged with N2 for 30 min or subjected to 3 freeze-pump-thaw cycles) under Ar atmosphere.The mixture was heated to 100 °C for 15 min, after which the resulting greenish solution was cooled to 0 °C and the respective aldehyde (0.29 mmol) in MeOH (0.3 mL) was added.The reaction mixture was stirred at room temperature for 3-5 days at rt, diluted with 5 mL water, and extracted with EtOAc (3 × 15 mL).The combined organic layers were dried over Na2SO4 and concentrated.
Purification by flash chromatography (FC) yielded the desired hemi-indigo product.

H
atom of the 3-oxindole fragment [N(H) … N distance = 2.81 Å].In the protonated form, the pyridinium nitrogen rotates away from the N-H hydrogen bond donor and now a stabilizing hydrogen bond can only be formed in the E isomer, between the pyridinium proton and the carbonyl oxygen atom [N(H) … O distance = 2.64 Å].

Fig. 2 |
Fig. 2 | DFT-minimized geometries and plots of the relative Gibbs free energies.The most stable

Fig. 3 |
Fig. 3 | Isomer interconversion controlled by acid/base.Selected regions in the 1 H NMR spectrum