Photochromism of pyridine-substituted merocyanine through reversible C-N bond formation

Domain: chemistry
Published: 2025-12-26
Editorial outcome: minor_revision

Abstract
The reversible interconversion between spiropyran and merocyanine constitutes one of the most intensively investigated photochromic systems, enabling diverse applications in stimuli-responsive materials and optical imaging. Here, we report pyridine-substituted merocyanines (PMCs) as a class of photochromic molecules. Upon visible light irradiation, these compounds undergo an uncommon photoinduced C-N bond formation to yield spiroindolizine (SIZ) structures, accompanied by the color fading. The SIZs can thermally revert to the colored PMC form. This photochromism is marked by rapid response, high photo-switching ratio, robust water compatibility, and fatigue resistance. Furthermore, we have synthesized quinoline-substituted merocyanines and found that they can be bidirectionally switched by 660 nm and 500 nm light to form SIZ and PMC structures. Finally, we showed the photochromism of these molecules in solid matrices and demonstrated a light-controlled sequential switching function, highlighting their potential for advanced photonic applications.

Introduction
Photochromic molecules represent a versatile class of smart materials capable of reversibly altering color and physicochemical properties upon light exposure. Owing to their distinctive photo-responsive characteristics, they have been applied across various fields of chemical and biological research, including photo-patterning, activity modulation and fluorescence switching1–6. To date, significant progress in photochromic molecules research has led to the development of diverse organic molecular photoswitches, such as azobenzenes7,8, spiropyrans9,10, diarylethenes11, indigoids12, hydrazones13,14 and Stenhouse compounds15–17. They can be classified into two main categories according to their structural changes: Z-E isomerization (e.g., azobenzene, indigoid) and covalent bond formation/cleavage (e.g., spiropyran, diarylethene, Stenhouse compound). These systems exhibit distinct advantages and have been applied in various fields. For instance, azobenzenes possess simple and diverse chemical structures, making them widely used in supramolecular chemistry and light-controlled biological regulation7,8. Spiropyrans undergo more pronounced structural changes upon light stimulation, thereby leading to higher contrast on spectral and physicochemical properties10. In addition, the isomerization of spiropyrans can be regulated by pH and metal ions, enabling their use in the design of complicated molecular logic gates9. Diarylethenes, on the other hand, exhibit excellent fatigue resistance and thermal bistability, which makes them highly suitable for multiple switching cycles and particularly the applications in molecular electronics and photochromic devices11. However, these molecules still exhibit certain limitations, such as relying predominantly on ultraviolet (UV) light for photoisomerization, insufficient water solubility, or ineffective switching and poor fatigue resistance in aqueous media. Thus, the development of additional photochromic molecules continues to be of substantial scientific and practical significance. Ideally, the desirable photochromic molecules should feature facile synthesis, high switching ratio, responsiveness to visible light, robust fatigue resistance and broad-spectrum matrix (aqueous and solid) compatibility.

From the perspective of chemical transformations, spiropyrans undergo reversible C-O bond cleavage to form merocyanine whereas diarylethenes rely on reversible C-C bond transition in the photo-cyclization processes (Fig. 1A). Both systems have been extensively explored as prototypical molecular photoswitches. In contrast, photoinduced reversible C-N bond transformations remain exceedingly rare (Fig. 1B), and most of these examples have only demonstrated photochromic behavior in organic solutions18–25. The discovery of additional photoinduced C-N bond transformations can expand the structural diversity of photoswitches.

In this work, we report a distinct class of photochromic molecules featuring reversible C-N bond formation (Fig. 1C). These compounds adopt a pyridyl-substituted cyanine (PMC) scaffold, in which light irradiation induces the pyridyl nitrogen to attack the C2 atom of the indolium moiety, leading to a cyclization reaction and formation of a spiroindolizine (SIZ) structure, accompanied by a drastic spectral variation. This transformation exhibits fast kinetics, high switching ratio, excellent fatigue resistance and water compatibility. Furthermore, we also synthesized quinoline-substituted derivatives and characterized their photochromic behavior under two wavelengths. Finally, we demonstrated the photo-responsiveness of PMCs in various solid-state matrices and constructed a light-controlled sequential switch. This study expands the scope of molecular photoswitches and provides a molecular platform for diverse applications.

Results
Pyridine-substituted merocyanines
PMC1-5 were synthesized by coupling 2-formylpyridine analogues to an indolium moiety (Figure S2). Density functional theory (DFT) calculation showed that the amino-substitution significantly decreased the energy gap between HOMO and LUMO of PMC1-3 (~4.5–4.8 eV, Figure S3) compared to that of PMC4 and PMC5 (~5.5 eV). Time dependent DFT revealed distinct intramolecular charge transfer (ICT) features during excitation process. For PMC1-3, charge density difference (CDD) on the amino-substituted pyridine is around 43%, implying a significant electron depletion during excitation (S0 → S1). In contrast, CDDs on the pyridine moieties of PMC4 and PMC5 are only −5.9% and 1.4%, respectively (Figure S4). Therefore, the pyridine moieties can not act as the electronic donor in PMC4 and PMC5. Experimentally, PMC1-3 exhibited absorption peaks at 462-530 nm (Fig. 1D) and fluorescence at 566-607 nm; whereas both PMC4 and PMC5 having relatively weak charge transfer effects displayed shorter absorption and fluorescence wavelengths (Table 1 and Figures S5–S9).

Unlike imidazole-biradicals and heterocycle-substituted diarylethenes which only showed the photochromic property in organic solvent, PMCs displayed photochromic behaviors in water and rapidly faded upon irradiation with white LED (Fig. 1D), generating blue-shifted absorption peaks at 280-419 nm. This spectroscopic change was consistent with our envision that PMCs may undergo cyclization to form spiroindolizine structures under light irradiation. The photostationary state distributions (PSD) of the SIZ products were all above 95%, indicating almost completed transitions (Table 1). Photoisomerization quantum yields of PMCs were between 5.4% and 12% (Table 1), which is comparable to those of spiropyrans and other photochromic systems (Table S1).

In the absence of light stimulation, the spectra of SIZs gradually returned to those of PMCs in a temperature-dependent manner, from which we calculated the activation energy between 15–21 kcal/mol (Table 1). Spectral tests in organic solvents also supported PMCs as T-type (thermally reversible type) photochromism (Figures S5–S9). Notably, PMC1-3 exhibited almost complete thermal recovery in all solvents (Figures S5–S7). In contrast, PMC4 showed a weak absorption peak at 372 nm and a stronger peak at 280 nm, suggesting that it can spontaneously convert to SIZ4 in solutions (71% SIZ4 in CH3OH/H2O by HPLC; 47% SIZ4 in CD2Cl2 by 1H NMR assay, Figure S10). It was noted that PMC4 and PMC5 can only retrieve half of their initial absorbance in water in the first recovery process (Figures S8F and S9F), suggesting their relatively low stability in water (Figure S11). We attributed this to the side reaction of PMC4 and PMC5 without the electron-donating amino groups, because the lack of electron-donating groups (e.g., amino) usually leads to instability26,27.

As mentioned above, the simple molecular structure of PMCs endows their photochromism with good water compatibility, which may be advantageous for various applications in aqueous environments. Then, taking PMC2 as an example, we studied its photochromic property in water. As shown in Fig. 2, under the irritation of 500 nm LED, PMC2 converts to SIZ2 in less than 10 s with PSD of 99.4% and the photoisomerization quantum yield is 8.4%. Then, SIZ2 fully returns to PMC2 (98.9%) at room temperature after about 500 min (Fig. 2A–C). Temperature increase can accelerate this transformation from half-life of 250 min at 25 °C to 12.4 min at 50 °C, with the corresponding rate constants of 0.0076 min−1 and 0.056 min−1 (Fig. 2C). Although irradiation with short wavelengths (310 nm, 365 nm or 405 nm) can accelerate the SIZ-to-PMC conversion, the resulting photostationary state remains far from complete recovery (<10%) of PMC2 (Figure S12), which may be attributed to the simultaneous absorption of PMC and SIZ at the stimulating wavelength. Considering the structure differences between PMC and SIZ, we measured their partitions in octanol-water system. It was found that 98% of PMC2 existed in water phase (oil-water partition coefficient, log P ~ −1.75), further verifying the good water solubility of PMC2; after irritation, only 36% of SIZ2 existed in water phase (log P ~ 0.25), suggesting a typical photo-induced phase-transfer behavior (Fig. 2E).

Then, we compared the photochromic properties of PMC2 and the most intensively studied spiropyran 1,3,3-trimethylindoline-6-nitrobenzopyran (6-nitro-BIPS) in water. Fatigue resistance is a crucial property for photochromic molecules. We tested the fatigue resistance of 6-nitro-BIPS in water by repeated irradiations at 365 nm and 500 nm. It was found that 6-nitro-BIPS degraded greatly after several cycles (Figure S13), which may be ascribed to the involvement of the triplet state under the UV 365 nm excitation28,29. In contrast, PMC2 exhibited excellent reversibility and fatigue resistance (the absorbance at 517 nm retains above 95% after 10 cycles) in water under repeated irradiation and thermal recovery (Fig. 2D). The possible explanation may be that the photoisomerization of PMC2 is driven by 500 nm light, which possesses lower photon energy and therefore reduces the likelihood of photochemical degradation. Whether this photoreaction involves a triplet-state pathway remains to be clarified. Additionally, previous reports have shown that the ring-opening/closing switching of spiropyrans is strongly influenced by pH and the presence of metal ions30–32. However, the photoisomerization of our PMC2 molecule could proceed efficiently within the pH range of 4–10 (Fig. 2F, Figure S14) and was not affected by common metal ions (Figure S15), demonstrating its good resistance to environmental interferences. Even in more acidic media (pH < 4), PMC2 with a slightly blue-shifted absorption peak still exhibited efficient photoisomerization, though strongly basic media (pH > 10) largely decreased the photoisomerization. The above results indicated that PMC represents a robust photochromic system with various advantages over traditional spiropyran systems.

Photochromic mechanism
The structures of pyridine-substituted merocyanines (PMC1, PMC2, PMC3 and PMC5) were unambiguously determined to be a planar molecule through single-crystal X-ray diffraction (Figure S16). We attempted to prepare single crystals of the corresponding SIZs but were unsuccessful. On the other hand, we used DFT calculations to determine the geometries of the SIZ isomers (Figure S3). The optimized structures of SIZs reveal a C-N bond length of ~1.53 Å, which is slightly longer than a typical C-N bond20,23,24.

Further, we employed NMR spectroscopy to investigate the structural information on the PMC2-SIZ2 transformation (Figures S17 and S18). As shown in Fig. 3A, the proton signals at 8.28 ppm (H4, doublet, J = 15.3 Hz) and 7.72 ppm (H5, doublet, J = 15.3 Hz) confirm that the predominant species is the trans-alkenyl merocyanine. These protons shift to 7.35 ppm (H4, d, J = 6.5 Hz) and 6.85 ppm (H5, d, J = 6.4 Hz) upon irradiation, and are assigned as cis-alkenyl hydrogens, indicating the photoinduced trans-cis isomerization of the ethylene bond. Meanwhile, the chemical shift of the C2 (i.e., Cp in Figures S17 and S18) in the indole moiety shifts from 180.7 ppm to 111.4 ppm (Fig. 3B), indicating a transformation from an imine carbon to a saturated spiro-carbon (Cspiro). Notably, in SIZ2, the resonance peaks of indole 1-methylene protons (H10) broaden into very wide peaks (Fig. 3A), possibly suggesting the formation of a chiral center at the spiro carbon33. More definitive evidence is provided by the heteronuclear multiple bond correlation (HMBC) spectra, which typically detects 2J or 3J correlation. In 1H-13C HMBC spectra of SIZ (Fig. 3B), Cspiro displays a correlation with the H1 proton in pyridine. In 1H-15N HMBC spectra (Fig. 3C), a strong coupling appears between the Npyridine and H5 in SIZ2. The emergence of these two correlations strongly supports the formation of the Cspiro-N bond, enabling 3JHC and 3JHN correlations in SIZ.

Quinoline-substituted merocyanines
Encouraged by the above findings, we anticipated that quinoline-substituted merocyanine may undergo photochromic reaction under longer wavelength irradiation due to extended conjugation. Hence, we studied the photochromic behavior of two quinoline-substituted merocyanines (PMC6 and PMC7) theoretically and experimentally. DFT calculations reveal that PMC6 and PMC7 possess relatively low HOMO-LUMO gap (~4.1 eV) and exhibit pronounced excited-state charge transfer characteristics (CDD > 48%, Figure S19). Under 660 nm LED irradiation, the absorption peaks of the PMCs around 610 nm fade rapidly and weak peaks around 436 nm emerge, implying the generation of SIZ6 and SIZ7 (Fig. 4A, B). The PSD of SIZ6 and SIZ7 is 90.1% and 95.8% (Table 1), respectively. Unlike SIZ1-5 that can thermally return to PMCs, SIZ6 and SIZ7 show thermostability. No significant absorption peak of PMCs was observed even when SIZ6 and SIZ7 were kept in dark for 72 h (Fig. 4C, D). However, exposure to 365 nm, 500 nm and white LED light could retrieve PMC6 and PMC7 rapidly, indicating that quinoline-substituted merocyanines can function as a P-type photochromic system (Fig. 4C, D and Figures S20–23). Thus, here we can manipulate the molecular transition between PMC and SIZ forms using 500 nm and 660 nm light. Moreover, PMC6 and PMC7 exhibited fast response, >90% switching ratio and impressive fatigue resistance (Fig. 4E, F).

To elucidate the distinct photochromic behaviors of PMC2 and PMC6 (T-type and P-type, respectively), the reaction energy profiles associated with the PMC-SIZ isomerization were investigated using DFT calculations. The Gibbs free energies of PMCs are lower than those of their corresponding SIZs (Fig. 5 and Figures S24-S28), which is consistent with their negative photochromic character. The only exception is PMC4, which exhibits a higher Gibbs free energy than SIZ4, explaining its spontaneous conversion to SIZ4 as observed in chromatographic and NMR analyses (Figure S10). The photoisomerization process from PMC to SIZ can be simplified into two steps: the C=C bond trans-cis isomerization and the subsequent C-N bond formation. The former step involves a higher energy barrier and proceeds through an excited state to a cis-configured intermediate, whereas the latter has a relatively low barrier and can occur on the ground state (Fig. 5). In the reverse process, the isomerization begins with the cleavage of the C-N bond to yield a cis-configured intermediate, followed by cis-trans isomerization of the C=C bond to form the PMC structure. As illustrated in the reverse process in Fig. 5, the cis-trans isomerization step for SIZ2 proceeds with a relatively low energy barrier (~24.2 kcal/mol), allowing for efficient thermal recovery to PMC2 (Fig. 5A). In contrast, SIZ6 encounters a significantly higher energy barrier (~32.1 kcal/mol) during the cis-trans isomerization (Fig. 5B), which drastically slows the thermal relaxation rate, calculated to be ~105-fold slower than that of SIZ2. Consequently, the SIZ6-to-PMC6 transformation predominantly requires photochemical activation rather than occurs spontaneously via thermal recovery. Similarly, the energy barrier calculations also provided a good explanation for the isomerization mechanisms of PMC1, PMC3, and PMC7 (Figures S24, S25 and S28). However, for PMC4 and PMC5, the transition states of trans-cis isomerization were difficult to converge, suggesting that these compounds may follow an alternative reaction pathway, probably due to the absence of ICT effects in PMC4 and PMC5 (Figures S26 and S27).

Photochromism in solid matrices
The applicability of molecular photoswitches critically depends on their ability to undergo photochromic transitions in the solid matrices1,34,35. Photo-responsive materials are particularly important for applications in optoelectronic devices, data storage, and smart coatings. Accordingly, we investigated the photochromic behavior of PMC2 embedded in polymethyl methacrylate (PMMA) as well as absorbed in silica gel, and filter paper (Fig. 6A and Figure S29). Upon irradiation with 500 nm light, the red color of PMC2 faded, and the complete recovery of the red color was observed at room temperature within 2 h; the recovery process with an apparent activation energy of 13.2 kcal/mol (Figure S30) can further be accelerated by heating. This reversible color change could be repeated over many cycles without any noticeable degradation (Figure S31). Furthermore, PMC7 embedded in PMMA exhibited P-type photochromism modulated by 500 and 660 nm light, consistent with its behavior in solution (Fig. 6B and Figures S32 and S33).

Finally, we demonstrated the application of PMCs by constructing a light-controlled sequential switch. As shown in Fig. 6C, a PMMA sheet co-loaded with PMC2 and PMC7 (PMC2: 0.5‰, PMC7: 1‰; w/w) initially appears brown (PMC2-PMC7). Sequential irradiation first with 500 nm light and then with 660 nm light resulted in an orange color (SIZ2-SIZ7), which is attributed to successive conversion of PMC2 to SIZ2 and PMC7 to SIZ7. In contrast, reversing the order of irradiation (first with 660 nm followed by 500 nm) produced a dark purple color (SIZ2-PMC7) via an orange transition (PMC2-SIZ7), as 500 nm light not only converted PMC2 to SIZ2 but also reverted SIZ7 back to PMC7 (Fig. 6C). The same switching sequence was observed in solution (Figure S34), corroborating the photochromic pathway. This behavior constitutes a sequential switch, in which the sequence of two input signals dictates the system’s output36. Such a strategy could be extended to the development of more sophisticated photo-controlled materials.

Methods
Syntheses
For all syntheses, and characterization, please see Supplementary Information.

Preparation of single crystals
Single crystals for X-ray diffraction analysis were obtained by dissolving the solid in CH2Cl2, followed by dropwise addition of low-solubility solvents (ether, n-hexane, or ethyl acetate) to induce crystal formation via solvent diffusion. The mixture was left undisturbed at room temperature for 2-3 days to form single crystal.

Fluorescence quantum yield measurements
Fluorescence quantum yields were determined by the relative method41. PMC solutions were prepared at a series of concentrations (ensuring absorbance below 0.05). Both absorption and emission spectra were recorded in 1 cm quartz cuvettes. Parallelly, the corresponding reference fluorophores were measured: fluorescein (0.85 in 0.1 M NaOH) for PMC142, resorufin (0.74 in H2O) for PMC2 and PMC343, quinine sulfate (0.54 in 0.05 M H2SO4) for PMC4 and PMC544, and Cy5 (0.27 in pH 7.4 phosphate buffer) for PMC6 and PMC745. The integrated fluorescence intensity was plotted against the absorbance (I vs. A) linearly, and the slope was compared with that of the reference fluorophore. The relative fluorescence quantum yields were calculated by:

Φs = Φr (Ss/Sr) (ns2/nr2) (1)

where S is the slope of the I vs. A linear regression, Φ is the quantum yield, and n is the refractive index of the solvent. The subscripts s and r denote sample and reference respectively.

Photochromism measurements
Generally, 3 mL of PMC solution (10 μM) in a 1-cm quartz cell was irradiated with LED light for 10-30 s and the UV-vis absorption spectra were determined before and after irradiation.

Determination of photoisomerization quantum yield
Because of the wide distribution of the absorption spectra of PMC1-7, it is hard to use single photochromic standard to determine the relative quantum yield. Hence, the photoisomerization quantum yield was determined by combining kinetic analysis with photon flux calculation. A 3-mL of PMC solution was placed in 1×1 cm quartz cuvette and fully irradiated with an LED light and continuous stirring. The time-dependent absorption spectra were recorded and fitted with a single-exponential decay function to obtain the isomerization reaction rate constant kiso. The absorbed photon flux per unit area (Iabs) was calculated by differentiating the incident and transmitted photon fluxes determined using a power meter. The photoisomerization quantum yield (Φiso) was then calculated using the following equation:

Φiso = (kiso * C0 * V * NA) / (I0 * Aabs) (2)

where C0 is the initial concentration, V is the solution volume, NA is Avogadro’s constant, and Aabs is the illuminated area.

Thermal recovery kinetics
The thermal recovery process was carried out by keeping the SIZ solution either under normal laboratory lighting or in a dark enclosure. During the study, no observable effect was detected for the normal laboratory lighting compared with dark condition. The absorbance evolution of the thermal recovery was analyzed using a first-order kinetic model:

At = A0 (1 - e-kt) (3)

where A0 is the initial absorbance, k is the rate constant, and t is elapsed time.

Determination of apparent activation energy
The apparent activation energy was determined using the Arrhenius equation. Sample solutions were kept in dark at different temperatures (25, 37 and 50 °C), and the absorption spectra were recorded at defined time intervals. The time-dependent spectral changes were fitted to obtain the rate constant k at each temperature. A plot of ln k vs. 1/T was linearly fitted, from which the apparent activation energy (Ea) was derived by:

ln k = ln A - (Ea / RT) (4)

where A is a pre-exponential factor, R is the gas constant and T is the absolute temperature.

Density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations
All calculations were carried out using Gaussian 16 (Rev. C.01) at the M06-2X/def2-TZVP level46,47. Solvent effect (CH2Cl2) was included using the solvent model based on density. In the vertical transition calculations, we calculated 10 excited states. During the excited state structure optimization, the lowest three excited states were computed. Frequency analyses confirmed that all optimized ground- and excited-state minima exhibited no imaginary frequencies. Transition states (TS1 and TS2) were optimized using opt(ts, calcfc, noeigentest) and verified by a single imaginary frequency together with intrinsic reaction coordinate calculations. Charge-density difference (CDD) analyses were performed with Multiwfn48,49.

Solid matrix preparation
Silica gel plates and filter papers were immersed in 100 μM PMC2 solution of acetonitrile for 10 min, and then dried to afford the functionalized materials.

PMMA (500 mg) was dissolved in CHCl3 (5 mL) under continuous stirring at 50 °C for 30 min. PMC2 stock solution was then added dropwise to the PMMA solution to achieve a PMC2:PMMA mass ratio of 1:1000 (w/w). The mixture was subsequently sonicated for 30 min to ensure homogeneous dispersion. Then the solution was cast into a glass beaker, and allowed to evaporate in oven at 50 °C for 48 h. Finally, the dried polymer film was further annealed at 40 °C under vacuum for 24 h to remove any residual solvents.

Preparation of the composite film: PMMA (500 mg) was dissolved in CHCl3 (5 mL) under continuous stirring at 50 °C for 30 min. Subsequently, PMC2 and PMC7 stock solutions were added dropwise into the PMMA solution to achieve a mass ratio of PMC2:PMC7:PMMA = 0.5:1:1000 (w/w). The resulting mixture was then sonicated for 30 min to ensure homogeneous dispersion. The solution was poured into a glass beaker and allowed to evaporate in an oven at 50 °C for 48 h. Finally, the dried polymer film was annealed at 40 °C under vacuum for 24 h to remove any residual solvents.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript presents the synthesis and characterizations of a new family of photochromic molecules, the pyridine-substituted merocyanine dyes, inspired from the spiropyran photochromic molecules where the color change is due to a bond formation/breaking. This manuscript is well written and interesting to read. This new molecular family is interesting however, I have the feeling that this work is on some aspects superficial and very phenomenological and do not provide a large range of characterizations that could help to figure out the strength and weaknesses of the system.\nHere is a list of more detailed remarks:\n1- The author should perform a photochemical investigation to determine the quantum efficiency of the photo-isomerization and ideally, compare the obtained values to other photochromic systems.\n2- There is no information about the fluorescence of the molecules (emission wavelength and fluorescence efficiency).\n3- In term of quantum chemistry, the investigation of the nature of the transitions (orbitals involved or better the variation of the electron density) must be done as long as the characterization of the nature of the transitions (using one the charge transfer index available in the literature or implemented on the codes).\n4- The reaction path of the isomerization must be computed for all systems both at the ground state and at the excited state. The molecules are not too large for this work.\n5- Surprisingly, the authors determine the kinetic of the return reaction SIZ -> PCM as a function of the temperature but do not provide the activation energy. I did the calculation myself with the data of the manuscript and found an activation energy ~15 kcal/mol. It is a bit weaker than the one obtained by DFT (~24 kcal/mol) but still acceptable since DFT is not very good to simulate double bound rotations. Actually, the authors should determine the activation energy for this reaction for all compounds and compare it to DFT values. This is a very important parameter. The authors can also determine the activation energy in a matrix such as PMMA to see how it affects the reactivity.\nHere is a list of minor remarks:\n1- In the introduction, the author says that “An ideal photochromic molecule should feature…pronounced structural change”. I do not agree. It depends on the application and on the matrix. If the change is very large, the photochromic effect can be very sensitive to the environment and not wanted.\n2- Figure 1, the notation N(CH2)4 for PMC3 is not clear.\n3- The expression “The optimized structures of SIZs reveal the formation of a new C-N bond” is not adapted. DFT calculations do not predict a bond formation since the authors built the starting geometries with this bond. The authors should say “The optimized structures of SIZs reveal a C-N bond length of…”", "reviewer_2": "The study by Shi and colleagues presents a well-executed investigation into a novel class of pyridine-substituted merocyanines, which differ mechanistically from conventional systems in that they undergo photoinduced C-N bond formation. This characteristic switching mechanism yields several advantageous properties, including a high photoswitching ratio, excellent water compatibility and remarkable photofatigue resistance. The authors further demonstrate the integration of these photoswitches into solid-state matrices while maintaining reversible functionality. The dual-wavelength switching and light-sequence-dependent optical logic highlight the application potential in photonic materials. While still at a proof-of-concept stage, the work expands the design space for molecular photoswitches and opens avenues for future development in optically responsive materials.\nIn general, the study appears well-rounded and technically sound within the methods applied. However, several important questions remain unaddressed, particularly regarding the positioning and impact of the work within the broader field of molecular photoswitches. A key weakness is the lack of contextual relevance to the broader photoswitching community. As written, the manuscript does not clearly convey how this class of photochromic systems outperforms or complements existing merocyanines or other photochromes. The authors should substantially strengthen the discussion of relevance and novelty, for example, by highlighting specific application domains (e.g., aqueous switching, logic devices) where classical merocyanines fall short. Moreover, a deeper comparison with existing merocyanine literature, including switching mechanisms, fatigue behavior, and environmental responsiveness, is largely missing. The current manuscript would benefit from integrating a more comprehensive literature context to better define the contribution. These general remarks concern the overall framing of the paper.\nSpecific comments:\n1) The spectral data presented for PMC2 in Figure S3 are noticeably less complete than for other PMCs shown in Figures S2 and S4. The authors should either include the missing data or explicitly justify why they are absent. Consistency across compound characterization is important for comparative interpretation.\n2) Table 1 reports relatively low thermal recovery yields for PMC4 and PMC5. While the authors briefly mention the reduced nucleophilicity of the pyridine nitrogen (especially for PMC5), this explanation remains superficial. Could spectral overlap, incomplete thermal recovery, or side reactions also play a role? Was recovery tested only thermally, or was photochemical back-conversion attempted? This aspect unfortunately remains unclear from the table and discussion.\n3) The authors claim that pH and metal ions do not affect the isomerization. However, Figure 2F shows notable changes in absorbance intensity of the PMC form across the pH range. While the SIZ appears stable, the changes in the PMC absorption suggest pH-dependent shift, protonation, or aggregation. These effects are not addressed. Also, reporting only the absorbance at 517 nm limits interpretability as shifts can’t be detected at all. Complete UV–vis spectra across the different pH/salt conditions would provide a clearer picture of environmental robustness.\n4) The claimed chemical shift for C2 in the indole unit (from 180.7 to 111.4 ppm) is difficult to verify in Figure 3B, as the scaling is with only one given number unclear, and the axis range is inconsistent between before and after spectra. The authors should replot these spectra with matched scaling and more informative axis labeling.\n5) The manuscript shows improved fatigue resistance over classical merocyanines, but provides no mechanistic explanation. The authors should discuss why their new PMC/SIZ system performs better. For example, could the C–N bond switching mechanism avoid triplet-state involvement, making it less susceptible to oxygen quenching? Are the SIZ states less prone to acid/base catalyzed degradation compared to classical MC forms? A brief discussion of potential photophysical or structural reasons for this enhanced stability would strengthen the impact of this result.\nThis is an interesting and promising study. However, the authors need to significantly improve the contextual discussion, especially the classification of their work within the broader field of photochromism and provide clearer and more complete data in several indicated places. The mechanistic interpretation, especially with regard to the observed improvements in photofatigue resistance, also requires more in-depth consideration. A follow-up mechanistic study, potentially involving transient absorption spectroscopy, would be highly valuable to better understand how these new systems differ from classical merocyanines in their excited-state behavior. Overall, I recommend extensive revisions to address these issues. The core of the work is strong and promising. If the authors address the points mentioned thoroughly and carefully, the manuscript could reach the level expected for a publication in Nature Communications.", "reviewer_3": "The authors report on a novel class of molecular photoswitches (Pyridine-Substituted Merocyanine, referred to as PMC). This class of compounds share structural similarities with the well-known spiropyran/merocyanine photoswitch. PMC, however, lacks the phenolate oxygen, preventing ring-closing to yield the O-C spirocyclic bond. Instead, PMC:s are equipped with a pyridine, so that the ring-closing reaction yields a C-N bond. This isomer is referred to as the SIZ form. T-type negative photochromism is observed.\nWhen pyridine is replaced with a quinoline, absorption is redshifted and the thermal isomerization reactions are dramatically slowed down. This, this class display P-type photochromism.\nThe photoconversion is efficient (at least 90% conversion to the SIZ form) and the thermal recovery as well (near quantitative thermal isomerization to PMC, at least for PMC1-3). Photostability is OK. The ability to use all-visible light is highly appealing.\nWhat is most appealing (at least to this reviewer) with this class of compounds (the T-type negative ones) is that they can be used in aqueous solution. As opposed to the previously reported most common classes DASA and the phenyxyl/imidazolyl radical complexes reported by Abe, the PMC:s maintain the photochromic behaviour in water. This is extremely useful for biological applications. The authors also showed compatibility with solid matrices. The material presented is well suited for publication in Nat. Commun. Before this can happen, the authors should address the following points.\n1) Isomerization quantum yields should be reported. I’m not saying this to be mean, but instead I believe that when reporting a novel class on photoswitches that will be very useful, these numbers must be presented.\n2) I’m not sure that I agree with the following statement:\n“Photochromic mechanisms are primarily divided into two categories: Z-E isomerization (e.g., azobenzene, indigoid) and covalent bond formation/cleavage (e.g., spiropyran, diarylethene, Stenhouse compound). The latter usually involves more pronounced structure change upon light stimulation…”\nCis-trans isomerizations give rise to structural changes that are often more pronounced compared to DAE:s etc.\n3) In Figure 1D, is the residual absorption between 500 nm and 600 nm after light exposure due to absorption from the PMC isomers? I guess so. Alternatively, it could be due to weak transitions for the SIZ form. I’m a bit surprised by the residual absorption observed, in particular if SIZ does not absorb light between 500 nm and 600 nm. The thermal back-isomerizations are so slow that it should be possible to photoisomerize everything to the SIZ form (irradiation for 10 s, whereas the thermal isomerizations are much slower). This matter is also accentuated in the following sentence:\n“The photostationary state distributions (PSD) of the SIZ products were all above 95%, indicating almost completed transitions (Table 1)…”.\nThe authors should explain better why they do not observe 100% photoconversion to the SIZ form.\n4) In Table 1, the authors put numbers on the “Recovery”. I believe that “Recovery” is no good, as it signals that these numbers are only observed after photoisomerization and subsequent thermal back-isomerization to yield PMC. “Thermal equilibrium position” sounds better, as this equilibrium position should not be dependent on if the compounds was exposed to light prior to the equilibrium establishment or not.\n5) In Table 1, I suggest the inclusion of also the thermal half-lives of the SIZ isomer. This facilitates for the reader to get a grip on how fast this process is without a calculator.\n6) Last, but not least, I must comment on DG (delta G) in Table 1. These numbers do NOT agree with the experimentally determined thermal equilibrium positions (reported as “Recovery”). From the “Recovery”, the authors can easily determine the equilibrium constant for the thermal equilibrium, K. With K known, DG is easily determined. 50% Recovery would imply DG=0 (in the absence of degeneracy). This is NOT aligned with the numbers for PMC5. I suggest that the authors calculate DG from the “Recovery”-values and compare these values to the theoretically (computationally) determined dittos. Comment on any discrepancies."}, "author_response": "Responses to reviewers’ comments\nDear Reviewers,\nOn behalf of the authorship group, I would like to thank you for taking the time to evaluate\nour manuscript entitled “Photochromism of Pyridine-Substituted Merocyanine through\nReversible C-N Bond Formation”. We are deeply grateful for your appreciation on our\nwork. Your valuable comments enabled us to greatly improve our manuscript. For your\nconvenience, all the revisions made in our revised manuscript are highlighted in blue, and\ntheir necessary explanations with point-by-point responses are listed as follows:\nTo Reviewer 1:\nThis manuscript presents the synthesis and characterizations of a new family of\nphotochromic molecules, the pyridine-substituted merocyanine dyes, inspired from the\nspiropyran photochromic molecules where the color change is due to a bond\nformation/breaking. This manuscript is well written and interesting to read. This new\nmolecular family is interesting. However, I have the feeling that this work is on some\naspects superficial and very phenomenological and do not provide a large range of\ncharacterizations that could help to figure out the strength and weaknesses of the system.\nHere is a list of more detailed remarks:\n1. The author should perform a photochemical investigation to determine the quantum\nefficiency of the photo-isomerization and ideally, compare the obtained values to other\nphotochromic systems.\nResponse: We appreciate your valuable suggestion. According to this constructive advice,\nwe have determined the photoisomerization quantum yields of PMCs (the determination\nmethod is given in the Methods section, page 13). The results indicated that PMC1-5\ndisplayed the photoisomerization quantum yields between 5.4%-12% in water, which is\ncomparable to the quantum yields of classical spiropyran and other photochromic systems.\nThese results have been added and described in the text (see paragraph 2, page 4) and\nTable 1. For comparison, photoisomerization quantum yields of some typical\nphotochromic compounds are also listed in Table S1 in Supplementary Information.\n2. There is no information about the fluorescence of the molecules (emission\nwavelength and fluorescence efficiency).\nResponse: Following your kind suggestion, we have measured the emission spectra and\nfluorescence quantum yields of PMCs, which have been added to Figures S4-S8, Figure\nS19, Figure S21, and Table 1. The relatively low fluorescence quantum yields might be\nattributed to increased non-radiative deactivation channels, for instance, photochemical\nprocesses and internal conversion during light irradiation.\n1\n3. In term of quantum chemistry, the investigation of the nature of the transitions\n(orbitals involved or better the variation of the electron density) must be done as long as\nthe characterization of the nature of the transitions (using one the charge transfer index\navailable in the literature or implemented on the codes).\nResponse: According to your valuable suggestion, we have calculated the molecular\norbitals of the PMCs in ground state and excited state. PMC1-3, 6 and 7 displayed a\nsignificantly lower energy gap between HOMO-LUMO compared to PMC4 and PMC5,\nprobably due to the conjugation effect of the nitrogen substitution. Then, we calculated\nthe charge density difference for PMC1-PMC5 during the S →S transition to\n0 1\ncharacterize the electronic transfer effect. The results indicated that the pyridine moieties\nof PMC1-3 showed significant electron depletion (~43%); however, the pyridine moiety\nof PMC4 showed small and negative charge density difference (-5.9%, electron\naccumulation on pyridine) and that of PMC5 displayed small charge density difference\n(1.4%) during the excitation, which accounts for the pronounced charge transfer\ndifferences between PMC1-3 and PMC4-5. According to the kind advice of the reviewer,\nwe have included these results and discussions in the manuscript (the bottom on page 3;\nsee also Figures S2 and S3, the numbers of other figures are adjusted accordingly).\n4. The reaction path of the isomerization must be computed for all systems both at the\nground state and at the excited state. The molecules are not too large for this work.\nResponse: Thank you for your constructive suggestion. According to the isomerization\npathway of classical spiropyrans, the reaction path of PMC may also undergo two key\nsteps: trans-cis isomerization and C-N bond formation/cleavage, and the trans-cis\nisomerization step should involve the excited state. Here, we calculated the first excited\nstate (S ) of PMCs, which then underwent trans-cis isomerization to the intermediate\n1\nthrough a speculated transition state (S ’ in Figure 5). The calculated pathways can be\n1\nconverged in PMC1-3, 6 and 7, and their reverse process. However, after multiple\nattempts, we failed in converging the transition states of the trans-cis isomerization of\nPMC4 and PMC5. The possible reason is that PMC4 and PMC5 do not exhibit obvious\ncharge transfer effects in the transition process (vide supra), which might significantly\nchange the reaction path in excited state. We have added this discussion in the text\n(paragraph 2 on page 9), and the computational results of all compounds are displayed in\nFigure 5 and Figures S23-S27.\n5. Surprisingly, the authors determine the kinetic of the return reaction SIZ -> PCM as\na function of the temperature but do not provide the activation energy. I did the calculation\nmyself with the data of the manuscript and found an activation energy ~15 kcal/mol. It is\na bit weaker than the one obtained by DFT (~24 kcal/mol) but still acceptable since DFT\nis not very good to simulate double bound rotations. Actually, the authors should\ndetermine the activation energy for this reaction for all compounds and compare it to DFT\n2\nvalues. This is a very important parameter. The authors can also determine the activation\nenergy in a matrix such as PMMA to see how it affects the reactivity.\nResponse: Following this helpful suggestion, we have determined the apparent activation\nenergy of thermal conversion of SIZ-to-PMC in both water and PMMA. As shown in\nTable 1, the apparent activation energies of PMC1-3 are 20.4, 17.9 and 16.4 kcal/mol,\nrespectively. These values are roughly consistent with the activation barriers obtained\nfrom DFT calculations (27.2, 24.2 and 23.6 kcal mol-1). Additionally, the apparent\nactivation energy of PMC2 in PMMA is 13.2 kcal/mol, which supports the faster thermal\nrecovery in PMMA. These descriptions have been added to the text (the bottoms on page\n4 and page 10), and the related data have been provided in Table 1 and Figure S29.\nHere is a list of minor remarks:\n6. In the introduction, the author says that “An ideal photochromic molecule should\nfeature…pronounced structural change”. I do not agree. It depends on the application and\non the matrix. If the change is very large, the photochromic effect can be very sensitive\nto the environment and not wanted.\nResponse: We fully agree with the reviewer’s opinion, and have deleted “pronounced\nstructural change” in the last sentence of paragraph 1 on page 2.\n7. Figure 1, the notation N(CH2)4 for PMC3 is not clear.\nResponse: Thank you for your helpful comment. Now we have re-drawn the specific\nstructure of PMC3 in Figure 1 for clarity.\n8. The expression “The optimized structures of SIZs reveal the formation of a new C-\nN bond” is not adapted. DFT calculations do not predict a bond formation since the\nauthors built the starting geometries with this bond. The authors should say “The\noptimized structures of SIZs reveal a CN bond length of…”\nResponse: Thank you for pointing out this mistake. We have used the description\nsuggested by the reviewer: “The optimized structures of SIZs reveal a C-N bond length\nof ~1.53 Å”. See the last sentence on page 6.\nReviewer #2 (Remarks to the Author):\nThe study by Shi and colleagues presents a well-executed investigation into a novel class\nof pyridine-substituted merocyanines, which differ mechanistically from conventional\nsystems in that they undergo photoinduced C-N bond formation. This characteristic\nswitching mechanism yields several advantageous properties, including a high\nphotoswitching ratio, excellent water compatibility and remarkable photofatigue\nresistance. The authors further demonstrate the integration of these photoswitches into\nsolid-state matrices while maintaining reversible functionality. The dual-wavelength\n3\nswitching and light-sequence-dependent optical logic highlight the application potential\nin photonic materials. While still at a proof-of-concept stage, the work expands the design\nspace for molecular photoswitches and opens avenues for future development in optically\nresponsive materials.\nResponse: We thank the reviewer for the positive and encouraging evaluation of our\nmanuscript and for recognizing the novelty and several advantageous properties of our\nphotoswitches.\nIn general, the study appears well-rounded and technically sound within the methods\napplied. However, several important questions remain unaddressed, particularly\nregarding the positioning and impact of the work within the broader field of molecular\nphotoswitches. A key weakness is the lack of contextual relevance to the broader\nphotoswitching community. As written, the manuscript does not clearly convey how this\nclass of photochromic systems outperforms or complements existing merocyanines or\nother photochromes. The authors should substantially strengthen the discussion of\nrelevance and novelty, for example, by highlighting specific application domains (e.g.,\naqueous switching, logic devices) where classical merocyanines fall short. Moreover, a\ndeeper comparison with existing merocyanine literature, including switching mechanisms,\nfatigue behavior, and environmental responsiveness, is largely missing. The current\nmanuscript would benefit from integrating a more comprehensive literature context to\nbetter define the contribution. These general remarks concern the overall framing of the\npaper.\nResponse: We sincerely thank the reviewer for the constructive comments. Following the\nkind suggestions, the contextual relevance of our study within the broader photoswitching\ncommunity has been strengthened. Specifically, we have substantially revised the\nIntroduction section (page 2), and added a separate Discussion section (discussing the\nnovelty of the C-N transformation, the substituent effect, advantage over spiropyran, and\napplication perspective of PMCs; page 11) in the revised manuscript. Moreover, we have\nprovided Table S1 in the Supplementary Information for a deeper comparison with\nexisting photochromic systems (especially classical spiropyran/merocyanines and\nemerging photoswitches), showing the overall advantageous performance of PMCs.\nSpecific comments:\n1. The spectral data presented for PMC2 in Figure S3 are noticeably less complete than\nfor other PMCs shown in Figures S2 and S4. The authors should either include the\nmissing data or explicitly justify why they are absent. Consistency across compound\ncharacterization is important for comparative interpretation.\nResponse: Thank you for pointing out this issue. The mentioned missing data (including\nabsorption spectra change, kinetic curve and thermal recovery curve of PMC2 in water)\nin original Figure S3 had been already shown in Figure 2 (in the main text) as a\n4\nrepresentative PMC. To avoid redundancy, these data are not shown again in the original\nFigure S3 (now Figure S5).\n2. Table 1 reports relatively low thermal recovery yields for PMC4 and PMC5. While\nthe authors briefly mention the reduced nucleophilicity of the pyridine nitrogen\n(especially for PMC5), this explanation remains superficial. Could spectral overlap,\nincomplete thermal recovery, or side reactions also play a role? Was recovery tested only\nthermally, or was photochemical back-conversion attempted? This aspect unfortunately\nremains unclear from the table and discussion.\nResponse: Thank you for your constructive comment. As suggested, we have tested the\nreversibility of PMC4 and PMC5 in water and found that both PMC4 and PMC5\ndisplayed poor reversibility, implying their poor stability (see Figure R1 below). It is\nknown that increasing electron density on double bond by introducing an electron-\ndonating group (e.g., amino) would improve the stability whereas an electron-withdraw\ngroup would decrease the stability. Thus, we attributed the low thermal recovery yields\nfor PMC4 and PMC5 without the electron-donating amino groups to the side reaction.\nAccording to the kind advice of the reviewer, these results and discussions have been\nadded to the text (the top on page 5) and the Supplementary Information (Figure S10; the\nnumbers of other figures are adjusted accordingly).\nA B\nC D\nm\nn\n40.1\n8 3\nta\n.s\nb\nA\n0.0\n0 1 2\nCycle number\nFigure R1. Multiple recovery tests of PMC4 and PMC5. (A) Absorption spectral change\nof PMC4 in two recovery processes. (B) Absorbance change of PMC4 at 372 nm during\nthe repeated irradiation and thermal recovery. (C) Absorption spectral change of PMC5\nin two recovery processes. (D) Absorbance change of PMC5 at 384 nm during the\nrepeated irradiation and thermal recovery.\n5\necnabrosbA\n0\n0\n0\n0\n.0\n.0\n.0\n.0\n9\n6\n3\n0\n3 0 0W\na v e le\n4n\nin\n1 s\n2 n\n5 0\ng th\nitia l\nt re c o v e ry\nd re c o v e ry\n6 0 0\n( n m )\nmn\n273\nta\n.sbA\n0\n0\n0\n.1\n.0\n.0\n0\n5\n0\n1\nn u m b e r\n2\n0.2 initial\n1st recovery\ne 2nd recovery\nc\nn\na b0.1\nr\no\ns\nb\nA\n0.0\n300 400 500 600\nWavelength (nm)\nAdditionally, we also tested if irradiation at 310 nm could increase the recovery yield\nof PMC4 and PMC5 (Figure R2 below). The results showed that this operation cannot\nimprove the recovery yield of PMC4, but indeed increase that of PMC5 (recovery yield\nfrom ~50% to 90%). The possible reason for this difference may be that these two\nmolecules exhibited different photostationary states at 310 nm. The result is not included\nin the manuscript because our research focused on the amino-substituted PMCs (PMC1-\n3,6,7) with better photochromic properties.\nFigure R2. Time-dependent absorption spectra (A) and absorbance change at 372 nm (B)\nof SIZ4 in water upon 310 nm irradiation. Time-dependent absorption spectra (C) and\nabsorbance change at 384 nm (D) of SIZ5 in water upon 310 nm irradiation.\n3. The authors claim that pH and metal ions do not affect the isomerization. However,\nFigure 2F shows notable changes in absorbance intensity of the PMC form across the pH\nrange. While the SIZ appears stable, the changes in the PMC absorption suggest pH-\ndependent shift, protonation, or aggregation. These effects are not addressed. Also,\nreporting only the absorbance at 517 nm limits interpretability as shifts can’t be detected\nat all. Complete UV–vis spectra across the different pH/salt conditions would provide a\nclearer picture of environmental robustness.\nResponse: We thank the reviewer for this valuable comment, which helped us to more\naccurately clarify the pH effect. Now we have specified the pH effect as “the\nphotoisomerization of our PMC2 molecule could proceed efficiently within the pH range\nof 4-10 (Figure 2F, Figure S13)” (paragraph 1, page 6). Following the kind suggestion of\nthe reviewer, we have also provided the complete absorption spectra of PMC2 under\ndifferent interference conditions (Figure S13 for pH and Figure S14 for metal ions), which\necnabrosbA\nC\necnabrosbA\n0\n0\n0\n0\n0\n0\n0\n.1\n.0\n.0\n.1\n.1\n.0\n.0\n0\n5\n0\n5\n0\n5\n0\n3 0W\n3 0W\n0a\n0a\nv\nv\ne\ne\n4 0 0\nle n g\n4 0 0\nle n g\nth\nth\n4 s\n0\n5 0 0\n( n m\n4 s\n0\n5 0 0\n( n m\n)\n)\n6\n6\n0\n0\n0\nD\nmn\n273\nta\n.sbA\nmn\n483\nta\n.sbA\n0\n0\n0\n0\n0\n0\n0\n0\n.1\n.1\n.0\n.0\n.1\n.1\n.0\n.0\n5\n0\n5\n0\n5\n0\n5\n0\n0\n0\n1\nT\n2\nim\n2\nim\ne\ne\n(s\n(s\n3)\n3)\n4\n4\nindicated that the photoisomerization of PMC2 was not significantly affected by pH in\nthe pH range of 4-10 and common metal ions, demonstrating its good resistance to\nenvironmental interferences. Even in more acidic media (pH < 4), PMC2 with a slightly\nblue-shifted absorption peak still exhibited remarkable photoisomerization, though\nextremely basic media (pH > 10) largely decreased the photoisomerization. These results\nhave been added to the text (paragraph 1, page 6) and the Supplementary Information\n(Figures S13 and S14).\n4. The claimed chemical shift for C2 in the indole unit (from 180.7 to 111.4 ppm) is\ndifficult to verify in Figure 3B, as the scaling is with only one given number unclear, and\nthe axis range is inconsistent between before and after spectra. The authors should replot\nthese spectra with matched scaling and more informative axis labeling.\nResponse: Thank you for pointing out this issue. According to your helpful advice, we\nhave replotted the spectra with more axis labels for clarity (see the revised Figure 3B).\n5. The manuscript shows improved fatigue resistance over classical merocyanines, but\nprovides no mechanistic explanation. The authors should discuss why their new PMC/SIZ\nsystem performs better. For example, could the C-N bond switching mechanism avoid\ntriplet-state involvement, making it less susceptible to oxygen quenching? Are the SIZ\nstates less prone to acid/base catalyzed degradation compared to classical MC forms? A\nbrief discussion of potential photophysical or structural reasons for this enhanced stability\nwould strengthen the impact of this result.\nResponse: Thank you for the constructive comments. We agree with the reviewer’s\nopinions that the fatigue resistance may be related to both photochemistry and base\ncatalyzed degradation. Specifically, the isomerization of classical spiropyrans is usually\ndriven by short wavelength light of 365 nm, whose high energy may lead to significant\nphotobleaching through triplet state (the newly added ref. 29). On the other hand, it has\nbeen reported that the base catalyzed degradation of spiropyrans may proceed through\nnucleophilic reaction on the double bond (ref. 52). Furthermore, it is known that the\nintroduction of an electron-donating group can increase the electron density on the double\nbond, thereby enhancing stability (ref. 26); in contrast, introducing an electron-\nwithdrawing group (e.g., nitro) can decrease the stability against base (ref. 27). Compared\nto the classical spiropyran, our PMC2 molecule contains an electron-donating amino\ngroup and uses longer wavelength light of 500 nm for photoisomerization, therefore it\nshows improved fatigue resistance. This discussion has been added to the text (the bottom\non page 5 and the Discussion section). Additionally, we have added the corresponding\nreferences (refs, 26, 27, 29 and 52) on the stability of merocyanine/spiropyran.\n6. This is an interesting and promising study. However, the authors need to significantly\nimprove the contextual discussion, especially the classification of their work within the\n7\nbroader field of photochromism and provide clearer and more complete data in several\nindicated places. The mechanistic interpretation, especially with regard to the observed\nimprovements in photofatigue resistance, also requires more in-depth consideration. A\nfollow-up mechanistic study, potentially involving transient absorption spectroscopy,\nwould be highly valuable to better understand how these new systems differ from classical\nmerocyanines in their excited-state behavior. Overall, I recommend extensive revisions\nto address these issues. The core of the work is strong and promising. If the authors\naddress the points mentioned thoroughly and carefully, the manuscript could reach the\nlevel expected for publication in Nature Communications.\nResponse: We thank the reviewer for encouraging assessment and constructive\nsuggestions. In the revised manuscript, we have substantially improved the contextual\ndiscussion by placing the PMC/SIZ system within the broader field of photochromism,\nincluding comparison with spiropyrans, diarylethenes, and some emerging\nphotochromism systems (Table S1), and added a separate Discussion section (page 11)\non the property and perspective of PMCs. We have also provided more mechanistic\ndiscussion and interpretation on the photo-fatigue resistance (page 5). We believe that\nthese revisions address the reviewer’s concerns and significantly strengthen the\nmanuscript.\nAdditionally, we agree that a follow-up mechanistic study, such as transient\nabsorption spectroscopy, would provide more valuable information on the excited-state\nbehavior of the PMC system. However, the PMC/SIZ system is a negative photochromic\nsystem, in which the detection wavelength (400-800 nm) overlaps with the responsive\nwavelength (white light or 500 nm). Hence, a tailor-made hardware is needed to\nhomogenize the sample solution before pump pulse, which is not available for us at\npresent. If these conditions are available in the future, we plan to carry out this experiment.\nIn response to this helpful comment of the reviewer, we have added a necessary note\n“Whether this photoreaction involves a triplet-state pathway remains to be clarified” in\nthe text (the bottom on page 5).\nReviewer #3 (Remarks to the Author):\nThe authors report on a novel class of molecular photoswitches (Pyridine-Substituted\nMerocyanine, referred to as PMC). This class of compounds share structural similarities\nwith the well-known spiropyran/merocyanine photoswitch. PMC, however, lacks the\nphenolate oxygen, preventing ring-closing to yield the O-C spirocyclic bond. Instead,\nPMCs are equipped with a pyridine, so that the ring-closing reaction yields a C-N bond.\nThis isomer is referred to as the SIZ form. T-type negative photochromism is observed.\nisomerization reactions are dramatically slowed down. This class display P-type\nphotochromism.\nThe photoconversion is efficient (at least 90% conversion to the SIZ form) and the\nthermal recovery as well (near quantitative thermal isomerization to PMC, at least for\nPMC1-3). Photostability is OK. The ability to use all-visible light is highly appealing.\nWhat is most appealing (at least to this reviewer) with this class of compounds (the\nT-type negative ones) is that they can be used in aqueous solutions. As opposed to the\npreviously reported most common classes DASA and the phenyxyl/imidazolyl radical\ncomplexes reported by Abe, the PMCs maintain the photochromic behaviour in water.\nThis is extremely useful for biological applications. The authors also showed\ncompatibility with solid matrices. The material presented is well suited for publication in\nNat. Commun. Before this can happen, the authors should address the following points.\nResponse: We are grateful to the reviewer for the thoughtful and encouraging comments,\nand for acknowledging the novelty and potential impact of our work. We are also pleased\nthat the reviewer finds the manuscript well suited for publication in Nature\nCommunications. We have carefully addressed all the reviewer’s comments and revised\nthe manuscript accordingly as outlined below.\n1. Isomerization quantum yields should be reported. I’m not saying this to be mean, but\ninstead I believe that when reporting a novel class on photoswitches that will be very\nuseful, these numbers must be presented.\nResponse: Following your kind suggestion, we have determined the photoisomerization\nquantum yield of PMCs. The results indicated that PMC1-5 displayed the\nphotoisomerization quantum yield between 5.4%-12% in water, which is comparable to\nthe quantum yields of classical spiropyran and other photochromic systems. These results\nhave been added and described in the text (see paragraph 2, page 4) and Table 1. For\ncomparison, photoisomerization quantum yields of some typical photochromic\ncompounds are also listed in Table S1 in Supplementary Information.\n2. I’m not sure that I agree with the following statement:\n“Photochromic mechanisms are primarily divided into two categories: Z-E\nisomerization (e.g., azobenzene, indigoid) and covalent bond formation/cleavage (e.g.,\nspiropyran, diarylethene, Stenhouse compound). The latter usually involves more\npronounced structure change upon light stimulation…”\nCis-trans isomerizations give rise to structural changes that are often more\npronounced compared to DAEs etc.\nResponse: We fully agree with the reviewer’s opinion. Now the improper or misleading\nstatement “The latter usually involves more pronounced structure change upon light\nstimulation” has been deleted in the revised manuscript.\n9\n3. In Figure 1D, is the residual absorption between 500 nm and 600 nm after light\nexposure due to absorption from the PMC isomers? I guess so. Alternatively, it could be\ndue to weak transitions for the SIZ form. I’m a bit surprised by the residual absorption\nobserved, in particular if SIZ does not absorb light between 500 nm and 600 nm. The\nthermal back-isomerizations are so slow that it should be possible to photoisomerize\neverything to the SIZ form (irradiation for 10 s, whereas the thermal isomerizations are\nmuch slower). This matter is also accentuated in the following sentence.\n“The photostationary state distributions (PSD) of the SIZ products were all above\n95%, indicating almost completed transitions (Table 1)…”.\nThe authors should explain better why they do not observe 100% photoconversion to the\nSIZ form.\nResponse: Thank you for your constructive suggestion. We have redetermined the time-\nlapse absorption evolution of PMC3 during the thermal recovery process. Because the\nspectral measurement was conducted approximately 20 seconds (limited by the scan rate\nof optical grating) after the end of irradiation, a slight thermal recovery occurred during\nthis interval, resulting in a small fraction (~0.9%, determined by absorbance at 530 nm)\nof SIZ3 converting back to PMC3. This observation is consistent with the calculated value\n(0.8% recovery after 20 s) from the reaction rate constant of the thermal process at 25 oC\n(PMC3 recovered faster than PMC2). As for the issue that the photoconversion rate\ncannot reach 100%, we think it may be caused by the combined effect of molecular\nstructure and instrumentation.\n4. In Table 1, the authors put numbers on the “Recovery”. I believe that “Recovery” is\nno good, as it signals that these numbers are only observed after photoisomerization and\nsubsequent thermal back-isomerization to yield PMC. “Thermal equilibrium position”\nsounds better, as this equilibrium position should not be dependent on if the compounds\nwas exposed to light prior to the equilibrium establishment or not.\nResponse: Thank you for your helpful comment. To better describe the data, now we used\n“Recovery yield (RY) of the SIZ-to-PMC conversion after the first cycle” as the title of\nthe column in Table 1. The term “thermal equilibrium position” may not well describe\nthe following two situations (degradation resulting from chemical instability, and photo-\ninduced transformation). For example, PMC4 and PMC5 displayed low recovery yield\nprobably caused by degradation; SIZ6 and SIZ7 recovered through the photo-induced\ntransformation rather than thermal equilibrium.\n5. In Table 1, I suggest the inclusion of also the thermal half-lives of the SIZ isomer.\nThis facilitates for the reader to get a grip on how fast this process is without a calculator.\nResponse: Following your kind advice, we have added the thermal half-lives of the SIZ\nisomer in Table 1 for the readers.\n10\n6. Last, but not least, I must comment on ΔG (delta G) in Table 1. These numbers do\nNOT agree with the experimentally determined thermal equilibrium positions (reported\nas “Recovery”). From the “Recovery”, the authors can easily determine the equilibrium\nconstant for the thermal equilibrium, K. With K known, ΔG is easily determined. 50%\nRecovery would imply ΔG=0 (in the absence of degeneracy). This is NOT aligned with\nthe numbers for PMC5. I suggest that the authors calculate ΔG from the “Recovery”-\nvalues and compare these values to the theoretically (computationally) determined dittos.\nComment on any discrepancies.\nResponse: We thank the reviewer for this insightful comment. Figure S9 shows that\nPMC4 exhibits solvent-dependent thermal equilibrium positions; Figure S10 reveals that\nPMC4 and PMC5 undergo partial decomposition in water. Thus, The Gibbs free energies\nof PMC4 and PMC5 might be less reliable, as DFT calculations may not fully capture\nthese effects. To avoid this misunderstanding, we have removed the ΔG column in Table\n1.\nIf you have any questions, please do not hesitate to contact us. Thank you, and we are\nlooking forward to hearing from you.\nSincerely yours,\nWen Shi\n11"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors positively answered to many of my comments. The new version of the manuscript, also containing the modifications made to consider the remarks of the other reviewers, is much better.\nI have some minor remarks about the new information provided:\n1- For the orbital energies (HOMO-LUMO gap, Figure S18, Figure S2), the values are very surprising. I guess the author used the raw data from Gaussian. These values are in Hartree and not in eV. Thus, the authors must multiply the values by 27.21 to convert Hartree in eV.\n2- In the computational details, no information is provided on the TD-DFT. How many states were considered? Did the authors performed the frequency calculation to validate that the transition states found at the ground state and at the S1 state is truly a transition state?", "reviewer_2": "The extensively revised manuscript by Shi and colleagues fully addresses the reviewers’ questions and suggestions from the first revision stage. The authors have significantly strengthened the clarity, rigor and depth of the work, and the revised version now highlights the value and potential of this new class of molecules much more effectively. In its current form, the manuscript leaves little room for remaining concerns. I therefore recommend the manuscript for publication as it stands.", "reviewer_3": "The authors have addressed all my concerns. However, I’m a bit surprised why they chose the described method for isomerization quantum yield determination, instead of a relative method using a photoswitch with known isomerization quantum yield as a standard (which is more reliable). The authors should comment on this. Moreover, the added method section about the isomerization quantum yield determination is too sparse. For example, was the whole sample volume irradiated? Was the sample stirred during irradiation?"}, "author_response": "Responses to reviewers’ comments\nDear Reviewers,\nWe are deeply grateful for your appreciation on our work. We have made additional\nrevisions according to your valuable comments. For your convenience, all the revisions\nmade in our revised manuscript are highlighted in blue, and their necessary explanations\nwith point-by-point responses are listed as follows:\nTo Reviewer 1:\nThe authors positively answered to many of my comments. The new version of the\nmanuscript, also containing the modifications made to consider the remarks of the other\nreviewers, is much better.\nI have some minor remarks about the new information provided:\n1. For the orbital energies (HOMO-LUMO gap, Figure S18, Figure S2), the values are\nvery surprising. I guess the author used the raw data from Gaussian. These values are in\nHartree and not in eV. Thus, the authors must multiply the values by 27.21 to convert\nHartree in eV.\nResponse: We thank the reviewer for pointing this out. You are correct. The\nHOMO/LUMO energies in the originally submitted Supporting Information were\ninadvertently reported using the raw Hartree output. We have corrected all energy values\nby converting from Hartree to electronvolt. The Figures in the Supplementary\nInformation have been updated accordingly. We sincerely appreciate the reviewer’s\ncareful reading.\nstates were considered? Did the authors performed the frequency calculation to validate\nthat the transition states found at the ground state and at the S1 state is truly a transition\nstate?\nResponse: We are grateful for this helpful comment. In the vertical transition calculations,\nwe considered 10 excited states. During the excited state structure optimization, the\nlowest three excited states were optimized. Frequency analyses confirmed that all\noptimized ground- and excited-state minima exhibited no imaginary frequencies.\nTransition states (TS1 and TS2) were optimized using opt=(ts,calcfc,noeigentest) and\nverified by a single imaginary frequency together with intrinsic reaction coordinate\ncalculations.\nS₁’ was obtained by using the twisted alkene geometry from TS1 as an initial guess and\nperforming a full excited-state optimization. This optimization directly converged to a\nstable excited-state minimum, corresponding to a twisted C=C conformation. Frequency\ncalculations confirmed the absence of imaginary modes, indicating that S₁’ is a stationary\n1\npoint on the S₁ surface. It represents the relaxed geometry on the excited-state potential\nenergy surface prior to further structural evolution.\nTo Reviewer 3:\nThe authors have addressed all my concerns. However, I’m a bit surprised why they\nchose the described method for isomerization quantum yield determination, instead of a\nrelative method using a photoswitch with known isomerization quantum yield as a\nstandard (which is more reliable). The authors should comment on this. Moreover, the\nadded method section about the isomerization quantum yield determination is too sparse.\nFor example, was the whole sample volume irradiated? Was the sample stirred during\nirradiation?\nResponse: Because of the wide distribution of the absorption spectra of PMC1-7, it is\nhard to use single photochromic standard to determine the relative quantum yield. Hence,\nwe determined the quantum yields by absolute method. In the experiment, 3 mL sample\nvolume was fully irradiated (irradiated area 3 cm², 1 cm optical path) under continuous\nstirring to ensure homogeneous excitation. These discussions have been added into the\nmethod section.\nIf you have any questions, please do not hesitate to contact us. Thank you, and we are\nlooking forward to hearing from you.\nSincerely yours,\nWen Shi\n2"}]

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