Molecular communication system using Zn²⁺ selective supramolecular nanochannel to induce photoregulated catalysis

Abstract

We developed synthetic ionophores that self-assemble into supramolecular nanochannels for the transport of Zn²⁺ across lipid bilayers. The translocated Zn²⁺ forms a photoactive PPIX–Zn²⁺ complex that generates ¹O₂ to oxidize tryptophan, establishing a Zn²⁺-mediated cascade reaction network within confined spaces, which is crucial for developing photoregulated molecular communication systems.

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Cellular communication, facilitated by transport proteins and ion channels, is essential for coordinating cellular activities and maintaining homeostasis. Several key biological functions, such as differentiation, apoptosis, and proliferation through ligand-receptor interactions and secondary messengers, are regulated by these transport proteins and ion channels.^1,2 Inspired by natural processes, molecular communication systems have emerged as an innovative approach to mimic cellular pathways using synthetic biomolecule-based nanochannels capable of transmitting and amplifying chemical signals across membranes. Such artificial ion transport-based systems not only enable precise spatiotemporal control of information exchange but also offer intelligent platforms for targeted therapy, biosensing, and synthetic cell-to-cell communication, thereby enhancing our understanding of stimulus-driven biochemical processes signalling.^3–7

Recent studies have demonstrated the development of inter-vesicle and transmembrane signalling systems capable of transmitting signals with or without directly transferring ions or signalling molecules.^6,8 However, a significant limitation of these systems is the lack of controllability once mass transport across the membrane triggers a downstream chemical reaction.⁹ Hence, introducing a stimulus-responsive communication system that regulates ion transport-mediated chemical reaction through external cues is crucial and presents a unique opportunity for understanding complex cellular signalling processes.^5,7 Transition metal ions, such as Zn²⁺, play a pivotal role in many signalling processes, including enzyme activation, DNA synthesis, and immune regulation.¹⁰ Hence, Zn²⁺ acts as an ideal cofactor for developing a controllable, biomimetic communication system. Inspired by the importance of Zn²⁺ in cellular environments, we developed a photo-responsive molecular communication system by integrating a synthetic ion transporter with a protoporphyrin IX (PPIX) (Fig. 1). The 2-(pyridin-2-yl)-1H-benzo[d]imidazol-6-amine-based tripodal compound (3d) self-assembled to form supramolecular nanochannels within the lipid bilayers to selectively transport Zn²⁺ from the extravesicular to the intravesicular environment. The complexation of transported Zn²⁺ with the PPIX resulted in the formation of a catalytic system to promote singlet oxygen (¹O₂)-mediated oxidation of tryptophan (Trp) amino acid to N-formyl kynurenine (Nfk) exclusively in the presence of light within the intravesicular environment. Therefore, this molecular communication system utilizes synthetic ion transporters for transferring encrypted chemical signals and employs PPIX and red light for decryption through biochemical transformations, thereby providing a modular strategy for designing programmable protocells.

Fig. 1 A schematic representation of the molecular communication process via Zn²⁺ transport, facilitating in situ complexation of Zn²⁺ with PPIX, which leads to reactive oxygen species (ROS) generation and the subsequent oxidation of Trp to Nfk.

The 2-(pyridin-2-yl)-1H-benzo[d]imidazole is a known sensor of Zn²⁺, particularly due to the interaction between the imidazole and pyridyl nitrogen atoms with the Zn²⁺.¹¹ However, due to its lower lipophilicity (log P 1.95), it cannot be directly used as a transmembrane transporter of Zn²⁺. Hence, to develop potent transmembrane Zn²⁺ transporters, we synthesized tripodal and dipodal thiourea derivatives of 2-(pyridin-2-yl)-1H-benzo[d]imidazole. The reactions of 1,3,5-triethyl-2,4,6-tris(isothiocyanatomethyl)benzene with 2-(pyridin-2-yl)-1H-benzo[d]imidazol-6-amine and other aryl amines yielded 3a–3d (Fig. 2). The reactions of 1,3-bis(isothiocyanatomethyl)benzene with 2-(pyridin-2-yl)-1H-benzo[d]imidazol-6-amine and 2-(1H-pyrrol-2-yl)-1H-benzo[d]imidazol-6-amine resulted in the formation of dipodal 5a. Compounds 3a, 3b, and 5a were synthesized to investigate the role of thiourea, 2-(pyridin-2-yl)-1H-benzo[d]imidazole, and tripodal moieties in Zn²⁺ transport.

Fig. 2 Structure of the synthesized compounds.

The ion transport aptitude of the synthesized compounds was evaluated using the large unilamellar vesicles (LUVs) of egg yolk phosphatidylcholine (EYPC) and cholesterol (Chol) (8 : 2 molar ratio) by encapsulating pentapotassium salt of magnesium green (MgG) (Fig. 3A).¹² The LUVs were prepared in 10 mM HEPES buffer containing 100 mM NaCl, 50 µM MgG, and 100 µM EDTA at pH 7.0. The compound screening assay showed higher Zn²⁺ transport efficacy for 3d (∼75%) compared to its related derivatives, 3a–3c and 5a, highlighting it as a promising candidate for further investigation and potential applications (Fig. 3B). The lower Zn²⁺ transport efficacy of 3c and 5a in comparison to 3d suggests the importance of the 2-(pyridin-2-yl)-1H-benzo[d]imidazole and tripodal moieties in Zn²⁺ recognition and transport.

Fig. 3 (A) Evaluation of the Zn²⁺ transport activity of the synthesized compounds (40 nM) across EYPC/Chol-LUVs⊃MgG (B) Concentration-dependent Zn²⁺ transport activities of 3d. (C) Cation and (D) anion transport selectivity of 3d (40 nM) across EYPC/Chol-LUVs⊃MgG. (E) Ion conductance measurements across the BLM at +150 mV and −150 mV in the presence of 3d in a symmetrical solution of ZnCl₂ (0.5 M) in water.

The concentration-dependent MgG assay of 3d showed a half-maximal effective concentration (EC₅₀) of 44.83 ± 1.74 nM (3d: lipid = 1 : 5 × 10⁵) and the Hill coefficient (n) value of 1.87 ± 0.09 (Fig. S1). The Hill coefficient value (∼2) suggests that 3d exhibits positive cooperativity in forming supramolecular channels within the lipid bilayers.¹³ The cation selectivity study performed for 3d revealed the selectivity for Zn²⁺ among the tested cations (Fig. 3C). Additionally, for monovalent cations, the transport study was performed using EYPC/CHOL-LUVs⊃HPTS, which showed negligible transport by 3d (Fig. S3). However, no difference in Zn²⁺ transport efficacy was observed in anion selectivity studies (Fig. 3D). Minimal but measurable Cl⁻ transport activity (∼20%) was observed, but this activity is considerably lower than its Zn²⁺ transport activity (Fig. S4). The sulfur atom of the thiourea moiety is known to interact with Zn²⁺ and other metal ions.¹⁴ The UV-Vis-based titration study revealed the binding constant of 7439 ± 3 M⁻¹, suggesting a moderate affinity of 3d for Zn²⁺ (Fig. S5). The ¹H NMR-based interaction studies clearly showed that the benzimidazole NH proton showed a strong interaction with the Zn²⁺, and a partial shift of thiourea NH suggests that Zn²⁺ weakly interacts with the thiourea group (S atom of the thiourea group) (Fig. S6 and S7).

Mechanistic studies using MgG-encapsulated liposomes were conducted in the presence of FCCP (H⁺ carrier) and valinomycin (K⁺ uniporter).⁹ No notable change in MgG fluorescence was observed with or without FCCP, suggesting H⁺ participation in 3d-mediated Zn²⁺ transport (Fig. S8). Further, pH-sensitive fluorescein assays showed increased fluorescence across EYPC/CHOL-LUV⊃fluorescein, confirming H⁺ efflux (Fig. S9). Thus, both FCCP and fluorescein assays established the Zn²⁺/H⁺ antiport activity of 3d. Valinomycin-based studies showed unchanged transport efficiency with or without the K⁺ uniporter, indicating no OH⁻ involvement (Fig. S10). The carboxyfluorescein leakage assay confirmed no vesicle disruption (Fig. S11). To probe the transport pathway, Chol-dependent and U-tube assays were performed. LUVs with EYPC: CHOL ratios of 6 : 4 and 8 : 2 showed no change in activity, supporting a channel-based mechanism (Fig. S12). In the U-tube assay, 3d failed to transport Zn²⁺ across the nonpolar phase, unlike clioquinol, a known Zn²⁺ carrier (Fig. S13).

The FESEM images revealed that 3d formed nanowire-like structures in a hydrophobic environment (chloroform) and exhibited spherical agglomeration in a hydrophilic medium (water), indicating solvent-dependent self-assembly (Fig. S14). The 2D-NOESY measurement of 3d revealed a strong spatial correlation between the pyridyl aromatic proton (δ 7.51 ppm) and the thiourea N–H proton (δ 9.49 ppm) (Fig. S15). Additionally, cross-peaks were observed between the benzylic proton (δ 4.68 ppm) and the ethyl group proton (δ 2.80 ppm) with the pyridyl aromatic proton (δ 7.49 ppm). These interactions suggest a self-assembled architecture in which pyridyl arms interlock via noncovalent interactions, while the remaining arms form a channel-like cavity.

To further investigate the channel-forming ability, the real-time change in current at a constant voltage in the presence of 3d was monitored through an electrophysiological experiment.¹⁵ After the addition of 3d (4 µM) to both chambers, we observed repetitive opening-closing events at various holding potentials, confirming the formation of an ion channel within the planar lipid bilayer (Fig. 3E and Fig. S16). Additional measurements of current against voltage (I–V plot) were further monitored using a symmetrical ZnCl₂ (0.5 M) solution (Fig. S17). A linear increase in current was noted with increasing voltage, indicating the ohmic behavior. The measured single-channel conductance (G) of 54 ± 11 pS indicates the formation of ion channels with a pore diameter of approximately 1.5 Å.

Metalloporphyrins are essential biomolecules with catalytic properties that play vital roles in physiological functions within living systems. Metalloporphyrins are also used in molecular catalysis, water splitting, and photodynamic therapy.^16,17 The zinc-porphyrin has been identified as a ROS generator under photo-irradiation,¹⁸ which can be utilized for carrying out reactions within the compartmentalized structures, such as LUVs. We observed that the complexation between PPIX and Zn²⁺ leads to the generation of ROS when exposed to red light. The enhancement of fluorescence intensity at 522 nm due to the conversion of 7′-dichlorofluorescin diacetate (H₂DCFDA) to 2′,7′-dichlorofluorescein (DCF) was only observed in the presence of both PPIX and Zn²⁺ upon photo irradiation (Fig. S18). No significant DCF fluorescence enhancement was observed with PPIX alone, indicating that Zn²⁺ binding significantly enhances the ROS generation aptitude of PPIX. The UV-Vis and fluorescence-based titration studies of PPIX with Zn²⁺ showed no metalation of PPIX (Fig. S19 and S20). All four Q bands (500–700 nm) of PPIX remained intact, suggesting no Zn²⁺ incorporation into the porphyrin ring of PPIX.¹⁹ However, a partial decrease in the Soret band (400 nm) and a significant reduction in fluorescence intensity at 640 nm indicated either complexation of Zn²⁺ with the carboxylate groups of PPIX or adopting a weakly bound, 'sitting-atop' geometry above the porphyrin ring, without coordination to the core nitrogen atoms. These Zn²⁺ binding modes may significantly alter the electronic structure of the porphyrin ring, thereby enhancing the ability to generate ROS upon photoactivation.¹⁸

To investigate the ROS-mediated catalytic activities of PPIX-Zn²⁺ complex, the reactions were performed in the presence of Trp amino acid, both with and without light, in a 20 mM HEPES buffer at pH 7.0 (Fig. S21). Upon optimization, we observed that red light achieves better photo-oxidation in just 3–4 h, whereas white light requires approximately 24 h to reach similar efficiency, highlighting the superior effectiveness of wavelength-specific excitation for Zn²⁺-PPIX-mediated photo-oxidation. Additionally, exposure to white light can promote increased nonspecific photodegradation of Zn²⁺-PPIX, reducing its effectiveness, as confirmed by HPLC analysis (Fig. S22).

Interestingly, after 4 h of red-light exposure, the HRMS analysis detected the formation of Nfk ([M + H]⁺ = 237.0905) only in the reaction mixture containing Trp and PPIX-Zn²⁺. Nfk is known to undergo hydrolysis to kynurenine (Kyn) in the presence of trichloroacetic acid (TCA).^20,21 The resulting Kyn was subsequently quantified using UV-Vis spectroscopy following derivatization with p-dimethylaminobenzaldehyde (p-DMAB). The UV-Vis spectral analysis revealed a significant enhancement of the peak at 480 nm, which corresponds to the formation of the imine complex of p-DMAB with Kyn (Fig. S18).²⁰ The time-dependent HPLC analysis confirmed increasing Kyn formation with light exposure, while no significant amount of Kyn was detected without light (Fig. S23).

The HRMS and NMR analysis of the eluted solution confirm the identity of Kyn (Fig. S24 and S25). Additional reactions of Trp with the PPIX-Zn²⁺ complex in the presence of different ROS quenchers were performed to investigate the Trp-to-Kyn formation mechanism. DABCO, known as a ¹O₂ scavenger, suppressed both HPLC and absorbance peaks, suggesting the conversion of Trp to Kyn proceeds via¹O₂ under light (Fig. S26).²² Additional photo-regulated oxidation studies using the PPIX-Zn²⁺ complex with tyrosine revealed its dimerization, while with methionine, it demonstrated mono-oxidation to methionine sulfoxide (Table S2 and Fig. S27).

This finding provided a unique opportunity to develop a photo-regulated, Zn²⁺-dependent molecular communication system. The EYPC/Chol-LUV⊃PPIX/Trp were prepared in 20 mM HEPES buffer at pH 7.0. The LUVs were suspended in 20 mM HEPES buffer at pH 7.0, and 3d, along with Zn²⁺, was added to the extravesicular solution (Fig. 4A). After prolonged exposure (4 h) to the red lights, the LUVs were lysed using 20% Triton X-100 (20 µL) (Fig. S28). The HPLC and UV-Vis spectral analysis suggested the conversion of Trp to Kyn under the vesicular environment. Control experiments without 3d, Zn²⁺, Tyr, or light showed no significant formation of Kyn. The time-resolved HPLC analysis confirmed a gradual increase in the Kyn formation, supporting Kyn formation via transmembrane Zn²⁺ transport by 3d and ¹O₂-mediated oxidation under light within a confined vesicular environment (Fig. 4B). We also conducted a similar reaction under alternating 60 min light and dark cycles for a total duration of 300 min. The HPLC and UV-Vis spectral analysis showed that light-dependent Trp to Kyn conversion is associated with the photo-induced ¹O₂ generation by the in situ generated PPIX-Zn²⁺ complex within the vesicles (Fig. 4C and Fig. S29). The indoleamine-2,3-dioxygenase (IDO1) enzyme-mediated catabolism of Trp to Nfk through the kynurenine pathways is involved in various physiological processes, including the immune response.^20,21 Hence, this photo-regulated molecular communication system functions as an artificial enzyme to mimic the catalytic conversion of Trp to Nfk.

Fig. 4 (A) A schematic illustration shows the in situ complexation of Zn²⁺ with PPIX, initiating light-regulated catalysis that significantly increases the generation of ROS and leads to the oxidation of Trp to Nfk. (B) HPLC analysis at different time intervals demonstrates the production of Kyn within the EYPC/Chol-LUVs⊃Trp/PPIX system after photo-irradiation in the presence of 3d and Zn²⁺. (C) Stepwise red-light irradiation ON/OFF experiment using EYPC/Chol-LUVs⊃Trp/PPIX in 20 mM HEPES buffer, pH 7.0, with 3d and Zn²⁺ to detect Kyn generation.

In conclusion, we developed an ion transport-mediated photo-responsive molecular communication system. The self-assemblies of 3d within the bilayer form supramolecular channels that transport Zn²⁺ along with H⁺ in an antiport manner. Due to the selective Zn²⁺ transport ability of 3d, Zn²⁺ was utilized as a chemical signal to form Zn²⁺-PPIX complex, a photosensitizer, within the vesicular environment. The PPIX-Zn²⁺ complex showed ¹O₂-mediated enzyme-like oxidation of Trp to Nfk within the vesicles. Hence, this molecular communication system presents a unique biomimetic approach where Zn²⁺ serves not only as a transportable ion but also as a functional chemical signal that triggers an enzyme-like oxidation reaction exclusively under red light.

Conflicts of interest

There are no conflicts to declare.

Data availability

Supplementary information (SI): detailed experimental procedures, compound characterization, and ion transport activity data, and catalytic activity data were included. See DOI: https://doi.org/10.1039/d5cc06434a.

Acknowledgements

The authors acknowledge the SERB (CRG/2021/000306) and DBT (BT/PR52583/NER/95/2189/2024) New Delhi, India, for financial support, New Delhi, India, for financial support. The authors also recognize the instrumental facilities provided by the Central Instrument Facility (CIF) and the Department of Chemistry at IIT Guwahati. DM acknowledges DAAD for the Research Stays fellowship.

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Footnote

† S. S. and B. M. P. contributed equally.

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