Highly Selective Bioinspired Peptide Channels for Rapid Water Transport

ABSTRACT

Innovative water‐purification technologies are urgently needed to address the global scarcity of clean water. In nature, aquaporin proteins enable rapid and highly selective water transport across membranes. Replicating their exceptional water flux and ion‐rejection capabilities in synthetic systems, however, remains a major challenge. Herein, minimalistic peptide‐based water‐channels inspired by the conserved aquaporin Asn‐Pro‐Ala (NPA) motif are reported. These designer peptides are suitably functionalized to enable the formation of water‐channels by self‐assembly. The crystal structures of these water channels highlight how inward‐facing polar groups facilitate water transport, while hydrophobic exteriors enable membrane interactions. Channels derived from functionalized PA dipeptides and a tripeptide NPA analog with a pendant cyano group exhibit exceptional single‐channel permeabilities (≈10⁸ H₂O s⁻¹ channel⁻¹). While both peptides achieve complete Na⁺, Cl⁻, and proton exclusion, the NPA tripeptide shows marginal (∼10%) K⁺ transport. Molecular dynamics simulations, x‐ray structures, and experimentally determined activation energies suggest a water‐transport mechanism involving hydrogen‐bonding interactions with the peptide‐backbone. This work illustrates how structurally simple peptides can accomplish highly efficient and selective water transport, paving the way for compact and tunable molecular platforms for water‐purification and biomedical applications.

Minimalistic, nature‐inspired peptides self‐assemble to form water channels that conduct ∼10⁸ water molecules per second per channel while excluding ions and protons.

1. Introduction

Freshwater scarcity impacts billions of people worldwide [1]. Population growth, climate change, industrial expansion, and pollution compound this problem by depleting and contaminating natural water resources [2]. Seawater desalination might be the most viable option for securing clean water in the future. Reverse osmosis is highly efficient for desalination but remains energy intensive [3]. Alternative strategies to deliver clean water are being actively explored to address the aforementioned challenges [4].

Nature achieves efficient water transport across the cellular membranes of plants, animals, bacterial, and fungi through aquaporin proteins (AQPs) [5]. AQPs facilitate water transport across a concentration gradient by the basic principles of osmosis. These protein channels conduct over 10⁹ water molecules per second per channel while excluding salts and protons [6]. AQPs have been incorporated into synthetic membranes for water desalination [7, 8, 9]. Simpler artificial water channels (AWCs) are desirable for scalable and cost‐effective industrial applications. A persistent challenge for AWCs is attaining the hallmark water selectivity of AQPs without compromising the rate of water flux [10, 11, 12]. Channels with strong water‐wall interactions, such as those featuring hydrogen‐bonding, enhance water‐over‐ion selectivity at the cost of reduced water flux [12]. In contrast, channels with weak water‐wall interactions, such as hydrophobic pores, allow rapid water transport at the cost of cation or proton leakage [13]. Recent studies have demonstrated that modulating pore electrostatics [14] and hydrophobicity [15], or controlling supramolecular self‐assembly [16], can alleviate this trade‐off between water flux rate and selectivity.

Suitably substituted pillararene macrocycles [17, 18] and carbon nanotubes form unimolecular water‐transporting channels [13]. Diverse molecular architectures, including organic cages [19], imidazole‐quartets [20], aromatic foldamers [21], fluorous macrocycles [22], phenylalanine‐derived hydroxyls [23], and aromatic hydrazides, form AWCs by self‐assembly [16]. Carbon nanotubes exhibit water transport rates up to six times higher than AQP1, whereas the aromatic foldamer, pillararene, and hydrazide‐based channels display permeabilities of approximately 10⁹ H₂O s⁻¹ channel⁻¹, comparable to biological benchmarks.

Reported herein are peptide‐derived water transporters inspired by the AQP1 protein [24]. AQP1 has six transmembrane helices that result in an “hour‐glass” shape that is 2.8 Å at its narrowest region (Figure 1a), allowing only single water molecules to move across [25]. The two loops, B and E, each containing Asn‐Pro‐Ala (NPA) motifs, form the central aqueous pore [25]. These NPA motifs flanking the narrow central constriction precisely orient water molecules as they traverse the pore. This unique structure disrupts the continuous hydrogen‐bonded chain network of water molecules, and blocks proton transfer via the Grotthuss mechanism [26]. Inspired by the NPA motif in aquaporin, we wished to probe the ability of functionalized NPA tripeptides to mediate water transport in the absence of the entire protein manifold (Figure 1b). Considering the turn‐inducing ability of proline, the backbone amide bonds available for hydrogen‐bonding and the use of suitable aromatic protecting groups, one can envisage the formation of tubular assemblies that could potentially transport water. While pursuing the minimalistic NPA core of aquaporin, we discovered efficient water transport mediated by PA dipeptides and NPA analogs, while rejecting ions.

FIGURE 1.

(a) Structural organization of aquaporins with six transmembrane α‐helices arranged to form a pore. Two conserved NPA motifs align symmetrically at the narrow central constriction. (b) Minimalistic synthetic peptide designed to self‐assemble into water‐conducting channels.

2. Results and Discussion

To synthesize a suitably protected NPA motif, the Boc‐Pro‐Ala‐OBn dipeptide 1 precursor was synthesized in 80% yield using standard solution phase synthesis [27]. Boc deprotection of dipeptide 1 and subsequent coupling with Boc‐protected asparagine (1.2 equiv) using HBTU (1.2 equiv), DIEA (3.5 equiv), and a catalytic amount of DMAP (0.1 equiv) did not result in the expected NPA tripeptide 2 (Scheme 1a). Interestingly, Boc‐Ala(β‐CN)‐Pro‐Ala‐OBn tripeptide 3 was obtained in 32% yield via an intramolecular reaction involving the amide side‐chain [28]. Upon using 2.5 equiv HBTU, the yield for the cyano tripeptide 3 increased to 68%. To access the NPA motif, the Asn side chain was protected. Aromatic protecting groups were chosen to enhance the ability of these peptides to self‐assemble. Fmoc‐trityl‐protected asparagine 4 (1.2 equiv) was coupled with NH‐Pro‐Ala‐OBn (1 equiv) to afford Fmoc‐Asn(Trt)‐Pro‐Ala‐OBn 5 in 75% yield (Scheme 1b). Trityl deprotection of 5 (1 equiv) with TFA (2 equiv), gave Fmoc‐Asn‐Pro‐Ala‐OBn 6 in 94% yield. Purity of the peptides was confirmed using spectroscopy and mass spectrometry (HRMS). Single‐crystal x‐ray diffraction further confirmed the structures of peptides 1 and 3.

SCHEME 1.

Synthetic route for peptides (a) 3 and (b) 6.

As per reported protocols [14], large unilamellar vesicles (LUVs) were used as model systems for studying water transport [27]. LUVs of DOPC lipids and 8 mol% of peptides 1, 3, 5, and 6 were prepared in 100 mM NaCl at pH 7. The vesicles were exposed to an outward osmotic gradient of 200 mM sucrose at pH 7 (Figure 2a). Under hyperosmotic conditions, peptide‐mediated water efflux was monitored by measuring the light scattered (450 nm) as a consequence of the vesicle shrinkage. Rapid transport kinetics were captured using stopped‐flow measurements on the millisecond timescale. The changes in light scattering over time were fitted to a first‐order exponential equation to determine the rate of vesicle shrinkage (k) (Figure 2b). Peptides Boc‐PA‐OBn 1 (k = 28.9 s⁻¹), Boc‐A(β‐CN)PA‐OBn 3 (k = 29.3 s⁻¹), and Fmoc‐NPA‐OBn 6 (k = 25.2 s⁻¹) exhibited substantially higher rates (Figure 2b and Figure S2) of shrinkage than the control (k = 13.4 s⁻¹), indicating efficient water transport by peptide‐derived pores. In contrast, Fmoc‐N(Trt)PA‐OBn 5, despite its high hydrophobicity, showed no activity (k = 13.9 s⁻¹, Figure S2), most likely due to presence of bulky Trt groups that prevent the formation of ordered hydrogen‐bonded stacks or pores to facilitate water transport. Precipitation of peptide 5 was not observed during vesicle preparation confirming efficient insertion. A steady increase in water transport rates was observed for peptides Boc‐A(β‐CN)PA‐OBn 3 and Fmoc‐NPA‐OBn 6 when their concentrations with respect to lipid were varied from 2 to 8 mol% (Figure 2c), reflecting effective channel formation. Boc‐PA‐OBn 1 showed a sharp rate increase between 6 and 8 mol%, suggesting that higher concentrations were required for channel formation.

FIGURE 2.

(a) Schematic representation of a water transport assay using stopped flow spectroscopy. (b) Light scattering intensity of vesicles under hyperosmotic conditions in the presence of peptide 1 (8 mol% relative to lipid) compared with the control (no peptide). (c) Bar graph showing the rate of vesicle shrinkage with varying concentrations of peptides 1, 3, and 6; data are presented as mean ± standard deviation (SD) from three independent experiments. (d) Net water permeability as a function of peptide concentration, calculated using van't Hoff (Equation 1) and Horner (Equation 2) methods.

The osmotic permeability (µm/s), that is, the rate of vesicle shrinkage normalized to a unit surface area, was determined using the widely applied van't Hoff method (Equation 1, Figure 2d) [6] as well as the osmolyte concentration corrected Horner method (Equation 2, Figure 2d). Both methods were used to enable comparison with reported AWCs that typically use one or both of these methods [29]. The Horner method yields lower water permeability values than the van't Hoff method [18] because it accounts for the progressive change in internal osmolyte concentration with decrease in vesicle volume [29]. The van't Hoff method estimates osmotic permeability, assuming a constant osmotic gradient across the membrane. Dynamic light scattering (DLS) was used to determine the initial vesicle size. Net osmotic permeabilities (Net P_f ) of the peptide channels at different concentrations were determined by subtracting the osmotic permeability of the control from that of the peptide samples (Figure 2d). The osmotic permeability linearly increased with an increase in peptide concentration. The maximum net permeabilities for peptides 1, 3, and 6 were found to be 112, 96, and 78 µm s⁻¹, respectively, using Method 1. Method 2, gave maximum net osmotic permeabilities of 31, 27, and 22 µm s⁻¹ for peptides 1, 3, and 6, respectively. For Boc‐A(β‐CN)PA‐OBn 3 a saturation in permeability was observed between 6 and 8 mol%. For Boc‐PA‐OBn 1 and Fmoc‐NPA‐OBn 6, saturation was not achieved at 8 mol% and a further increase in peptide concentration was found to compromise peptide solubility and vesicle insertion. Dipeptide 1 and the cyano tripeptide 3 showed best rates of water transport.

The single‐crystal x‐ray structures of Boc‐PA‐OBn 1 and Boc‐A(β‐CN)PA‐OBn 3 obtained from ethyl acetate/petroleum ether provided structural insights into their mode of self‐assembly [30]. Boc‐PA‐OBn 1 adopted a distinctive double‐barrel architecture (Figure 3a–c). Each barrel had a cavity size measuring ∼3 Å that can readily accommodate water molecules (∼2.5 Å) but is narrow for hydrated sodium ions (∼9 Å). The self‐assembly was driven by intermolecular hydrogen bonding between the alanine NH_amide and the C = O_Boc group (Figure 3b). The rigid proline scaffold adjacent to the hydrogen‐bonding network provided a robust central framework or “spine” for the assembly (Figure 3c).

FIGURE 3.

(a) Top view of the XRD assembly of Boc‐PA‐OBn 1 showing the double‐barrel architecture with ∼3 Å pore. (b) Front view showing the hydrogen‐bonding network. (c) Front view: schematic representation of the peptide assembly across the lipid bilayer (∼5 nm). (d) Average water permeability of Boc‐PA‐OBn 1 calculated for the mCLR range of 0.0025 to 0.01, using Method 1 (van't Hoff) and Method 2 (Horner). Data are presented as mean ± standard deviation (SD) from three independent experiments.

The curved molecular extensions projecting from the central spine in opposite directions resulted in the formation of the double barrel architecture. This molecular packing oriented the polar groups inward, favoring water conduction and the hydrophobic benzyl_OBn, CH_3(alanine), and t‐but_Boc groups outward, enhancing membrane insertion. With a calculated cross‐sectional area of ∼6.8 nm² per channel, a vertical stack of eight dipeptide units can span the ∼5 nm thick lipid bilayer (Figure 3c). Based on this assumption, the molar channel‐to‐lipid ratio (mCLR), the number of channels per vesicle and the water permeability in cm³/s per channel were calculated (Figure 3d, Equation S4 and Table S7). The average osmotic water permeability calculated for the mCLR range of 0.0025–0.01 was 4 × 10⁻¹⁵ cm³ s⁻¹ based on the van't Hoff method. Converting volume flux into molecular counts the average single‐channel water transport rate mediated by dipeptide 1 was found to be 1.3 × 10⁸ H₂O s⁻¹ channel⁻¹ (Figure 3d). Using the Horner method (Method 2), the permeability was found to be 1.1 × 10⁻¹⁵ cm³ s⁻¹, yielding a single‐channel water flux of 0.4 × 10⁸ H₂O s⁻¹ channel⁻¹.

The peptide Boc‐A(β‐CN)PA‐OBn 3 crystallized with water molecules, providing insights into the mode of channel formation as well as its interaction with water molecules (Figure 4a–c). The channel cavity measured ∼4 Å in diameter (Figure 4a) sufficient to accommodate water molecules while excluding hydrated ions. The channel architecture arose from intermolecular hydrogen bonding between the alanine_carbonyl and the β‐cyano‐alanine NH_amide and aromatic stacking (Figure 4b,c). This tunnel architecture had carbonyl groups adjacent to proline as hydrogen bond acceptors and the alanine amide NH as a hydrogen bond donor (Figure 4b). The carbonyls on a single peptide formed hydrogen bonds with two water molecules, while the amide NH from the peptide on the opposite side formed an additional hydrogen bond with water. Within the membrane, such an architecture can facilitate water transport by a hopping mechanism and can potentially suppress the Grotthuss mechanism of proton conduction. The channel seen in the crystal structure also presented a hydrophobic outer surface that would facilitate membrane insertion. A stack of 16 peptide units can span the ∼5 nm bilayer thickness (Figure 4c), with a calculated cross‐sectional area of ∼3.2 nm² per channel. This translated to an average osmotic water permeability of approximately 6.1 × 10⁻¹⁵ cm³ s⁻¹ calculated for the mCLR range of 0.0013–0.005 (Method‐1) and a single‐channel water transport rate of 2.0 × 10⁸ H₂O s⁻¹ channel⁻¹ (Figure 3d). When analyzed using Method ‐ 2, the permeability was found to be 1.7 × 10⁻¹⁵ cm³ s⁻¹, yielding a single‐channel water flux of 0.6 × 10⁸ H₂O s⁻¹ channel⁻¹.

FIGURE 4.

(a) Top view showing cavity of 4 Å observed in the crystal structure of Boc‐A(β‐CN)PA‐OBn 3. (b) Top view showing the hydrogen bonding interactions between peptides and water. (c) Front view of the peptide assembly across the lipid bilayer (∼5 nm). (d) Average water permeability of Boc‐A(β‐CN)PA‐OBn 3 calculated for the mCLR range of 0.0025 to 0.01, using Method 1 (van't Hoff) and Method 2 (Horner). Data are presented as mean ± standard deviation (SD) from three independent experiments.

Both peptides display comparable net osmotic permeability at the same concentration (Figure 2d). However, given that at a given concentration the number of channels derived from Boc‐A(β‐CN)PA‐OBn 3 (16 mer) is lower than the number of channels derived from peptide 1 (8 mer), the tripeptide shows a superior water transport per‐channel as compared to the dipeptide. It is notable that peptides 1 and 3 demonstrate exceptional single‐channel permeability values approaching 10⁸ H₂O s⁻¹ channel⁻¹ at 25°C, which is only an order of magnitude lower than AQP1. Reported water channels derived from self‐assembly of acyclic molecules have achieved water flux values of 10⁶–10⁸ H₂O s⁻¹ channel⁻¹, whereas optimized foldamers or unimolecular macrocycles show a higher water flux of 10⁹–10¹⁰ H₂O s⁻¹ channel⁻¹ (Table S14). Within this context, our minimalistic NPA‐peptide channel exhibits water transport in the upper range of self‐assembled acyclic systems and is comparable to macrocyclic channels reported.

Given the ease of synthesizing these structurally simple peptides, they are attractive scaffolds for scalable applications. The peptides were stable in solution across a pH range of 2–9 (Figure S8). The water transporting ability of the peptides was retained when exposed to 600 mM NaCl (1200 mOsm; ∼35 g L⁻¹), which is comparable to the salinity of natural seawater (Figure 5a) [31]. The activity was also retained over 6 days, indicating good functional stability (Figure S6). Dynamic light scattering (DLS) further confirmed that the vesicle systems remained stable without significant aggregation or structural disruption over time (Figure S7).

FIGURE 5.

(a) Comparison of water‐transport rates when subjected to 600 mM NaCl over a period of 6 days. (b) Comparison of reflection coefficients based on the water permeability of peptides 1, 3, and 6 (5 mol% relative to lipid) in the presence of different external salts of the same osmolarity. An activity ratio (MCl/sucrose) close to 1 indicates high salt rejection. (c) EYPC‐LUVs⊃Lucigenin assay (pH 7.0) and corresponding graph, confirming no chloride transport by the peptides. (d) pH‐sensitive HPTS‐based LUV assays under high ionic gradients showing that peptides do not transport Na⁺ or K⁺, whereas the known cation transporter gramicidin A exhibits high activity. (e) EYPC‐LUVs⊃HPTS assays in the presence of FCCP, showing no proton transport in the presence of a pH gradient (pH 7.0 inside, pH 8.0 outside). Error bars represent standard deviation (SD) from three independent experiments. (f) Arrhenius plots of osmotic permeability as a function of temperature for gA (0.5 mol% relative to lipid), peptides 1, 3, and 6 (5 mol% relative to lipid), and blank (DOPC only), used to determine activation energies (E_a).

As aforementioned, a significant challenge for AWCs is the ability to selectively transport water and reject ions. To probe ion selectivity, osmotic permeability (P_f ) measurements were performed by creating identical osmotic gradients at pH 7 while varying the extravesicular environment [21] using NaCl (300 mM), a mixed salt solution containing KCl‐NaCl (200 and 100 mM) and non‐permeable sucrose‐NaCl (400 and 100 mM) as shown in Figure 5b. The experiment with sucrose was used as a reference. For all three peptides investigated, the reflection coefficients, defined as P_{f (salt)}/P_{f (sucrose)} were found to be close to unity. The fact that the water permeability was not affected by the nature of the extravesicular ions signifies robust water flux while effectively excluding ions.

The anion transport capabilities of the peptides were evaluated using lucigenin, a chloride‐sensitive fluorescent dye [14, 32]. Unilamellar vesicles composed of EYPC lipids were prepared with an internal solution containing 225 mM NaNO₃ and an external solution containing 225 mM NaCl (Figure 5c). No quenching of the lucigenin dye was observed upon the addition of peptides, even at 10 mol% relative to lipid (Figure S12), further confirming their anion‐rejection capability. Large unilamellar vesicles (LUVs) loaded with the pH‐sensitive HPTS dye (8‐hydroxypyrene‐1,3,6‐trisulfonic acid trisodium salt) were used to evaluate the cation transport capabilities of the peptides. For the assay, vesicles were prepared with an intravesicular buffer at pH 7 containing 1 mM HPTS dye (Figure 5d). These vesicles were then suspended in an extravesicular buffer with 200 mM M₂SO₄ (M = K, Na) at pH 8 [21]. Upon the addition of peptides (10 mol% w.r.t. lipid; 3 mM), cation transport, if present, would be expected to dissipate the pH gradient. No detectable ion transport was observed, suggesting that the active water channels effectively rejected Na⁺ and K⁺ ions (Figure 5d and Figure S10). Only in the case of Boc‐A(β‐CN)PA‐OBn 3, approximately 10% K⁺ leakage was exhibited indicating marginal ion permeability under the experimental conditions. In contrast, the well‐known cation‐selective gramicidin A (gA) facilitated robust transport of both Na⁺ and K⁺ (∼65%) even at a lower concentration (1 µM). To evaluate proton transporting capabilities of the peptides, the protonophore FCCP (carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone, 1 µM) was introduced in the vesicles. The vesicles were prepared with the same intra‐ and extravesicular Na⁺ concentration, an anion‐gradient and a pH gradient of 1 (Figure 5e) [33]. When tested alone, Boc‐PA‐OBn 1 and Fmoc‐NPA‐OBn 6 exhibited no significant pH gradient dissipation (Figure 5e and Figure S11), while Boc‐A(β‐CN)PA‐OBn 3 showed minimal transport. In the presence of FCCP, the transport activity showed little or no change, suggesting no cooperativity between peptide and FCCP for proton transport. Overall, the ion leakage study explains that these peptides did not facilitate anion, cation, and proton transport.

To gain further mechanistic and thermodynamic insights, the activation energy (E_a ) for water transport was determined from Arrhenius analysis of osmotic permeability (P_f ) across a temperature range of 5‐28°C, using six data points for each system (Figure 5f) [6]. The E_a values for Boc‐PA‐OBn 1 and Boc‐A(β‐CN)PA‐OBn 3, at 5 mol% relative to lipid, were calculated to be 8.1 and 9.4 kcal mol⁻¹, respectively, which are substantially lower than that of the pure DOPC lipid bilayer (12.1 kcal mol⁻¹). Tripeptide Fmoc‐NPA‐OBn 6 that was not as efficient as the other peptides showed a higher E_a of 10.6 kcal mol⁻¹ (Figure S10). The naturally occurring gramicidin A (gA), at 0.5 mol% relative to lipid, exhibited an E_a of 5.9 kcal mol⁻¹. It is well established that gA conducts water through a single‐file transport mechanism, where water molecules are hydrogen‐bonded in a linear chain. The relatively higher E_a values observed for the peptides studied here suggest that single‐file water transport is unlikely and additional energetic barriers associated with hydrogen‐bond rearrangement are involved during transport. Across reported artificial water channels, activation energies below approximately 5 kcal mol⁻¹ are generally associated with single‐file water transport, whereas wider channel cavities tend to favor multi‐water or bulk‐like transport. The narrow pore diameter of 3–4 Å of our peptidic channels as evidenced by the crystallographic analysis disfavors bulk or continuous chain transport. Furthermore, pH‐gradient fluorescence experiments showed no detectable proton leakage, indicating that proton conduction through a Grotthuss‐type mechanism is unlikely. Together, these observations suggest that water transport is proceeding through a process involving multiple interactions with the peptide backbone, rather than through a single‐file hydrogen‐bonded water wire or bulk transport.

Molecular dynamics (MD) simulations showed that the synthesized peptide, when embedded within a DOPC bilayer, enabled water transport (Figure 6 and Figure S16). After an initial equilibration of 5 ns, during which the peptide assembly was held rigid, a pressure difference of 97 MPa [34] was applied along the Z − dimension of the simulation box to drive water permeation through the nanopore (simulation details are presented in the Supporting Information). Following the application of the pressure gradient, multiple water molecules approached and traversed the channel simultaneously (Figure 6a,b), indicating that the synthetic pore forms a pathway to facilitate trans‐membrane water flux. Intra‐channel dynamics revealed that permeating water molecules interacted closely with peptide residues through hydrogen bonding (Figure 6c). The number of peptide‐water hydrogen bonds remained approximately constant throughout the simulation. In contrast, the number of hydrogen bonds between confined water molecules increased markedly after the pressure gradient was applied. This behavior suggests the formation of a hydrogen‐bonded water network that facilitates transport through the pore. The intermolecular contacts between peptide residues remained constant throughout permeation events, as seen by the time evolution of intra‐channel hydrogen bonds (Figure 6c). Inspection of the hydrogen‐bonding distribution revealed an asymmetric interaction profile within the channel. A higher density of hydrogen bonding was observed on one side of the channel (Figure 6a). This indicated that water molecules preferentially interacted with a localized region of the pore interior. This asymmetry is reflected in the average hydrogen‐bonding profile across the tri‐peptide assembly (Figure 6d), which shows non‐uniform contributions from individual peptide units. Residue‐wise analysis (Figure 6e) revealed that modified asparagine (Asm: A(β‐CN)) and proline contributed comparable numbers of peptide–water hydrogen bonds, whereas alanine participated only weakly. Despite its nonpolar character, proline, therefore, contributed to water‐peptide interactions, likely due to its position within the channel. Interaction energy analysis further supported this interpretation. Electrostatic interactions dominated over van der Waals interactions in stabilizing water within the pore (Figure 6f). Collectively, these findings indicate stable peptide–water interactions and a hydrogen‐bonded network enable efficient water transport through the pore.

FIGURE 6.

(a) Structural representation of the peptide assembly highlighting the stacked arrangement of tripeptide units (P1–P16) forming the transmembrane channel with top and side views of the channel illustrating the spatial organization of residues and the asymmetric distribution of peptide‐water interaction sites along the pore axis. (b) Time‐dependent variation in the number of water molecules within the channel, indicating continuous tri‐peptide water interactions. (c) Time evolution of hydrogen bonds illustrating contributions from water–water, peptide–water, and peptide–peptide interactions, confirming structural stability of the channel. The highlighted region indicates the application of a 97 MPa pressure gradient after 5 ns. (d) Average number of hydrogen bonds formed between water molecules and individual tripeptide units, revealing an asymmetric interaction profile along the channel. (e) Residue‐wise analysis of hydrogen bonding reveals that modified asparagine (Asm: A(β‐CN)) and proline contribute nearly equal numbers of tri‐peptide–water hydrogen bonds, whereas alanine shows minimal involvement. (f) Interaction energy profile between the peptide and confined water molecules, showing that electrostatic interactions dominate over van der Waals contributions throughout the simulation.

3. Conclusion

Inspired by the natural aquaporin pore, the NPA and PA scaffolds were functionalized with aromatic protecting groups to enable self‐assembly. The peptides Boc‐A(β‐CN)PA‐OBn 3 and Boc‐PA‐OBn 1 transported water very efficiently with single‐channel water permeabilities of 2.0 × 10⁸ H₂O s⁻¹ channel⁻¹ and 1.3 × 10⁸ H₂O s⁻¹ channel⁻¹, respectively. The x‐ray structures of these peptides revealed that they assemble into well‐defined water channels through an extended network of hydrogen bonds. The Boc‐PA‐OBn 1 showed achieved complete proton and ion exclusion, while the Boc‐A(β‐CN)PA‐OBn 3, showed very marginal ion‐transport. Overall, these peptide channels efficiently excluded Na⁺, K⁺, and Cl⁻, exhibiting desalination‐like selectivity without compromising water flux, an exceptional property for such minimalistic motifs. The experimentally determined activation energies, x‐ray structures, and MD simulations suggest that hydrogen bonding interactions between the peptides and water play an important role in the water transport mechanism. These minimalistic nature inspired peptides demonstrate how simple scaffolds can emulate the hallmark efficiency of biological aquaporins, marking a promising step toward water purification technologies.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Experimental procedures, synthetic details, spectroscopic characterization data and x‐ray crystallographic details of peptide channels, experimental methods for vesicle‐based transport assays, single‐channel permeability evaluation, and ion exclusion measurements are available. Details on the MD simulation study are available. Additional references are cited within the Supporting Information [35, 36, 37, 38, 39, 40, 41, 42, 43].

Supporting File: anie72786‐sup‐0001‐SuppMat.pdf.

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.