Dynamically assembled photochromic cages operational in water with visible light

Domain: biology
Published: 2026-03-15
Editorial outcome: major_revision

Abstract
Producing chemical nanostructures that can mimic the efficient adaptability of complicated biological systems to environment changes is among the main goals of nanotechnology. Progress in this area requires understanding of the adaptation mechanisms towards external stimuli at the molecular level. Due to rapid and precise spatiotemporal addressability, light-driven dynamic systems are particularly attractive for such mechanistic studies. Here, we show efficient formation of dynamic covalent cages that undergo a series of reversible constitutional changes driven by visible light. Their complex, yet predictable and often quantitative response to irradiation and other external stimuli (metal ions, pH) reveals design principles that can be applied to assemble adaptable molecular machines showing life-like behavior. Upon reduction of the dynamic imine bonds, stable covalent cages are isolated. Their response to red light, also in aqueous media, indicates the potential for in vivo applicability, as red light can deeply penetrate human tissues.

Introduction
Supramolecular self-assembly of functional building blocks enables preparation of diverse nanostructures, such as cages or macrocycles, with a broad range of functions directly related to the nanoconfinement—like allosteric receptors1, regulated catalysis2, or modulation of photophysical properties of guest molecules3. The nanostructures can respond to external stimuli, such as pH, temperature, or chemical input, in order to change their configuration or selectivity towards guest molecules4. Particularly advantageous are nanostructures resulting from dynamic covalent self-assembly, as they show self-sorting behavior in response to external stimuli or reagents—the initial complex mixtures of intermediates can often be directed into well-defined heteroleptic or homoleptic species5. Light is a highly advantageous stimulus to be applied in such systems, as it does not permanently contaminate the reaction, and can be used with high spatiotemporal precision. The commonly used antenna to convert the light energy into effects at the molecular level are molecular photoswitches6, such as azobenzenes7, indigoids8, DASA, norbornadienes, dihydropyrenes, or hemipiperazines9–13. A variety of molecular photoswitches has been incorporated into nanocages and macrocycles14,15, yielding photoresponsive nanostructures, including diarylethenes16, sterically crowded alkenes, diazocines17, and azobenzenes. Yet, most of the demonstrated nanostructures were operational in organic solvents, but not in water—due to lack of solubility, hydrolysis, or incompatibility of the switching mechanism. Moreover, switches triggered with red and near-infrared light are highly demanded, as these wavelengths (>630 nm, therapeutic window of light) can efficiently penetrate soft human tissues rich in hemoglobin, and thus have broad applicability potential in human photopharmacology18–21 or as in vivo receptors22.

In 2021, we demonstrated23 a tetra-ortho-fluoroazobenzene bis-aldehyde 1 as a photoswitch triggered with red light (660 nm) and a useful intermediate in soft material preparation. This compound has been used by the Schmidt group in an elegant work to dynamically assemble macrocycles that change their constitution upon irradiation24,25. Around that time, the Feringa group demonstrated UV-light-driven (340 nm) destabilization of a dynamically assembled cage26—the stable (E)-configured cage based on a non-halogenated azobenzene bis-aldehyde resists an imine exchange, while the more strained (Z)-configured cage can be decomposed using an external reagent. This process can be inverted upon violet light-induced (420 nm) (Z)-to-(E) back-isomerization followed by thermal equilibration, showing the dynamic nature of imine-based photochromic cages. Interestingly, only the meta-configured azobenzene architecture produced well-defined cages, while the para- and ortho-isomers did not form cage-shaped species. That work was further expanded to reversible cage-ring phototransformations27. Other interesting systems based on photoreactive cages bearing azobenzene functions have been recently reported by the groups of Beves28, Greenaway29, Kataev30, Wang31, and Clever17.

Results
We were intrigued whether the compound 1 can be used for assembly—via dynamic covalent chemistry—of well-defined supramolecular cages sensitive to red light, as well as responsive to other external stimuli. We were also curious whether modifications of the switch structure (replacing fluorine with chlorine atoms) may yield a chromophore switchable with higher wavelengths (such as near-infrared light), and still capable of forming photochromic cages. Other interesting opportunities were the formation of heteroleptic species and the implementation of other triggers that may shift the equilibria in such dynamic systems, such as transition metal ions. Upon demonstrating all that, we fixed the dynamic outcome of our assembly by irreversible formation of stable covalent bonding via imine reduction. The resulting stable cage can be reversibly transferred between the organic and aqueous phase upon protonation—even CO2 is sufficiently acidic—and can be reversibly photoswitched in water with visible light frequencies.

Formation of a stable cage via dynamic covalent chemistry
We mixed (in the 3:2 ratio) the photochromic bis-aldehyde 1 (FA) in the thermodynamically stable (E)-configuration with the tris(2-aminoethyl)-amine 2 (EN)—a flexible trivalent linker that enables dynamic imine bond formation with aldehydes substrates—in chloroform. After 24 h incubation, we isolated a well-defined dynamic covalent cage 3 (FA3EN2) with 60% yield (Fig. 1a, Supplementary Fig. 4A). Its stability was sufficient to grow crystals and characterize 3 via X-ray diffraction (Fig. 2a). If the ratio of 1 was slightly decreased, we observed—in addition to 3—the incompletely formed cage 4 (FA2EN2), with one missing FA pillar. Yet, 3 resolubilized in chloroform after purification (filtration, re-dissolving, and trituration with ethyl acetate) did not produce 4 nor other intermediates. It demonstrates that 3 is the most stable form in that system. The isolated (E,E,E)-3 cage has been almost quantitatively (>95%) photoisomerized to the (Z,Z,Z)-3 cage with red light (660 nm). We believe that our system shows a pronounced templating effect, as we did not observe any meaningful amounts of the mixed (E,Z,Z) or (E,E,Z) forms at 660 nm. Incorporation of the switch into the cage geometry slightly reduced its thermal stability (from 312 h to 123 h at 25 °C in CDCl3, Supplementary Figs. 7 and 8) in comparison with the free aldehyde 1. The cage has shown well-pronounced photochromism in solution (Fig. 1b). The forms (E,E,Z)-3 and (E,Z,Z)-3 could be observed as minor products upon irradiation with green light (523 nm), but their lifetime is much shorter (<7 h) than the (Z,Z,Z)-3 (Supplementary Fig. 6, 9–10). Interestingly, we could also form the (Z,Z,Z)-3 cage with good yields upon incubation of the bis-aldehyde 1 pre-irradiated with red light (660 nm, containing >85% of the (Z)-1 form under these conditions) with the triamine 2 upon 24 h (Supplementary Figs. 4B, 11, and 12).

Reduction of 3 yields a stable cage 5, reversibly switchable in water
The (E,E,E)-configured dynamic cage 3 was treated with NaBH4 and produced a stable covalent cage (E,E,E)-5, where the imine bonds have been reduced to secondary amines. Both species formed crystals and their structures could be determined via X-ray diffraction (Fig. 2a). The (E,E,E)-5 can be photoisomerized with amber (590 nm) or green (523 nm) light, resulting in almost quantitative formation of the (Z,Z,Z)-5. This isomer upon exposure to violet light (430–410 nm) is fully converted. The majority (>70%) of it forms the (E,E,E)-isomer. The remaining 30% is distributed between the (E,E,Z)- and (E,Z,Z)-form (Supplementary Fig. 13 and 14). Due to the diminished π-electron system, 5 is not sensitive to 660 nm light anymore (and with 620 nm, it produced a mixture of isomers). While 5 is insoluble in water, protonation with TFA quantitatively transfers it into the aqueous phase (Fig. 2b). The resulting 5*H+ cage can be reversibly switched between two states. One containing predominantly (>60%) (Z,Z,Z)-isomer (590–523 nm) and the other >70% (E,E,E)-isomer (410 nm; Supplementary Table 2 and Supplementary Figs. 16–21). Even carbonic acid is strong enough to fully transfer 5 from chloroform to the aqueous phase saturated with CO2. Upon stirring overnight in an open vial, CO2 merely evaporates, and 5 fully returns to chloroform (Fig. 2c). We have also investigated the formation of inclusion complexes between the cage 5*H+ and cucurbiturils. Based on the diameter of 5 obtained by X-ray diffraction analysis (~8 Å, estimated using Mercury©), we chose cucurbit[8]uril (CB8) as host (inner diameter of the cavity = 8.8 Å), and we observed spectral shifts which may attest to supramolecular interactions, such as inclusion complex formation (Supplementary Figs. 22 and 23)30. The complexes were irradiated with 523 nm (green light) and 410 nm (violet light), and then treated with 10 eq. excess of amantadine (protonated 1-aminoadamantane), which shows high affinity (>1012 M–1) to CB8, and thus completely removes the cage from the complex (Supplementary Figs. 24–27 and Supplementary Table 3). We have noticed that irradiation of 5 inside the inclusion complex increases the preference for the forms containing (Z)-configured isomers, as compared with photoisomerization in free aqueous solution (Supplementary Fig. 26 and Supplementary Table 3).

To further assess the biocompatibility of our system operational in water with visible light wavelengths, we have performed a colorimetric assay for cell metabolic activity in the presence of our cage (so-called MTT assay used as standard for cytotoxicity determination). The (E,E,E)-5*H+ isomer showed the value of IC50 = 0.58 μM, while the mixture of isomers obtained at the photodynamic equilibrium with irradiation of 523 nm light (with the majority of (Z,Z,Z)-isomer) showed a slightly higher value of IC50 = 1 μM (Supplementary Fig. 30 and Supplementary Table 4). While the general toxicity at the sub-micromolar level can be explained, e.g., by the physical, detergent-like interactions of the cationic species with the anionic phospholipid components of cellular membranes, its photomodulation (less than an order of magnitude) seems too little to be useful in any practical application. Here, we can also see that the cage 5*H+ can be safely used in biological setups at concentrations below 0.5 μM.

Dynamic covalent chemistry of the cage 3 with monovalent reagents
Next, we examined the stability of cage 3. We treated the stable isomer (E,E,E)-3 with 3 equiv. of benzaldehyde or benzylamine—two monovalent reagents competing for the imine bond. After 7 days of incubation at 20 °C, no significant exchange has been observed. Yet, upon generation of the (Z,Z,Z)-3 isomer and its incubation with these reagents over the same time period, we have seen significant exchange with benzaldehyde, and almost quantitative with benzylamine (Supplementary Figs. 31–33). It demonstrates that the (Z,Z,Z)-configured cage 3 is less stable than the (E,E,E) isomer, and that benzylamine is more reactive in the replacement process than benzaldehyde.

In contrary, the free aldehyde (E)-1 reacts with benzylamine 18 quantitatively, forming the (E)-bis-imine (E)-17 (Supplementary Fig. 35), which upon irradiation with red light (660 nm) yields 89% of the (Z)-17 (Supplementary Fig. 36)—photoconversion similar to the bis-aldehyde 1, but lower than for the 3. The t1/2 of the (Z)-17 was determined to be 239 h (25 °C in CDCl3) (Supplementary Fig. 37)—slightly reduced in comparison to the bis-aldehyde (Z)-1 (t1/2 = 312 h), but still significantly higher than for the (Z,Z,Z)-3 cage (t1/2 = 123 h). Thus, both the enhanced photoconversion and the decrease in thermal stability for the photochromic cage seem to derive mainly from the discussed template effect in the cage. This is corroborated with the fact that during thermal back-isomerization of (Z,Z,Z)-3, no (E,Z,Z)- or (E,E,Z)-isomers were detected, but only the (E,E,E)-3 product.

Exchange of cage 3 with non-halogenated photochromic bis-aldehyde
Then, we exposed cage 3 to a bis-aldehyde 6 containing the non-halogenated azobenzene chromophore (AZ). Again, the (E,E,E)-3 did not undergo significant reaction upon at least 15 days of incubation. Yet, the (Z,Z,Z)-3 obtained upon exposure of the sample to red light (660 nm) demonstrated a significant exchange. According to NMR spectra, multiple species have been generated at this stage (Supplementary Fig. 40). But upon back-isomerization of the mixture with 430 nm light (which restores the (E)-configuration on both fluorinated and nonfluorinated azobenzenes (see Table 1 and Supplementary Table 6), we have isolated only the unchanged cage (E,E,E)-3 and a compound (E,E,E)-7 together with the fluorinated bis-aldehyde (E)-1. The compound 7 occurred to be a cage analog of 3, where one of the three azobenzene pillars has been exchanged from the tetra-fluorinated to the nonfluorinated block (Fig. 3 and Supplementary Fig. 41).

We did not isolate any cage products of further exchange in measurable quantities (although respective ESI traces could be registered, Supplementary Fig. 45), also upon elongated periods of incubation (Supplementary Fig. 46). This result corroborates the report of the Feringa group26 where the para-configured bis-aldehyde 6 does not form well-defined cages with the tris-amine 2, but forms precipitation, likely containing linear polymers. The structure of cage 7 has been confirmed by DOSY (Supplementary Fig. 41B). We have also reduced the reaction mixture containing the 7 with NaBH4 and identified the mass of the corresponding reduced heteroleptic cage 8 (Supplementary Fig. 49).

Tetra-ortho-chlorinated azobenzene bis-aldehyde addressable with near-IR light—its photochromic properties and dynamic exchange with the cage 3
The bis-aldehyde 1 can be triggered with red light due to the expansion of its π-electron system in comparison with the unmodified tetra-ortho-fluoroazobenzene chromophore (which is inert to >600 nm light)23. We were interested, if the extension of π–electron system in a tetra-ortho-chlorinated chromophore (already addressable with red light18) will expand the range of its light response even further. To check that, we have synthesized the chlorinated bis-aldehyde 9 (CA) (Fig. 4a) and characterized its photochromism with various wavelengths of light. While the fluorinated aldehyde 1 photoisomerizes from (E)- to (Z)-form with green or red light (523–660 nm), the chlorinated 9 is photoconverted with red light (623 or 660 nm) from the thermally stable (E)-isomer to the mixture containing the majority of the (Z)-form. Yet, the reverse photoconversion (to mixtures containing >75% of the (E)-9) occurs either upon green/blue light irradiation (455–523 nm) or upon exposure to the near-IR light (730 nm) with additional cut-off filter for wavelengths below 715 nm, (see Table 1). This observation makes 9 a rare switch that can be bidirectionally addressable with wavelengths within the therapeutic window of light (650–900 nm). That property might be used in the future for precise temporary in vivo photocontrol of photopharmacological systems, or photomodulation in complex biological setups.

We used 9 to generate another cage system. Upon mixing of 9 with the triamine 2 over 20 hours at elevated temperature (65 °C), we observed complete substrate consumption and formation of two new species (Fig. 4b), later identified as a fully formed cage 10 with three chlorinated azobenzene pillars and a partially opened cage 11 (ca. 15% of the isolated product, see Supplementary Fig. 54)—the assembly where three chlorinated CA pillars are bound with the two triamines EN with total 5 imine bonds (leaving one aldehyde and one amine group unreacted). The ESI-MS identification is supported with DOSY NMR spectra that show similar hydrodynamic radii of both species (Supplementary Fig. 55). This result indicates that formation of the complete cage 10 is more sterically demanding than in the case of fluorinated cage 3, due to increased steric hindrance and changed geometry of the CA pillars.

The cage (E,E,E)-10 can be isolated upon repetitive trituration with ethyl acetate. It was irradiated with red light (660 nm), to cause the (E)→(Z) photoisomerization of the chlorinated azobenzene fragments. The resulting (Z,Z,Z)-10 cage is likely unstable—due to even larger steric demands of the (Z)-tetrachloroazobenzene-containing systems in comparison to the respective (E)-isomers. Thus, multiple species are produced. Yet, irradiation of that mixture with light in the range 523–430 nm (Supplementary Fig. 57) quickly restores the original distribution of the cage (E,E,E)-10 and the minor component (E,E,E)-11 (collectively denoted as Mix-E10), proving the highly dynamic properties of our system.

We performed five full red/blue switching cycles (660 nm/455 nm), which restored the original Mix-E10 product distribution at the end (see Supplementary Figs. 58 and 89). Then, we wanted to use NIR light (730 nm) or thermal equilibration at room temperature to reconstitute the cage (E,E,E)-10 previously dissipated with red light. However, this was not successful—most of the soluble fractions contained the bis-aldehyde 9, while the rest of the material was converted into insoluble linear polymerization products (likely because the cage reformation was too slow due to steric hindrance, Supplementary Fig. 59). Yet, if the thermal equilibration was performed at 50 °C, the original Mix-E10 distribution was fully restored within 4 hours (Supplementary Fig. 60). In another experiment, we partially destabilized the Mix-E10 with NIR light (730 nm). Upon irradiation of the resulting composition with blue light (455 nm), we recovered the Mix-E10 (Supplementary Fig. 61) upon two switching cycles. In that experiment, the disruption of the cage is less pronounced—in line with the lower ratio of (Z)-9 produced at 730 nm (25% at the PSS730nm) than at 660 nm (68%, see Table 1).

To further explore the dynamic nature of our system, we incubated the pre-formed fluorinated cage 3 with the chlorinated pillar 9. When performed with the (E)-isomers in the dark, very slow conversion (<10%) was observed within 1 week, eventually equilibrating after 1 month, with ~20% of a new species, which was identified as the heteroleptic cage 12 (with one chlorinated and two fluorinated pillars) (Fig. 5 and Supplementary Fig. 62). If the reaction is initiated by irradiating the sample with 660 nm (>95% (Z,Z,Z)-3 and >60% (Z)-9), immediate formation of several new species was observed (Supplementary Fig. 63) and after 1 week, >30% of 12 formed. Yet, thermal equilibration (1 month) of that system in darkness resulted in partial reestablishment of the cage 3 after (E)-configuration of both azobenzene pillars is thermally restored, resulting in the same final component ratio as in the process in the absence of light.

Covalent cage addressable with red light in water
Encouraged by the efficient formation and green-light-induced switching of the covalently fixed cage 5 based on fluorinated azobenzene, described above, we attempted to prepare an analogous cage with the chlorinated bis-aldehyde 9. For that, we tried to reduce the dynamic cage system Mix-E10. The attempts with NaBH4 or BH3*THF were unsuccessful, yielding an inseparable mixture of many substances. Yet, reduction with NaBH(OAc)3 showed sufficient selectivity to isolate the desired covalent cage 13 in fair yield (15% over two steps from the bis-aldehyde (E)-9 and TREN 2; Fig. 6 and Supplementary Figs. 64 and 65).

The cage 13 (which contains predominantly one isomer in the dark state, which we assume to be the (E,E,E)-13) can be also protonated with TFA, and in this state shows good solubility in water. We have irradiated the protonated (E,E,E)-13*H+ in aqueous solution with 660 nm (red light within the therapeutic window range), and the (E,E,E)-isomer was fully converted to the mixture of photoisomers. Green light irradiation (523 nm) produced isomer mixture with different component distribution, while the blue light (455 nm) restored the original isomer distribution—predominantly the (E,E,E)-13 isomer—observed in the non-irradiated sample (NMR experiments, see the Supplementary Fig. 68). These results were corroborated with UV–Vis spectra of the dark and irradiated states (Supplementary Fig. 66), where the absorbance of the dark sample is almost fully restored upon 455 nm irradiation. Due to the insufficient amount of the isolated cage, we could not perform the cytotoxicity tests.

Exchange of the cage 3 with a non-photochromic bipyridine bis-aldehyde and in situ formation of a multivalent ligand for transition metals
In the last section, we demonstrate that our dynamic cage can react with metal ions as a stimulus orthogonal to the effects of irradiation. In the presence of the principles analogous to the aforementioned cage 3, the triamine 2 (EN) can form a non-photoresponsive homoleptic cage 15 with 3,3’-bipyridine-4,4’-bis-aldehyde 14. The process is slow and incomplete in organic solvents, but the well-defined product has been identified with an approximate yield of 40% (judged by 1H NMR and MS) (Supplementary Figs. 69–70 and 71A). For further characterization, we were also able to isolate 15 on a larger scale by precipitation (supporting information).

As the cage 15 seems to be less stable than the photochromic cage 3, there is only partial interconversion observed, if 3 is mixed with the respective bipyridine pillar 14 in chloroform (Supplementary Fig. 72). Both the expected heteroleptic cages—the product of single (16a) and double (16b) exchange—are formed as minor products (Fig. 7a and Supplementary Figs. 71B and 73). Interestingly, while the cage 16a with two azobenzene pillars can be dissipated with red light and restored with blue light irradiation, the cage 16b (one remaining azobenzene) is inert to both light frequencies, probably due to the steric congestion (Supplementary Fig. 71C).

Upon formation of the homoleptic cage 15 from 2 and 14, we added water to the reaction mixture in chloroform. Under these conditions, none of the resulting components is extracted into aqueous phase (Supplementary Fig. 75). Yet, if we replace water with a solution of transition metal salts, like Fe, Co, Ni, or Cu, the cage 15 forms multivalent colorful complexes with the metal ions, which are instantly and quantitatively transferred into the aqueous phase (only the unreacted pyridine bis-aldehyde 14 used in excess remains in the organic phase, as it does not form complexes with the used metal ions), (Fig. 7b and Supplementary Fig. 76A). The efficient formation of the cage 15 in presence of metal ions32–34, as well as similar molecular cages33,34, has been already described in literature, whereby a 1:2 binding ratio between 15 and metal ions like Zn2+ and Fe2+ was reported. Following these results, we determined the association constants Ka1 (15*Fe2+) = 806 (±13) M–1 and Ka2 (15*2Fe2+) = 1789 (±67) M–1 (Supplementary Figs. 125–127).

Also if we mix the pre-formed cage (E,E,E)-3 with the bipyridine bis-aldehyde 14 in presence of aqueous iron(II) chloride solution, the heteroleptic bipyridine-containing cages 16a and 16b are quantitatively transferred into the aqueous phase with concomitant complexation of the iron ion, leaving only residual amounts of the cage 3 and the photochromic fluorinated bis-aldehyde 1 as the exchange by-product (Fig. 7b, sample V).

Prior research has demonstrated numerous photochromic dynamic covalent assemblies with diverse topologies and assembly efficiency (Fig. 8 and Table 2). We have shown that halogenated azobenzene-derived bis-aldehydes can efficiently form cages, which undergo dynamic and reversible exchange in response to various external stimuli (other bis-aldehydes, metal ions) in a light-dependent manner, upon exposure to visible light frequencies. The dynamic outcome can be fixed upon reducing the dynamic imine bonds to permanent covalent amine bonds. The resulting stable cages can be bidirectionally and reversibly switched with a pair of colors in the visible light range—green/violet for the fluorinated system, and red/blue for the chlorinated one—in organic solvents, as well as upon protonation in water. The fluorinated stable cage works as a simple CO2 sensor within a biphasic setup.

We also reported that one of our further cage components—the chlorinated azobenzene bis-aldehyde—shows bidirectional addressability with light frequencies within the therapeutic window (660 nm (E)→(Z), 730 nm (Z)→(E)), and thus is a promising chromophore for in vivo applications, including photopharmacology. We demonstrated that our dynamic cages undergo formation and reversible exchange with high efficiency and a predictable outcome. We believe, that especially our cages operational in aqueous media with red light make a step forward toward the construction of biocompatible molecular machines—also via self-assembly, which will be applicable for cargo storage, or stimuli-dependent selective catalysis.

Methods
Synthesis of the cage (E,E,E)-3
The fluorinated bis-aldehyde (E)-1 (150 mg, 484 μmol, 1.00 eq.) was dissolved in anhydrous chloroform (220 mL) under an argon atmosphere. To the solution, the triamine (TREN) 2 (47.2 mg, 48.3 μl, 323 μmol, 0.667 eq.) in 5 mL chloroform was added dropwise. The resulting mixture was heated to 62 °C for 24 h. Afterwards, the solution was cooled down to 20 °C, filtered to remove insoluble material, and the filtrate was dried in vacuo. The resulting red powder was purified by trituration with ethyl acetate (5 × 5 mL), redissolved in chloroform, filtered again, and dried under reduced pressure. A final washing of the solid with ethyl acetate (5 mL) yielded the cage (E,E,E)-3 (99.0 mg, 88.8 μmol, 55%) as a dark red solid.

Synthesis of the cage (E,E,E)-5 upon reduction of the dynamic cage (E,E,E)-3
To a solution of (E,E,E)-3 (60.0 mg, 53.8 μmol, 1.00 eq.) in chloroform (11 mL) and methanol (11 mL) was added sodium borohydride (NaBH4, 42.8 mg, 1.13 mmol, 21.0 eq.) under argon counter flow. The mixture was stirred for 24 h at 20 °C, during which the color of the solution changed from red to orange. Afterwards, the solvents were removed under reduced pressure. The resulting solids were redissolved in a biphasic mixture of chloroform (15 mL) and saturated aq. NaHCO3 solution (15 mL) and vigorously stirred for 30 min, whereupon a clear orange organic layer and a turbid aqueous layer formed. The layers were separated and the aq. layer was extracted with chloroform (3 × 15 mL). The solvent of the combined organic layers was removed under reduced pressure, yielding the cage (E,E,E)-5 (53.4 μmol, 60.2 mg, 99%) as orange powder.

Synthesis of the cage (E,E,E)-10
The chlorinated bis-aldehyde (E)-9 (40.0 mg, 106 μmol, 1.00 eq.) was dissolved in anhydrous chloroform (55 mL) under an argon atmosphere. To the solution, the triamine (TREN) 2 (10.4 mg, 10.6 μl, 71.0 μmol, 0.667 eq.) in 5 mL of chloroform was added dropwise. The resulting mixture was heated to 62 °C for 12 h. Afterward, the solution was cooled down to 20 °C, filtered to remove insoluble material, and the filtrate was dried in vacuo. The resulting brown powder was purified by trituration with ethyl acetate (5 × 5 mL), redissolved in chloroform, filtered again and dried under reduced pressure. A final washing of the solid with ethyl acetate (5 mL) yielded the cage (E,E,E)-10 (35 mg, 26.7 μmol, 76%) as a brown powder.

Synthesis of the cage (E,E,E)-13 upon reduction of the dynamic cage (E,E,E)-10
The chlorinated bis-aldehyde (E)-9 (30.0 mg, 79.8 μmol, 1.00 eq.) was dissolved in 40 mL of anhydrous CHCl3 under an argon atmosphere. N’,N’-Bis(2-aminoethyl)ethane-1,2-diamine (7.78 mg, 7.97 μL, 53.2 μmol, 0.667 eq.) was dissolved in 2 mL of anhydrous CHCl3 and added dropwise over the course of 1 h. The resulting mixture was stirred at 23 °C for 7 days. Then, the solution was filtered (0.2 μm), cooled to 0 °C, and solid NaBH(OAc)3 (118 mg, 559 μmol, 21.0 eq.) was added under argon counter flow. The mixture was slowly warmed up to 23 °C and stirred for 20 h. Afterwards, it was again cooled to 0 °C and 3 mL of MeOH were added slowly. After stirring for another 2 h, 15 mL H2O were added, followed by 50 mL of saturated NaHCO3 solution. The aqueous layer was extracted with 50 mL of CHCl3, the combined organic layers were washed with H2O (2 × 30 mL), dried over Na2SO4, and the solvent was evaporated under reduced pressure. The crude product was dissolved in a MeOH/MeCN/TFA mixture (70/20/10) and purified using preparative HPLC (MeCN/H2O, 0.1% TFA; gradient = MeCN 20% → 70% over 60 min), yielding 13 (9 mg, 15%, overall yield from cage components) as a dark orange solid.

Solvents and reagents
Solvents (per analysis quality) were commercially purchased (Acros Organics, Fisher Scientific, Sigma-Aldrich, Eurisotop, VWR) and used without further purification if not stated otherwise. Reagents were commercially purchased from industry companies (ABCR, Acros, Alfa Aesar, Fisher Scientific, Merck, BLDpharm, VWR) and used without further purification if not stated otherwise.

Graphs and spectra
Graphs were plotted and fitted in OriginPro, Version 2023 (Copyright © 1991–2022 OriginLab Corporation, Northampton, MA, USA). NMR spectra were processed using MestReNova, Version 14.1.2 (Copyright © 2005–2020 Mestrelab Research S.L., Santiago de Compostela, Spain).

Graphics
Chemical structures, molecular graphics and crystal structures were created and visualized using ChemDraw (Version 20.1, Copyright © 1985–2020 PerkinElmer Informatics, Inc., Waltham, MA, USA), the PyMOL Molecular Graphics System (Version 3.1.4, Copyright © Schrödinger, LLC, New York, NY, USA), and Mercury (Version 3.6, Copyright © 2001–2015 Cambridge Crystallographic Data Centre, Cambridge, UK), respectively.

Analytical balance
Sartorius Basic (PreZion: 0.001 g), Sartorius M2P Micro Balance (0.001 mg) and Mettler Toledo AE163 (0.01 mg) were used.

Nuclear magnetic resonance spectroscopy
1H-NMR and 2D-spectra were recorded using Bruker AM 400 (400 MHz) and Bruker Avance DRX 500 (500 MHz). 13C-NMR spectra were recorded using Bruker AM 400 (101 MHz) and Bruker Avance DRX 500 (126 MHz). 19F-NMR spectra were recorded using Bruker AM 400 (376 MHz) with a 1H–19F composite pulse decoupling sequence. All measurements were carried out at 25 °C. The spectra were analyzed according to the first order. All coupling constants are absolute values and expressed in Hertz (Hz). The following solvents from Eurisotop, Thermo Fisher Scientific, and Sigma-Aldrich were used: chloroform-d, dichloromethane-d2, acetonitrile-d3, trifluoroacetic acid-d and D2O. Chemical shifts δ were expressed in parts per million (ppm) and referenced to chloroform-d (1H NMR: δ = 7.26 ppm, 13C NMR: δ = 77.16 ppm), dichloromethane-d2 (1H NMR: δ = 5.32 ppm), acetonitrile-d3 (1H NMR: δ = 1.94 ppm), D2O (1H NMR: δ = 4.79 ppm). The signal structure is described as follows: s = singlet, d = doublet, t = triplet, q = quartet, p = quintet, b = broad, m = multiplet.

Infrared spectroscopy
IR spectra were recorded on a Bruker IFS 88 using ATR (attenuated total reflection) for solids. The absolute intensities of the peaks are given as follows: vs = very strong 0–9%, s = strong 10–39%, m = medium 40–69%, w = weak 70–89%, vw = very weak 90–100%.

Mass spectrometry
The mass spectra were measured on a Finnigan MAT 95 (70 eV) for EI-MS (electron ionization mass spectrometry) or FAB-MS (fast atom bombardment mass spectrometry) (with 3-nitrobenzyl alcohol as matrix). ESI-MS (electron spray ionization mass spectrometry) was conducted on a Thermo Scientific Q Exactive Plus. As an abbreviation for the ionized molecule [M]+ or [M+H]+ was used. Calibration was carried out using premixed calibration solutions (Thermo Fisher Scientific). The molecular fragments are stated as a ratio of mass per charge m/z.

UV–Vis spectroscopy
UV–Vis spectra were recorded on a Lambda 750 (PerkinElmer) UV–Vis spectrophotometer, an UV/Vis/NIR spectrometer Cary 500 (Varian), and a Cary 3500 Multicell UV–Vis spectrophotometer. The absorbance was measured from 200 to 800 nm using a quartz cuvette of 2, 5, or 10 mm optical path length at 20 °C. According to the samples, the respective solvent was used as background and subtracted. If the sample contained metal ions, the metal ion was added to the blank in the same concentration as it was present in the sample. To dilute the samples, Eppendorf Research® plus pipets ranging from 2 to 20 μL, 20 to 200 μL, and 100 to 1000 μL and Hamilton glass syringes were used. The samples were prepared by weighing in the solid compounds and dissolving them in the appropriate solvents, followed by filtration using Fisherbrand PTFE syringe filters, 0.2 µm.

High-performance liquid chromatography
Analytical high-performance liquid chromatography (HLPC) was performed using a Thermofisher UltiMate 3000 system containing a degasser, pump, autosampler, column compartment, and diode array detector. The flow rate was 1 mL/min on a stationary YMC-Triart C18 ExRS plus column (8 nm, S-5 µm, 250 mm × 4.6 mm) or a VDSpher 100 C18-M-SE column (5 µm, 250 mm × 4.6 mm). Chromeleon 7 software was used for data extraction. Preparative HPLC was performed with the Puriflash® 4125 system from Interchim, equipped with InterSoft V5.1.08 software and a UV diode array detector (200–600 nm). The stationary phase was a preparative C18 column (VDSpher 100 C18-M-SE, C18, 10 μm, 250 mm × 20 mm) at a flow rate of 15 mL/min. The precolumn was a small C18 column (VDSpher 100 C18-M-SE, 10 μm, 20 × 10 mm).

Procedure for irradiation
For irradiation experiments, the samples were dissolved in the respective solvent and placed in a translucent glass vial (clear glass crimp neck vial, borosilicate glass NMR tube, or Quartz cuvette). The vessel was then placed in a customized metal block, which has openings on the sides where the required LEDs are attached to irradiate the sample. Constant temperature of 22 ± 3 °C was maintained. Irradiation time, wavelengths, and power of the applied LEDs are listed in the supplementary information (Supplementary Table 1).

Thermal stability
The thermal stability was determined for the respective photoisomers at 25 °C. After sample preparation, the solutions were irradiated with a wavelength that gives a high photoisomer content and then kept at 25 °C in the dark. The isomer ratio was determined by integration of the respective (E)- and (Z)-isomer signals in 1H NMR in intervals. The data was processed by calculating ln(X0/Xt), where X is the percentage of the respective (Z)-isomer, followed by linear fitting (Eq. (1)). The calculated slope corresponds to the degradation rate constant k which is used to calculate the half-life t1/2.

Photostationary states
The photostationary states were calculated according to Eq. (2), where IE is the integral of signals assigned to the (E)-isomer, and IZ the integral of respective signals of the (Z)-isomer.

Photostability measurements
To evaluate photostability, the compound was irradiated at an appropriate wavelength to induce isomerization until the photostationary state was reached (evident by no further changes in the absorption spectrum), yielding a high proportion of the photoisomer. Subsequent irradiation at a shorter wavelength reversed the process, regenerating a high (E)-content. This photoswitching cycle was repeated ten times for each cage compound and the absorbance at λmax,abs was compared after the first and the last irradiation cycle. Experimental details for each compound are described in the respective section in the manuscript. Irradiation intensities and irradiation time are summarized in Supplementary Table 1.

LED irradiation
Sample irradiation for photoisomerization was performed using LEDs from Avonec, Mouser Electronics Inc., and OSRAM GmbH with emission maxima of 730, 700, 660, 623, 590, 523, 490, 470, 455, 430, and 365 nm. Standard time of irradiation for NMR and UV–Vis experiments are summarized in Supplementary Table 1. Any deviations from these standards are noted in the corresponding experiment. For the time of irradiation, samples were maintained at constant temperature (22 ± 3 °C) using a metal cooling block and a fan. Irradiation intensities of the respective LEDs were determined using the PowerMax USB (type PS19Q) sensor device (Coherent®) in five independent measurements. The detector (diameter 19 mm) was located at a distance of 55 mm from the light source, identical to the position of irradiated samples. The results are presented in Supplementary Table 1. “Max Power” refers to the smallest possible distance of the irradiated sample to the LED and “Min Power” to the largest possible distance between sample and LED.

CO2-mediated extraction of the cage 5 into water
The cage 5 is insoluble in water in its neutral form, but upon protonation, it becomes well-soluble in water and can be fully transferred into the aqueous phase. 5 was dissolved in chloroform to form 1 mM solution, and then mixed with D2O, resulting in no extraction into the water phase. A constant stream of CO2 bubbled through the solution for 10 min resulted in the transfer of protonated 5 (5*H+) into the water phase. Stirring that system for 30 h in an open vessel reversed the process (due to CO2 evaporation into the atmosphere), bringing the deprotonated 5 back into the organic layer.

Crystallographic study
Single crystals were obtained by a vapor-diffusion system of chloroform/n-hexane, whereby the compound was dissolved in chloroform at c = 0.5–2 mM in an open vial and then placed in a larger vessel containing n-hexane. The outer vessel was sealed and placed in the fridge at 3 °C. Single crystal X-ray diffraction data were collected on a STOE STADI VARI diffractometer with monochromated Ga Kα (1.34143 Å) radiation generated by an EXCILLIUM METAL JET D2+ at low temperature. Using Olex235, the structures were solved with the ShelXT36 structure solution program using Intrinsic Phasing and refined with the ShelXL37 refinement package using Least Squares minimization. Refinement was performed with anisotropic temperature factors for all non-hydrogen atoms; hydrogen atoms were calculated on idealized positions.

MTT assays
HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin solution (Gibco®, 10000 units/mL penicillin and 10000 µg/mL streptomycin) in a humid incubator at 37 °C and 5% CO2. To suspend the cells, they were first washed with PBS (Gibco®) and then treated with trypsin-EDTA (0.25%, Gibco®) for 5 min to detach them from the surface. Flat-bottom 96-well plates were prepared by adding 200 µL/well PBS to the outer border wells to maintain constant vaporization in all wells. The cells were seeded into the inner wells (3000 cells/well) and incubated for 24 h. Stock solutions (500 µM) of compound 5*H+ were prepared in H2O, and aliquots of the solutions were irradiated with 523 nm for 1 h (10 W LED from LEDENGIN; maximum power = 7.08 mW/cm2, minimum power = 6.56 mW/cm2; see Supplementary Table 1). The samples were subsequently diluted with DMEM to the final concentrations (0.091 µM, 0.5 µM, 1 µM, 2 µM, 4 µM, 6 µM, 8 µM, and 10 µM, 2% H2O, v/v). The medium was replaced with medium supplemented with 5*H+ (dark or irradiated) in different concentrations (100 µL/well, six technical replicates), and the cells were incubated for another 48 h in the dark. To ensure uniform treatment of the cells, the medium of the controls was replaced with DMEM supplemented with 2% H2O (v/v, 100 µL/well). After the 48-h incubation period, 5 µL/well TritonTM X-100 detergent (10% solution in PBS (w/v)) was added to the positive control for at least 5 min to induce cell death. Subsequently, 10 µL/well MTT dye solution in water (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Cell Proliferation Kit I from Roche) was added to all sample wells and cells were incubated for 3 h. The reduction of the dye to formazan was stopped by adding 100 µL/well of solubilization buffer (Cell Proliferation Kit I from Roche), and the plates were placed in the incubator overnight to ensure solubilization of formazan. The absorbance of each well was measured at 595 nm using a CLARIOstar Plus plate reader (BMG Labtech). The absorbance data were averaged across the technical replicates, the positive control was subtracted as background, and the data were then normalized to the number of viable cells in the negative control as 100%. Data were plotted against the log of agonist concentration (log [agonist], M) with mean and SD in GraphPad Prism Version 9.5.1 for Windows, GraphPad Software, San Diego, California, USA. The number of individual experiments n performed for the dark and irradiated samples was n = 3. The HeLa cell line was obtained from DSMZ (Leibniz-Institut Deutsche Sammlung von Mikroorganismen und Zellkulturen), DSMZ no. ACC 57; deposited by ATCC (CCL 2), Rockville, Maryland, USA.

Positive control. Cells that were specifically killed by treating them with 5 µL/well TritonTM X-100 detergent (10% solution in PBS (w/v)) for at least 5 min before adding MTT dye.

Negative control. Cells that remained alive (treated with neither 5*H+ nor TritonTM X-100), corresponding to 100% cell viability.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Schäfer, Fuhr and Pianowski report a series of photoswitchable cages that are able to operate in water.\nThe authors make use of one of their previously reported photochromic motifs (a tetra-ortho-fluoroazobenzene bis-aldehyde)\nas well as a new motif being tetra-ortho-chloroazobenzene (aiming to further induce a red-shift in the E-isomer UV/vis\nspectra). The tetra-ortho-fluorinated switch was able to be assembled with TREN to form a [3+2] capsule. The authors could\ncovert this EEE-capsule to the ZZZ almost quantitatively with 660 nm light with no mixed intermediate EEZ or EZZ states\nbeing observed (attributed to templating effect), but with a reduced thermal stability (although measured in CDCl3 where\ntrace amounts of acid could catalyse Z-to-E isomerism). The authors could reduce their dynamic-covalent imine bonds to\nsecondary amines, affording more robust capsules which could also be photoisomerized, but using higher energy light due\nto the loss in conjugation. Protonation of this reduced cage renders the system water soluble, and the authors can display\nphase-transfer of the system between CDCl3 and D2O.\nWhat is quite surprising is that the protonated form is also photoswitchable from the E to the Z-isomer. This enabled the\nauthors to use a transient stimulus (carbonic acid) to develop a cycle that protonates the capsule (transferring it from CDCl3\nto D2O), and then CO2 is given off over time resetting the system back to the chlorinated phase. This could be particularly\ninteresting if any Host-Guest chemistry could be achieved, allowing cargo transport between phases.\nThe authors further looked at the dynamic-covalent chemistry associated with the ZZZ stage of the imine capsule. They\nnoted that exchange of a tetra-fluorinated bisaldehyde with benzaldehyde was possible. This is interesting as such\ntransimination is typically observed for an amine reacting with the imine, but here it appears that the aldehyde component\ncan also do this. The authors should clarify their proposed mechanism for this, or if they think there is some disassembly and\nreassembly taking place.\nThe thermal relaxation of ZZZ-3 to EEE-3 does not show any observable mixed ZZE or ZEE states, this is also quite an\ninteresting result that sets it apart from previous work of Greenaway. It would be useful if the authors could further comment\non this aspect of templation and why it is more significant for this tetra-ortho-fluorinated ligand. Further study into the use of\nbisaldehydes have been reported, possibly affording oligomeric structures.\nThe chlorinated photoswitch derivative shows reduced thermal half-life but ability to operate with near IR light. The resulting\ncapsule appears unstable when converted to the ZZZ state, affording a mixture of species, but this could be regenerated by\nswitching them back to the EEE state. This chlorinated cage could not be reduced with NaBH4 (perhaps BH3.THF would\nwork?)\nFinally, they investigate binding metal ions by using a bipyridine pillar. They can form complexes with various metal ions.\nThe experimental details are accurately reported, and the authors discuss the previous work in this area citing the most\nrelevant papers.\nOverall, I think this is a fantastic and full piece of work. I strongly congratulate the authors on their efforts, and I strongly\nsupport publication of this manuscript in Nature Communications, pending answers to some of the questions raised above\nand the minor points below.\nMinor:\n1. How does the thermal half-life of the cage 3 change when in DCM? Trace amounts of acid in chloroform has been known\nto catalyse Z-to-E isomerism.\n2. For the NIR irradiation, the absorbance of the chlorinated switch above 700 nm looks minimal, how do the authors know\nthat this is a switching effect and not a heating effect under such prolonged irradiation? Especially with such a short thermal\nhalf-life.", "reviewer_2": "This manuscript describes the design, synthesis, and characterization of photoresponsive dynamic covalent cages\nassembled from azobenzene-derived bis-aldehydes and a trivalent amine linker. The authors demonstrate cages that\nundergo constitutional changes in response to light, pH, and metal ions. While the synthetic chemistry and characterization\nefforts are technically sound, the manuscript falls significantly short of the novelty, depth, and rigor expected for Nature\nCommunications. I recommend rejection based on the following critical issues:\nA) The manuscript reads as a disjointed series of experiments rather than a well-packed study. It jumps between unrelated\nsystems (fluorinated cage 3, reduced cage 5, chlorinated cage 10, bipyridine cage 14) without establishing a clear\noverarching hypothesis. The connection between individual results and the purported goal of \"biocompatible molecular\nmachines\" is poorly articulated, weakening the conceptual impact.\nB) The core concept—light-driven dynamic covalent cages—and many specific methodologies (e.g., imine-based cage\nassembly, photoisomerization-triggered transformation, reduction to stabilize cages) have been extensively reported by\nothers (e.g., Feringa [Ref 26], Clever [Ref 17], Schmidt [Ref 24], Wang [Ref 31]). The new chlorinated switch 9 is promising\nbut underdeveloped; its integration into functional cages (10) is inefficient and lacks demonstration of significant advantages\nover prior systems. Therefore, the overall novelty of this work does not meet the general standard of Nature\nCommunications.\nC) The title prominently claims cages \"operational in water\" implying broad applicability. However, detailed water-\ncompatible studies are only demonstrated for the single, non-dynamic cage 5 (derived from 3 via reduction). The primary\ndynamic cages (3, 10, heteroleptic cages) are synthesized, switched, and characterized exclusively in organic solvents.\nSwitching in water is only shown for cage 5 (after protonation). Even here, the \"biocompatibility\" claim is unsupported (no\nstability data in physiologically relevant media, no biological testing). The CO₂-triggered phase transfer of 5 is a simple pH\neffect, not a demonstration of \"operation\" as a functional machine in water. The title and abstract significantly overstate the\nactual aqueous results presented. In addition, protonation of azobenzene can induce Z to E transition; however, there is no\ncomment for this effect (see ref J. Am. Chem. Soc. 2024, 146, 10246−10250).\nD) Key conclusions rely heavily on qualitative data (e.g., color changes, simple extractions, basic NMR spectra) and lack\nrigorous quantitative support. Claims of efficient self-assembly, exchange, and reversibility lack detailed kinetic data,\nthermodynamic parameters (e.g., binding constants for guest/metal interaction), or robust quantification of species\ndistribution during stimuli-responsive processes (beyond limited NMR integrations). While X-ray structures of 3 and 5 are\nvaluable, solution-state structural integrity (e.g., under switching conditions, in water) is not thoroughly established (e.g., lack\nof detailed DOSY for key species in relevant solvents/conditions).", "reviewer_3": "The manuscript of Pianowski et al. focuses on the synthesis and investigation of (non-)photoresponsive supramolecular\ncage made of substituted azobenzenes, imine/amine and bipyridine units. The achieved photoswitching within the\ntherapeutic window of light along with the performed structural modifications towards water soluble cage are interesting.\nAfter reading the manuscript, we have raised the following questions that should be addressed prior to publication.\nPage 1, the term “more recently developed structures“ sounds awkward and should be replaced with concrete derivatives.\nFigure 1A, the structures of 1-4 shown should be completed with the elements to increase the chemical clarity. The NMR\nspectra should be completed with the integral intensities to properly distinguish the ration of 1 and 4\nPage 4, Besides power of the used LEDs (given in the ESI), the authors should also specify the time of irradiation.\nA comparison of the achieved fundamental parameters (e.g., half life of the Z isomeric form, used operational light\nwavelengths for photoswitching or maximal reached E/Z ratio) with known and structurally-related cages is missing.\nPage 4, The text states that (Z,Z,Z) 5 returns to (E,E,E) 5 upon irradiation with 430 or 410 nm light. But the “430 nm” and “410\nnm” NMR spectra in Figure S7 clearly contain more signals than the dark adapted photostationary states.\nPage 4, second paragraph, Some references are missing or matched incorrectly, please check.\nWhy are not the photoswitching processes of 5 demonstrated by UV Vis spectroscopy as shown for 3 (Figure S43) and 10\n(Figure S44)?\nFigure 3, The double-arrow is used incorrectly.\nPage 7, Replace “frequencies” with wavelengths, especially when discussing in nm.\nFigure S34, The obtained value of 441.22 Da below the illustration of cage 14 does not match the theoretical value.\nPage 11, The complexation of 14 with metals is referenced to Figure 6B but 3/13 are mentioned in the caption of Figure 6B.\nExtended data Figure 1A, A continuous irradiation with 660 nm light is mentioned, which contradicts Figure S5 mentioning\n“short intervals”, what is correct?\nExtended data Figure 2, How is the reaction of (Z,Z,Z) 3 with monovalent benzaldehyde or benzylamine affected by\nirradiation? Would the same result be delivered upon pre-irradiating cage 3 and subsequent reaction with monovalent\nreagents?\nThe fatigue resistance of the investigated photoresponsive cages should be completed, e.g. after 10 cycles.\nNothing is disclosed on how the trans. metal cations are bonded to the bipyridine moieties of 14?\nThe text should uniformly use stereochemical descriptors, e.g. (E,E,E)-5 as compared to often used E,E,E-5 (round brackets\nmissing).", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "In the name of all authors, we would like to thank all the reviewers for their time and attention\nin reading our manuscript. We are grateful for all the comments, also the critical ones, which\nmotivated us to increase the quality of our initial submission. The comments are individually\naddressed below.\nReviewer #1 (Remarks to the Author):\nIn this manuscript, Schäfer, Fuhr and Pianowski report a series of photoswitchable cages that\nare able to operate in water. The authors make use of one of their previously reported\nphotochromic motifs (a tetra-ortho-fluoroazobenzene bis-aldehyde) as well as a new motif\nbeing tetra-ortho-chloroazobenzene (aiming to further induce a red-shift in the E-isomer\nUV/vis spectra). The tetra-ortho-fluorinated switch was able to be assembled with TREN to\nform a [3+2] capsule. The authors could covert this EEE-capsule to the ZZZ almost\nquantitatively with 660 nm light with no mixed intermediate EEZ or EZZ states being observed\n(attributed to templating effect), but with a reduced thermal stability (although measured in\nCDCl3 where trace amounts of acid could catalyse Z-to-E isomerism). The authors could\nreduce their dynamic-covalent imine bonds to secondary amines, affording more robust\ncapsules which could also be photoisomerized, but using higher energy light due to the loss in\nconjugation. Protonation of this reduced cage renders the system water soluble, and the authors\ncan display phase-transfer of the system between CDCl3 and D2O.\n1) What is quite surprising is that the protonated form is also photoswitchable from the E to\nthe Z-isomer.\nAd 1) Indeed. However, we have confirmed it by 1H and 19F NMR and multiple switching\ncycles (NMR, UV-Vis) (Table S2, Figure S12-S14, Figure S72, Figure S77-S78).\n2) This enabled the authors to use a transient stimulus (carbonic acid) to develop a cycle that\nprotonates the capsule (transferring it from CDCl3 to D2O), and then CO2 is given off over\ntime resetting the system back to the chlorinated phase. This could be particularly interesting\nif any Host-Guest chemistry could be achieved, allowing cargo transport between phases.\nAd 2) We have tried to encapsulate over 20 potential guest molecules (such as phenols,\nnaphthols, aromatic sulphonic acids and halogenated aromatic compounds). However, we\ncould not see any convincing proof of encapsulation, nor of its photomodulation, probably due\nto small cavity size. On the other hand, as the shape of the cage changes upon\nphotoisomerization, the protonated cage 5 can interact as a guest with cucurbit[8]uril (CB8).\nResponse to reviewers Page 1\nIn the resulting inclusion complex we could see an influence on the isomer distribution in\ncomparison to the cage dissolved in water in the absence of CB8, especially after irradiation at\n410 nm (Figures S20-S25 and Table S3).\n3) The authors further looked at the dynamic-covalent chemistry associated with the ZZZ stage\nof the imine capsule. They noted that exchange of a tetra-fluorinated bisaldehyde with\nbenzaldehyde was possible. This is interesting as such transimination is typically observed for\nan amine reacting with the imine, but here it appears that the aldehyde component can also do\nthis. The authors should clarify their proposed mechanism for this, or if they think there is some\ndisassembly and reassembly taking place.\nAd 3) As depicted in Figure 3, we believe that due to the presence of residual humidity in the\nsystem (water was not actively eliminated from the reaction mixtures), the more strained\n(Z,Z,Z)-3 isomer partially opens to the form that contains a free aldehyde and an amine groups,\nwhich both can then interact with externally added reactive partners and result in the aldehyde\n/ imine exchange. Additionally, the experiments were performed in CDCl , so the minor acidity\n3\nof chloroform may also help catalyze this process.\n4) The thermal relaxation of ZZZ-3 to EEE-3 does not show any observable mixed ZZE or\nZEE states, this is also quite an interesting result that sets it apart from previous work of\nGreenaway. It would be useful if the authors could further comment on this aspect of templation\nand why it is more significant for this tetra-ortho-fluorinated ligand.\nAd 4) As this result is indeed interesting, the behavior of 3 upon irradiation with various light\nwavelengths has been thoroughly investigated. The interesting result, shown in the Figure 1B,\nhas been confirmed in several independent experiments, to minimize the risk of error. Of\ncourse, the formation of other isomers is not impossible, but under the conditions applied by\nus, the fully converted isomers are evidently dominating. While 660 nm results in near-\nquantitative (Z,Z,Z)-3 formation and 430 nm restores the (E,E,E)-isomer, 523 nm forms small\nquantities of the (E,E,Z)- and (E,Z,Z)-isomer, while the (Z,Z,Z)-isomer remains the main\nphotoproduct (Figure S2). Yet, the intermediary forms have much shorter lifetimes (probably\ncaused by the enhanced strain within the cage) than the (Z,Z,Z)-isomer (123 h at 25 ̊C,\nFigure S3). While after 523 nm irradiation, signals assigned to (E,E,Z)- and (E,Z,Z)-3 are\nvisible immediately after irradiation, they disappear within 1 day at 25 °C (Figure S5). While\nunequivocally assigning the signals to either (E,E,Z) or (E,Z,Z) was not possible, we have\ndetermined their lifetimes to be t = 2.16 h and t = 6.54 h (Figure S6).\n1/2 1/2\nResponse to reviewers Page 2\nAnother recently published (H. Shan et al. J. Am. Chem. Soc. 2025, 147, 14960－14965,\nhttps://doi.org/10.1021/jacs.5c04399 ) cage system, based on azobenzenes, showed different\nswitching behavior (either concerted ZZZ-to-EEE transition, or stepwise ZZZ-EZZ-EEZ-EEE\nswitching) depending on the anion that is complexed in the adjacent ligand. Therefore, we find\nsuch a behavior rare, but not unprecedented.\nIn our case, we believe that the presence of fluorine atoms might additionally stabilize the\n(E,E,E)-3 and (Z,Z,Z)-3 isomers due to the favorable overlap of the C-F bonds or the fluorinated\naryl residues, and thus sequester the intermediary forms from the system. As stated below, the\nanalogous chlorinated cage (where the C-Cl bonds are less polarized) does not show such\neffect, forming various species and transient isomers upon irradiation (although the geometry\nin the latter case is also more strained due to the presence of bulk chlorine atoms, so this effects\ncannot be seen separately).\n5) Further study into the use of bisaldehydes have been reported, possibly affording oligomeric\nstructures.\nThe chlorinated photoswitch derivative shows reduced thermal half-life but ability to operate\nwith near IR light. The resulting capsule appears unstable when converted to the ZZZ state,\naffording a mixture of species, but this could be regenerated by switching them back to the\nEEE state. This chlorinated cage could not be reduced with NaBH4 (perhaps BH3.THF would\nwork?)\nAd 5) We thank the reviewer for motivating us to explore the issue of chlorinated cages even\nfurther. Although the BH *THF did not work in our system either (various products observed,\n3\nbut not the desired one), upon screening on alternative reaction conditions we discovered that\nother reducing agent - NaBH(OAc) – can provide us with the desired product (the reduced,\n3\ncovalently bound and stable chlorinated cage 13), although in moderate yield (we have isolated\nthe expected product with 15 % overall yield, starting from the assembly of the dynamic cage\n10 out of the component – the tetra-chloro-azobenzene bisaldehyde 9 and TREN 2, and\napplying the reducing agent in situ).(Figures S56-S57). This cage 13 was switchable with red\nlight (660 nm) and characterized further (Figure S58-S60; see below).\n6) Finally, they investigate binding metal ions by using a bipyridine pillar. They can form\ncomplexes with various metal ions.\nResponse to reviewers Page 3\nThe experimental details are accurately reported, and the authors discuss the previous work in\nthis area citing the most relevant papers.\nOverall, I think this is a fantastic and full piece of work. I strongly congratulate the authors on their efforts,\nand I strongly support publication of this manuscript in Nature Communications, pending answers to some of the questions raised\nabove and the minor points below.\nAd 6) we highly appreciate the opinion of the reviewer on the novelty and interesting aspects\nof our manuscript\nMinor:\n7) How does the thermal half-life of the cage 3 change when in DCM? Trace amounts of acid\nin chloroform has been known to catalyse Z-to-E isomerism.\nAd 7) Indeed we observed a difference in thermal stability of the cage (Z,Z,Z)-3, with t in\n1/2\ndichloromethane being 321 h (Figure S32), compared to 123 h in CDCl (Figure S3). In the\n3\nnon-acidic solvent, the life-time of the cage (Z,Z,Z)-3 is now similar to the linear imine (Z)-17\n(307 h, Figure S33), but we still do not see the formation of intermediary isomers EEZ / EZZ,\nwhich supports our remarks above on the templating effect.\n8) For the NIR irradiation, the absorbance of the chlorinated switch above 700 nm looks\nminimal, how do the authors know that this is a switching effect and not a heating effect under\nsuch prolonged irradiation? Especially with such a short thermal half-life.\nAd 8) especially with such minimal effects, we tried to perform careful control of the system.\nPrimarily, for the 730 nm irradiation we have applied a 715-nm-cutoff filter, to make sure that\nwe do not irradiate our sample with the emission peak shoulder in the red light range below\n715 nm. To exclude the photothermal effect, which is known and often reported, particularly\nfor the NIR-irradiated systems, we wrapped a control sample with aluminum foil and irradiated\nunder the same conditions as our main sample. In the tube covered with aluminium foil, we did\nnot observe any isomerization under the same irradiation parameters (time, frequency of the\nused LED light emitter. Thus, we attribute the observed isomerization purely to the photon\nabsorption by the sample (Figure S44, including a photo of our experimental setup).\nReviewer #2 (Remarks to the Author):\nThis manuscript describes the design, synthesis, and characterization of photoresponsive\ndynamic covalent cages assembled from azobenzene-derived bis-aldehydes and a trivalent\namine linker. The authors demonstrate cages that undergo constitutional changes in response\nResponse to reviewers Page 4\nto light, pH, and metal ions. While the synthetic chemistry and characterization efforts are\ntechnically sound, the manuscript falls significantly short of the novelty, depth, and rigor\nexpected for Nature Communications.\n-------------\nWe appreciate the careful reading of our manuscript by the Reviewer 2 and his critical opinion\non our work. It became a motivation for us to increase the quality of our manuscript, which we\nwill discuss in details in our comments on particular critical issues below. In particular, we\nhave provided additional experiments to demonstrate the range of biocompatibility of our\ncompounds. Additionally, the upgraded version of our manuscript contains a novel stable\ncovalent cage “13” based on chlorinated bis-aldehyde components, which undergoes reversible\nphotoisomerization in water under irradiation with 660 nm light – the wavelength that\nefficiently penetrates (to the depth of several cm, according to the available literature) soft\nhuman tissues.\n-------------\nI recommend rejection based on the following critical issues:\nA) The manuscript reads as a disjointed series of experiments rather than a well-packed study.\nIt jumps between unrelated systems (fluorinated cage 3, reduced cage 5, chlorinated cage 10,\nbipyridine cage 14) without establishing a clear overarching hypothesis. The connection\nbetween individual results and the purported goal of \"biocompatible molecular machines\" is\npoorly articulated, weakening the conceptual impact.\nAd A) The clear overarching hypothesis in the manuscript is that we can synthesize - with good\nyields and good control over their constitution - complex photochromic molecular cages using\nsimple methodology demonstrated in the manuscript. The resulting dynamic cages can undergo\nthe exchange with numerous components in a light-controlled fashion. Yet, upon reduction we\nobtain stable compounds (in the revised version, two cages “5” and “13”) capable of\nphotoisomerization in aqueous conditions with biocompatible light frequencies (see below).\nAll the presented dynamic systems have similar topology and therefore cannot be treated as\ndisjointed. The latter series of experiments build up on the initial observations regarding the\nsynthesis and behavior of the dynamic cage “3” and are directly related/comparable with its\ndesign and properties. The latter series intent to demonstrate the vast flexibility and stimuli-\nresponsiveness of the initial setup. The additional highlight is the power of supramolecular\nResponse to reviewers Page 5\nself-assembly, as the stepwise synthesis of cage “5” and its chlorinated analogue “13” would\nbe extremely challenging.\nTo make the connection of our results to the overarching concept more clear, and at the same\ntime to underline that our system does not constitute yet a biocompatible molecular machine,\nbut is an important step towards realizing that goal, the following sentence in conclusions has\nbeen given as follows: “We believe, that especially our cages operational in aqueous media\nwith red light make a step forward towards construction of biocompatible molecular machines”\nB) The core concept—light-driven dynamic covalent cages—and many specific methodologies\n(e.g., imine-based cage assembly, photoisomerization-triggered transformation, reduction to\nstabilize cages) have been extensively reported by others (e.g., Feringa [Ref 26], Clever [Ref\n17], Schmidt [Ref 24], Wang [Ref 31]). The new chlorinated switch 9 is promising but\nunderdeveloped; its integration into functional cages (10) is inefficient and lacks demonstration\nof significant advantages over prior systems. Therefore, the overall novelty of this work does\nnot meet the general standard of Nature Communications.\nAd B) We tried our best to present the state-of-the-art and credit our predecessors properly (as\nlisted by the Reviewer 2 above in “reported by others”). We sincerely hope that we did not\nmiss any important contribution to the area. However, in our manuscript a novel quality\nemerged out of existing methodologies in several aspects listed below.\nIn the first version, we reported an unprecedented observation of clean photoconversion\nbetween two oppositely configured photoisomers: (Z,Z,Z)-3 and the (E,E,E)-3, while our\nforerunners typically report pretty complex mixtures of isomers (geometry and constitutional)\nat photoequilibria, often including significant fractions of linear polymers which irreversibly\nprecipitate out of the solution. While we were certainly lucky with the choice of substrates and\nthe reaction conditions (as this was one of our first experiments, and not the result of lengthy\noptimization), this does not diminish the novelty of our observation and the progress it makes\nto the methodology.\nWe agree with the Reviewer 2, that the cage formation from the chlorinated switch “9” was\npromising, but underdeveloped in the first version of the manuscript. We are happy to report\nthat in the revised version we managed to provide a covalent cage “13” containing the\nchlorinated switch 9. This cage is reversibly switchable in water with red light (660 nm) (see\nFigures S58 and S60) – a frequency that can deeply penetrate soft human tissues. A cage\nreversibly switchable in water with red light was – to our best knowledge - not demonstrated\nby others, and we believe that this increases the novelty of our report even further, particularly\nResponse to reviewers Page 6\nin its aspect of biocompatibility. We believe that it shows the way (to us and others) to design\nmore complex systems with enhanced functionalities – and, in perspective, true molecular\nmachines - that will be operational in vivo.\nC) The title prominently claims cages \"operational in water\" implying broad applicability.\nHowever, detailed water-compatible studies are only demonstrated for the single, non-dynamic\ncage 5 (derived from 3 via reduction). The primary dynamic cages (3, 10, heteroleptic cages)\nare synthesized, switched, and characterized exclusively in organic solvents. Switching in\nwater is only shown for cage 5 (after protonation). Even here, the \"biocompatibility\" claim is\nunsupported (no stability data in physiologically relevant media, no biological testing). The\nCO₂-triggered phase transfer of 5 is a simple pH effect, not a demonstration of \"operation\" as\na functional machine in water.\nAd C) In the revised manuscript, we provided a second non-dynamic cage “13” – the\nchlorinated analogue of 5 – which is also switchable in water, now with 660 nm red light. Of\ncourse, as the reviewer correctly remarked, the term “biocompatibility” was in our case related\nmainly to the used wavelength of light, and – in the broader sense, it encompasses multiple\nfactors. For that reason, we have tested the stability of the protonated cage “5” in\nphysiologically relevant media - in water and in acetate buffer pH 3.6 (Figures S77 and S78,\nrespectively). After 10 switching cycles, we have seen less than 1% degradation, which attests\nto very good stability of “5” in aqueous media.\nWe have also tested the cytotoxicity of the cages 5 towards HeLa cells using a standard\ntechnique (MTT assays) (Figure S26, Tables S4). It demonstrated, that a) the cage 5 can be\nused in biological context at the concentration below 0.5 uM, and b) that photoisomerization\ndoes not significantly alter the cytotoxicity of this cage. Due to the insufficient amount of\nmaterial, we could not perform the MTT assays for the cage “13”, but we have no reason to\nsuppose that the toxicity would drastically differ from the fluorinated derivative “5”, as the\ncage “13” does not have additional H-bonding sites, charged or ionizable groups such as\namines/carboxylates, nor pharmacophore-related motifs.\nRegarding the last issue (“operation of the machine in water”), while the cages do not form\nhost-guest complexes with small cargo molecules (as far as the over 20 compounds screened\nby us indicated), we have observed that upon formation an inclusion complex of the cage 5 in\nwater with cucurbit(8)uril, the photoisomerization output (upon irradiation with violet light)\ndiffers from the outcome in free solution. These results were added in the revised version\n(Figures S18-S25) and expand the scope of functionality of the demonstrated system.\nResponse to reviewers Page 7\nC1) The title and abstract significantly overstate the actual aqueous results presented. In\naddition, protonation of azobenzene can induce Z to E transition; however, there is no comment\nfor this effect (see ref J. Am. Chem. Soc. 2024, 146, 10246−10250).\nAd C1) We have analyzed the stability of the cage “5” in chloroform (in the neutral form) and\nin water (as the protonated system) (Figures S15-S16). In both cases, we followed by NMR\nthe samples over the course of several weeks. We have not noticed any drastic impact of\nprotonation on the lifetime of the (Z,Z,Z)-5 cage.\nD) Key conclusions rely heavily on qualitative data (e.g., color changes, simple extractions,\nbasic NMR spectra) and lack rigorous quantitative support. Claims of efficient self-assembly,\nexchange, and reversibility lack detailed kinetic data, thermodynamic parameters (e.g., binding\nconstants for guest/metal interaction), or robust quantification of species distribution during\nstimuli-responsive processes (beyond limited NMR integrations). While X-ray structures of 3\nand 5 are valuable, solution-state structural integrity (e.g., under switching conditions, in water)\nis not thoroughly established (e.g., lack of detailed DOSY for key species in relevant\nsolvents/conditions).\nAd D) we are happy to report that additional data (DOSY and other relevant 2D-NMR\nexperiments, additional quantification of species’ distribution during stimuli-responsive\nprocesses) provided in the revised manuscript fully support our previous conclusions. We have\nprovided detailed insight into the photoisomerization results under various conditions, and the\ntime course of certain processes (instead of the start and end point provided in the initial version\nof the manuscript).\nTo be more detailed: we provided COSY, NOESY, and DOSY, spectra of the dark-state cage\nmolecules and their photoisomers under various conditions (Figures S80-S111), including\nshort discussion on hydrodynamic radii of the particular cages, which are in agreement with\nthe observable properties in other analytical methods. In particular, the Figures S110-S111\nclearly supports our earlier discussion on partial disassembly of the dynamic chlorinated cage\n“10” upon irradiation with 660 nm. We quantified photostability of the cages under various\nconditions over 20 irradiation steps (Figure S74-S79), specified the photostationary states of\n5*H+ in water (Table S2), and determined k and t for the other isomers of 3 ((E,E,Z) and\n1/2\n(E,Z,Z)) and for (Z,Z,Z)-3 and (Z)-17 in DCM. A summary of fundamental parameters is given\nin Extended Data Table 1.\nResponse to reviewers Page 8\nDue to the low amount of the material, we were not able to determine the metal ion binding\nconstants for the cage “15”. However, as such non-photochromic complexes (and similar ones)\nhave been previously reported in literature, we have added related references (refs. [32-34]) for\nthe readers who are interested in such architecture in more details.\nReviewer #3 (Remarks to the Author):\nThe manuscript of Pianowski et al. focuses on the synthesis and investigation of (non-\n)photoresponsive supramolecular cage made of substituted azobenzenes, imine/amine and\nbipyridine units. The achieved photoswitching within the therapeutic window of light along\nwith the performed structural modifications towards water soluble cage are interesting.\n-----------\nWe thank the Reviewer 3 for his time, questions and suggestions which help us to improve the\nquality of our manuscript.\n-----------\nAfter reading the manuscript, we have raised the following questions that should be addressed\nprior to publication.\n11) Page 1, the term “more recently developed structures“ sounds awkward and should be\nreplaced with concrete derivatives.\nAd 11) Concrete derivatives have been placed in the manuscript\n12) Figure 1A, the structures of 1-4 shown should be completed with the elements to increase\nthe chemical clarity.\nAd 12) We have added the chemical structures to Figure 1A and Figure 2 for more clarity.\n13) The NMR spectra should be completed with the integral intensities to properly distinguish\nthe ration of 1 and 4\nAd 13) The integrations in Figure 1 A have been added\n14) Page 4, Besides power of the used LEDs (given in the ESI), the authors should also specify\nthe time of irradiation.\nAd 14) We have specified irradiation times explicitly for each figure in the main part. In\nsupporting information, standardized protocols include the irradiation times have been added\n(Table S1). Any deviations have been marked in the respective experiment descriptions.\nOtherwise they follow the general protocols.\nResponse to reviewers Page 9\n15) A comparison of the achieved fundamental parameters (e.g., half life of the Z isomeric\nform, used operational light wavelengths for photoswitching or maximal reached E/Z ratio)\nwith known and structurally-related cages is missing.\nAd 15) We have added parameters for several structurally related cages from literature (half-\nlife times, absorbance maxima and photoconversion) and summarized our results for\ncomparison in Extended Data Table 1);\n16) Page 4, The text states that (Z,Z,Z) 5 returns to (E,E,E) 5 upon irradiation with 430 or 410\nnm light. But the “430 nm” and “410 nm” NMR spectra in Figure S7 clearly contain more\nsignals than the dark adapted photostationary states.\nAd 16) We thank to the reviewer for this remark. Indeed, the conversion is not quantitative for\nthe reduced cage 5. We have rephrased the respective sentences to accurately reflect the isomer\ndistribution, listed in the Table S2 and on the Figure S9-S10 and Figure S12-S13) (in\nchloroform and – protonated – in water).\n17) Page 4, second paragraph, Some references are missing or matched incorrectly, please\ncheck.\nAd 17) we will appreciate, if the reviewer can tell us more specifically which references need\nto be changed/added, as the second paragraph of the page 4 (the section entitled “Reduction of\n3 yields a stable cage 5 reversibly switchable in water”) contains no references at all. Maybe\nthe reviewer had in mind another section. If so, we will be happy to correct it. After preventive\nchecking, we have found no incorrectly matched references among the existing ones.\n18) Why are not the photoswitching processes of 5 demonstrated by UV Vis spectroscopy as\nshown for 3 (Figure S43) and 10 (Figure S44)?\nAd 18) We have provided the UV-Vis spectra illustrating the photoswitching process of 5 and\n5*H+ (Figures S71-S72).\n19) Figure 3, The double-arrow is used incorrectly.\nAd 19) Indeed, by mistake we have used the wrong arrow- the arrow has been corrected and\nthe required water molecule has been added.\n20) Page 7, Replace “frequencies” with wavelengths, especially when discussing in nm.\nAd 20) The word frequencies was exchanged to wavelengths whenever in context with nm.\n21) Figure S34, The obtained value of 441.22 Da below the illustration of cage 14 does not\nmatch the theoretical value.\nResponse to reviewers Page 10\nAd 21) We have checked the Figure S34 (now Figure S61) and it seems to contain the correct\nvalue 411.22 Da.\n22) Page 11, The complexation of 14 with metals is referenced to Figure 6B but 3/13 are\nmentioned in the caption of Figure 6B.\nAd 22) Due to the addition of an extra cage (the chlorinated covalent cage, now with the\nnumber “13”), the numbers above have been shifted by one. In the old version, the cage 14 is\nformed insitu upon mixing of the pillar 13 with TREN or with the cage 3. Therefore, the\ncomponents of the experiment (3 / 13) are mentioned in the caption. We have double-checked\nthe content of the caption, and it corresponds to the composition of respective experiments, to\nthe best of our knowledge. Now the bipyridine-cage will have the number 15, and the respective\npillar – number 14. So in the caption it will be cage 15 and the components 3/14.\n23) Extended data Figure 1A, A continuous irradiation with 660 nm light is mentioned, which\ncontradicts Figure S5 mentioning “short intervals”, what is correct?\nAd 23) The correct description is “short intervals”. We have adapted the Extended data\nFigure 1B accordingly.\n24) Extended data Figure 2, How is the reaction of (Z,Z,Z) 3 with monovalent benzaldehyde\nor benzylamine affected by irradiation? Would the same result be delivered upon pre-\nirradiating cage 3 and subsequent reaction with monovalent reagents?\nAd 24) We conducted the experiment with pre-irradiated cage 3 which resulted in a decreased\nconversion, especially for the reaction with benzaldehyde. This may be reasoned with the\nincreasing formation of unreactive (Z,Z,Z)-3 over the course of the experiment. Details have\nbeen added Figure S27-S28 and Table S5.\n25) The fatigue resistance of the investigated photoresponsive cages should be completed, e.g.\nafter 10 cycles.\nAd 25) The fatigue resistances have been measured and provided in Figure S74-S79.\n26) Nothing is disclosed on how the trans. metal cations are bonded to the bipyridine moieties\nof 14?\nAd 26) The binding of metals cations to 14 (now 15) have been clarified in the literature\nreference added by us to the revised version (ref.[32])\n27) The text should uniformly use stereochemical descriptors, e.g. (E,E,E)-5 as compared to\noften used E,E,E-5 (round brackets missing)\nResponse to reviewers Page 11\nAd 27) We thank for this remark. Stereochemical descriptors have been unified across the\npaper.\nResponse to reviewers Page 12"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have carefully and fully considered my comments. I appreciate their willingness to constructively engage and I\nam pleased with the revisions made. I have no outstanding comments and I congratulate the authors on a nice and thorough\nstudy. I fully support publication of this manuscript in Nature Communications.", "reviewer_3": "The authors have addressed our comments and, therefore, we recommend publication.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the\nNature Communications initiative to facilitate training in peer review and to provide appropriate recognition\nfor Early Career Researchers who co-review manuscripts.", "reviewer_5": "In this manuscript, Pianowski and coworkers report a series of photoswitchable cages assembled from the\nbisaldehydes substituted azobenzene and a trivalent amine linker. It describes the design, synthesis,\ncharacterization of these photoresponsive cages and their response to the stimuli of light, pH, and metal\nions. From my point of view, great improvement has been made in the revised manuscript: 1) most of the\nissues raised by the three previous reviewers have been carefully addressed. 2) some additional\nexperiments have been performed, including the synthesis of a novel stable covalent cage “13” by replacing\nthe corresponding fluorinated bis-aldehyde component with their corresponding chlorinated one. This new\ncage can undergo a reversible photoisomerization in water under a 660 nm light irradiation condition with\ngood biocompatibilities. Therefore, I would like to support the publication of this manuscript in Nature\nCommunications after further addressing some of the following issues.\n1) When addressing the 4th question raised by reviewer 1 (regarding the high selective thermal relaxation\nof ZZZ-3 to EEE-3 without any observable mixed ZZE or ZEE states), the authors attributed it to the much\nshorter lifetimes of (E,E,Z)- and (E,Z,Z)-3 in comparison with the corresponding (E,E,E)- and (Z,Z,Z)-3\nisomers. Since the formation of minor amounts of other isomers have been detected, I would suggest the\nauthors to perform additional theoretical calculations (energy of the intermediates and/or final products and\nthe favorite conformations, etc.) for a thorough explanation of the selectivity and stability difference of these\nisomers.\nS-1\nAd 1) we appreciate the reviewer’s insight into this issue. However, as we are a synthetic organic group\nwith some capacities in molecular biology, we cannot guarantee that our theoretical calculations are\nperformed correctly. The proper performance of that task would require additional collaboration or an effort\nthat exceeds the scope of the current manuscript. The Editor supported this point of view and notified us\nthat “additional theoretical calculations are not required at this time.”\n2) When addressing the 8th question raised by reviewer 1 (regarding the heating effect on the switching of\nthe isomers under a prolonged NIR irradiation condition, especially when considering that the absorbance\nof the chlorinated switch above 700 nm looks minimal), the authors used an aluminum-foil-wrapped sample\nas a control and irradiated under the same conditions as their main sample. In the tube covered with\naluminum foil, they did not observe any isomerization under the same irradiation parameters (time,\nfrequency of the used LED light emitter. It is highly suspicious to use an aluminum-foil-wrapped sample as\na control, since this aluminum foil might reflect the NIR light, resulting in the lack enough energy to heat the\nsample. Some additional experiments should be provided to eliminate the thermal effect on the switching.\nAd 2) We thank the reviewer for bringing potential NIR-reflection problems with our aluminum foil control\nsample to our attention. Thus we revised the original setup and used a black vial (absorbing visible and\nNIR light) as a control sample instead of one wrapped in aluminum foil during 730 nm irradiation\n(Figure S44A). We also monitored the temperature of a sample of 9 during 730 nm irradiation and we\nmonitored the isomeric ratio of 9 at 30 °C, 40 °C and 50 °C (Figure S44B). Since neither the sample in the\nblack vial, nor the samples at elevated temperatures formed any (Z)-isomer, we concluded that indeed,\n730 nm absorption is responsible for the observed (E)→(Z)-isomerization (see Figure S44 and respective\ndescription for a more detailed discussion).\n3) When addressing Question D raised by reviewer 2 and the 26th question raised by reviewer 3 (regarding\nthe binding constants for guest/metal interaction), the authors only provided some related literature and\ndeclared that they were not able to determine the binding constants for the cage “15” due to the limited\namount of the compound. I would recommend the absorption titration to ascertain the binding mechanism\nof metal ions complexes with guest molecules, since this absorption titration only needs small amount of\nsample to get the binding constants.\nAd 3) We would like to thank the reviewer for encouraging us to examine cage 15 and its interaction with\nFe2+ in more detail. We were able to isolate 15 in good yield (see added procedure in Synthetic procedures\nand characterization in the supporting info). Subsequently, 15 was characterized using 2D NMR\ntechniques (Figure S112–S114) and UV-Vis absorption spectroscopy (Figure S116). Its interaction with\nFe2+ was investigated using 1H NMR titration (Figure S115) revealing a low association constant (<105 M-1).\nAdditionally, absorption titration was performed (Figure S117), which could be used to determine\nassociation constants K and K for a 1 : 2 complex (15*2Fe2+). The program “BindFit” was then used to\na1 a2\ncalculate K = 806 (±13) M–1 and K = 1789 (±67) M–1 (see newly introduced references 44–45 for further\na1 a2\ninformation).\nS-2"}, "author_response": "In the name of all authors, we would like to thank all the reviewers for their time and attention in reading\nour manuscript. We are grateful for all the comments, also the critical ones, which motivated us to increase\nthe quality of our initial submission. The comments from reviewers 1-4 have been addressed in the previous\nrevision round. The comments of the reviewer 5 are individually addressed below.\nReviewer #1 (Remarks to the Author):\nThe authors have carefully and fully considered my comments. I appreciate their willingness to\nconstructively engage and I am pleased with the revisions made. I have no outstanding comments and I\ncongratulate the authors on a nice and thorough study. I fully support publication of this manuscript in\nNature Communications.\nReviewer #3 (Remarks to the Author):\nThe authors have addressed our comments and, therefore, we recommend publication.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the\nNature Communications initiative to facilitate training in peer review and to provide appropriate recognition\nfor Early Career Researchers who co-review manuscripts.\nReviewer #5 (Remarks to the Author):\nIn this manuscript, Pianowski and coworkers report a series of photoswitchable cages assembled from the\nbisaldehydes substituted azobenzene and a trivalent amine linker. It describes the design, synthesis,\ncharacterization of these photoresponsive cages and their response to the stimuli of light, pH, and metal\nions. From my point of view, great improvement has been made in the revised manuscript: 1) most of the\nissues raised by the three previous reviewers have been carefully addressed. 2) some additional\nexperiments have been performed, including the synthesis of a novel stable covalent cage “13” by replacing\nthe corresponding fluorinated bis-aldehyde component with their corresponding chlorinated one. This new\ncage can undergo a reversible photoisomerization in water under a 660 nm light irradiation condition with\ngood biocompatibilities. Therefore, I would like to support the publication of this manuscript in Nature\nCommunications after further addressing some of the following issues.\n1) When addressing the 4th question raised by reviewer 1 (regarding the high selective thermal relaxation\nof ZZZ-3 to EEE-3 without any observable mixed ZZE or ZEE states), the authors attributed it to the much\nshorter lifetimes of (E,E,Z)- and (E,Z,Z)-3 in comparison with the corresponding (E,E,E)- and (Z,Z,Z)-3\nisomers. Since the formation of minor amounts of other isomers have been detected, I would suggest the\nauthors to perform additional theoretical calculations (energy of the intermediates and/or final products and\nthe favorite conformations, etc.) for a thorough explanation of the selectivity and stability difference of these\nisomers.\nS-1\nAd 1) we appreciate the reviewer’s insight into this issue. However, as we are a synthetic organic group\nwith some capacities in molecular biology, we cannot guarantee that our theoretical calculations are\nperformed correctly. The proper performance of that task would require additional collaboration or an effort\nthat exceeds the scope of the current manuscript. The Editor supported this point of view and notified us\nthat “additional theoretical calculations are not required at this time.”\n2) When addressing the 8th question raised by reviewer 1 (regarding the heating effect on the switching of\nthe isomers under a prolonged NIR irradiation condition, especially when considering that the absorbance\nof the chlorinated switch above 700 nm looks minimal), the authors used an aluminum-foil-wrapped sample\nas a control and irradiated under the same conditions as their main sample. In the tube covered with\naluminum foil, they did not observe any isomerization under the same irradiation parameters (time,\nfrequency of the used LED light emitter. It is highly suspicious to use an aluminum-foil-wrapped sample as\na control, since this aluminum foil might reflect the NIR light, resulting in the lack enough energy to heat the\nsample. Some additional experiments should be provided to eliminate the thermal effect on the switching.\nAd 2) We thank the reviewer for bringing potential NIR-reflection problems with our aluminum foil control\nsample to our attention. Thus we revised the original setup and used a black vial (absorbing visible and\nNIR light) as a control sample instead of one wrapped in aluminum foil during 730 nm irradiation\n(Figure S44A). We also monitored the temperature of a sample of 9 during 730 nm irradiation and we\nmonitored the isomeric ratio of 9 at 30 °C, 40 °C and 50 °C (Figure S44B). Since neither the sample in the\nblack vial, nor the samples at elevated temperatures formed any (Z)-isomer, we concluded that indeed,\n730 nm absorption is responsible for the observed (E)→(Z)-isomerization (see Figure S44 and respective\ndescription for a more detailed discussion).\n3) When addressing Question D raised by reviewer 2 and the 26th question raised by reviewer 3 (regarding\nthe binding constants for guest/metal interaction), the authors only provided some related literature and\ndeclared that they were not able to determine the binding constants for the cage “15” due to the limited\namount of the compound. I would recommend the absorption titration to ascertain the binding mechanism\nof metal ions complexes with guest molecules, since this absorption titration only needs small amount of\nsample to get the binding constants.\nAd 3) We would like to thank the reviewer for encouraging us to examine cage 15 and its interaction with\nFe2+ in more detail. We were able to isolate 15 in good yield (see added procedure in Synthetic procedures\nand characterization in the supporting info). Subsequently, 15 was characterized using 2D NMR\ntechniques (Figure S112–S114) and UV-Vis absorption spectroscopy (Figure S116). Its interaction with\nFe2+ was investigated using 1H NMR titration (Figure S115) revealing a low association constant (<105 M-1).\nAdditionally, absorption titration was performed (Figure S117), which could be used to determine\nassociation constants K and K for a 1 : 2 complex (15*2Fe2+). The program “BindFit” was then used to\na1 a2\ncalculate K = 806 (±13) M–1 and K = 1789 (±67) M–1 (see newly introduced references 44–45 for further\na1 a2\ninformation).\nS-2"}, {"round": 3, "reviewer_comments": {"reviewer_5": "The authors have carefully addressed my comments. Therefore, I would like to support the publication of this manuscript in\nNature Communications."}, "author_response": ""}]

Decision letter