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Activity Control of a Synthetic Transporter by Photodynamic Modulation of Membrane Mobility and Incorporation
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Cite this: J. Am. Chem. Soc. 2024, 146, 45, 31085–31093
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https://doi.org/10.1021/jacs.4c10952
Published November 1, 2024

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Abstract

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Artificial transmembrane transport systems are receiving a great deal of attention for their potential therapeutic application. A major challenge is to switch their activity in response to environmental stimuli, which has been achieved mostly by modulating the binding affinity. We demonstrate here that the activity of a synthetic anion transporter can be controlled through changes in the membrane mobility and incorporation. The transporters─equipped with azobenzene photoswitches─poorly incorporate into the bilayer membrane as their thermally stable (E,E,E)-isomers, but incorporation is triggered by UV irradiation to give the (Z)-containing isomers. The latter isomers, however, are found to have a lower mobility and are therefore the least active transporters. This opposite effect of E-Z isomerization on transport capability offers unique photocontrol as is demonstrated by in situ irradiation studies during the used transport assays. These results help to understand the behavior of artificial transporters in a bilayer and are highly important to future designs, with new modes of biological activity and with the possibility to direct motion, which may be crucial toward achieving active transport.

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Introduction

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Membrane-embedded proteins are essential for many important cellular functions. (1) Among them are transporters, which mediate the passage of ions and solutes across the lipid bilayer, and are involved in processes such as signal transduction, ion homeostasis, and regulation of osmotic pressure. Many of these proteins are able to switch between high- and low-affinity modes─as a means to control their transport activity─in response to environmental stimuli. (2) Other proteins are known to undergo changes in mobility as a way to control their biological function. Such mobility changes are often caused by local fluctuations in lipid composition and can also be due to protein aggregation and/or shape changes. (3)
Over the past decades, much effort has been devoted to the development of artificial anion transport systems, (4) owing to their therapeutic potential. (5) It is of interest to make such systems capable of responding to environmental stimuli, (6) for example, to allow local (de)activation. The predominant strategy to achieve this is based on (dynamic) control of binding affinity using pH change, (7) light, (8) or redox agents, (9) where light has the benefit that it is noninvasive. Another frequently used approach is to influence membrane partitioning via light- or chemically cleavable water-soluble groups that are installed to known anion transporters. (10) In addition, the group of Langton recently showed that photocleavage of a membrane-anchoring group (i.e., long alkyl chains) leads to activation of anion transport. (11) Cleavage of these groups, however, is an irreversible process. So far, to the best of our knowledge, there have been no designs of photodynamic transporters that deliberately target control of membrane mobility and incorporation, while such control could enable directed motion and would offer an alternative approach to alter biological properties.
To fill this gap, we considered functionalizing tren-based tris-thiourea transporters [tren = tris(2-aminoethyl)amine], which were previously developed by Gale and co-workers (12) (and for which the transport mechanism is well understood) with azobenzene photoswitches. In such a transporter, the impact of E/Z isomerization on the binding properties would be minimal. Conversely, owing to the large change in dipole moment of azobenzene upon isomerization, and resultantly a large difference in solubility between the E and the Z-isomer, (13) membrane mobility and incorporation were expected to be largely influenced. An additional benefit of the tren-based scaffold is that it allows appendage of up to three azobenzene groups to enhance these expected effects.
Herein, we describe the azobenzene-appended tren-based tris-thiourea transporters 1 and 2 (Scheme 1A), whose isomers can be interconverted by UV and visible light. The half-lives of the photogenerated, metastable state of these two compounds are different. That is, the thiourea substitution in the benzylic position in 2 is known to afford higher thermal stability of azobenzene as compared to the direct substitution in 1. We show here that the photogenerated Z-containing isomers of these transporters are better incorporated into the lipid bilayer membrane, while their mobility─and with that their transport activity─is lower than the corresponding (E,E,E)-isomers (Scheme 1B). These opposing effects of isomerization on the transport capability are shown to give unprecedented control of the transport process. Furthermore, the observed dynamic modulation of membrane mobility will be key toward directed motion and to achieve active transport in the future.

Scheme 1

Scheme 1. Photoisomerization of Azobenzene-Based Tris-Thioureas 1 and 2 (A) and Schematic Representation of the Photocontrol over Membrane Incorporation and Mobility (B)
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Results and Discussion

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Synthesis and Isomerization Behavior

Transporters 1 and 2 were synthesized in one step by reacting previously reported tris(2-isothiocyanatoethyl)amine (14) with commercially available 4-aminoazobenzene and known 4-aminomethylazobenzene, (15) respectively. After purification by column chromatography, followed by recrystallization, the compounds were isolated exclusively as their (E,E,E)-isomers (see the Supporting Information for synthetic details and characterization). For compound 1, single crystals suitable for X-ray structure determination were obtained from a mixture of CHCl3/MeOH. The solid-state structure, depicted in Figure 1, further confirms the isolation of the (E,E,E)-isomer. Interestingly, it shows involvement of two of the thiourea groups in intramolecular hydrogen bonding with the sulfur atom of a neighboring thiourea group [N(H) ··· S distance: 3.5585(12)–3.3622(13) Å].

Figure 1

Figure 1. Side view (A) and top view (B) of (E,E,E)-1 as found in the crystal structure, shown in stick representation. Disorder in the azobenzene moieties was omitted for clarity.

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Next, the photoswitching properties of the obtained compounds were investigated with UV–vis and 1H NMR spectroscopy. Early in our studies, we noted that mixing MeCN with DMSO increased the thermal stability of the photogenerated isomers of 1, when compared to DMSO alone, likely due to aggregation effects. (16) To avoid quick thermal decay, the switching behavior was characterized in DMSO/MeCN (1:1 v/v). In this mixture, tren-based tris-thiourea (E,E,E)-1 displayed an absorption maximum around 375 nm (Figure 2A). Upon irradiation with 385 nm light, this maximum decreased, while a new absorption maximum appeared at λ = 450 nm, characteristic of E-to-Z isomerization. An isosbestic point was observed at around λ = 301 and 440 nm (Figure S5), which indicates independent switching of the azobenzene units. In the dark, the original spectrum of the (E,E,E)-isomer was regained as a result of thermally promoted back isomerization, and a half-life of 49 min at 293 K was determined for the photogenerated isomers (Figure S6).

Figure 2

Figure 2. UV–vis spectral changes measured at 293 K of (E,E,E)-1 (A) and (E,E,E)-2 (B) in DMSO/MeCN (1:1 v/v, 1 × 10–5 m) upon irradiation with 385, 365 or 455 nm light, as well as upon thermal equilibration.

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Compared to (E,E,E)-1, the absorption of (E,E,E)-2 was blue-shifted and a maximum was observed around λ = 330 nm (Figure 2B). Therefore, a shorter wavelength of light, i.e., 365 nm, was used here to promote E-to-Z isomerization, resulting again in a decrease of the overall absorption and the appearance of a new maximum around λ = 430 nm, where isosbestic points were observed at λ = 276 and 392 nm (Figure S7). As anticipated, the thermal Z-to-E isomerization was much slower in this case than that for 1. It was therefore followed at 333 K, and the half-life at this temperature was determined as 120 min (Figure S8). This back isomerization could be accelerated by irradiation with 455 nm light (Figures 2B and S9). In contrast to compound 1, at thermal equilibrium, the initial spectrum of the (E,E,E)-isomer was not fully recovered for compound 2, indicating that some of the Z-containing isomers remained present in solution. As usual for azobenzene, (13b) also 455 nm irradiation did not give full conversion back to the (E,E,E)-isomer, but led to the formation of a photostationary state (PSS) mixture.
These isomerization processes were then followed by 1H NMR spectroscopy in DMSO-d6/MeCN-d3 (1:1 v/v) in order to determine the PSS ratios. Irradiation of solutions containing either 1 or 2 with 385 and 365 nm light, respectively, led to the appearance of new sets of signals belonging to the Z-azobenzene-containing isomers (Figures S16 and S17). It should be noted that no separate sets of signals were observed for the possible different photogenerated isomers [i.e., (Z,Z,Z), (Z,E,E), (Z,Z,E)]. As by 1H NMR spectroscopy these isomers could thus not be distinguished, the overall E/Z ratio for the PSS mixtures was determined only (see Table 1). Nevertheless, by assuming that each azobenzene moiety switches independently, which is supported by the observation of clear isosbestic points (vide supra), we calculated that the PSSUV mixture for 1 and 2, from here on denoted as ZPSS, consists of at least 70% of the (Z,Z,Z)-isomer and less than 5% as the (Z,E,E)- or (E,E,E)-isomers (Figure S19). Finally, similar to what was observed in the UV–vis experiments, the initial signals of the (E,E,E)-isomer were recovered over time in the dark. In the case of 2, back isomerization was again also induced by irradiation with 455 nm light (see Figure S18 and see Table 1 for all PSS and thermal equilibrium ratios).
Table 1. Photoswitching, Chloride Binding, and Transport Properties of 1 and 2
compoundequil. ratio (E/Z)PSSUV (E/Z)PSSVis (E/Z)Ka(E,E,E)a (m–1)Ka(Zpss)a (m–1)EC50(Zpss)b (mol %)EC50(E,E,E)b (mol %)F (E/Z)d
1100:011:89n.d.2.7 × 102n.d.0.0100.0868.6
291:97:9377:232.9 × 1021800.037>10c>270
a

Determined by 1H NMR titrations using the tetrabutylammonium chloride salt in DMSO-d6/0.5%H2O; errors are estimated to be no more than 15%.

b

EC50 is defined as the effective concentration needed to reach 50% of the maximum activity at t = 360 s; values are reported in transporter-to-lipid molar ratio.

c

Poor transport activity prevented full Hill analysis.

d

Factor of enhancement in chloride transport activity between the (E,E,E)-isomer and (ZPSS)-isomers (F(E/Z) = EC50(E,E,E)/EC50(Zpss)); when EC50 > 10, a value of 10 was used in the calculation.

Binding and Transport Properties

We then determined the strength of chloride binding starting with the (E,E,E)-isomers using 1H NMR titrations in DMSO-d6/0.5% H2O. This solvent mixture was chosen in order to compare the binding constant to other tren-based tris-thioureas reported in the literature. (12b) A downfield shift in the thiourea NH signals was observed upon the stepwise addition of tetrabutylammonium chloride ([Bu4N]+[Cl]−), in addition to relatively small chemical shift changes in the aromatic and aliphatic signals (Figures S20–21). The titration data was fitted to a 1:1 binding model using HypNMR software (Figures S23–24), giving similar binding constants for (E,E,E)-1 and (E,E,E)-2 (Table 1). These constants are of the same order of magnitude as those reported previously for aromatically substituted tren-based tris-thiourea compounds (e.g., Ka = 191 m–1 for the analogue containing phenyl instead of azobenzene groups). (12b)
We then also determined the (apparent) binding affinity of the ZPSS mixture of 2. It should be noted that a titration using (ZPSS)-1 could not be performed due to fast thermal back isomerization in the solvent mixture used for titrations; however, its chloride binding affinity is expected to be similar to (ZPSS)-2. As also observed for the (E,E,E)-isomer, the addition of tetrabutylammonium chloride to (ZPSS)-2 led to a gradual downfield shift of the thiourea NH signals, alongside small changes in the aromatic and aliphatic signals (Figure S22). Fitting to a 1:1 binding model afforded a Ka value that shows minimal difference with (E,E,E)-2 (i.e., 1.6-fold, see Figure S25 and Table 1). As anticipated, photoswitching thus has only a small effect on the binding strength of the azobenzene-appended tren-based tris-thiourea transporter.
After having established that the binding affinity is minimally affected by photoswitching, we set out to investigate whether the isomers of 1 and 2 have distinct transmembrane chloride transport properties. Initial assays for screening were conducted with large unilamellar vesicles (LUVs) from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), loaded with the pH-sensitive dye 8-hydroxypyrene-1,3,6-trisulfonate (HPTS), and suspended in a NaCl solution buffered to pH 7.0 with HEPES. (17) After the addition of the compounds in DMSO/MeCN (1:1 v/v) to the vesicle solution, a pH gradient was applied across the membrane through addition of a base pulse (NaOH). The ability of the compounds to dissipate this pH gradient through H+/Cl– symport (or OH–/Cl– antiport) was then monitored via the change in HPTS emission.
In this HPTS assay, the photogenerated (ZPSS)-isomer mixture of both 1 and 2 proved to be effective in anion transport, whereas the corresponding (E,E,E)-isomers showed poor transport behavior when measured at the same transporter-to-lipid ratios (Figure 3A,D). The half-maximal effective concentrations (EC50, expressed in mol % with respect to lipids) were determined by Hill analysis (Figures 3B,E and S28–33, as well as Table 1), and showed much higher activity for the (ZPSS)-isomer mixtures than for the respective (E,E,E)-isomers. The largest enhancement upon irradiation was found for 2 (>270 times, see Table 1), while 1 proved to be the more active transporter. Owing to this marked difference in activity between isomers, it was possible to activate transport using either 1 or 2 in situ. That is, addition of a solution of the (E,E,E)-isomer, to the vesicle solution, followed by irradiation for 30 s, led to the enhancement of chloride efflux (Figure 3C,F).

Figure 3

Figure 3. Plots of chloride efflux against time facilitated by the (E,E,E)-isomers (blue line) and (ZPSS)-isomers (red line) of 1 (A) and 2 (D). Hill plots of activity versus concentration of the (E,E,E)-isomers (blue squares) and (ZPSS)-isomers (red circles) of 1 (B) and 2 (E). Change in chloride efflux upon irradiation of 0.1 mol % (E,E,E)-1 at 385 nm (C) and 1 mol % (E,E,E)-2 at 365 nm (F).

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Based on these results, the logical conclusion would be that the (ZPSS)-isomers are the more active transporters, with the in situ activation of transport as compelling evidence. Nevertheless, it assumes that both isomers, when postadded from DMSO/MeCN to the vesicle solution, are incorporated into the bilayer to the same extent. The Hill plots obtained for the (E,E,E)-isomers, however, show that full efflux is never reached, also not at the highest loadings, which can be an indication of poor incorporation. (8g,17a) Preincorporation of the transporters into the bilayer membrane, by hydrating a lipid film already containing the compounds to form the vesicles, would mitigate this issue. However, it requires the E/Z ratio to remain constant during the transport run; i.e., no significant thermal decay should occur. Alternatively, the PSS (E/Z) ratio could be kept constant by continuous irradiation during the experiment; however, since the HPTS dye absorbs UV light, irradiation with the wavelengths used for isomerization would additionally cause its excitation, leading to false results.
To check the thermal stability of the photogenerated Z-containing isomers in the lipid bilayer, we monitored the UV–vis absorption over time of vesicles that had 1 and 2 preincorporated (see Figures S10–S15). After photochemically induced isomerization, compound 2 did not show significant thermal isomerization during the measurement time frame (1 h). However, the half-life of 1 was drastically shortened (35 s) compared to what was measured in DMSO/MeCN solution (49 min). Such a difference in half-life is not uncommon for azobenzene as its thermal stability is known to be highly dependent on solvent polarity. (18) For the HPTS assay described above, this short half-life means that the (E,E,E)-isomer of 1 is regained at the onset of the experiment. To determine the true activity of the (ZPSS)-isomers of 1, it was, therefore, necessary to irradiate the vesicle solution continuously during the transport run.
As this could not be done in the HPTS assay due to excitation of the dye with the UV irradiation wavelength used to promote E/Z isomerization (vide supra), we turned to a cationophore-coupled osmotic response assay. (12e) For this assay, LUVs are created containing buffered KCl, suspended in potassium gluconate (KGlu) and, to maintain the charge balance, a transporter will mediate chloride efflux only in combination with a cationophore that can transport K+ ions. This net KCl efflux leads to shrinkage of the vesicle, which can be followed by changes in the 90° light scattering intensity. By evaluating the difference between coupling with the cationophores valinomycin (a selective K+ transporter) and monensin (a K+/H+ exchanger), it can be deduced if a transporter preferentially operates as an electroneutral Cl– transporter or as an H+/Cl– (or OH–/Cl–) cotransporter. (17)
We first verified that irradiation during the transport run using the osmotic assay does not lead to any undesired effects or damage to the vesicle integrity when compounds 1 and 2 are incorporated (Figures S34 and S35). Next, we addressed the potential issue with their incorporation into the membrane by comparing the (apparent) activity (in combination with valinomycin) between preincorporation and postaddition from DMSO/MeCN (1:1 v/v) at the same transporter-to-lipid ratio (Figure 4). Upon postaddition, as also observed in the HPTS assay, the (ZPSS)-isomers showed higher activity than the respective (E,E,E)-isomers. Yet, when preincorporated, the activity of both the (ZPSS)-isomers and (E,E,E)-isomers was enhanced. Strikingly, the difference in activity between these isomers was reversed compared to the postaddition studies, i.e., now the (E,E,E)-isomers turned out to be the most active transporters. This result clearly shows that the isomers of 1 and 2 have distinct membrane incorporation ability. In fact, the low transport rate observed for the (E,E,E)-isomers upon postaddition from organic solution must be due to very poor membrane incorporation.

Figure 4

Figure 4. Plots of chloride efflux over time of the isomers of 1 (A) and 2 (B) facilitated by preincorporated transporter compared with postaddition from DMSO-MeCN (1:1 v/v).

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It has been shown previously that the incorporation ability of a transporter relates to its lipophilicity, with more lipophilic transporters incorporating less well into the membrane. (19) To gain insight into the relative lipophilicity of the isomers of 2, we determined their retention time on a C18 RP-HPLC column (Table S1 and Figures S26–S27). Here, (E,E,E)-2 proved to be the most lipophilic isomer, showing a large difference in lipophilicity with the (Z,Z,Z)-isomer. Lipophilicity thus seems to be the major factor explaining the difference in membrane incorporation, although potential aggregation of the (E,E,E)-isomer could also limit its incorporation ability.
Preincorporation into the bilayer thus reveals that these (E,E,E)-isomers are actually more active transporters than the (ZPSS)-isomers. It is important to note that, without such preincorporation studies, the inherent transport ability of the (E,E,E)-isomers would have been overlooked and that the activity changes upon photoswitching would have been misinterpreted. To summarize, irradiation with UV light [to generate the (ZPSS)-isomers] triggers membrane incorporation but, on the other hand, switches the transporters to a less active state. Such an opposite effect on transport capability upon isomerization has, to our best knowledge, not been reported for photoactive transporters and enables a new level of control over transport (vide infra).
To quantify the difference in inherent transport activity and to gain insight into the preferred mechanism of transport, we again performed concentration-dependent (Hill) analysis, now using the osmotic assay and vesicles in which the transporters were preincorporated in the bilayer. When promoting operation as an electrogenic Cl– transporter through coupling with valinomycin, the (E,E,E)-isomers of 1 and 2 turned out to be up to 5.4 times more active than the (Zpss)-isomer mixture (Figures 5A,B and S36–S47, and Table 2). Surprisingly, when coupled with monensin allowing H+/Cl– symport (or OH–/Cl– antiport), virtually no difference in activity was observed as becomes very clear when comparing the concentration-dependent curves in Figure 5D,E.

Figure 5

Figure 5. Hill plots generated using the isomers of 1 and 2 in combination with valinomycin (A, B) and the corresponding mechanism (C), and in combination with monensin (D, E) and the corresponding mechanism (F).

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Table 2. Transport Properties as Determined Using the Osmotic Assay of 1 and 2
compoundEC50(Zpss)a Vln (mol %)EC50(E)a Vln (mol %)F (Z/E)bEC50(Zpss)a Mon (mol %)EC50(E)a Mon (mol %)F (Z/E)b
10.270.0495.440.0310.0330.95
21.130.502.250.510.570.89
a

EC50 defined as the effective concentration needed to reach 50% of the maximal activity at t = 658 s; values reported in transporter to lipid molar ratio.

b

Factor of enhancement in chloride transport activity between the (ZPSS)-isomer and (E)-isomers (F(Z/E) = EC50(Zpss)/EC50(E)).

This remarkable observation can be understood by taking a closer look at the rate-limiting steps in these two different transport mechanisms (Figure 5C,F). In the Cl– uniport, after binding an anion at the inner leaflet of the membrane, the complexed transporter moves to the outer leaflet where the anion is released. The free transporter subsequently has to move back to the inner leaflet to pick up another anion and repeat the same cycle (Steps 1–4 in Figure 5C). For H+/Cl– symport (or OH–/Cl– antiport), the initial step is the same; however, additionally, the transport of a proton needs to be facilitated. For these types of transporters based on the tren scaffold, this proton transport is known to occur either through thiourea (de)protonation or by a fatty acid-assisted pathway. (12e) In the case of the latter, fatty acids present in the lipid bilayer cannot repetitively transfer protons on their own, as in their carboxylate form (after deprotonation) they are not able to diffuse through the bilayer. Nevertheless, when a transporter binds the carboxylate headgroup, it can mediate the diffusion (flip-flop) of the fatty acid, resulting in net proton transport (Steps 5–10 in Figure 5F). Fatty acids are present as impurities in commercially available POPC, (20) and several tren-based anion transporters have been shown to be most efficient when operating through such a fatty acid-assisted pathway. (12k) These transporters were shown to not diffuse well through the membrane when in unbound form, yet, binding with a fatty acid gives a complex that has increased interactions with the phospholipid tails and therefore diffuses easier. (12k) Hence, we investigated the influence of fatty acid-assisted proton transport for (E,E,E)-1 and (E,E,E)-2, by either removal of fatty acids using BSA treatment or external addition of oleic acid (Figures S48–51). As can be expected based on the proposed mechanism, fatty acid concentration-dependent activity was observed when coupling to monensin but not when using valinomycin. Furthermore, the use of monensin and large amounts of fatty acid resulted in the highest transport activity, which reveals that during H+/Cl– symport diffusion and formation of the fatty acid-transporter complex across the membrane is rate-limiting (steps 5–7).
Coming back to the Cl– uniport process, here the transporter needs to move back to the inner leaflet in the unbound form. Since less activity was observed as compared to the fatty acid-assisted pathway described above, diffusion of the free transporter through the membrane must be the rate-limiting step (step 4). As in this case the (ZPSS)-isomers showed less activity than the (E,E,E)-isomers, and diffusion of their unbound form is rate limiting, it can be concluded that the former isomers have lower mobility in the bilayer than the latter isomers. The observed activity difference upon isomerization can thus be ascribed to a mobility change. To the best of our knowledge, this represents the first example in which reversible control over the mobility of a transporter is demonstrated.

Single- and Dual-Wavelength Control

While the (E,E,E)-isomers are thus the more active transporters owing to their higher mobility, they do not incorporate well into the bilayer. Therefore, no transport is observed once they have been postadded to the vesicle solution. Yet, their incorporation into the membrane can be triggered by UV irradiation to give the (ZPSS)-isomers, which are the least active transporters. As a result, the activation of transmembrane transport becomes a two-step process, which we set out to demonstrate by performing in situ irradiation studies in the osmotic assay. For example, when (E,E,E)-1 was postadded to a vesicle solution, initially no significant transport activity was observed due to the poor incorporation into the membrane (Figures 6A and S52). Irradiation with 385 nm light did not immediately lead to an appreciable increase in activity; however, when irradiation was halted, the transport process started. This is explained by the fact that, while promoting membrane incorporation, UV irradiation produces the less mobile (ZPSS)-form and therefore, not until it isomerizes back to (E,E,E)-1 in the dark, significant chloride efflux is observed. During irradiation, the transport process is thus suppressed, either at the beginning of the run or in a later stage, by exposing the solution to the same wavelength that was initially used to promote membrane incorporation. Using the same light source, the transport process can thus be initiated (to trigger membrane incorporation) as well as deactivated (to reduce the transport activity), which is a unique feature.

Figure 6

Figure 6. Plots of electrogenic Cl– transport against time facilitated by 0.02 mol % (E,E,E)-1 (A) and 1 mol % dark-adapted 2 (B) in combination with valinomycin, and activation by in situ irradiation. The shaded area denotes the irradiation time span using the mentioned wavelengths. For control experiments without irradiation or only a single irradiation step, see Figures S52 and S53.

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In addition, to achieve higher spatiotemporal precision, it would be beneficial to activate transport with two separate wavelengths, which can be achieved with transporter 2. As this compound does not rapidly isomerize back to the (E,E,E)-isomer in the dark, visible light (455 nm) irradiation is needed to generate the more mobile (E,E,E)-isomer and activate transport, after the UV irradiation step that promotes incorporation (Figures 6B and S53). These in situ experiments nicely illustrate control of transport by altering both membrane incorporation ability and mobility, using one or two irradiation wavelengths.

Conclusions

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In conclusion, functionalization of tren-based tripodal tris-thiourea with azobenzene photoswitches enables the dynamic modulation of transport activity. We showed that light- and thermally induced isomerization causes only a minimal change in chloride binding affinity, while membrane mobility and incorporation are majorly affected. That is, the (E,E,E)-isomers poorly incorporate into the bilayer when postadded (from organic solvent) to the vesicle solution, while the (ZPSS)-isomers incorporate well. Yet, when preincorporated, the (E,E,E)-isomers turned out to be more active in transport than the respective (ZPSS)-isomers owing to a higher mobility. We demonstrated that these opposite effects of photoisomerization on transport capability offer unique possibilities to activate and deactivate transport in situ, either by using single- or dual-wavelength irradiation, depending on the thermal stability of the azobenzene motif. Importantly, this work demonstrates the first example of dynamic control over the mobility of a transporter, (21) instead of control over the usually targeted binding affinity. Our results will prove important in the future design and development of photoswitchable transport systems. We envision that mobility control will allow, for example, local (im)mobilization of synthetic transporters in membranes, with consequences for their biological functioning. In addition, it may enable directed motion and active transport, which will be the subject of future studies in our laboratory.

Data Availability

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The data that support the findings of this study are available within the manuscript and its Supporting Information. Crystallographic data can be obtained free of charge from https://www.ccdc.cam.ac.uk/ under CCDC deposition numbers.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.4c10952.

  • Experimental procedures for the synthesis of 1 and 2 and full characterization, 1H NMR and UV–vis studies, transport assays, and X-ray analysis (CCDC 2371925) can be found in the Supporting Information (PDF)

Accession Codes

Deposition Number 2371925 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via the joint Cambridge Crystallographic Data Centre (CCDC) and Fachinformationszentrum Karlsruhe Access Structures service.

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Author Information

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  • Corresponding Author
  • Authors
    • Jasper E. Bos - Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands
    • Maxime A. Siegler - Department of Chemistry, Johns Hopkins University, 3400 N. Charles St., Baltimore, Maryland 21218, United States;  Orcidhttps://orcid.org/0000-0003-4165-7810
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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The authors gratefully acknowledge financial support from the European Research Council (Starting Grant no. 802830 to S.J.W.) and the Dutch Research Council (NWO-ENW, Vidi Grant no. VI.Vidi.192.049 to S.J.W.).

References

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    Nature (London, United Kingdom) (2014), 507 (7490), 68-72CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    The NRT1/PTR family of proton-coupled transporters are responsible for nitrogen assimilation in eukaryotes and bacteria through the uptake of peptides. However, in most plant species members of this family have evolved to transport nitrate as well as addnl. secondary metabolites and hormones. In response to falling nitrate levels, NRT1.1 is phosphorylated on an intracellular threonine that switches the transporter from a low-affinity to high-affinity state. Here we present both the apo and nitrate-bound crystal structures of Arabidopsis thaliana NRT1.1, which together with in vitro binding and transport data identify a key role for His 356 in nitrate binding. Our data support a model whereby phosphorylation increases structural flexibility and in turn the rate of transport. Comparison with peptide transporters further reveals how the NRT1/PTR family has evolved to recognize diverse nitrogenous ligands, while maintaining elements of a conserved coupling mechanism within this superfamily of nutrient transporters.
    (b) Sun, J.; Bankston, J. R.; Payandeh, J.; Hinds, T. R.; Zagotta, W. N.; Zheng, N. Crystal Structure of the Plant Dual-Affinity Nitrate Transporter NRT1.1. Nature 2014, 507, 73– 77,  DOI: 10.1038/nature13074
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    2b
    Crystal structure of the plant dual-affinity nitrate transporter NRT1.1
    Sun, Ji; Bankston, John R.; Payandeh, Jian; Hinds, Thomas R.; Zagotta, William N.; Zheng, Ning
    Nature (London, United Kingdom) (2014), 507 (7490), 73-77CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Nitrate is a primary nutrient for plant growth, but its levels in soil can fluctuate by several orders of magnitude. Previous studies have identified Arabidopsis NRT1.1 as a dual-affinity nitrate transporter that can take up nitrate over a wide range of concns. The mode of action of NRT1.1 is controlled by phosphorylation of a key residue, Thr 101; however, how this post-translational modification switches the transporter between two affinity states remains unclear. Here we report the crystal structure of unphosphorylated NRT1.1, which reveals an unexpected homodimer in the inward-facing conformation. In this low-affinity state, the Thr 101 phosphorylation site is embedded in a pocket immediately adjacent to the dimer interface, linking the phosphorylation status of the transporter to its oligomeric state. Using a cell-based fluorescence resonance energy transfer assay, we show that functional NRT1.1 dimerizes in the cell membrane and that the phosphomimetic mutation of Thr 101 converts the protein into a monophasic high-affinity transporter by structurally decoupling the dimer. Together with analyses of the substrate transport tunnel, our results establish a phosphorylation-controlled dimerization switch that allows NRT1.1 to uptake nitrate with two distinct affinity modes.
  3. 3
    (a) Trimble, W. S.; Grinstein, S. Barriers to the Free Diffusion of Proteins and Lipids in the Plasma Membrane. J. Cell Biol. 2015, 208, 259– 271,  DOI: 10.1083/jcb.201410071
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    3a
    Barriers to the free diffusion of proteins and lipids in the plasma membrane
    Trimble, William S.; Grinstein, Sergio
    Journal of Cell Biology (2015), 208 (3), 259-271, 13 pp.CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)
    Biol. membranes segregate into specialized functional domains of distinct compn., which can persist for the entire life of the cell. How sepn. of their lipid and (glyco)protein components is generated and maintained is not well understood, but the existence of diffusional barriers has been proposed. Remarkably, the phys. nature of such barriers and the manner whereby they impede the free diffusion of mols. in the plane of the membrane has rarely been studied in depth. Moreover, alternative mechanisms capable of generating membrane inhomogeneity are often disregarded. Here we describe prototypical biol. systems where membrane segregation has been amply documented and discuss the role of diffusional barriers and other processes in the generation and maintenance of their structural and functional compartmentalization.
    (b) Heine, M.; Ciuraszkiewicz, A.; Voigt, A.; Heck, J.; Bikbaev, A. Surface Dynamics of Voltage-Gated Ion Channels. Channels 2016, 10, 267– 281
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    (c) Jacobson, K.; Liu, P.; Lagerholm, B. C. The Lateral Organization and Mobility of Plasma Membrane Components. Cell 2019, 177, 806– 819,  DOI: 10.1016/j.cell.2019.04.018
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    3c
    The Lateral Organization and Mobility of Plasma Membrane Components
    Jacobson, Ken; Liu, Ping; Lagerholm, B. Christoffer
    Cell (Cambridge, MA, United States) (2019), 177 (4), 806-819CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    A review. Over the last several decades, an impressive array of advanced microscopic and anal. tools, such as single-particle tracking and nanoscopic fluorescence correlation spectroscopy, has been applied to characterize the lateral organization and mobility of components in the plasma membrane. Such anal. can tell researchers about the local dynamic compn. and structure of membranes and is important for predicting the outcome of membrane-based reactions. However, owing to the unresolved complexity of the membrane and the structures peripheral to it, identification of the detailed mol. origin of the interactions that regulate the organization and mobility of the membrane has not proceeded quickly. This Perspective presents an overview of how cell-surface structure may give rise to the types of lateral mobility that are obsd. and some potentially fruitful future directions to elucidate the architecture of these structures in more mol. detail.
  4. 4
    (a) Davis, A. P.; Sheppard, D. N.; Smith, B. D. Development of Synthetic Membrane Transporters for Anions. Chem. Soc. Rev. 2007, 36, 348– 357,  DOI: 10.1039/B512651G
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    4a
    Development of synthetic membrane transporters for anions
    Davis, Anthony P.; Sheppard, David N.; Smith, Bradley D.
    Chemical Society Reviews (2007), 36 (2), 348-357CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    A review. The development of low-mol.-wt. anion transporters is an emerging topic in supramol. chem. The major focus of this tutorial review is on synthetic Cl- transport systems that operate in vesicle and cell membranes. The transporters alter transmembrane concn. gradients, and thus they have applications as reagents for cell biol. research and as potential chemotherapeutic agents. The mol. designs include monomol. channels, self-assembled channels, and mobile carriers. Also discussed are exptl. assays that measure transport rates across model bilayer membranes.
    (b) Davis, J. T.; Okunola, O.; Quesada, R. Recent Advances in the Transmembrane Transport of Anions. Chem. Soc. Rev. 2010, 39, 3843,  DOI: 10.1039/b926164h
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    4b
    Recent advances in the transmembrane transport of anions
    Davis, Jeffery T.; Okunola, Oluyomi; Quesada, Roberto
    Chemical Society Reviews (2010), 39 (10), 3843-3862CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    A review. Anions cannot diffuse passively through biol. membranes and membrane-bound proteins mainly govern the transmembrane movement of these charged species. The use of synthetic compds. that are able to facilitate the transmembrane transport of anions is a fascinating and burgeoning topic. The study of facilitated anion transport across lipid bilayers is an emerging field in supramol. and bioorg. chem. In this crit. review we describe the recent research progress in this area, focusing on literature published during the years 2007-2009. An overview of the assays that are used in the transmembrane transport of anions is also included (158 refs.).
    (c) Valkenier, H.; Davis, A. P. Making a Match for Valinomycin: Steroidal Scaffolds in the Design of Electroneutral, Electrogenic Anion Carriers. Acc. Chem. Res. 2013, 46, 2898– 2909,  DOI: 10.1021/ar4000345
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    4c
    Making a Match for Valinomycin: Steroidal Scaffolds in the Design of Electroneutral, Electrogenic Anion Carriers
    Valkenier, Hennie; Davis, Anthony P.
    Accounts of Chemical Research (2013), 46 (12), 2898-2909CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
    A review. The natural product Valinomycin is a well-known transmembrane cation carrier. Despite being uncharged, this mol. can ext. potassium ions from water without counterions and ferry them through a membrane interior. Because it only transports pos. ions, it is electrogenic, mediating a flow of charge across the membrane. Equivalent agents for anions would be valuable research tools and may have therapeutic applications, esp. in the treatment of "channelopathies" such as cystic fibrosis. However, no such mols. have been found in nature. In this Account, we describe our research toward synthetic and rationally designed "anti- Valinomycins". As our core approach to this problem, we used the steroid nucleus, provided by cholic acid, as a scaffold for the assembly of anion receptors. By positioning H-bond donors on this framework, esp. urea and thiourea groups in conformationally constrained axial positions, we created binding sites capable of exceptionally high affinities (up to 1011 M-1 for R4N+Cl- in chloroform). The extended hydrocarbon surface of the steroid helped to maintain compatibility with nonpolar media. When we tested these "cholapods" for chloride transport in vesicles, they provided the first evidence for electrogenic anion transport mediated by electroneutral org. carriers: in other words, they are the first authenticated anti-Valinomycins. They also proved active in live cells that we grew and assayed in an Ussing chamber. In subsequent work, we have shown that the cholapods can exhibit very high activities, with transport obsd. down to carrier/lipid ratios of 1:250 000. We also understand some of the effects of structure on the activity of these mols. For example, in most cases, powerful transporters also act as powerful receptors. On the other hand, some modifications which favor binding do not promote transport. We gained functional advantages by cyclizing the cholapod architecture, which encloses the anion binding site. We could also simplify the structure without compromising function. A steroid-inspired trans-decalin framework has proved highly effective and may lead to agents with practical advantages. Changing an ester side-chain in this system revealed a surprising effect, whereby increased length and/or lipophilicity resulted in substantially raised activity. Although much remains to be discovered about these anionophores, their high activities and intrinsic tuneabilities bode well for applications. In future work, we plan to develop and exploit these mols. as tools for biophys. research and to explore the possibility of useful biol. activity.
    (d) Akhtar, N.; Biswas, O.; Manna, D. Biological Applications of Synthetic Anion Transporters. Chem. Commun. 2020, 56, 14137– 14153,  DOI: 10.1039/D0CC05489E
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    4d
    Biological applications of synthetic anion transporters
    Akhtar, Nasim; Biswas, Oindrila; Manna, Debasis
    Chemical Communications (Cambridge, United Kingdom) (2020), 56 (91), 14137-14153CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    A review. The use of synthetic ion transporters for alteration of the concn. of ions across cell membranes has drawn attention from scientists over the last two decades. This ion transport property has been sensibly used to reduce the viability of cancer cells mainly due to the disruption of their ion homeostasis, leading to the perturbation of their abnormal pH gradient. The use of the proanionophore strategy has been recently adopted to increase cellular deliverability and reduce unwanted cytotoxicity towards normal cells. Meanwhile, various anionophores exhibiting non-toxic behavior in epithelial cells have shown a great propensity to be used as a putative treatment for channelopathies like cystic fibrosis. Anionophores with Cl- ion transport mediated antibacterial activities have also been successfully used against clin. relevant bacterial strains, many of which are tolerant towards com. antibiotics. Recent developments in the chloride ion transport mediated biol. activities of anionophores have been mainly discussed in this article. It also highlights other aspects, such as design criteria, targeted delivery options, and others, which can be useful for the further development of selective anionophores for specific biol. applications.
  5. 5
    (a) Ko, S.-K.; Kim, S. K.; Share, A.; Lynch, V. M.; Park, J.; Namkung, W.; Van Rossom, W.; Busschaert, N.; Gale, P. A.; Sessler, J. L.; Shin, I. Synthetic Ion Transporters Can Induce Apoptosis by Facilitating Chloride Anion Transport into Cells. Nat. Chem. 2014, 6, 885– 892,  DOI: 10.1038/nchem.2021
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    5a
    Synthetic ion transporters can induce apoptosis by facilitating chloride anion transport into cells
    Ko, Sung-Kyun; Kim, Sung Kuk; Share, Andrew; Lynch, Vincent M.; Park, Jinhong; Namkung, Wan; Van Rossom, Wim; Busschaert, Nathalie; Gale, Philip A.; Sessler, Jonathan L.; Shin, Injae
    Nature Chemistry (2014), 6 (10), 885-892CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
    Anion transporters based on small mols. have received attention as therapeutic agents because of their potential to disrupt cellular ion homeostasis. However, a direct correlation between a change in cellular chloride anion concn. and cytotoxicity has not been established for synthetic ion carriers. Here the authors show that two pyridine diamide-strapped calix[4]pyrroles induce coupled chloride anion and sodium cation transport in both liposomal models and cells, and promote cell death by increasing intracellular chloride and sodium ion concns. Removing either ion from the extracellular media or blocking natural sodium channels with amiloride prevents this effect. Cell expts. show that the ion transporters induce the sodium chloride influx, which leads to an increased concn. of reactive oxygen species, release of cytochrome c from the mitochondria and apoptosis via caspase activation. However, they do not activate the caspase-independent apoptotic pathway assocd. with the apoptosis-inducing factor. Ion transporters, therefore, represent an attractive approach for regulating cellular processes that are normally controlled tightly by homeostasis.
    (b) Li, H.; Valkenier, H.; Thorne, A. G.; Dias, C. M.; Cooper, J. A.; Kieffer, M.; Busschaert, N.; Gale, P. A.; Sheppard, D. N.; Davis, A. P. Anion Carriers as Potential Treatments for Cystic Fibrosis: Transport in Cystic Fibrosis Cells, and Additivity to Channel-Targeting Drugs. Chem. Sci. 2019, 10, 9663– 9672,  DOI: 10.1039/C9SC04242C
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    5b
    Anion carriers as potential treatments for cystic fibrosis: transport in cystic fibrosis cells, and additivity to channel-targeting drugs
    Li, Hongyu; Valkenier, Hennie; Thorne, Abigail G.; Dias, Christopher M.; Cooper, James A.; Kieffer, Marion; Busschaert, Nathalie; Gale, Philip A.; Sheppard, David N.; Davis, Anthony P.
    Chemical Science (2019), 10 (42), 9663-9672CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    Defective anion transport is a hallmark of the genetic disease cystic fibrosis (CF). One approach to restore anion transport to CF cells utilizes alternative pathways for transmembrane anion transport, including artificial anion carriers (anionophores). Here, we screened 22 anionophores for biol. activity using fluorescence emission from the halide-sensitive yellow fluorescent protein. Three compds. possessed anion transport activity similar to or greater than that of a bis-(p-nitrophenyl)ureidodecalin previously shown to have promising biol. activity. Anion transport by these anionophores was concn.-dependent and persistent. All four anionophores mediated anion transport in CF cells, and their activity was additive to rescue of the predominant disease-causing variant F508del-CFTR using the clin.-licensed drugs lumacaftor and ivacaftor. Toxicity was variable but minimal at the lower end. The results provide further evidence that anionophores, by themselves or together with other treatments that restore anion transport, offer a potential therapeutic strategy for CF.
    (c) Davis, J. T.; Gale, P. A.; Quesada, R. Advances in Anion Transport and Supramolecular Medicinal Chemistry. Chem. Soc. Rev. 2020, 49, 6056– 6086,  DOI: 10.1039/C9CS00662A
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    5c
    Advances in anion transport and supramolecular medicinal chemistry
    Davis, Jeffery T.; Gale, Philip A.; Quesada, Roberto
    Chemical Society Reviews (2020), 49 (16), 6056-6086CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    Advances in anion transport by synthetic supramol. systems are discussed in this article. Developments in the design of discrete mol. carriers for anions and supramol. anion channels are reviewed followed by an overview of the use of these systems in biol. systems as putative treatments for diseases such as cystic fibrosis and cancer.
  6. 6
    (a) Bickerton, L. E.; Johnson, T. G.; Kerckhoffs, A.; Langton, M. J. Supramolecular Chemistry in Lipid Bilayer Membranes. Chem. Sci. 2021, 12, 11252– 11274,  DOI: 10.1039/D1SC03545B
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    6a
    Supramolecular chemistry in lipid bilayer membranes
    Bickerton, Laura E.; Johnson, Toby G.; Kerckhoffs, Aidan; Langton, Matthew J.
    Chemical Science (2021), 12 (34), 11252-11274CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    Lipid bilayer membranes form compartments requisite for life. Interfacing supramol. systems, including receptors, catalysts, signal transducers and ion transporters, enables the function of the membrane to be controlled in artificial and living cellular compartments. In this perspective, we take stock of the current state of the art of this rapidly expanding field, and discuss prospects for the future in both fundamental science and applications in biol. and medicine.
    (b) de Jong, J.; Bos, J. E.; Wezenberg, S. J. Stimulus-Controlled Anion Binding and Transport by Synthetic Receptors. Chem. Rev. 2023, 123, 8530– 8574,  DOI: 10.1021/acs.chemrev.3c00039
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    6b
    Stimulus-Controlled Anion Binding and Transport by Synthetic Receptors
    de Jong, Jorn; Bos, Jasper E.; Wezenberg, Sander J.
    Chemical Reviews (Washington, DC, United States) (2023), 123 (13), 8530-8574CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. Anionic species are omnipresent and involved in many important biol. processes. A large no. of artificial anion receptors has therefore been developed. Some of these are capable of mediating transmembrane transport. However, where transport proteins can respond to stimuli in their surroundings, creation of synthetic receptors with stimuli-responsive functions poses a major challenge. Herein, we give a full overview of the stimulus-controlled anion receptors that have been developed thus far, including their application in membrane transport. In addn. to their potential operation as membrane carriers, the use of anion recognition motifs in forming responsive membrane-spanning channels is discussed. With this review article, we intend to increase interest in transmembrane transport among scientist working on host-guest complexes and dynamic functional systems in order to stimulate further developments.
  7. 7
    (a) Santacroce, P. V.; Davis, J. T.; Light, M. E.; Gale, P. A.; Iglesias-Sánchez, J. C.; Prados, P.; Quesada, R. Conformational Control of Transmembrane Cl - Transport. J. Am. Chem. Soc. 2007, 129, 1886– 1887,  DOI: 10.1021/ja068067v
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    7a
    Conformational Control of Transmembrane Cl- Transport
    Santacroce, Paul V.; Davis, Jeffery T.; Light, Mark E.; Gale, Philip A.; Iglesias-Sanchez, Jose Carlos; Prados, Pilar; Quesada, Roberto
    Journal of the American Chemical Society (2007), 129 (7), 1886-1887CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Preorganization of the isophthalamide cleft in N,N'-dibutyl-4,6-dihydroxyisophthalamide (2) by means of intramol. hydrogen bonds results in an enhanced anion receptor that displays significant chloride transport activity into EYPC liposomes. This function is easily switchable by controlling the pH.
    (b) Howe, E. N. W.; Busschaert, N.; Wu, X.; Berry, S. N.; Ho, J.; Light, M. E.; Czech, D. D.; Klein, H. A.; Kitchen, J. A.; Gale, P. A. pH-Regulated Nonelectrogenic Anion Transport by Phenylthiosemicarbazones. J. Am. Chem. Soc. 2016, 138, 8301– 8308,  DOI: 10.1021/jacs.6b04656
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    7b
    pH-Regulated Nonelectrogenic Anion Transport by Phenylthiosemicarbazones
    Howe, Ethan N. W.; Busschaert, Nathalie; Wu, Xin; Berry, Stuart N.; Ho, Junming; Light, Mark E.; Czech, Dawid D.; Klein, Harry A.; Kitchen, Jonathan A.; Gale, Philip A.
    Journal of the American Chemical Society (2016), 138 (26), 8301-8308CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Gated ion transport across biol. membranes is an intrinsic process regulated by protein channels. Synthetic anion carriers (anionophores) have potential applications in biol. research; however, previously reported examples are mostly nonspecific, capable of mediating both electrogenic and electroneutral (nonelectrogenic) transport processes. Here we show the transmembrane Cl- transport studies of synthetic phenylthiosemicarbazones mimicking the function of acid-sensing (proton-gated) ion channels. These anionophores have remarkable pH-switchable transport properties with up to 640-fold increase in transport efficacy on going from pH 7.2 to 4.0. This "gated" process is triggered by protonation of the imino nitrogen and concomitant conformational change of the anion-binding thiourea moiety from anti to syn. By using a combination of two cationophore-coupled transport assays, with either monensin or valinomycin, we have elucidated the fundamental transport mechanism of phenylthiosemicarbazones which is shown to be nonelectrogenic, inseparable H+/Cl- cotransport. This study demonstrates the first examples of pH-switchable nonelectrogenic anion transporters.
    (c) Shinde, S. V.; Talukdar, P. A Dimeric Bis(Melamine)-Substituted Bispidine for Efficient Transmembrane H+/Cl– Cotransport. Angew. Chem., Int. Ed. 2017, 56, 4238– 4242,  DOI: 10.1002/anie.201700803
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    7c
    A Dimeric Bis(melamine)-Substituted Bispidine for Efficient Transmembrane H+/Cl- Cotransport
    Shinde, Sopan Valiba; Talukdar, Pinaki
    Angewandte Chemie, International Edition (2017), 56 (15), 4238-4242CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A 3,7-diazabicyclo[3.3.1]nonane linking to two melamines is a unique transmembrane H+/Cl- carrier. In the solid state, the V-shaped compd. forms a HCl-bound zig-zag network through cooperative protonation and hydrogen bond interactions. In the lipid membrane, the receptor forms a dimeric self-assembly involving multiple H+ and Cl- leading to the efficient transport of the acid. The pH-dependent Cl- efflux obsd. for the compd. was rationalized based on a gradual protonation model that confers an active transmembrane carrier at physiol. pH.
  8. 8
    (a) Choi, Y. R.; Kim, G. C.; Jeon, H.-G.; Park, J.; Namkung, W.; Jeong, K.-S. Azobenzene-Based Chloride Transporters with Light-Controllable Activities. Chem. Commun. 2014, 50, 15305– 15308,  DOI: 10.1039/C4CC07560A
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    8a
    Azobenzene-based chloride transporters with light-controllable activities
    Choi, Ye Rin; Kim, Gyu Chan; Jeon, Hae-Geun; Park, Jinhong; Namkung, Wan; Jeong, Kyu-Sung
    Chemical Communications (Cambridge, United Kingdom) (2014), 50 (97), 15305-15308CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    Synthetic chloride transporters contg. two urea groups linked through a diazobenzene spacer have been prepd. and the trans-to-cis isomerization by light stimulation results in dramatic changes in the chloride transport activities across lipid and cell membranes.
    (b) Ahmad, M.; Metya, S.; Das, A.; Talukdar, P. A Sandwich Azobenzene–Diamide Dimer for Photoregulated Chloride Transport. Chem. - Eur. J. 2020, 26, 8703– 8708,  DOI: 10.1002/chem.202000400
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    8b
    A Sandwich Azobenzene-Diamide Dimer for Photoregulated Chloride Transport
    Ahmad, Manzoor; Metya, Surajit; Das, Aloke; Talukdar, Pinaki
    Chemistry - A European Journal (2020), 26 (40), 8703-8708CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    There has been a tremendous evolution for artificial ion transport systems, esp. gated synthetic systems, which closely mimic their natural congeners. Herein, we demonstrate a trans-azobenzene-based photoregulatory anionophoric system that transports chloride by forming a sandwich dimeric complex. Further studies confirmed a carrier-mediated chloride-anion antiport mechanism, and the supramol. interactions involved in chloride recognition within the sandwich complex were revealed from theor. studies. Reversible trans-cis photoisomerization of the azobenzene was achieved without any significant contribution from the thermal cis→trans isomerization at room temp. Photoregulatory transport activity across the lipid bilayer membrane inferred an outstanding off-on response of the azobenzene photoswitch.
    (c) Kerckhoffs, A.; Langton, M. J. Reversible Photo-Control over Transmembrane Anion Transport Using Visible-Light Responsive Supramolecular Carriers. Chem. Sci. 2020, 11, 6325– 6331,  DOI: 10.1039/D0SC02745F
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    8c
    Reversible photo-control over transmembrane anion transport using visible-light responsive supramolecular carriers
    Kerckhoffs, Aidan; Langton, Matthew J.
    Chemical Science (2020), 11 (24), 6325-6331CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    Ion transport across lipid bilayer membranes in biol. is controlled by membrane proteins, which in turn are regulated in response to chem.-, phys.- and photo-stimuli. The design of synthetic supramol. ion transporters able to be precisely controlled by external signals, in particular bio-compatible wavelengths of visible light, is key for achieving spatio-temporal control over function. Here we report two-color responsive mol. photo-switches that act as supramol. transmembrane anion carriers. Reversible switching of the photo-switch within the lipid bilayer membrane is achieved using biocompatible visible wavelengths of light, such that temporal control over transmembrane anion transport is achieved through alternating irradn. with red and blue light.
    (d) Ahmad, M.; Chattopadhayay, S.; Mondal, D.; Vijayakanth, T.; Talukdar, P. Stimuli-Responsive Anion Transport through Acylhydrazone-Based Synthetic Anionophores. Org. Lett. 2021, 23, 7319– 7324,  DOI: 10.1021/acs.orglett.1c02249
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    8d
    Stimuli-Responsive Anion Transport through Acylhydrazone-Based Synthetic Anionophores
    Ahmad, Manzoor; Chattopadhayay, Sandip; Mondal, Debashis; Vijayakanth, Thangavel; Talukdar, Pinaki
    Organic Letters (2021), 23 (19), 7319-7324CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)
    Photoswitchable acylhydrazone-based synthetic anionophores are reported. Single-crystal X-ray structure and 1H NMR titrn. studies confirmed the chloride binding in solid and soln. states. The ion transport activity of 1a was greatly attenuated through a phototriggered E to Z photoisomerization process, and the photoisomerized deactivated state showed high kinetic stability due to an intramol. hydrogen bond. Switchable "OFF-ON" transport activity was achieved by the application of light and acid-catalyzed reactivation process.
    (e) Kerckhoffs, A.; Bo, Z.; Penty, S. E.; Duarte, F.; Langton, M. J. Red-Shifted Tetra-Ortho-Halo-Azobenzenes for Photo-Regulated Transmembrane Anion Transport. Org. Biomol. Chem. 2021, 19, 9058– 9067,  DOI: 10.1039/D1OB01457A
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    8e
    Red-shifted tetra-ortho-halo-azobenzenes for photo-regulated transmembrane anion transport
    Kerckhoffs, Aidan; Bo, Zonghua; Penty, Samuel E.; Duarte, Fernanda; Langton, Matthew J.
    Organic & Biomolecular Chemistry (2021), 19 (41), 9058-9067CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    Photo-responsive synthetic ion transporters are of interest as tools for studying transmembrane transport processes and have potential applications as targeted therapeutics, due to the possibility of spatiotemporal control and wavelength-dependent function. Here we report the synthesis of novel sym. and non-sym. red-shifted tetra-ortho-chloro- and tetra-ortho-fluoro azobenzenes, bearing pendant amine functionality. Functionalisation of the photo-switchable scaffolds with squaramide hydrogen bond donors enabled the prepn. of a family of anion receptors, which act as photo-regulated transmembrane chloride transporters in response to green or red light. The subtle effects of chlorine/fluorine substitution, meta/para positioning of the anion receptors, and the use of more flexible linkers are explored. NMR titrn. expts. on the structurally diverse photo-switchable receptors reveal cooperative binding of chloride in the Z, but not E isomer, by the two squaramide binding sites. These results are supported by mol. dynamics simulations in explicit solvent and model membranes. We show that this intramol. anion recognition leads to effective switching of transport activity in lipid bilayer membranes, in which optimal Z isomer activity is achieved using a combination of fluorine substitution and para-methylene spacer units.
    (f) Wezenberg, S. J.; Chen, L.-J.; Bos, J. E.; Feringa, B. L.; Howe, E. N. W.; Wu, X.; Siegler, M. A.; Gale, P. A. Photomodulation of Transmembrane Transport and Potential by Stiff-Stilbene Based Bis(Thio)Ureas. J. Am. Chem. Soc. 2022, 144, 331– 338,  DOI: 10.1021/jacs.1c10034
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    8f
    Photomodulation of Transmembrane Transport and Potential by Stiff-Stilbene Based Bis(thio)ureas
    Wezenberg, Sander J.; Chen, Li-Jun; Bos, Jasper E.; Feringa, Ben L.; Howe, Ethan N. W.; Wu, Xin; Siegler, Maxime A.; Gale, Philip A.
    Journal of the American Chemical Society (2022), 144 (1), 331-338CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Membrane transport proteins fulfill important regulatory functions in biol. with a common trait being their ability to respond to stimuli in the environment. Various small-mol. receptors, capable of mediating transmembrane transport, have been successfully developed. However, to confer stimuli-responsiveness on them poses a fundamental challenge. Here we demonstrate photocontrol of transmembrane transport and elec. potential using bis(thio)ureas derived from stiff-stilbene. UV-vis and 1H NMR spectroscopy are used to monitor E-Z photoisomerization of these bis(thio)ureas and 1H NMR titrns. reveal stronger binding of chloride to the (Z)-form than to the (E)-form. Addnl. insight into the binding properties is provided by single crystal X-ray crystallog. anal. and DFT geometry optimization. Importantly, the (Z)-isomers are much more active in transmembrane transport than the resp. (E)-isomers as shown through various assays. As a result, both membrane transport and depolarization can be modulated upon irradn., opening up new prospects toward light-based therapeutics as well as physiol. and optopharmacol. tools for studying anion transport-assocd. diseases and to stimulate neuronal activity, resp.
    (g) Villarón, D.; Bos, J. E.; Kohl, F.; Mommer, S.; de Jong, J.; Wezenberg, S. J. Photoswitchable Bis(Amidopyrroles): Modulating Anion Transport Activity Independent of Binding Affinity. J. Org. Chem. 2023, 88, 11328– 11334,  DOI: 10.1021/acs.joc.3c01018
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  9. 9
    Docker, A.; Johnson, T. G.; Kuhn, H.; Zhang, Z.; Langton, M. J. Multistate Redox-Switchable Ion Transport Using Chalcogen-Bonding Anionophores. J. Am. Chem. Soc. 2023, 145, 2661– 2668,  DOI: 10.1021/jacs.2c12892
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    9
    Multistate Redox-Switchable Ion Transport Using Chalcogen-Bonding Anionophores
    Docker, Andrew; Johnson, Toby G.; Kuhn, Heike; Zhang, Zongyao; Langton, Matthew J.
    Journal of the American Chemical Society (2023), 145 (4), 2661-2668CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Synthetic supramol. transmembrane anionophores have emerged as promising anticancer chemotherapeutics. However, key to their targeted application is achieving spatiotemporally controlled activity. Herein, we report a series of chalcogen-bonding diaryl tellurium-based transporters in which their anion binding potency and anionophoric activity are controlled through reversible redox cycling between Te oxidn. states. This unprecedented in situ reversible multistate switching allows for switching between ON and OFF anion transport and is crucially achieved with biomimetic chem. redox couples.
  10. 10
    (a) Choi, Y. R.; Lee, B.; Park, J.; Namkung, W.; Jeong, K.-S. Enzyme-Responsive Procarriers Capable of Transporting Chloride Ions across Lipid and Cellular Membranes. J. Am. Chem. Soc. 2016, 138, 15319– 15322,  DOI: 10.1021/jacs.6b10592
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    10a
    Enzyme-Responsive Procarriers Capable of Transporting Chloride Ions across Lipid and Cellular Membranes
    Choi, Ye Rin; Lee, Bom; Park, Jinhong; Namkung, Wan; Jeong, Kyu-Sung
    Journal of the American Chemical Society (2016), 138 (47), 15319-15322CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Adopting the concept of procarrier for the first time, we demonstrated the controlled transport of chloride ions across lipid and cellular membranes. Procarriers contg. highly hydrophilic appendages were initially inactive due to the lack of their partitioning into lipophilic membranes but were activated to transport chloride ions in the presence of specific enzymes that were able to hydrolyze off the appendages to generate an active carrier under specific conditions. Namely, the procarrier with an ester-bond-linked appendage was most activated by an esterase (PLE) at pH = 7.4, whereas the procarrier with a glycosyl-bond-linked appendage was activated only by a glycosylase (AOG) under slightly acidic conditions (pH = 5.5-6). In addn. to controlling chloride transport, hydrophilic appendages greatly increase the water soly. of the procarrier, which may improve the deliverability of a hydrophobic active carrier into a plasma membrane.
    (b) Salunke, S. B.; Malla, J. A.; Talukdar, P. Phototriggered Release of a Transmembrane Chloride Carrier from an O-Nitrobenzyl-Linked Procarrier. Angew. Chem., Int. Ed. 2019, 58, 5354– 5358,  DOI: 10.1002/anie.201900869
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    10b
    Phototriggered Release of a Transmembrane Chloride Carrier from an o-Nitrobenzyl-Linked Procarrier
    Salunke, Swati Bansi; Malla, Javid Ahmad; Talukdar, Pinaki
    Angewandte Chemie, International Edition (2019), 58 (16), 5354-5358CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    While there have been many studies on synthetic chloride carriers and a recent application for apoptotic cell death, so far, the proposed huge potential of these systems in targeting cancer has not been realized due to their cytotoxicity to healthy cells. Herein, we describe the development of an indole-2-carboxamide receptor as an efficient membrane chloride carrier while the corresponding o-nitrobenzyl-linked deriv. is a procarrier of the ion. Photoirradn. of the procarrier in liposomes results in release of the active carrier with up to 90% transport efficiency. Such photorelease of the carrier also works within cancer cells, resulting in efficient cell killing. Such photocleavable procarriers have great potential as a photodynamic therapy to combat various types of cancers.
    (c) Akhtar, N.; Pradhan, N.; Saha, A.; Kumar, V.; Biswas, O.; Dey, S.; Shah, M.; Kumar, S.; Manna, D. Tuning the Solubility of Ionophores: Glutathione-Mediated Transport of Chloride Ions across Hydrophobic Membranes. Chem. Commun. 2019, 55, 8482– 8485,  DOI: 10.1039/C9CC04518J
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    10c
    Tuning the solubility of ionophores: glutathione-mediated transport of chloride ions across hydrophobic membranes
    Akhtar, Nasim; Pradhan, Nirmalya; Saha, Abhishek; Kumar, Vishnu; Biswas, Oindrila; Dey, Subhasis; Shah, Manisha; Kumar, Sachin; Manna, Debasis
    Chemical Communications (Cambridge, United Kingdom) (2019), 55 (58), 8482-8485CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    A stimuli-responsive anion transport strategy was successfully employed to regulate the water soly. of ionophores and promote controlled transport of Cl- ions across lipid bilayers. A sulfonium-based proanionophore was efficiently activated by glutathione to regenerate the active anionophore, which allowed controlled transport of Cl- ions in a cellular environment with lower cytotoxicity.
    (d) Fares, M.; Wu, X.; Ramesh, D.; Lewis, W.; Keller, P. A.; Howe, E. N. W.; Pérez-Tomás, R.; Gale, P. A. Stimuli-Responsive Cycloaurated “OFF-ON” Switchable Anion Transporters. Angew. Chem., Int. Ed. 2020, 59, 17614– 17621,  DOI: 10.1002/anie.202006392
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    10d
    Stimuli-Responsive Cycloaurated "OFF-ON" Switchable Anion Transporters
    Fares, Mohamed; Wu, Xin; Ramesh, Deepthi; Lewis, William; Keller, Paul A.; Howe, Ethan N. W.; Perez-Tomas, Ricardo; Gale, Philip A.
    Angewandte Chemie, International Edition (2020), 59 (40), 17614-17621CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Anion transporters have shown potential application as anti-cancer agents that function by disrupting homeostasis and triggering cell death. In this research article we report switchable anion transport by gold complexes of anion transporters that are "switched on" in situ in the presence of the reducing agent GSH by decomplexation of gold. GSH is found in higher concns. in tumors than in healthy tissue and hence this approach offers a strategy to target these systems to tumors.
    (e) Park, G.; Gabbaï, F. P. Redox-Controlled Chalcogen and Pnictogen Bonding: The Case of a Sulfonium/Stibonium Dication as a Preanionophore for Chloride Anion Transport. Chem. Sci. 2020, 11, 10107– 10112,  DOI: 10.1039/D0SC04417B
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    10e
    Redox-controlled chalcogen and pnictogen bonding: the case of a sulfonium/stibonium dication as a preanionophore for chloride anion transport
    Park, Gyeongjin; Gabbai, Francois P.
    Chemical Science (2020), 11 (37), 10107-10112CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    Our interest in the chem. of tunable chalcogen and pnictogen bond donors as Lewis acidic platforms for the complexation and transport of anions has led us to investigate examples of such compds. that can be activated by redox events. Here, we describe the synthesis of [o-MePhS(C6H4)SbPh3]2+ ([3]2+) and [o-MePhS(C6H4)Sb(p-Tol)3]2+ ([4]2+), two dicationic stibonium/sulfonium bifunctional Lewis acids which were obtained by methylation of the phenylthioether derivs. [o-PhS(C6H4)SbPh3]+ ([1]+) and [o-PhS(C6H4)Sb(p-Tol)3]+ ([2]+), resp. An evaluation of the chloride anion transport properties of these derivs. using chloride-loaded POPC unilamellar vesicles shows that the activity of the monocations [1]+ and [2]+ greatly exceeds that of the dications [3]2+ and [4]2+, a phenomenon that we assign to the higher lipophilicity of the monocationic compds. Harnessing this large transport activity differential, we show that [4]2+ can be used as a prechloridophore that is readily activated by redn. of the sulfonium moiety. Indeed, [4]2+ reacts with GSH to afford [2]+ as an active transporter. This activation, which has been monitored in aq. soln., can also be carried out in situ, in the presence of the chloride-loaded POPC unilamellar vesicles.
    (f) Das, S.; Biswas, O.; Akhtar, N.; Patel, A.; Manna, D. Multi-Stimuli Controlled Release of a Transmembrane Chloride Ion Carrier from a Sulfonium-Linked Procarrier. Org. Biomol. Chem. 2020, 18, 9246– 9252,  DOI: 10.1039/D0OB00938E
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    10f
    Multi-stimuli controlled release of a transmembrane chloride ion carrier from a sulfonium-linked procarrier
    Das, Sribash; Biswas, Oindrila; Akhtar, Nasim; Patel, Anjali; Manna, Debasis
    Organic & Biomolecular Chemistry (2020), 18 (45), 9246-9252CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    In recent times, anion transporters have received substantial consideration due to their ability to disrupt the ionic equil. across membrane bilayers. While numerous Cl- ion transporters were developed for channelopathies, unfortunately, poor aq. soly. precluded their bioapplicability. Herein, we demonstrate the development of a multi-stimuli activatable anion transport approach to induce regulated transport of Cl- ions across membranes under specific conditions. The sulfonium-based procarrier was initially inactive, but the transmembrane transport of Cl- ions was activated in the presence of stimuli such as glutathione (GSH), reactive oxygen species (ROS) and light. The release of the hydrophobic anionophore from the aq.-sol. procarrier under specific conditions leads to the successful transport of Cl- ions. Under physiol. conditions, these anion carriers follow an antiport exchange mechanism to transport Cl- ions across lipid bilayers. Such multi-stimuli activatable procarriers have great potential to combat various types of channelopathies, including cancer, cystic fibrosis, kidney stones, myotonia, and others.
    (g) Akhtar, N.; Biswas, O.; Manna, D. Stimuli-Responsive Transmembrane Anion Transport by AIE-Active Fluorescent Probes. Org. Biomol. Chem. 2021, 19, 7446– 7459,  DOI: 10.1039/D1OB00584G
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    10g
    Stimuli-responsive transmembrane anion transport by AIE-active fluorescent probes
    Akhtar, Nasim; Biswas, Oindrila; Manna, Debasis
    Organic & Biomolecular Chemistry (2021), 19 (34), 7446-7459CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    Anticancer drug resistance implicates multifunctional mechanisms, and hypoxia is one of the key factors in therapeutic resistance. Hypoxia-specific therapy is considered an extremely effective strategy to fight against cancer. The development of small mol.-based synthetic anion transporters has also recently drawn attention for their potential therapeutic applications against several ion-transport-assocd. diseases, such as cancer and others. Herein, we describe the development of a hypoxia-responsive proanionophore to trigger controlled transport of anions across membranes under pathogenic conditions. Herein, we report the development of tetraphenylethene (TPE)-based anion transporters. The sulfonium-linked p-nitrobenzyl contg. TPE-based proanionophore could be converted into a lipophilic fluorescent Cl- ion carrier in a hypoxic or reductive environment. Stimuli such as nitroreductase (NTR) and glutathione (GSH) mediated regeneration of the TPE-based active Cl- ion transporter also showed aggregation-induced emission (AIE) properties. We hypothesize that such hypoxia and reductive stimuli activatable proanionophores have tremendous potential to fight against channelopathies, including cancer.
    (h) Ahmad, M.; Johnson, T. G.; Flerin, M.; Duarte, F.; Langton, M. J. Responsive Anionophores with AND Logic Multi-Stimuli Activation. Angew. Chem., Int. Ed. 2024, 63, e202403314  DOI: 10.1002/anie.202403314
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    There is no corresponding record for this reference.
  11. 11
    Bickerton, L. E.; Langton, M. J. Controlling Transmembrane Ion Transport via Photo-Regulated Carrier Mobility. Chem. Sci. 2022, 13, 9531– 9536,  DOI: 10.1039/D2SC03322D
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    11
    Controlling transmembrane ion transport via photo-regulated carrier mobility
    Bickerton, Laura E.; Langton, Matthew J.
    Chemical Science (2022), 13 (33), 9531-9536CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    Stimuli-responsive transmembrane ion carriers allow for targeted and controllable transport activity, with potential applications as therapeutics for channelopathies and cancer, and in fundamental studies into ion transport phenomena. These applications require OFF-ON activation from a fully inactive state which does not exhibit background activity, but this remains challenging to achieve with synthetic transport systems. Here we introduce a novel mechanism for photo-gating mobile ion carriers, which involves modulating the mobility of the carriers within the lipid bilayer membrane. By appending a membrane-targeting anchor to the carrier using a photo-cleavable linker, the carriers ion transport activity is fully switched off by suppressing its ability to shuttle between the two aq.-membrane interfaces of the bilayer. The system can be reactivated rapidly in situ within the membrane by photo-triggered cleavage of the anchor to release the mobile ion carrier. This approach does not involve direct functionalization of the ion binding site of the carrier, and so does not require the de novo design of novel ion binding motifs to implement the photo-caging of activity. This work demonstrates that controlling the mobility of artificial transport systems enables precise control over activity, opening up new avenues for spatio-temporally targeted ionophores.
  12. 12
    (a) Busschaert, N.; Gale, P. A.; Haynes, C. J. E.; Light, M. E.; Moore, S. J.; Tong, C. C.; Davis, J. T.; Harrell, W. A., Jr. Tripodal Transmembrane Transporters for Bicarbonate. Chem. Commun. 2010, 46, 6252,  DOI: 10.1039/c0cc01684e
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    12a
    Tripodal transmembrane transporters for bicarbonate
    Busschaert, Nathalie; Gale, Philip A.; Haynes, Cally J. E.; Light, Mark E.; Moore, Stephen J.; Tong, Christine C.; Davis, Jeffery T.; Harrell, William A., Jr.
    Chemical Communications (Cambridge, United Kingdom) (2010), 46 (34), 6252-6254CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    Easy-to-make tripodal tris-thiourea receptors based upon tris(2-aminoethyl)amine are capable of chloride/bicarbonate transport and as such represent a new class of bicarbonate transport agent.
    (b) Busschaert, N.; Wenzel, M.; Light, M. E.; Iglesias-Hernández, P.; Pérez-Tomás, R.; Gale, P. A. Structure–Activity Relationships in Tripodal Transmembrane Anion Transporters: The Effect of Fluorination. J. Am. Chem. Soc. 2011, 133, 14136– 14148,  DOI: 10.1021/ja205884y
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    12b
    Structure-Activity Relationships in Tripodal Transmembrane Anion Transporters: The Effect of Fluorination
    Busschaert, Nathalie; Wenzel, Marco; Light, Mark E.; Iglesias-Hernandez, Paulina; Perez-Tomas, Ricardo; Gale, Philip A.
    Journal of the American Chemical Society (2011), 133 (35), 14136-14148CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A series of easy-to-make fluorinated tripodal anion transporters contg. urea and thiourea groups have been prepd. and their anion transport properties studied. Vesicle anion transport assays using ion-selective electrodes show that this class of compd. is capable of transporting chloride through a lipid bilayer via a variety of mechanisms, including chloride/H+ co-transport and chloride/nitrate, chloride/bicarbonate, and to a lesser extent an unusual chloride/sulfate antiport process. Calcns. indicate that increasing the degree of fluorination of the tripodal transmembrane transporters increases the lipophilicity of the transporter and this is shown to be the major contributing factor in the superior transport activity of the fluorinated compds., with a max. transport rate achieved for clog P = 8. The most active transporter 5 contained a urea functionality appended with a 3,5-bis(trifluoromethyl)phenyl group and was able to mediate transmembrane chloride transport at receptor to lipid ratios as low as 1:250,000. Proton NMR titrn. and single crystal X-ray diffraction revealed the ability of the tripodal receptors to bind different anions with varying affinities in a 1:1 or 2:1 stoichiometry in soln. and in the solid state. Evidence is also provided that the most potent anion transporters are able to induce apoptosis in human cancer cells by using a selection of in vitro viability and fluorescence assays.
    (c) Marques, I.; Colaço, A. R.; Costa, P. J.; Busschaert, N.; Gale, P. A.; Félix, V. Tris–Thiourea Tripodal-Based Molecules as Chloride Transmembrane Transporters: Insights from Molecular Dynamics Simulations. Soft Matter 2014, 10, 3608,  DOI: 10.1039/c3sm52140k
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    12c
    Tris-thiourea tripodal-based molecules as chloride transmembrane transporters: insights from molecular dynamics simulations
    Marques, Igor; Colaco, Ana R.; Costa, Paulo J.; Busschaert, Nathalie; Gale, Philip A.; Felix, Vitor
    Soft Matter (2014), 10 (20), 3608-3621CODEN: SMOABF; ISSN:1744-683X. (Royal Society of Chemistry)
    The interaction of six tripodal synthetic chloride transmembrane transporters with a POPC bilayer was investigated by means of mol. dynamics simulations using the general Amber force field (GAFF) for the transporters and the LIPID11 force field for phospholipids. These transporters are structurally simple mols., based on the tris(2-aminoethyl)amine scaffold, contg. three thiourea binding units coupled with three Bu (1), Ph (2), fluorophenyl (3), pentafluorophenyl (4), trifluoromethylphenyl (5), or bis(trifluoromethyl)phenyl (6) substituents. The passive diffusion of 1-6 ∋ Cl- was evaluated with the complexes initially positioned either in the water phase or inside the bilayer. In the first scenario the chloride is released in the water soln. before the synthetic mols. achieve the water-lipid interface and permeate the membrane. In the latter one, only when the chloride complex reaches the interface is the anion released to the water phase, with the transporter losing the initial ggg tripodal shape. Independently of the transporter used in the membrane system, the bilayer structure is preserved and the synthetic mols. interact with the POPC mols. at the phosphate headgroup level, via N-H···O hydrogen bonds. Overall, the mol. dynamics simulations' results indicate that the small tripodal mols. in this series have a low impact on the bilayer and are able to diffuse with chloride inside the lipid environment. Indeed, these are essential conditions for these mols. to promote the transmembrane transport as anion carriers, in agreement with exptl. efflux data.
    (d) Wu, X.; Judd, L. W.; Howe, E. N. W.; Withecombe, A. M.; Soto-Cerrato, V.; Li, H.; Busschaert, N.; Valkenier, H.; Pérez-Tomás, R.; Sheppard, D. N.; Jiang, Y.-B.; Davis, A. P.; Gale, P. A. Nonprotonophoric Electrogenic Cl– Transport Mediated by Valinomycin-like Carriers. Chem 2016, 1, 127– 146,  DOI: 10.1016/j.chempr.2016.04.002
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    12d
    Nonprotonophoric Electrogenic Cl- Transport Mediated by Valinomycin-like Carriers
    Wu, Xin; Judd, Luke W.; Howe, Ethan N. W.; Withecombe, Anne M.; Soto-Cerrato, Vanessa; Li, Hongyu; Busschaert, Nathalie; Valkenier, Hennie; Perez-Tomas, Ricardo; Sheppard, David N.; Jiang, Yun-Bao; Davis, Anthony P.; Gale, Philip A.
    Chem (2016), 1 (1), 127-146CODEN: CHEMVE; ISSN:2451-9294. (Cell Press)
    Synthetic transmembrane anion transporters (anionophores) have potential as tools for biomedical research and as therapeutic agents for diseases assocd. with anion-channel dysfunction. However, the possibility of H+ or OH- transport by anionophores has received little attention, and an anionophore selective for Cl- over H+/OH- is currently unavailable. Here, we show that depending on anionophore acidity, many anionophores facilitate electrogenic H+ or OH- transport, potentially leading to toxicity. Nevertheless, using several liposome-membrane-based assays, we identified two newly developed small mols. that promote electrogenic Cl- transport without effectively dissipating the transmembrane pH gradient, essentially mimicking the electrogenic cationophore valinomycin. The Cl- > H+/OH- selectivity of anionophores showed a consistent pos. correlation with the degree of Cl- encapsulation and a neg. correlation with the acidity of hydrogen-bond donors. Our study demonstrates that a valinomycin equiv. for Cl--selective transport is achievable.
    (e) Wu, X.; Gale, P. A. Small-Molecule Uncoupling Protein Mimics: Synthetic Anion Receptors as Fatty Acid-Activated Proton Transporters. J. Am. Chem. Soc. 2016, 138, 16508– 16514,  DOI: 10.1021/jacs.6b10615
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    12e
    Small-Molecule Uncoupling Protein Mimics: Synthetic Anion Receptors as Fatty Acid-Activated Proton Transporters
    Wu, Xin; Gale, Philip A.
    Journal of the American Chemical Society (2016), 138 (50), 16508-16514CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Uncoupling proteins (UCPs) regulate energy expenditure in living cells by inducing proton leakage across the mitochondrial inner membrane, thereby uncoupling ADP phosphorylation from nutrient oxidn. The proton transport activity of UCP1 and UCP2 requires activation by fatty acids. Here, the authors report the 1st examples of synthetic small mols. performing this biol. important fatty acid-activated function. The authors previously showed that a tripodal thiourea possesses poor H+/OH- transport activity without fatty acids, but in the presence of long-chain fatty acids is "switched on" as a proton transporter with an activity close to a commonly used protonophore. Fatty acid-enhanced proton transport was also obsd. for other H- and halogen-bond-based synthetic anion transporters. The authors propose that these compds. induce proton permeability by catalyzing transbilayer movement ("flip-flop") of anionic fatty acids, so allowing the fatty acids to complete a proton transport cycle. Several lines of evidence have been provided to support such a fatty acid cycling mechanism. These findings open up new applications of anion receptor chem., and provide important clues for understanding biol. activities of synthetic anion transporters and potentially the uncoupling mechanism of naturally occurring membrane proteins.
    (f) Spooner, M. J.; Gale, P. A. A Tripodal Tris-Selenourea Anion Transporter Matches the Activity of Its Thio- Analogue but Shows Distinct Selectivity. Supramol. Chem. 2018, 30, 514– 519,  DOI: 10.1080/10610278.2018.1431394
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    (g) Jowett, L. A.; Howe, E. N. W.; Wu, X.; Busschaert, N.; Gale, P. A. New Insights into the Anion Transport Selectivity and Mechanism of Tren-based Tris-(Thio)Ureas. Chem. - Eur. J. 2018, 24, 10475– 10487,  DOI: 10.1002/chem.201801463
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    12g
    New Insights into the Anion Transport Selectivity and Mechanism of Tren-based Tris-(thio)ureas
    Jowett, Laura A.; Howe, Ethan N. W.; Wu, Xin; Busschaert, Nathalie; Gale, Philip A.
    Chemistry - A European Journal (2018), 24 (41), 10475-10487CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The anion transport properties of a series of previously reported tren-based anionophores have been revisited using new assays designed to measure anion uniport. This study provides new insights into the transport mechanism and selectivity of this important class of transporters. Specifically, we report the chloride and nitrate transport selectivity of these systems and quantify sulfate transport to det. EC50 values for sulfate transport for the first time. Two new assays were developed to study bicarbonate transport allowing accurate quantification of chloride/bicarbonate exchange.
    (h) Jowett, L. A.; Ricci, A.; Wu, X.; Howe, E. N. W.; Gale, P. A. Investigating the Influence of Steric Hindrance on Selective Anion Transport. Molecules 2019, 24, 1278,  DOI: 10.3390/molecules24071278
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    (i) Howe, E. N. W.; Gale, P. A. Fatty Acid Fueled Transmembrane Chloride Transport. J. Am. Chem. Soc. 2019, 141, 10654– 10660,  DOI: 10.1021/jacs.9b02116
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    12i
    Fatty Acid Fueled Transmembrane Chloride Transport
    Howe, Ethan N. W.; Gale, Philip A.
    Journal of the American Chemical Society (2019), 141 (27), 10654-10660CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Generation of chem. gradients across biol. membranes of cellular compartments is a hallmark of all living systems. Here the authors report a proof-of-concept prototype transmembrane pumping system in liposomes. The pump uses fatty acid to fuel chloride transport, thus generating a transmembrane chloride gradient. Addn. of fatty acid to phospholipid vesicles generates a transmembrane pH gradient (pHin < pHout), and this electrochem. H+ potential is harnessed by an anionophore to drive chloride efflux via H+/Cl- cotransport. Further addn. of fatty acid efficiently fuels the system to continuously drive chloride transport against the concn. gradient, up to [Cl-]in 65 mM | [Cl-]out 100 mM, and is 1400 times more efficient than using an external fuel. Based on the findings from dissecting the H+/Cl- flux process with the use of different liposomal fluorescence assays, and supported by addnl. liposome-based 13C NMR and DLS studies; the authors proposed that the presence of an anionophore can induce asym. distribution of fatty acid, and contribute to another Cl- flux mechanism in this system.
    (j) Wu, X.; Small, J. R.; Cataldo, A.; Withecombe, A. M.; Turner, P.; Gale, P. A. Voltage-Switchable HCl Transport Enabled by Lipid Headgroup–Transporter Interactions. Angew. Chem., Int. Ed. 2019, 58, 15142– 15147,  DOI: 10.1002/anie.201907466
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    12j
    Voltage-switchable HCl transport enabled by lipid headgroup-transporter interactions
    Wu, Xin; Small, Jennifer R.; Cataldo, Alessio; Withecombe, Anne M.; Turner, Peter; Gale, Philip A.
    Angewandte Chemie, International Edition (2019), 58 (42), 15142-15147CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Synthetic anion transporters that facilitate transmembrane H+/Cl- symport (cotransport) have anti-cancer potential due to their ability to neutralize pH gradients and inhibit autophagy in cells. However, compared to the natural product prodigiosin, synthetic anion transporters have low-to-modest H+/Cl- symport activity and their mechanism of action remains less well understood. We report a chloride-selective tetraurea macrocycle that has a record-high H+/Cl- symport activity similar to that of prodigiosin and most importantly demonstrates unprecedented voltage-switchable transport properties that are linked to the lack of uniport activity. By studying the anion binding affinity and transport mechanisms of four other anion transporters, we show that the lack of uniport and voltage-dependent H+/Cl- symport originate from strong binding to phospholipid headgroups that hampers the diffusion of the free transporters through the membrane, leading to an unusual H+/Cl- symport mechanism that involves only charged species. Our work provides important mechanistic insights into different classes of anion transporters and a new approach to achieve voltage-switchability in artificial membrane transport systems.
    (k) Spooner, M. J.; Li, H.; Marques, I.; Costa, P. M. R.; Wu, X.; Howe, E. N. W.; Busschaert, N.; Moore, S. J.; Light, M. E.; Sheppard, D. N.; Félix, V.; Gale, P. A. Fluorinated Synthetic Anion Carriers: Experimental and Computational Insights into Transmembrane Chloride Transport. Chem. Sci. 2019, 10, 1976– 1985,  DOI: 10.1039/C8SC05155K
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    12k
    Fluorinated synthetic anion carriers: experimental and computational insights into transmembrane chloride transport
    Spooner, Michael J.; Li, Hongyu; Marques, Igor; Costa, Pedro M. R.; Wu, Xin; Howe, Ethan N. W.; Busschaert, Nathalie; Moore, Stephen J.; Light, Mark E.; Sheppard, David N.; Felix, Vitor; Gale, Philip A.
    Chemical Science (2019), 10 (7), 1976-1985CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
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    The groups of Montenegro and Basílio recently demonstrated photocontrol of cationic peptide transport by an azobenzene-appended calixarene, where the higher activity observed for the (E)-isomer was ascribed to a change in lipophilicity (i.e., increased hydrophobicity). Yet, altered membrane mobility might offer an additional explanation, see:

    Martins, J. N.; Raimundo, B.; Rioboo, A.; Folgar-Cameán, Y.; Montenegro, J.; Basílio, N. Photoswitchable Calixarene Activators for Controlled Peptide Transport across Lipid Membranes. J. Am. Chem. Soc. 2023, 145, 13126– 13133,  DOI: 10.1021/jacs.3c01829
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    Photoswitchable Calixarene Activators for Controlled Peptide Transport across Lipid Membranes
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    Supramol. synthetic transporters are crucial to understand and activate the passage across lipid membranes of hydrophilic effector mols. Herein, we introduce photoswitchable calixarenes for the light-controlled transport activation of cationic peptide cargos across model lipid bilayers and inside living cells. Our approach was based on rationally designed p-sulfonatocalix[4]arene receptors equipped with a hydrophobic azobenzene arm, which recognize cationic peptide sequences at the nM range. Activation of membrane peptide transport is confirmed, in synthetic vesicles and living cells, for calixarene activators featuring the azobenzene arm in the E configuration. Therefore, this method allows the modulation of the transmembrane transport of peptide cargos upon Z-E photoisomerization of functionalized calixarenes using 500 nm visible light. These results showcase the potential of photoswitchable counterion activators for the light-triggered delivery of hydrophilic biomols. and pave the way for potential applications in remotely controlled membrane transport and photopharmacol. applications of hydrophilic functional biomols.

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  1. Sandip Chattopadhayay, Pinaki Talukdar. Stimuli-responsive synthetic ionophores for therapeutic applications. Current Opinion in Chemical Biology 2025, 85 , 102582. https://doi.org/10.1016/j.cbpa.2025.102582
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  • Abstract

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    Scheme 1

    Scheme 1. Photoisomerization of Azobenzene-Based Tris-Thioureas 1 and 2 (A) and Schematic Representation of the Photocontrol over Membrane Incorporation and Mobility (B)
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    Figure 1

    Figure 1. Side view (A) and top view (B) of (E,E,E)-1 as found in the crystal structure, shown in stick representation. Disorder in the azobenzene moieties was omitted for clarity.

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    Figure 2

    Figure 2. UV–vis spectral changes measured at 293 K of (E,E,E)-1 (A) and (E,E,E)-2 (B) in DMSO/MeCN (1:1 v/v, 1 × 10–5 m) upon irradiation with 385, 365 or 455 nm light, as well as upon thermal equilibration.

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    Figure 3

    Figure 3. Plots of chloride efflux against time facilitated by the (E,E,E)-isomers (blue line) and (ZPSS)-isomers (red line) of 1 (A) and 2 (D). Hill plots of activity versus concentration of the (E,E,E)-isomers (blue squares) and (ZPSS)-isomers (red circles) of 1 (B) and 2 (E). Change in chloride efflux upon irradiation of 0.1 mol % (E,E,E)-1 at 385 nm (C) and 1 mol % (E,E,E)-2 at 365 nm (F).

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    Figure 4

    Figure 4. Plots of chloride efflux over time of the isomers of 1 (A) and 2 (B) facilitated by preincorporated transporter compared with postaddition from DMSO-MeCN (1:1 v/v).

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    Figure 5

    Figure 5. Hill plots generated using the isomers of 1 and 2 in combination with valinomycin (A, B) and the corresponding mechanism (C), and in combination with monensin (D, E) and the corresponding mechanism (F).

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    Figure 6

    Figure 6. Plots of electrogenic Cl– transport against time facilitated by 0.02 mol % (E,E,E)-1 (A) and 1 mol % dark-adapted 2 (B) in combination with valinomycin, and activation by in situ irradiation. The shaded area denotes the irradiation time span using the mentioned wavelengths. For control experiments without irradiation or only a single irradiation step, see Figures S52 and S53.

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  • References

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      Biological applications of synthetic anion transporters
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      A review. The use of synthetic ion transporters for alteration of the concn. of ions across cell membranes has drawn attention from scientists over the last two decades. This ion transport property has been sensibly used to reduce the viability of cancer cells mainly due to the disruption of their ion homeostasis, leading to the perturbation of their abnormal pH gradient. The use of the proanionophore strategy has been recently adopted to increase cellular deliverability and reduce unwanted cytotoxicity towards normal cells. Meanwhile, various anionophores exhibiting non-toxic behavior in epithelial cells have shown a great propensity to be used as a putative treatment for channelopathies like cystic fibrosis. Anionophores with Cl- ion transport mediated antibacterial activities have also been successfully used against clin. relevant bacterial strains, many of which are tolerant towards com. antibiotics. Recent developments in the chloride ion transport mediated biol. activities of anionophores have been mainly discussed in this article. It also highlights other aspects, such as design criteria, targeted delivery options, and others, which can be useful for the further development of selective anionophores for specific biol. applications.
    5. 5
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      Nature Chemistry (2014), 6 (10), 885-892CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
      Anion transporters based on small mols. have received attention as therapeutic agents because of their potential to disrupt cellular ion homeostasis. However, a direct correlation between a change in cellular chloride anion concn. and cytotoxicity has not been established for synthetic ion carriers. Here the authors show that two pyridine diamide-strapped calix[4]pyrroles induce coupled chloride anion and sodium cation transport in both liposomal models and cells, and promote cell death by increasing intracellular chloride and sodium ion concns. Removing either ion from the extracellular media or blocking natural sodium channels with amiloride prevents this effect. Cell expts. show that the ion transporters induce the sodium chloride influx, which leads to an increased concn. of reactive oxygen species, release of cytochrome c from the mitochondria and apoptosis via caspase activation. However, they do not activate the caspase-independent apoptotic pathway assocd. with the apoptosis-inducing factor. Ion transporters, therefore, represent an attractive approach for regulating cellular processes that are normally controlled tightly by homeostasis.
      (b) Li, H.; Valkenier, H.; Thorne, A. G.; Dias, C. M.; Cooper, J. A.; Kieffer, M.; Busschaert, N.; Gale, P. A.; Sheppard, D. N.; Davis, A. P. Anion Carriers as Potential Treatments for Cystic Fibrosis: Transport in Cystic Fibrosis Cells, and Additivity to Channel-Targeting Drugs. Chem. Sci. 2019, 10, 9663– 9672,  DOI: 10.1039/C9SC04242C
      5b
      Anion carriers as potential treatments for cystic fibrosis: transport in cystic fibrosis cells, and additivity to channel-targeting drugs
      Li, Hongyu; Valkenier, Hennie; Thorne, Abigail G.; Dias, Christopher M.; Cooper, James A.; Kieffer, Marion; Busschaert, Nathalie; Gale, Philip A.; Sheppard, David N.; Davis, Anthony P.
      Chemical Science (2019), 10 (42), 9663-9672CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      Defective anion transport is a hallmark of the genetic disease cystic fibrosis (CF). One approach to restore anion transport to CF cells utilizes alternative pathways for transmembrane anion transport, including artificial anion carriers (anionophores). Here, we screened 22 anionophores for biol. activity using fluorescence emission from the halide-sensitive yellow fluorescent protein. Three compds. possessed anion transport activity similar to or greater than that of a bis-(p-nitrophenyl)ureidodecalin previously shown to have promising biol. activity. Anion transport by these anionophores was concn.-dependent and persistent. All four anionophores mediated anion transport in CF cells, and their activity was additive to rescue of the predominant disease-causing variant F508del-CFTR using the clin.-licensed drugs lumacaftor and ivacaftor. Toxicity was variable but minimal at the lower end. The results provide further evidence that anionophores, by themselves or together with other treatments that restore anion transport, offer a potential therapeutic strategy for CF.
      (c) Davis, J. T.; Gale, P. A.; Quesada, R. Advances in Anion Transport and Supramolecular Medicinal Chemistry. Chem. Soc. Rev. 2020, 49, 6056– 6086,  DOI: 10.1039/C9CS00662A
      5c
      Advances in anion transport and supramolecular medicinal chemistry
      Davis, Jeffery T.; Gale, Philip A.; Quesada, Roberto
      Chemical Society Reviews (2020), 49 (16), 6056-6086CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      Advances in anion transport by synthetic supramol. systems are discussed in this article. Developments in the design of discrete mol. carriers for anions and supramol. anion channels are reviewed followed by an overview of the use of these systems in biol. systems as putative treatments for diseases such as cystic fibrosis and cancer.
    6. 6
      (a) Bickerton, L. E.; Johnson, T. G.; Kerckhoffs, A.; Langton, M. J. Supramolecular Chemistry in Lipid Bilayer Membranes. Chem. Sci. 2021, 12, 11252– 11274,  DOI: 10.1039/D1SC03545B
      6a
      Supramolecular chemistry in lipid bilayer membranes
      Bickerton, Laura E.; Johnson, Toby G.; Kerckhoffs, Aidan; Langton, Matthew J.
      Chemical Science (2021), 12 (34), 11252-11274CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      Lipid bilayer membranes form compartments requisite for life. Interfacing supramol. systems, including receptors, catalysts, signal transducers and ion transporters, enables the function of the membrane to be controlled in artificial and living cellular compartments. In this perspective, we take stock of the current state of the art of this rapidly expanding field, and discuss prospects for the future in both fundamental science and applications in biol. and medicine.
      (b) de Jong, J.; Bos, J. E.; Wezenberg, S. J. Stimulus-Controlled Anion Binding and Transport by Synthetic Receptors. Chem. Rev. 2023, 123, 8530– 8574,  DOI: 10.1021/acs.chemrev.3c00039
      6b
      Stimulus-Controlled Anion Binding and Transport by Synthetic Receptors
      de Jong, Jorn; Bos, Jasper E.; Wezenberg, Sander J.
      Chemical Reviews (Washington, DC, United States) (2023), 123 (13), 8530-8574CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. Anionic species are omnipresent and involved in many important biol. processes. A large no. of artificial anion receptors has therefore been developed. Some of these are capable of mediating transmembrane transport. However, where transport proteins can respond to stimuli in their surroundings, creation of synthetic receptors with stimuli-responsive functions poses a major challenge. Herein, we give a full overview of the stimulus-controlled anion receptors that have been developed thus far, including their application in membrane transport. In addn. to their potential operation as membrane carriers, the use of anion recognition motifs in forming responsive membrane-spanning channels is discussed. With this review article, we intend to increase interest in transmembrane transport among scientist working on host-guest complexes and dynamic functional systems in order to stimulate further developments.
    7. 7
      (a) Santacroce, P. V.; Davis, J. T.; Light, M. E.; Gale, P. A.; Iglesias-Sánchez, J. C.; Prados, P.; Quesada, R. Conformational Control of Transmembrane Cl - Transport. J. Am. Chem. Soc. 2007, 129, 1886– 1887,  DOI: 10.1021/ja068067v
      7a
      Conformational Control of Transmembrane Cl- Transport
      Santacroce, Paul V.; Davis, Jeffery T.; Light, Mark E.; Gale, Philip A.; Iglesias-Sanchez, Jose Carlos; Prados, Pilar; Quesada, Roberto
      Journal of the American Chemical Society (2007), 129 (7), 1886-1887CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Preorganization of the isophthalamide cleft in N,N'-dibutyl-4,6-dihydroxyisophthalamide (2) by means of intramol. hydrogen bonds results in an enhanced anion receptor that displays significant chloride transport activity into EYPC liposomes. This function is easily switchable by controlling the pH.
      (b) Howe, E. N. W.; Busschaert, N.; Wu, X.; Berry, S. N.; Ho, J.; Light, M. E.; Czech, D. D.; Klein, H. A.; Kitchen, J. A.; Gale, P. A. pH-Regulated Nonelectrogenic Anion Transport by Phenylthiosemicarbazones. J. Am. Chem. Soc. 2016, 138, 8301– 8308,  DOI: 10.1021/jacs.6b04656
      7b
      pH-Regulated Nonelectrogenic Anion Transport by Phenylthiosemicarbazones
      Howe, Ethan N. W.; Busschaert, Nathalie; Wu, Xin; Berry, Stuart N.; Ho, Junming; Light, Mark E.; Czech, Dawid D.; Klein, Harry A.; Kitchen, Jonathan A.; Gale, Philip A.
      Journal of the American Chemical Society (2016), 138 (26), 8301-8308CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Gated ion transport across biol. membranes is an intrinsic process regulated by protein channels. Synthetic anion carriers (anionophores) have potential applications in biol. research; however, previously reported examples are mostly nonspecific, capable of mediating both electrogenic and electroneutral (nonelectrogenic) transport processes. Here we show the transmembrane Cl- transport studies of synthetic phenylthiosemicarbazones mimicking the function of acid-sensing (proton-gated) ion channels. These anionophores have remarkable pH-switchable transport properties with up to 640-fold increase in transport efficacy on going from pH 7.2 to 4.0. This "gated" process is triggered by protonation of the imino nitrogen and concomitant conformational change of the anion-binding thiourea moiety from anti to syn. By using a combination of two cationophore-coupled transport assays, with either monensin or valinomycin, we have elucidated the fundamental transport mechanism of phenylthiosemicarbazones which is shown to be nonelectrogenic, inseparable H+/Cl- cotransport. This study demonstrates the first examples of pH-switchable nonelectrogenic anion transporters.
      (c) Shinde, S. V.; Talukdar, P. A Dimeric Bis(Melamine)-Substituted Bispidine for Efficient Transmembrane H+/Cl– Cotransport. Angew. Chem., Int. Ed. 2017, 56, 4238– 4242,  DOI: 10.1002/anie.201700803
      7c
      A Dimeric Bis(melamine)-Substituted Bispidine for Efficient Transmembrane H+/Cl- Cotransport
      Shinde, Sopan Valiba; Talukdar, Pinaki
      Angewandte Chemie, International Edition (2017), 56 (15), 4238-4242CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A 3,7-diazabicyclo[3.3.1]nonane linking to two melamines is a unique transmembrane H+/Cl- carrier. In the solid state, the V-shaped compd. forms a HCl-bound zig-zag network through cooperative protonation and hydrogen bond interactions. In the lipid membrane, the receptor forms a dimeric self-assembly involving multiple H+ and Cl- leading to the efficient transport of the acid. The pH-dependent Cl- efflux obsd. for the compd. was rationalized based on a gradual protonation model that confers an active transmembrane carrier at physiol. pH.
    8. 8
      (a) Choi, Y. R.; Kim, G. C.; Jeon, H.-G.; Park, J.; Namkung, W.; Jeong, K.-S. Azobenzene-Based Chloride Transporters with Light-Controllable Activities. Chem. Commun. 2014, 50, 15305– 15308,  DOI: 10.1039/C4CC07560A
      8a
      Azobenzene-based chloride transporters with light-controllable activities
      Choi, Ye Rin; Kim, Gyu Chan; Jeon, Hae-Geun; Park, Jinhong; Namkung, Wan; Jeong, Kyu-Sung
      Chemical Communications (Cambridge, United Kingdom) (2014), 50 (97), 15305-15308CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      Synthetic chloride transporters contg. two urea groups linked through a diazobenzene spacer have been prepd. and the trans-to-cis isomerization by light stimulation results in dramatic changes in the chloride transport activities across lipid and cell membranes.
      (b) Ahmad, M.; Metya, S.; Das, A.; Talukdar, P. A Sandwich Azobenzene–Diamide Dimer for Photoregulated Chloride Transport. Chem. - Eur. J. 2020, 26, 8703– 8708,  DOI: 10.1002/chem.202000400
      8b
      A Sandwich Azobenzene-Diamide Dimer for Photoregulated Chloride Transport
      Ahmad, Manzoor; Metya, Surajit; Das, Aloke; Talukdar, Pinaki
      Chemistry - A European Journal (2020), 26 (40), 8703-8708CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      There has been a tremendous evolution for artificial ion transport systems, esp. gated synthetic systems, which closely mimic their natural congeners. Herein, we demonstrate a trans-azobenzene-based photoregulatory anionophoric system that transports chloride by forming a sandwich dimeric complex. Further studies confirmed a carrier-mediated chloride-anion antiport mechanism, and the supramol. interactions involved in chloride recognition within the sandwich complex were revealed from theor. studies. Reversible trans-cis photoisomerization of the azobenzene was achieved without any significant contribution from the thermal cis→trans isomerization at room temp. Photoregulatory transport activity across the lipid bilayer membrane inferred an outstanding off-on response of the azobenzene photoswitch.
      (c) Kerckhoffs, A.; Langton, M. J. Reversible Photo-Control over Transmembrane Anion Transport Using Visible-Light Responsive Supramolecular Carriers. Chem. Sci. 2020, 11, 6325– 6331,  DOI: 10.1039/D0SC02745F
      8c
      Reversible photo-control over transmembrane anion transport using visible-light responsive supramolecular carriers
      Kerckhoffs, Aidan; Langton, Matthew J.
      Chemical Science (2020), 11 (24), 6325-6331CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      Ion transport across lipid bilayer membranes in biol. is controlled by membrane proteins, which in turn are regulated in response to chem.-, phys.- and photo-stimuli. The design of synthetic supramol. ion transporters able to be precisely controlled by external signals, in particular bio-compatible wavelengths of visible light, is key for achieving spatio-temporal control over function. Here we report two-color responsive mol. photo-switches that act as supramol. transmembrane anion carriers. Reversible switching of the photo-switch within the lipid bilayer membrane is achieved using biocompatible visible wavelengths of light, such that temporal control over transmembrane anion transport is achieved through alternating irradn. with red and blue light.
      (d) Ahmad, M.; Chattopadhayay, S.; Mondal, D.; Vijayakanth, T.; Talukdar, P. Stimuli-Responsive Anion Transport through Acylhydrazone-Based Synthetic Anionophores. Org. Lett. 2021, 23, 7319– 7324,  DOI: 10.1021/acs.orglett.1c02249
      8d
      Stimuli-Responsive Anion Transport through Acylhydrazone-Based Synthetic Anionophores
      Ahmad, Manzoor; Chattopadhayay, Sandip; Mondal, Debashis; Vijayakanth, Thangavel; Talukdar, Pinaki
      Organic Letters (2021), 23 (19), 7319-7324CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)
      Photoswitchable acylhydrazone-based synthetic anionophores are reported. Single-crystal X-ray structure and 1H NMR titrn. studies confirmed the chloride binding in solid and soln. states. The ion transport activity of 1a was greatly attenuated through a phototriggered E to Z photoisomerization process, and the photoisomerized deactivated state showed high kinetic stability due to an intramol. hydrogen bond. Switchable "OFF-ON" transport activity was achieved by the application of light and acid-catalyzed reactivation process.
      (e) Kerckhoffs, A.; Bo, Z.; Penty, S. E.; Duarte, F.; Langton, M. J. Red-Shifted Tetra-Ortho-Halo-Azobenzenes for Photo-Regulated Transmembrane Anion Transport. Org. Biomol. Chem. 2021, 19, 9058– 9067,  DOI: 10.1039/D1OB01457A
      8e
      Red-shifted tetra-ortho-halo-azobenzenes for photo-regulated transmembrane anion transport
      Kerckhoffs, Aidan; Bo, Zonghua; Penty, Samuel E.; Duarte, Fernanda; Langton, Matthew J.
      Organic & Biomolecular Chemistry (2021), 19 (41), 9058-9067CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      Photo-responsive synthetic ion transporters are of interest as tools for studying transmembrane transport processes and have potential applications as targeted therapeutics, due to the possibility of spatiotemporal control and wavelength-dependent function. Here we report the synthesis of novel sym. and non-sym. red-shifted tetra-ortho-chloro- and tetra-ortho-fluoro azobenzenes, bearing pendant amine functionality. Functionalisation of the photo-switchable scaffolds with squaramide hydrogen bond donors enabled the prepn. of a family of anion receptors, which act as photo-regulated transmembrane chloride transporters in response to green or red light. The subtle effects of chlorine/fluorine substitution, meta/para positioning of the anion receptors, and the use of more flexible linkers are explored. NMR titrn. expts. on the structurally diverse photo-switchable receptors reveal cooperative binding of chloride in the Z, but not E isomer, by the two squaramide binding sites. These results are supported by mol. dynamics simulations in explicit solvent and model membranes. We show that this intramol. anion recognition leads to effective switching of transport activity in lipid bilayer membranes, in which optimal Z isomer activity is achieved using a combination of fluorine substitution and para-methylene spacer units.
      (f) Wezenberg, S. J.; Chen, L.-J.; Bos, J. E.; Feringa, B. L.; Howe, E. N. W.; Wu, X.; Siegler, M. A.; Gale, P. A. Photomodulation of Transmembrane Transport and Potential by Stiff-Stilbene Based Bis(Thio)Ureas. J. Am. Chem. Soc. 2022, 144, 331– 338,  DOI: 10.1021/jacs.1c10034
      8f
      Photomodulation of Transmembrane Transport and Potential by Stiff-Stilbene Based Bis(thio)ureas
      Wezenberg, Sander J.; Chen, Li-Jun; Bos, Jasper E.; Feringa, Ben L.; Howe, Ethan N. W.; Wu, Xin; Siegler, Maxime A.; Gale, Philip A.
      Journal of the American Chemical Society (2022), 144 (1), 331-338CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Membrane transport proteins fulfill important regulatory functions in biol. with a common trait being their ability to respond to stimuli in the environment. Various small-mol. receptors, capable of mediating transmembrane transport, have been successfully developed. However, to confer stimuli-responsiveness on them poses a fundamental challenge. Here we demonstrate photocontrol of transmembrane transport and elec. potential using bis(thio)ureas derived from stiff-stilbene. UV-vis and 1H NMR spectroscopy are used to monitor E-Z photoisomerization of these bis(thio)ureas and 1H NMR titrns. reveal stronger binding of chloride to the (Z)-form than to the (E)-form. Addnl. insight into the binding properties is provided by single crystal X-ray crystallog. anal. and DFT geometry optimization. Importantly, the (Z)-isomers are much more active in transmembrane transport than the resp. (E)-isomers as shown through various assays. As a result, both membrane transport and depolarization can be modulated upon irradn., opening up new prospects toward light-based therapeutics as well as physiol. and optopharmacol. tools for studying anion transport-assocd. diseases and to stimulate neuronal activity, resp.
      (g) Villarón, D.; Bos, J. E.; Kohl, F.; Mommer, S.; de Jong, J.; Wezenberg, S. J. Photoswitchable Bis(Amidopyrroles): Modulating Anion Transport Activity Independent of Binding Affinity. J. Org. Chem. 2023, 88, 11328– 11334,  DOI: 10.1021/acs.joc.3c01018
      There is no corresponding record for this reference.
    9. 9
      Docker, A.; Johnson, T. G.; Kuhn, H.; Zhang, Z.; Langton, M. J. Multistate Redox-Switchable Ion Transport Using Chalcogen-Bonding Anionophores. J. Am. Chem. Soc. 2023, 145, 2661– 2668,  DOI: 10.1021/jacs.2c12892
      9
      Multistate Redox-Switchable Ion Transport Using Chalcogen-Bonding Anionophores
      Docker, Andrew; Johnson, Toby G.; Kuhn, Heike; Zhang, Zongyao; Langton, Matthew J.
      Journal of the American Chemical Society (2023), 145 (4), 2661-2668CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Synthetic supramol. transmembrane anionophores have emerged as promising anticancer chemotherapeutics. However, key to their targeted application is achieving spatiotemporally controlled activity. Herein, we report a series of chalcogen-bonding diaryl tellurium-based transporters in which their anion binding potency and anionophoric activity are controlled through reversible redox cycling between Te oxidn. states. This unprecedented in situ reversible multistate switching allows for switching between ON and OFF anion transport and is crucially achieved with biomimetic chem. redox couples.
    10. 10
      (a) Choi, Y. R.; Lee, B.; Park, J.; Namkung, W.; Jeong, K.-S. Enzyme-Responsive Procarriers Capable of Transporting Chloride Ions across Lipid and Cellular Membranes. J. Am. Chem. Soc. 2016, 138, 15319– 15322,  DOI: 10.1021/jacs.6b10592
      10a
      Enzyme-Responsive Procarriers Capable of Transporting Chloride Ions across Lipid and Cellular Membranes
      Choi, Ye Rin; Lee, Bom; Park, Jinhong; Namkung, Wan; Jeong, Kyu-Sung
      Journal of the American Chemical Society (2016), 138 (47), 15319-15322CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Adopting the concept of procarrier for the first time, we demonstrated the controlled transport of chloride ions across lipid and cellular membranes. Procarriers contg. highly hydrophilic appendages were initially inactive due to the lack of their partitioning into lipophilic membranes but were activated to transport chloride ions in the presence of specific enzymes that were able to hydrolyze off the appendages to generate an active carrier under specific conditions. Namely, the procarrier with an ester-bond-linked appendage was most activated by an esterase (PLE) at pH = 7.4, whereas the procarrier with a glycosyl-bond-linked appendage was activated only by a glycosylase (AOG) under slightly acidic conditions (pH = 5.5-6). In addn. to controlling chloride transport, hydrophilic appendages greatly increase the water soly. of the procarrier, which may improve the deliverability of a hydrophobic active carrier into a plasma membrane.
      (b) Salunke, S. B.; Malla, J. A.; Talukdar, P. Phototriggered Release of a Transmembrane Chloride Carrier from an O-Nitrobenzyl-Linked Procarrier. Angew. Chem., Int. Ed. 2019, 58, 5354– 5358,  DOI: 10.1002/anie.201900869
      10b
      Phototriggered Release of a Transmembrane Chloride Carrier from an o-Nitrobenzyl-Linked Procarrier
      Salunke, Swati Bansi; Malla, Javid Ahmad; Talukdar, Pinaki
      Angewandte Chemie, International Edition (2019), 58 (16), 5354-5358CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      While there have been many studies on synthetic chloride carriers and a recent application for apoptotic cell death, so far, the proposed huge potential of these systems in targeting cancer has not been realized due to their cytotoxicity to healthy cells. Herein, we describe the development of an indole-2-carboxamide receptor as an efficient membrane chloride carrier while the corresponding o-nitrobenzyl-linked deriv. is a procarrier of the ion. Photoirradn. of the procarrier in liposomes results in release of the active carrier with up to 90% transport efficiency. Such photorelease of the carrier also works within cancer cells, resulting in efficient cell killing. Such photocleavable procarriers have great potential as a photodynamic therapy to combat various types of cancers.
      (c) Akhtar, N.; Pradhan, N.; Saha, A.; Kumar, V.; Biswas, O.; Dey, S.; Shah, M.; Kumar, S.; Manna, D. Tuning the Solubility of Ionophores: Glutathione-Mediated Transport of Chloride Ions across Hydrophobic Membranes. Chem. Commun. 2019, 55, 8482– 8485,  DOI: 10.1039/C9CC04518J
      10c
      Tuning the solubility of ionophores: glutathione-mediated transport of chloride ions across hydrophobic membranes
      Akhtar, Nasim; Pradhan, Nirmalya; Saha, Abhishek; Kumar, Vishnu; Biswas, Oindrila; Dey, Subhasis; Shah, Manisha; Kumar, Sachin; Manna, Debasis
      Chemical Communications (Cambridge, United Kingdom) (2019), 55 (58), 8482-8485CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      A stimuli-responsive anion transport strategy was successfully employed to regulate the water soly. of ionophores and promote controlled transport of Cl- ions across lipid bilayers. A sulfonium-based proanionophore was efficiently activated by glutathione to regenerate the active anionophore, which allowed controlled transport of Cl- ions in a cellular environment with lower cytotoxicity.
      (d) Fares, M.; Wu, X.; Ramesh, D.; Lewis, W.; Keller, P. A.; Howe, E. N. W.; Pérez-Tomás, R.; Gale, P. A. Stimuli-Responsive Cycloaurated “OFF-ON” Switchable Anion Transporters. Angew. Chem., Int. Ed. 2020, 59, 17614– 17621,  DOI: 10.1002/anie.202006392
      10d
      Stimuli-Responsive Cycloaurated "OFF-ON" Switchable Anion Transporters
      Fares, Mohamed; Wu, Xin; Ramesh, Deepthi; Lewis, William; Keller, Paul A.; Howe, Ethan N. W.; Perez-Tomas, Ricardo; Gale, Philip A.
      Angewandte Chemie, International Edition (2020), 59 (40), 17614-17621CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Anion transporters have shown potential application as anti-cancer agents that function by disrupting homeostasis and triggering cell death. In this research article we report switchable anion transport by gold complexes of anion transporters that are "switched on" in situ in the presence of the reducing agent GSH by decomplexation of gold. GSH is found in higher concns. in tumors than in healthy tissue and hence this approach offers a strategy to target these systems to tumors.
      (e) Park, G.; Gabbaï, F. P. Redox-Controlled Chalcogen and Pnictogen Bonding: The Case of a Sulfonium/Stibonium Dication as a Preanionophore for Chloride Anion Transport. Chem. Sci. 2020, 11, 10107– 10112,  DOI: 10.1039/D0SC04417B
      10e
      Redox-controlled chalcogen and pnictogen bonding: the case of a sulfonium/stibonium dication as a preanionophore for chloride anion transport
      Park, Gyeongjin; Gabbai, Francois P.
      Chemical Science (2020), 11 (37), 10107-10112CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      Our interest in the chem. of tunable chalcogen and pnictogen bond donors as Lewis acidic platforms for the complexation and transport of anions has led us to investigate examples of such compds. that can be activated by redox events. Here, we describe the synthesis of [o-MePhS(C6H4)SbPh3]2+ ([3]2+) and [o-MePhS(C6H4)Sb(p-Tol)3]2+ ([4]2+), two dicationic stibonium/sulfonium bifunctional Lewis acids which were obtained by methylation of the phenylthioether derivs. [o-PhS(C6H4)SbPh3]+ ([1]+) and [o-PhS(C6H4)Sb(p-Tol)3]+ ([2]+), resp. An evaluation of the chloride anion transport properties of these derivs. using chloride-loaded POPC unilamellar vesicles shows that the activity of the monocations [1]+ and [2]+ greatly exceeds that of the dications [3]2+ and [4]2+, a phenomenon that we assign to the higher lipophilicity of the monocationic compds. Harnessing this large transport activity differential, we show that [4]2+ can be used as a prechloridophore that is readily activated by redn. of the sulfonium moiety. Indeed, [4]2+ reacts with GSH to afford [2]+ as an active transporter. This activation, which has been monitored in aq. soln., can also be carried out in situ, in the presence of the chloride-loaded POPC unilamellar vesicles.
      (f) Das, S.; Biswas, O.; Akhtar, N.; Patel, A.; Manna, D. Multi-Stimuli Controlled Release of a Transmembrane Chloride Ion Carrier from a Sulfonium-Linked Procarrier. Org. Biomol. Chem. 2020, 18, 9246– 9252,  DOI: 10.1039/D0OB00938E
      10f
      Multi-stimuli controlled release of a transmembrane chloride ion carrier from a sulfonium-linked procarrier
      Das, Sribash; Biswas, Oindrila; Akhtar, Nasim; Patel, Anjali; Manna, Debasis
      Organic & Biomolecular Chemistry (2020), 18 (45), 9246-9252CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      In recent times, anion transporters have received substantial consideration due to their ability to disrupt the ionic equil. across membrane bilayers. While numerous Cl- ion transporters were developed for channelopathies, unfortunately, poor aq. soly. precluded their bioapplicability. Herein, we demonstrate the development of a multi-stimuli activatable anion transport approach to induce regulated transport of Cl- ions across membranes under specific conditions. The sulfonium-based procarrier was initially inactive, but the transmembrane transport of Cl- ions was activated in the presence of stimuli such as glutathione (GSH), reactive oxygen species (ROS) and light. The release of the hydrophobic anionophore from the aq.-sol. procarrier under specific conditions leads to the successful transport of Cl- ions. Under physiol. conditions, these anion carriers follow an antiport exchange mechanism to transport Cl- ions across lipid bilayers. Such multi-stimuli activatable procarriers have great potential to combat various types of channelopathies, including cancer, cystic fibrosis, kidney stones, myotonia, and others.
      (g) Akhtar, N.; Biswas, O.; Manna, D. Stimuli-Responsive Transmembrane Anion Transport by AIE-Active Fluorescent Probes. Org. Biomol. Chem. 2021, 19, 7446– 7459,  DOI: 10.1039/D1OB00584G
      10g
      Stimuli-responsive transmembrane anion transport by AIE-active fluorescent probes
      Akhtar, Nasim; Biswas, Oindrila; Manna, Debasis
      Organic & Biomolecular Chemistry (2021), 19 (34), 7446-7459CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      Anticancer drug resistance implicates multifunctional mechanisms, and hypoxia is one of the key factors in therapeutic resistance. Hypoxia-specific therapy is considered an extremely effective strategy to fight against cancer. The development of small mol.-based synthetic anion transporters has also recently drawn attention for their potential therapeutic applications against several ion-transport-assocd. diseases, such as cancer and others. Herein, we describe the development of a hypoxia-responsive proanionophore to trigger controlled transport of anions across membranes under pathogenic conditions. Herein, we report the development of tetraphenylethene (TPE)-based anion transporters. The sulfonium-linked p-nitrobenzyl contg. TPE-based proanionophore could be converted into a lipophilic fluorescent Cl- ion carrier in a hypoxic or reductive environment. Stimuli such as nitroreductase (NTR) and glutathione (GSH) mediated regeneration of the TPE-based active Cl- ion transporter also showed aggregation-induced emission (AIE) properties. We hypothesize that such hypoxia and reductive stimuli activatable proanionophores have tremendous potential to fight against channelopathies, including cancer.
      (h) Ahmad, M.; Johnson, T. G.; Flerin, M.; Duarte, F.; Langton, M. J. Responsive Anionophores with AND Logic Multi-Stimuli Activation. Angew. Chem., Int. Ed. 2024, 63, e202403314  DOI: 10.1002/anie.202403314
      There is no corresponding record for this reference.
    11. 11
      Bickerton, L. E.; Langton, M. J. Controlling Transmembrane Ion Transport via Photo-Regulated Carrier Mobility. Chem. Sci. 2022, 13, 9531– 9536,  DOI: 10.1039/D2SC03322D
      11
      Controlling transmembrane ion transport via photo-regulated carrier mobility
      Bickerton, Laura E.; Langton, Matthew J.
      Chemical Science (2022), 13 (33), 9531-9536CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      Stimuli-responsive transmembrane ion carriers allow for targeted and controllable transport activity, with potential applications as therapeutics for channelopathies and cancer, and in fundamental studies into ion transport phenomena. These applications require OFF-ON activation from a fully inactive state which does not exhibit background activity, but this remains challenging to achieve with synthetic transport systems. Here we introduce a novel mechanism for photo-gating mobile ion carriers, which involves modulating the mobility of the carriers within the lipid bilayer membrane. By appending a membrane-targeting anchor to the carrier using a photo-cleavable linker, the carriers ion transport activity is fully switched off by suppressing its ability to shuttle between the two aq.-membrane interfaces of the bilayer. The system can be reactivated rapidly in situ within the membrane by photo-triggered cleavage of the anchor to release the mobile ion carrier. This approach does not involve direct functionalization of the ion binding site of the carrier, and so does not require the de novo design of novel ion binding motifs to implement the photo-caging of activity. This work demonstrates that controlling the mobility of artificial transport systems enables precise control over activity, opening up new avenues for spatio-temporally targeted ionophores.
    12. 12
      (a) Busschaert, N.; Gale, P. A.; Haynes, C. J. E.; Light, M. E.; Moore, S. J.; Tong, C. C.; Davis, J. T.; Harrell, W. A., Jr. Tripodal Transmembrane Transporters for Bicarbonate. Chem. Commun. 2010, 46, 6252,  DOI: 10.1039/c0cc01684e
      12a
      Tripodal transmembrane transporters for bicarbonate
      Busschaert, Nathalie; Gale, Philip A.; Haynes, Cally J. E.; Light, Mark E.; Moore, Stephen J.; Tong, Christine C.; Davis, Jeffery T.; Harrell, William A., Jr.
      Chemical Communications (Cambridge, United Kingdom) (2010), 46 (34), 6252-6254CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      Easy-to-make tripodal tris-thiourea receptors based upon tris(2-aminoethyl)amine are capable of chloride/bicarbonate transport and as such represent a new class of bicarbonate transport agent.
      (b) Busschaert, N.; Wenzel, M.; Light, M. E.; Iglesias-Hernández, P.; Pérez-Tomás, R.; Gale, P. A. Structure–Activity Relationships in Tripodal Transmembrane Anion Transporters: The Effect of Fluorination. J. Am. Chem. Soc. 2011, 133, 14136– 14148,  DOI: 10.1021/ja205884y
      12b
      Structure-Activity Relationships in Tripodal Transmembrane Anion Transporters: The Effect of Fluorination
      Busschaert, Nathalie; Wenzel, Marco; Light, Mark E.; Iglesias-Hernandez, Paulina; Perez-Tomas, Ricardo; Gale, Philip A.
      Journal of the American Chemical Society (2011), 133 (35), 14136-14148CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A series of easy-to-make fluorinated tripodal anion transporters contg. urea and thiourea groups have been prepd. and their anion transport properties studied. Vesicle anion transport assays using ion-selective electrodes show that this class of compd. is capable of transporting chloride through a lipid bilayer via a variety of mechanisms, including chloride/H+ co-transport and chloride/nitrate, chloride/bicarbonate, and to a lesser extent an unusual chloride/sulfate antiport process. Calcns. indicate that increasing the degree of fluorination of the tripodal transmembrane transporters increases the lipophilicity of the transporter and this is shown to be the major contributing factor in the superior transport activity of the fluorinated compds., with a max. transport rate achieved for clog P = 8. The most active transporter 5 contained a urea functionality appended with a 3,5-bis(trifluoromethyl)phenyl group and was able to mediate transmembrane chloride transport at receptor to lipid ratios as low as 1:250,000. Proton NMR titrn. and single crystal X-ray diffraction revealed the ability of the tripodal receptors to bind different anions with varying affinities in a 1:1 or 2:1 stoichiometry in soln. and in the solid state. Evidence is also provided that the most potent anion transporters are able to induce apoptosis in human cancer cells by using a selection of in vitro viability and fluorescence assays.
      (c) Marques, I.; Colaço, A. R.; Costa, P. J.; Busschaert, N.; Gale, P. A.; Félix, V. Tris–Thiourea Tripodal-Based Molecules as Chloride Transmembrane Transporters: Insights from Molecular Dynamics Simulations. Soft Matter 2014, 10, 3608,  DOI: 10.1039/c3sm52140k
      12c
      Tris-thiourea tripodal-based molecules as chloride transmembrane transporters: insights from molecular dynamics simulations
      Marques, Igor; Colaco, Ana R.; Costa, Paulo J.; Busschaert, Nathalie; Gale, Philip A.; Felix, Vitor
      Soft Matter (2014), 10 (20), 3608-3621CODEN: SMOABF; ISSN:1744-683X. (Royal Society of Chemistry)
      The interaction of six tripodal synthetic chloride transmembrane transporters with a POPC bilayer was investigated by means of mol. dynamics simulations using the general Amber force field (GAFF) for the transporters and the LIPID11 force field for phospholipids. These transporters are structurally simple mols., based on the tris(2-aminoethyl)amine scaffold, contg. three thiourea binding units coupled with three Bu (1), Ph (2), fluorophenyl (3), pentafluorophenyl (4), trifluoromethylphenyl (5), or bis(trifluoromethyl)phenyl (6) substituents. The passive diffusion of 1-6 ∋ Cl- was evaluated with the complexes initially positioned either in the water phase or inside the bilayer. In the first scenario the chloride is released in the water soln. before the synthetic mols. achieve the water-lipid interface and permeate the membrane. In the latter one, only when the chloride complex reaches the interface is the anion released to the water phase, with the transporter losing the initial ggg tripodal shape. Independently of the transporter used in the membrane system, the bilayer structure is preserved and the synthetic mols. interact with the POPC mols. at the phosphate headgroup level, via N-H···O hydrogen bonds. Overall, the mol. dynamics simulations' results indicate that the small tripodal mols. in this series have a low impact on the bilayer and are able to diffuse with chloride inside the lipid environment. Indeed, these are essential conditions for these mols. to promote the transmembrane transport as anion carriers, in agreement with exptl. efflux data.
      (d) Wu, X.; Judd, L. W.; Howe, E. N. W.; Withecombe, A. M.; Soto-Cerrato, V.; Li, H.; Busschaert, N.; Valkenier, H.; Pérez-Tomás, R.; Sheppard, D. N.; Jiang, Y.-B.; Davis, A. P.; Gale, P. A. Nonprotonophoric Electrogenic Cl– Transport Mediated by Valinomycin-like Carriers. Chem 2016, 1, 127– 146,  DOI: 10.1016/j.chempr.2016.04.002
      12d
      Nonprotonophoric Electrogenic Cl- Transport Mediated by Valinomycin-like Carriers
      Wu, Xin; Judd, Luke W.; Howe, Ethan N. W.; Withecombe, Anne M.; Soto-Cerrato, Vanessa; Li, Hongyu; Busschaert, Nathalie; Valkenier, Hennie; Perez-Tomas, Ricardo; Sheppard, David N.; Jiang, Yun-Bao; Davis, Anthony P.; Gale, Philip A.
      Chem (2016), 1 (1), 127-146CODEN: CHEMVE; ISSN:2451-9294. (Cell Press)
      Synthetic transmembrane anion transporters (anionophores) have potential as tools for biomedical research and as therapeutic agents for diseases assocd. with anion-channel dysfunction. However, the possibility of H+ or OH- transport by anionophores has received little attention, and an anionophore selective for Cl- over H+/OH- is currently unavailable. Here, we show that depending on anionophore acidity, many anionophores facilitate electrogenic H+ or OH- transport, potentially leading to toxicity. Nevertheless, using several liposome-membrane-based assays, we identified two newly developed small mols. that promote electrogenic Cl- transport without effectively dissipating the transmembrane pH gradient, essentially mimicking the electrogenic cationophore valinomycin. The Cl- > H+/OH- selectivity of anionophores showed a consistent pos. correlation with the degree of Cl- encapsulation and a neg. correlation with the acidity of hydrogen-bond donors. Our study demonstrates that a valinomycin equiv. for Cl--selective transport is achievable.
      (e) Wu, X.; Gale, P. A. Small-Molecule Uncoupling Protein Mimics: Synthetic Anion Receptors as Fatty Acid-Activated Proton Transporters. J. Am. Chem. Soc. 2016, 138, 16508– 16514,  DOI: 10.1021/jacs.6b10615
      12e
      Small-Molecule Uncoupling Protein Mimics: Synthetic Anion Receptors as Fatty Acid-Activated Proton Transporters
      Wu, Xin; Gale, Philip A.
      Journal of the American Chemical Society (2016), 138 (50), 16508-16514CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Uncoupling proteins (UCPs) regulate energy expenditure in living cells by inducing proton leakage across the mitochondrial inner membrane, thereby uncoupling ADP phosphorylation from nutrient oxidn. The proton transport activity of UCP1 and UCP2 requires activation by fatty acids. Here, the authors report the 1st examples of synthetic small mols. performing this biol. important fatty acid-activated function. The authors previously showed that a tripodal thiourea possesses poor H+/OH- transport activity without fatty acids, but in the presence of long-chain fatty acids is "switched on" as a proton transporter with an activity close to a commonly used protonophore. Fatty acid-enhanced proton transport was also obsd. for other H- and halogen-bond-based synthetic anion transporters. The authors propose that these compds. induce proton permeability by catalyzing transbilayer movement ("flip-flop") of anionic fatty acids, so allowing the fatty acids to complete a proton transport cycle. Several lines of evidence have been provided to support such a fatty acid cycling mechanism. These findings open up new applications of anion receptor chem., and provide important clues for understanding biol. activities of synthetic anion transporters and potentially the uncoupling mechanism of naturally occurring membrane proteins.
      (f) Spooner, M. J.; Gale, P. A. A Tripodal Tris-Selenourea Anion Transporter Matches the Activity of Its Thio- Analogue but Shows Distinct Selectivity. Supramol. Chem. 2018, 30, 514– 519,  DOI: 10.1080/10610278.2018.1431394
      There is no corresponding record for this reference.
      (g) Jowett, L. A.; Howe, E. N. W.; Wu, X.; Busschaert, N.; Gale, P. A. New Insights into the Anion Transport Selectivity and Mechanism of Tren-based Tris-(Thio)Ureas. Chem. - Eur. J. 2018, 24, 10475– 10487,  DOI: 10.1002/chem.201801463
      12g
      New Insights into the Anion Transport Selectivity and Mechanism of Tren-based Tris-(thio)ureas
      Jowett, Laura A.; Howe, Ethan N. W.; Wu, Xin; Busschaert, Nathalie; Gale, Philip A.
      Chemistry - A European Journal (2018), 24 (41), 10475-10487CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The anion transport properties of a series of previously reported tren-based anionophores have been revisited using new assays designed to measure anion uniport. This study provides new insights into the transport mechanism and selectivity of this important class of transporters. Specifically, we report the chloride and nitrate transport selectivity of these systems and quantify sulfate transport to det. EC50 values for sulfate transport for the first time. Two new assays were developed to study bicarbonate transport allowing accurate quantification of chloride/bicarbonate exchange.
      (h) Jowett, L. A.; Ricci, A.; Wu, X.; Howe, E. N. W.; Gale, P. A. Investigating the Influence of Steric Hindrance on Selective Anion Transport. Molecules 2019, 24, 1278,  DOI: 10.3390/molecules24071278
      There is no corresponding record for this reference.
      (i) Howe, E. N. W.; Gale, P. A. Fatty Acid Fueled Transmembrane Chloride Transport. J. Am. Chem. Soc. 2019, 141, 10654– 10660,  DOI: 10.1021/jacs.9b02116
      12i
      Fatty Acid Fueled Transmembrane Chloride Transport
      Howe, Ethan N. W.; Gale, Philip A.
      Journal of the American Chemical Society (2019), 141 (27), 10654-10660CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Generation of chem. gradients across biol. membranes of cellular compartments is a hallmark of all living systems. Here the authors report a proof-of-concept prototype transmembrane pumping system in liposomes. The pump uses fatty acid to fuel chloride transport, thus generating a transmembrane chloride gradient. Addn. of fatty acid to phospholipid vesicles generates a transmembrane pH gradient (pHin < pHout), and this electrochem. H+ potential is harnessed by an anionophore to drive chloride efflux via H+/Cl- cotransport. Further addn. of fatty acid efficiently fuels the system to continuously drive chloride transport against the concn. gradient, up to [Cl-]in 65 mM | [Cl-]out 100 mM, and is 1400 times more efficient than using an external fuel. Based on the findings from dissecting the H+/Cl- flux process with the use of different liposomal fluorescence assays, and supported by addnl. liposome-based 13C NMR and DLS studies; the authors proposed that the presence of an anionophore can induce asym. distribution of fatty acid, and contribute to another Cl- flux mechanism in this system.
      (j) Wu, X.; Small, J. R.; Cataldo, A.; Withecombe, A. M.; Turner, P.; Gale, P. A. Voltage-Switchable HCl Transport Enabled by Lipid Headgroup–Transporter Interactions. Angew. Chem., Int. Ed. 2019, 58, 15142– 15147,  DOI: 10.1002/anie.201907466
      12j
      Voltage-switchable HCl transport enabled by lipid headgroup-transporter interactions
      Wu, Xin; Small, Jennifer R.; Cataldo, Alessio; Withecombe, Anne M.; Turner, Peter; Gale, Philip A.
      Angewandte Chemie, International Edition (2019), 58 (42), 15142-15147CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Synthetic anion transporters that facilitate transmembrane H+/Cl- symport (cotransport) have anti-cancer potential due to their ability to neutralize pH gradients and inhibit autophagy in cells. However, compared to the natural product prodigiosin, synthetic anion transporters have low-to-modest H+/Cl- symport activity and their mechanism of action remains less well understood. We report a chloride-selective tetraurea macrocycle that has a record-high H+/Cl- symport activity similar to that of prodigiosin and most importantly demonstrates unprecedented voltage-switchable transport properties that are linked to the lack of uniport activity. By studying the anion binding affinity and transport mechanisms of four other anion transporters, we show that the lack of uniport and voltage-dependent H+/Cl- symport originate from strong binding to phospholipid headgroups that hampers the diffusion of the free transporters through the membrane, leading to an unusual H+/Cl- symport mechanism that involves only charged species. Our work provides important mechanistic insights into different classes of anion transporters and a new approach to achieve voltage-switchability in artificial membrane transport systems.
      (k) Spooner, M. J.; Li, H.; Marques, I.; Costa, P. M. R.; Wu, X.; Howe, E. N. W.; Busschaert, N.; Moore, S. J.; Light, M. E.; Sheppard, D. N.; Félix, V.; Gale, P. A. Fluorinated Synthetic Anion Carriers: Experimental and Computational Insights into Transmembrane Chloride Transport. Chem. Sci. 2019, 10, 1976– 1985,  DOI: 10.1039/C8SC05155K
      12k
      Fluorinated synthetic anion carriers: experimental and computational insights into transmembrane chloride transport
      Spooner, Michael J.; Li, Hongyu; Marques, Igor; Costa, Pedro M. R.; Wu, Xin; Howe, Ethan N. W.; Busschaert, Nathalie; Moore, Stephen J.; Light, Mark E.; Sheppard, David N.; Felix, Vitor; Gale, Philip A.
      Chemical Science (2019), 10 (7), 1976-1985CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      A series of fluorinated tripodal tris-thioureas function as highly active anion transporters across lipid bilayers and cell membranes. Here, we investigate their mechanism of action using anion transport assays in cells and synthetic vesicles and mol. modeling of transporter-lipid interactions. When compared with non-fluorinated analogs, fluorinated compds. demonstrate a different mechanism of membrane transport because the free transporter cannot effectively diffuse through the membrane. As a result, in H+/Cl- cotransport assays, fluorinated transporters require the presence of oleic acid to form anionic oleate complexes for recycling of the transporter, whereas non-fluorinated analogs readily diffuse through the membrane as free transporters and show synergistic transport with the proton transporter gramicidin. Mol. dynamics simulations revealed markedly stronger transporter-lipid interactions for fluorinated compds. compared with non-fluorinated analogs and hence, higher energy barriers for fluorinated compds. to cross the membrane as free transporters. With use of appropriate proton transporters to ensure measurement of the correct rate-limiting steps, the transport rates detd. in synthetic vesicle assays show excellent agreement with the anion transport rates detd. in cell-based assays. We conclude that integration of computational and exptl. methods provides a strategy to optimize transmembrane anion transporter design for biomedical applications.
      (l) Wu, X.; Gale, P. A. Measuring Anion Transport Selectivity: A Cautionary Tale. Chem. Commun. 2021, 57, 3979– 3982,  DOI: 10.1039/D1CC01038G
      12l
      Measuring anion transport selectivity: a cautionary tale
      Wu, Xin; Gale, Philip A.
      Chemical Communications (Cambridge, United Kingdom) (2021), 57 (33), 3979-3982CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      PH-dependent liposomal assays are often used to det. anion selectivity in transmembrane anion transport expts. We discuss the validity and limitations of these assays, and provide guidelines for their use to avoid misleading results.
    13. 13
      (a) Shinkai, S.; Ogawa, T.; Kusano, Y.; Manabe, O. Selective Extraction of Alkali Metal Cations by a Photoresponsive Bis(crown ether). Chem. Lett. 1980, 9, 283– 286,  DOI: 10.1246/cl.1980.283
      There is no corresponding record for this reference.
      (b) Bandara, H. M. D.; Burdette, S. C. Photoisomerization in Different Classes of Azobenzene. Chem. Soc. Rev. 2012, 41, 1809– 1825,  DOI: 10.1039/C1CS15179G
      13b
      Photoisomerization in different classes of azobenzene
      Bandara, H. M. Dhammika; Burdette, Shawn C.
      Chemical Society Reviews (2012), 41 (5), 1809-1825CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      A review. Azobenzene undergoes trans cis isomerization when irradiated with light tuned to an appropriate wavelength. The reverse cis trans isomerization can be driven by light or occurs thermally in the dark. Azobenzene's photochromatic properties make it an ideal component of numerous mol. devices and functional materials. Despite the abundance of application-driven research, azobenzene photochem. and the isomerization mechanism remain topics of study. Addnl. substituents on the azobenzene ring system change the spectroscopic properties and isomerization mechanism. This crit. review details the studies completed to date on the 3 main classes of azobenzene derivs. Understanding the differences in photochem., which originate from substitution, is imperative in exploiting azobenzene in the desired applications.
    14. 14
      Zhang, X.-a.; Woggon, W.-D. A Supramolecular Fluorescence Sensor for Pyrovanadate as a Functional Model of Vanadium Haloperoxidase. J. Am. Chem. Soc. 2005, 127, 14138– 14139,  DOI: 10.1021/ja053209r
      14
      A Supramolecular Fluorescence Sensor for Pyrovanadate as a Functional Model of Vanadium Haloperoxidase
      Zhang, Xiao-an; Woggon, Wolf-D.
      Journal of the American Chemical Society (2005), 127 (41), 14138-14139CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A novel basket-shaped tris(pyrene guanidinium) receptor was synthesized which binds pyrovanadate and pyrophosphate with Ka > 107 M-1. The binding of both anions is assocd. with quenching of the excimer fluorescence of the pyrenes. The supramol. vanadate complex catalyzes the bromination of activated C-H bonds and hence is an enzyme mimic of vanadium haloperoxidases.
    15. 15
      Rodríguez-Soacha, D. A.; Steinmüller, S. A. M.; Işbilir, A.; Fender, J.; Deventer, M. H.; Ramírez, Y. A.; Tutov, A.; Sotriffer, C.; Stove, C. P.; Lorenz, K.; Lohse, M. J.; Hislop, J. N.; Decker, M. Development of an Indole-Amide-Based Photoswitchable Cannabinoid Receptor Subtype 1 (CB 1 R) “Cis-On” Agonist. ACS Chem. Neurosci. 2022, 13, 2410– 2435,  DOI: 10.1021/acschemneuro.2c00160
      There is no corresponding record for this reference.
    16. 16
      Qian, H.; Wang, Y.-Y.; Guo, D.-S.; Aprahamian, I. Controlling the Isomerization Rate of an Azo-BF 2 Switch Using Aggregation. J. Am. Chem. Soc. 2017, 139, 1037– 1040,  DOI: 10.1021/jacs.6b10982
      16
      Controlling the Isomerization Rate of an Azo-BF2 Switch Using Aggregation
      Qian, Hai; Wang, Yu-Ying; Guo, Dong-Sheng; Aprahamian, Ivan
      Journal of the American Chemical Society (2017), 139 (3), 1037-1040CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A visible-light activated azo-BF2 switch possessing a phenanthridinyl π-system has been synthesized, and its switching properties have been characterized as a function of concn. The switch self-aggregates through π-π interactions, and the degree of aggregation modulates the Z → E thermal isomerization rate. This property allows for the active tuning of the thermal relaxation half-life of the same switch from seconds to days.
    17. 17
      (a) Matile, S.; Sakai, N.; Hennig, A. Transport Experiments in Membranes. In Supramolecular Chemistry; Wiley: Chichester, UK, 2012.
      There is no corresponding record for this reference.
      (b) Gilchrist, A. M.; Wang, P.; Carreira-Barral, I.; Alonso-Carrillo, D.; Wu, X.; Quesada, R.; Gale, P. A. Supramolecular Methods: The 8-Hydroxypyrene-1,3,6-Trisulfonic Acid (HPTS) Transport Assay. Supramol. Chem. 2021, 33, 325– 344,  DOI: 10.1080/10610278.2021.1999956
      17b
      Supramolecular methods: the 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) transport assay
      Gilchrist, Alexander M.; Wang, Patrick; Carreira-Barral, Israel; Alonso-Carrillo, Daniel; Wu, Xin; Quesada, Roberto; Gale, Philip A.
      Supramolecular Chemistry (2021), 33 (7), 325-344CODEN: SCHEER; ISSN:1029-0478. (Taylor & Francis Ltd.)
      The liposomal membrane transport assay, which uses 8-hydroxypyrene-1,3,6-trisulfonic acid to monitor the internal pH of the vesicles (the HPTS assay), is a widely used technique for analyzing the activity of anionophore-facilitated transport across a phospholipid membrane. This paper describes the stepwise technique to conduct this transport assay, detailing both the perks and pitfalls of using this method to det. the activity of an anionophore and the transport mechanism.
    18. 18
      Serra, F.; Terentjev, E. M. Effects of Solvent Viscosity and Polarity on the Isomerization of Azobenzene. Macromolecules 2008, 41, 981– 986,  DOI: 10.1021/ma702033e
      18
      Effects of Solvent Viscosity and Polarity on the Isomerization of Azobenzene
      Serra, Francesca; Terentjev, Eugene M.
      Macromolecules (Washington, DC, United States) (2008), 41 (3), 981-986CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
      Isomerization of azobenzene was studied for many years, but some aspects are still unclear or controversial. This work provides further insight and clarifies the role that environmental parameters play in detg. the reaction kinetics of azobenzene derivs. in soln. Detailed spectroscopic measurements of the photoisomerization and thermal isomerization kinetics in mixts. of various solvents and polystyrene were carried out. The results indicate a strong dependence of both photoisomerization and thermal isomerization rates on the polarity, but not on the viscosity of the solvent.
    19. 19
      Dias, C. M.; Li, H.; Valkenier, H.; Karagiannidis, L. E.; Gale, P. A.; Sheppard, D. N.; Davis, A. P. Anion Transport by Ortho-Phenylene Bis-Ureas across Cell and Vesicle Membranes. Org. Biomol. Chem. 2018, 16, 1083– 1087,  DOI: 10.1039/C7OB02787G
      19
      Anion transport by ortho-phenylene bis-ureas across cell and vesicle membranes
      Dias, Christopher M.; Li, Hongyu; Valkenier, Hennie; Karagiannidis, Louise E.; Gale, Philip A.; Sheppard, David N.; Davis, Anthony P.
      Organic & Biomolecular Chemistry (2018), 16 (7), 1083-1087CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      Ortho-phenylene bis-ureas can serve as anionophores in cells expressing halide-sensitive yellow fluorescent protein, as well as in synthetic vesicles. The activities can reach high levels, and are strongly dependent on the deliverability of the transporters.
    20. 20
      Gensure, R. H.; Zeidel, M. L.; Hill, W. G. Lipid Raft Components Cholesterol and Sphingomyelin Increase H+/OH– Permeability of Phosphatidylcholine Membranes. Biochem. J. 2006, 398, 485– 495,  DOI: 10.1042/BJ20051620
      There is no corresponding record for this reference.
    21. 21

      The groups of Montenegro and Basílio recently demonstrated photocontrol of cationic peptide transport by an azobenzene-appended calixarene, where the higher activity observed for the (E)-isomer was ascribed to a change in lipophilicity (i.e., increased hydrophobicity). Yet, altered membrane mobility might offer an additional explanation, see:

      Martins, J. N.; Raimundo, B.; Rioboo, A.; Folgar-Cameán, Y.; Montenegro, J.; Basílio, N. Photoswitchable Calixarene Activators for Controlled Peptide Transport across Lipid Membranes. J. Am. Chem. Soc. 2023, 145, 13126– 13133,  DOI: 10.1021/jacs.3c01829
      21
      Photoswitchable Calixarene Activators for Controlled Peptide Transport across Lipid Membranes
      Martins, Joana N.; Raimundo, Beatriz; Rioboo, Alicia; Folgar-Camean, Yeray; Montenegro, Javier; Basilio, Nuno
      Journal of the American Chemical Society (2023), 145 (24), 13126-13133CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Supramol. synthetic transporters are crucial to understand and activate the passage across lipid membranes of hydrophilic effector mols. Herein, we introduce photoswitchable calixarenes for the light-controlled transport activation of cationic peptide cargos across model lipid bilayers and inside living cells. Our approach was based on rationally designed p-sulfonatocalix[4]arene receptors equipped with a hydrophobic azobenzene arm, which recognize cationic peptide sequences at the nM range. Activation of membrane peptide transport is confirmed, in synthetic vesicles and living cells, for calixarene activators featuring the azobenzene arm in the E configuration. Therefore, this method allows the modulation of the transmembrane transport of peptide cargos upon Z-E photoisomerization of functionalized calixarenes using 500 nm visible light. These results showcase the potential of photoswitchable counterion activators for the light-triggered delivery of hydrophilic biomols. and pave the way for potential applications in remotely controlled membrane transport and photopharmacol. applications of hydrophilic functional biomols.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.4c10952.

    • Experimental procedures for the synthesis of 1 and 2 and full characterization, 1H NMR and UV–vis studies, transport assays, and X-ray analysis (CCDC 2371925) can be found in the Supporting Information (PDF)

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    Deposition Number 2371925 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via the joint Cambridge Crystallographic Data Centre (CCDC) and Fachinformationszentrum Karlsruhe Access Structures service.


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