Non-traditional fluorescence in quadruple hydrogen bonded supramolecular polymers

Domain: physics
Published: 2026-01-20
Editorial outcome: minor_revision

Abstract
Non-traditional luminescent polymers exhibit significant advantages in bio-diagnostics and intelligent materials but suffer from low luminescence efficiency and limited functionality. Inspired by the excited-state proton transfer mechanism mediated by dense hydrogen bonds in jellyfish fluorescent proteins, we propose a strategy using dynamic quadruple hydrogen bonding ureidopyrimidinone motifs to create highly efficient luminescent polymers. By modulating the aggregated structure of the supramolecular units, proton transfer between paired motifs is activated, thereby achieving a high photoluminescent quantum yield up to 52% in supramolecular polyurethane. Ultrafast spectroscopy directly revealed this intermolecular proton transfer, while solid-state NMR spectroscopy confirmed the essential role of quadruple hydrogen bonds. The dynamically switchable hydrogen bonding structure endows the material with multifunctional integration, including strong fluorescence properties, high toughness, self-healing, reprocessability, and stimulus responsiveness. This research not only introduces a pioneering approach for advancing high-performance light-emitting materials but also enhances the prospects for their practical applications.

Introduction
Non-traditional luminescent polymers (NTLPs) hold significant promise for bioimaging1, anti-counterfeiting2–4, and sensing5–10, due to their advantages of good biocompatibility, film-forming ability, low cost, and facile synthesis. However, their practical adoption faces two fundamental constraints: (1) intrinsically strong non-radiative transitions arising from electronic localization and structural flexibility, leading to low photoluminescence quantum yields (PLQYs) compared to traditional conjugated systems; (2) the challenge of integrating high luminescence efficiency with multifunctionality for real-world applications10–12. Therefore, developing highly efficient NTLPs with integrated multifunctionality remains a pivotal challenge in light-emitting materials research.

Intriguingly, non-aromatic biomacromolecules widely present in nature, such as proteins and peptides devoid of any aromatic residues, have been shown to exhibit intrinsic fluorescence in the visible range13. This phenomenon provides critical insights into addressing the aforementioned challenges. Research demonstrates that the strong fluorescence of these bio-based NTLPs originates from the synergistic interplay between their precise high-density hydrogen bonding networks14 and the excited-state proton transfer (ESPT) mechanism15. In particular, since the discovery of green fluorescent protein (GFP) in 1962, it has revolutionized the field of life sciences and medicine16. The amino acid residues around the chromophore in GFP create a rigid microenvironment with water molecules through hydrogen bonds, which accelerates the ESPT process and significantly enhances the fluorescence efficiency by suppressing the excited-state conformational relaxation17,18 (Fig. 1a). ESPT is a photophysical process closely associated with fundamental life processes19. Luminescent materials based on ESPT have received extensive attention due to their good biocompatibility, large Stokes shifts, and stimulus responsiveness, and have been applied in fields such as fluorescence probes20 and sensors21. However, replicating the kinetic characteristics and efficiency of intricate ESPT observed in biological systems during the molecular engineering of NTLPs has not been achieved yet.

In the conventional structural design of NTLPs, hydrogen bond interactions between groups like amide bonds22,23 and urea bonds24 can effectively enhance n-π conjugation and promote conformational rigidification, leading to improved fluorescence intensity. For example, recent reports have stated that enhanced fluorescence emission25 and an extended spectral emission range26,27 can be achieved by introducing high-density hydrogen bonds, which has become a promising strategy for improving fluorescence efficiency. Despite advancements, NTLPs face persistent bottlenecks: deficient synthetic access to efficient luminophores and inadequate mechanistic insight into structure-property relationships, which jointly hinder their development. Achieving a high photoluminescence quantum yield via precise hydrogen bond engineering in non-conjugated systems remains a critical challenge to be addressed.

Ureidopyrimidinone (UPy), a star molecule that dimerizes via strong quadruple hydrogen bonding, is widely used to construct supramolecular systems with high toughness and self-healing ability28. Recently, UPy has been employed as an auxiliary unit to enhance traditional fluorophores29, or as an energy transfer medium to improve phosphorescence efficiency30. Additionally, photo-controlled hydrogen bonding UPy motifs enables reversible assembly processes31. However, its intrinsic luminescent properties and photoinduced keto–enol tautomerism have remained undiscovered until now.

Inspired by the multi-step proton transfer mediated by the hydrogen-bonding network confined within the β-barrel of GFP in Aequorea victoria, we propose a strategy of coupling ESPT with NTLPs containing high-density hydrogen bonding networks, as illustrated in Fig. 1b. This design enables successful preparation of an intrinsically highly luminescent polymer, utilizing a UPy-based supramolecular ESPT luminophore with programmable quadruple hydrogen bonds. The resulting supramolecular material (named PU-UPy) exhibits intense blue-violet photoluminescence with a remarkably high quantum yield of 52% among NTLPs reported to date. Through a combination of solid-state NMR, transient spectroscopy, and theoretical calculations, an ESPT mechanism is established as the origin of the strong emission. Incorporating UPy to polymer not only enhances mechanical properties, but also enabling multifunctional integration of efficient luminescence, self-healing, remoldability, and stimulus responsiveness. This polymeric material is well-suited for widely applications, including fluorescent coatings, cultural relic restoration, anti-counterfeiting, temperature/ion probes. This study not only establishes a paradigm for the molecular design of NTLPs but also develops a versatile material platform that integrates optical, mechanical, and intelligent response functionalities, paving the way for the next generation of bio-integrated devices and adaptive optoelectronics.

Results
Preparation and photophysical property of PU-UPy
The highly luminescent small molecule (UPy-DHDI) precursor and the supramolecular polymer (PU-UPy) incorporating UPy-DHDI were successfully prepared (Supplementary Figs. 1–6). The solid-state photoluminescence (PL) spectrum of PU-UPy (Fig. 2a) exhibits a consistent emission wavelength at 410 nm, independent of the excitation wavelength. This observation highlights the structural stability of its chromophore and serves as a distinguishing feature when compared to conventional clusterization-triggered emission (CTE) phenomena observed in NTLPs. The ultraviolet (UV) absorption and excitation spectra of the material (Fig. 2b) indicate that the unconventional emission is associated with weak UV absorption in the range of 320 nm to 370 nm. This absorption can be attributed to the n-π* transition of the UPy heterocycle, which was not observed in either the solution absorption spectrum of PU-UPy or the solid-state UV absorption spectrum of the blank PU sample (PU-b) (Supplementary Fig. 7). These findings suggest that the absorption in the range of 320–370 nm originates from aggregation-induced effects of UPy motifs. More importantly, PU-UPy exhibits a remarkably high PLQY of 52% in the solid state (Fig. 2c), surpassing most reported NTLPs (Fig. 2d)8,22,23,25,32–36. Due to the presence of multiple hydrogen bond interactions, PU-UPy exhibits high tensile stress (~20 MPa) and high stretchability (965%) (Supplementary Fig. 8). Compared to the blank PU sample, PU-UPy demonstrates significantly enhanced mechanical strength, toughness, and PLQY (Fig. 2e), thereby providing a basis for achieving multi-functional integration in polymeric materials. Circularly polarized fluorescence spectroscopy indicates a clear, albeit weak, left-handed polarization in the fluorescence emission of PU-UPy (Fig. 2f).

The luminescent properties of the PU-UPy were further investigated in DMF and CHCl₃ with various concentrations (Fig. 2g–i), and the absorption spectra (Supplementary Fig. 7) were measured. It was found that the trend of the emission intensity changing with concentration was inconsistent in the two solutions. Notably, in a 10−3 M CHCl₃ solution (Fig. 2g), the PL spectra of the sample transition from single-peak emission to double-peak emission. Solvent dependence and multi-peak emission are significant characteristics of ESPT26,32. Due to the existence of a high-density dynamic proton network between UPy dimers, proton transfer is facilitated efficiently. Therefore, it is speculated that the unique fluorescence properties of PU-UPy are related to the ESPT behavior facilitated by quadruple hydrogen bonds.

The luminescence mechanism of UPy motifs
To further uncover the underlying luminescence origin of the UPy motifs, we first investigated the fluorescence properties of the monomer UPy-DHDI used to construct the supramolecular polymer. It was found that UPy-DHDI not only exhibited emission characteristics and fluorescence quantum yield values highly consistent with those of the polymer material, but also demonstrated consistency in its excitation spectrum compared to the polymer (Supplementary Fig. 9a, c). Combined with the analysis of the solid-state UV absorption spectrum, it was found that the changes in the excitation behavior of both originated from the enhanced absorption in the range of 320–370 nm (Supplementary Fig. 10). This result directly confirms that the UPy motif is the fundamental structural unit responsible for the luminescent functionality of the PU-UPy. It has been reported in the literature that the existence of the inherent tautomerization of heterocyclic ring-based system, such as UPy derivatives37. And the structure control of intramolecular proton tautomerism has been an inspired and fruitful research area in supramolecular chemistry38. In fact, the equilibrium of proton tautomer of UPy is solvent and temperature dependent39, referring to a permanent conformational change in the ground state. Based on the experimental results presented above as well as the existence of the inherent tautomerization of UPy, we hypothesize that UPy undergo photoinduced tautomerism in the excited state. Thus, a luminescence mechanism for UPy based on this inference was proposed: in the ground state, the UPy dimer usually has a donor-donor-acceptor-acceptor (DDAA) hydrogen-bonding array with keto form; after transitioning to the excited state, the UPy dimer undergoes an intermolecular double proton transfer to form an enol form (Enol1) with a DADA hydrogen-bonding array, and may further isomerize to another enol form (Enol2) with an ADDA hydrogen-bonding array (Fig. 3a).

Systematic theoretical investigation using time-dependent density functional theory (TD-DFT) was performed to verify the aforementioned mechanism. For simplification of the calculations, UPy-DHDI was employed as a model molecule instead of PU-UPy. Structural optimization was conducted at the B3LYP/6-31G* level of theory, utilizing the Gaussian 16 and Multiwfn software packages40,41. The calculation results show that proton-transfer-driven tautomeric transformation in the excited state of UPy dimer could cause a red-shifted emission. The fluorescence emission wavelengths can be predicted by calculating the vertical excitation energies from S0 to S1 state of three tautomeric forms of UPy-DHDI, which are 3.34, 3.01, and 2.87 eV, respectively (Fig. 3b). It was found that the enol form has lower energy than the keto form, and the energies of the three tautomeric forms decrease progressively. This confirms that the proton transfer and tautomerization processes are thermodynamically favorable. The predicted maximum absorption wavelengths are 371.8, 411.5, and 431.3 nm for Keto, Enol1 and Enol2 form of UPy, respectively. Notably, the calculated wavelength of Enol1 exhibits excellent agreement with the emission wavelength obtained from the fluorescence spectrum (Fig. 2a). This suggests that the formation of Enol1 structure in the excited state is responsible for the efficient luminescence of UPy motifs. Moreover, the emission wavelength of Enol2 form is calculated as 431.3 nm, which corresponds to the long-wave emission peak of PU-UPy in CHCl3 (peak II in Fig. 2h), providing evidence for the existence of Enol2 form in the excited state. Potential energy curves (PECs) can effectively reveal the feasibility and energy barriers of proton transfer in both ground and excited states. The ground- and excited-state PECs for the proton-transfer product in the first-step were calculated (Supplementary Fig. 11a, b). The results show that the energy barrier for proton transfer in the excited state is significantly reduced compared to that in the ground state, thereby further demonstrating the feasibility of ESPT rather than intermolecular proton transfer in the ground state.

To obtain direct experimental evidence for proton transfer dynamics, femtosecond transient absorption (fs-TA) spectroscopy was employed to investigate the ultrafast photophysical behavior of PU-UPy in the excited state. The transient absorption optical path was pre-assembled to meet the testing needs (Supplementary Figs. 12 and 13). Following excitation at 380 nm (derived from a 1030 nm Pharos femtosecond laser), two distinct spectral features emerged within the first 20 ps of the transient absorption spectra (Fig. 3c). A short-wavelength signal attributed to the pre-transfer keto form decays rapidly on the sub-picosecond timescale. Simultaneously, a long-wavelength signal assigned to the post-transfer Enol1 rises and maximizes at ~1 ps. These temporal-spectral characteristics provide compelling evidence for ESPT42, unambiguously confirming that UPy units undergo multi-proton transfer in the excited state, accompanied by a keto → enol tautomerization. The decay kinetics of both signals reflect the relaxation of excited molecules to the ground state (Fig. 3d). Time-resolved fluorescence spectroscopy based on time-correlated single photon counting (Supplementary Fig. 14), was employed to capture the weak emission signal from Enol2 with a long delay time. The evolution of the emission spectra with delay time demonstrates the existence of the ESPT process and the existence of the Enol2 form. The energy transfer process of the excited state of PU-UPy is summarized with two ESPT processes (Fig. 3e). The foregoing experimentally resolved ESPT dynamics perfectly corroborates the theoretically predicted multi-proton transfer pathway confined by multiple hydrogen bonds, thereby establishing a comprehensive microscopic mechanism: “ESPT → tautomerization → fluorescence enhancement”. To more accurately describe the proton transfer process, we performed potential energy surface scans for both the intramolecular and intermolecular proton transfer pathways (Supplementary Fig. 15). The results suggested that direct intermolecular proton transfer from the keto configuration faces a high energy barrier (2.97 eV), making it unlikely under conventional photoexcitation. In contrast, the intramolecular pathway has a much lower barrier (1.12 eV), suggesting it occurs preferentially. Once the intramolecular transfer is complete, the resulting electronic rearrangement significantly reduces the barrier for subsequent intermolecular transfer, facilitating a concerted mechanism. Therefore, the ESPT of UPy dimers should be an intramolecular-intermolecular cooperative multi-proton transfer process.

The influence of UPy motif structure on luminescent properties
The photophysical properties of the luminescent unit are closely tied to its aggregated structure43,44. To investigate the structure-function relationship of emissive UPy motifs, a control sample named UPy-DHI was synthesized by replacing the HDI reagent with hexyl isocyanate. This modification yielded a weakly emissive sample (Fig. 4a). Spectral characterization revealed that UPy-DHI exhibited a quantum yield below 10% and displayed excitation wavelength-dependent fluorescence emission (Supplementary Fig. 9b, d). UPy-DHI exhibited fixed emission wavelength and distinct emission peaks in CHCl3 (Supplementary Fig. 18a, b), similar to those of UPy-DHDI in the solid state. These findings indicate that changing the aggregated structure of UPy-DHI can endow it with a luminescent behavior similar to that of UPy-DHDI. We further explored the differences in the aggregate structures of the two samples. X-ray diffraction results reveal that UPy-DHI exhibits a crystalline structure, whereas UPy-DHDI shows disordered amorphous structure (Fig. 4b). Subjecting the UPy-DHI sample to liquid nitrogen quenching produced an amorphous material (Supplementary Fig. 16) that still exhibited weak fluorescence (Supplementary Fig. 17), confirming that crystallization is not the origin of the fluorescence differences. Furthermore, one-dimensional profiles of small-angle X-ray scattering experiment show a distinct peak at q~1.33 Å−1, corresponding to a d-spacing of 4.72 Å, close to typical π-π stacking distances, indicating plane separation formed by UPy dimers. In contrast, no significant scattering peaks were observed in the wide-angle region for UPy-DHDI; only a very weak shoulder peak was present at q~1.24 Å−1 (d-spacing~5.06 Å). This indicates an increased interplanar distance and a disruption of ordered packing (Fig. 4c). DFT simulations revealed the tetrameric structures of UPy-DHI and UPy-DHDI (Supplementary Fig. 19a, b). The results indicate that UPy-DHDI tetramers exhibit a longer interplanar distance than those of UPy-DHI, consistent with the WAXS results. The increased molecular spacing and more disordered molecular orientation directly weaken intermolecular π–π stacking. This structural change enhances fluorescence efficiency through two distinct mechanisms. First, by reducing the number of π–π stacking structures that can act as exciton quenching sites, it ensures the efficient progression of the ESPT process. Second, the suppression of π–π stacking mitigates aggregation-caused quenching, thereby preventing the dissipation of ESPT-generated fluorescence via non-radiative pathways.

The 1H double-quantum/single-quantum (DQ/SQ) chemical shift correlation NMR spectrum is a powerful tool for elucidating the packing structure and hydrogen bonding interactions of molecules45. From the proton 2D DQ/SQ spectra (Fig. 4d, e), the presence of DQ correlation signal Hb-Hc indicates that both UPy-DHDI and UPy-DHI formed a DDAA-type hydrogen-bonding array, further supporting the correctness of above hypothesis regarding the mechanism of emission (Fig. 3a). Notably, the ¹H NMR spectrum of UPy-DHI—both crystalline and amorphous—shows a distinct signal for the isolated proton Hc’, which is not involved in UPy hydrogen bonding (Fig. 4e and Supplementary Fig. 20). This directly demonstrates the disruption of the quadruple hydrogen-bonding network in UPy-DHI, which we proposed suppresses the intermolecular proton transfer process, leading to its markedly weaker fluorescence. When dissolved in CHCl3, the emission behavior of UPy-DHI undergoes a significant change and closely resembles that of UPy-DHDI and PU-UPy in the solid state. The above experiments confirmed that the aggregated state structure can modulate the generation of UPy fluorescence by influencing hydrogen bond formation and stacking structure. Furthermore, the 13C NMR relaxation spectra (Supplementary Fig. 21 and Table 1) shows that the spin-lattice relaxation times (T1) of individual carbon atoms within the UPy-DHDI structure are shorter than those in UPy-DHI, which implies that the chain in UPy-DHDI are more mobile than those in UPy-DHI. This observation suggests that the commonly held belief that restricted molecular motion leading to enhanced luminescence is not the primary mechanism underlying UPy luminescence. Furthermore, these results provide an explanation for the highly efficient fluorescence of UPy-DHDI from the perspective of conformational freedom. The relatively amorphous structure offers greater conformational flexibility, thereby allowing dynamic rearrangement of the hydrogen-bonding network, which is crucial for the ESPT process.

Molecular dynamics (MD) simulations for aggregated structure of UPy dimer
MD simulations were used to study the aggregated structure of UPy-DHDI dimers and tetramers, revealing the influence of aggregated structure of chromophores on fluorescent properties. The results show that both the dimer and tetramer form stable quadruple hydrogen-bonding arrays (Fig. 5a). The tetramer exhibits irregular stacking between planes, which suppresses exciton annihilation and resonance energy transfer in the excited state. Hydrogen bond distributions during assembly were quantified: approximately 4 bonds in the dimer and 8–9 in the tetramer (Fig. 5c and f), confirming consistent formation of stable quadruple hydrogen-bonding arrays. Calculations of interplanar distances and dihedral angles within the tetramer (Fig. 5d–g) show that one pair of UPy motifs (UPy1-UPy2) has a dihedral angle <30° and an interplanar distance of 8 Å, while the other pair (UPy3-UPy4) has a smaller interplanar distance but a dihedral angle >30°. These results indicate irregular stacking between UPy dimer units with quadruple hydrogen bonding, which can ensure efficient ESPT and enhances luminescence emission46. This conclusion is also supported by SAXS and DFT simulation results previously presented. On the basis of the above discussion, a comprehensive understanding of the luminescence mechanism similar to that observed in jellyfish fluorescent protein (Fig. 1) of PU-UPy polymer can be achieved. Within this mechanism, the UPy dimer undergoes concerted intra- and intermolecular multiple proton transfer upon excitation. The resultant tautomer enables red-shifted visible-light emission. Crucially, the rigid end groups (or polymer chains engaged in hydrogen bonding) prevent the formation of regular interplanar stacking structures. This suppression occurs without hindering proton transfer, thereby preventing the efficiency losses caused by exciton annihilation and resonance energy transfer.

Application of strong emissive PU-UPy
Conventional fluorescent materials often suffer from functional limitations, with integration of luminescence and other essential properties proving challenging. In this work, this challenge is addressed by activating the intrinsic fluorescence of UPy motifs, achieving simultaneous integration of high toughness, self-healing, and multi-stimuli responsiveness in a UPy-based supramolecular polymer. This synergistic multifunctionality adapts to diverse applications, as demonstrated by comprehensive performance assessments that highlight cross-disciplinary potential. PU-UPy exhibits excellent optical properties. When spin-coated on glass, it causes only a 0.17% decrease in visible light transmittance (Supplementary Fig. 23a, b), making it suitable for applications requiring high light transmittance, such as surface coatings and optical device interfaces. It also shows good recyclability by hot-press molding (Fig. 6a) and solution casting. After being dissolved and remolded five times in DMF at 100 °C, the quantum yield remains stable (from 51.96% to 49.44%, Supplementary Figs. 25–27). The PLQY data from different batches also serves as evidence for the stability of the synthesis (Supplementary Fig. 24). At 40 °C, the fractured surface can heal efficiently within 10 min (Fig. 6b). The mild self-healing ability, high toughness, and reprocessability imparted by the dynamic hydrogen bonding network enhance the durability of the material under harsh conditions. Based on the reprocessability and self-healing properties of PU-UPy, it can be used as a fluorescent adhesive for restoring cultural relics. Under natural light, the cracks are concealed, not affecting the exhibition; under UV light, the cracks fluoresce, facilitating inspection and repair (Fig. 6c).

The fluorescence properties of the luminophore showing ESPT behavior are strongly correlated with hydrogen bond strength. These properties are influenced by solvent type and pH, demonstrating stimulus responsiveness25,32. Specifically, the fluorescence intensity of PU-UPy varies significantly between −40 and 120 °C (Fig. 6d, e), following a two-segment linear relationship. The inflection point corresponds to its melting temperatures (Supplementary Fig. 28), indicating that UPy fluorescence can sensitively monitor polymer network mobility changes. Under a fluorescence microscope, these variations are visible to the naked eye within this temperature range. Additionally, the changes exhibit rapid response kinetics and a broad operational range, making this material ideal for use as a fluorescent temperature probe (Fig. 6f). Treatment with trifluoroacetic acid (TFA) caused significant fluorescence quenching of PU-UPy (quantum yield reduced to 2.16%, Supplementary Fig. 29), accompanied by a red shift of the emission peak from 410 nm (characteristic of UPy dimer) to 440 nm (attributed to CTE from the urethane moiety) (Fig. 6g). The emission spectra exhibited CTE features and excitation wavelength dependence (Supplementary Fig. 30), indicating disruption of the multiple-hydrogen-bond-confined luminescent centers. Upon partial removal of TFA by heating, the fluorescence intensity partially recovered, and the emission peak blue-shifted back to 410 nm (Fig. 6g), demonstrating its potential as an acidity probe. Fourier transform infrared (FTIR) and solid-state nuclear magnetic resonance (SSNMR) spectroscopy (Supplementary Figs. 31 and 32) confirmed that protons from TFA disrupted the multiple hydrogen-bonding network, thereby hindering the ESPT process and resulting in fluorescence quenching. This result further validates the ESPT-based luminescence mechanism of PU-UPy confined by multiple hydrogen bonds.

Similarly, FeCl3 treatment induced significant fluorescence quenching (quantum yield <1%, Supplementary Fig. 33). NMR, FTIR (Supplementary Figs. 31 and 32), and Raman spectroscopy (Supplementary Fig. 34) revealed that Fe³⁺ introduction neither disrupted the multiple hydrogen-bonding structure nor induced new coordination. The formation of a new absorption band in the 400–600 nm range in the UV–vis spectrum (Supplementary Fig. 35), along with the broadening of the carbonyl peak in the solid-state ¹³C NMR spectrum (Supplementary Fig. 36), indicates the presence of excited-state charge transfer between Fe³⁺ and the carbonyl groups of UPy due to spatial proximity47. Leveraging this property, a 0.001 M FeCl3 aqueous solution can be utilized as “ink” for writing on PU-UPy “paper” producing clearly visible text under 365 nm UV light (Fig. 6i). The quenched fluorescence is nearly fully restored upon ethanol wiping, demonstrating its rewritable capability and applicability in fluorescent anti-counterfeiting materials. DSC and SAXS analyses confirmed that FeCl3 (and TFA) treatment did not alter the polymer’s microphase-separated structure or thermal stability (Supplementary Fig. 37), thereby excluding structural changes as the quenching origin and highlighting the material’s sensing stability. In a 20 μM PU-UPy DMF probe solution containing 200 μM of various ions (Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺), only Fe³⁺ induced substantial fluorescence quenching to below 10% of its original PL intensity (Fig. 6h and Supplementary Fig. 38), exhibiting exceptional selectivity and validating its potential as a specific Fe³⁺ probe. Collectively, the multifunctional stimulus-responsive behavior—integrating temperature monitoring, acidity sensing, specific detection of Fe³⁺, and reversible anti-counterfeiting—demonstrates PU-UPy’s capability as a versatile platform. We cultured L929 cells using an extract of PU-UPy and assessed its biocompatibility by measuring cell viability over a 1–5 days period. The results, presented in Supplementary Fig. 39, demonstrate that the extracts at various dilution ratios showed no cytotoxicity compared to the negative control, confirming the material’s potential for applications in the biomedical field. The synergistic interplay between its multiple-hydrogen-bond-confined ESPT mechanism and controllable charge transfer underpins this material’s foundation for developing intelligent fluorescent sensing systems.

In summary, inspired by the luminescent mechanism of GFP, this study proposes an approach to design and synthesize multifunctional fluorescent supramolecular polymers using dynamic quadruple hydrogen bonding of UPy motifs. By modulating the aggregated structure of UPy units, the ESPT process was activated, achieving a quantum yield of up to 52%, thereby overcoming the limitations of low luminescent efficiency and single functionality in NTLPs. Experimental and theoretical calculations revealed that the configurational transformation of UPy dimers through the ESPT process in the excited state is the fundamental reason for the material’s efficient luminescence. Additionally, SSNMR spectroscopy and time-resolved transient absorption spectroscopy further confirmed the critical role of quadruple hydrogen bonds of UPy in intermolecular proton transfer. The study also suggests that end groups with certain rigidity or polymer chains with hydrogen bonding interactions can improve the aggregated structure of UPy dimers, facilitate efficient ESPT, and suppress non-radiative transitions, thereby enhancing luminescent efficiency. The prepared fluorescent polymer material not only exhibits excellent luminescent properties but also integrates multiple functionalities such as high toughness, self-healing, reprocessability, and stimulus responsiveness. These multifunctional characteristics make the material promising for applications in fluorescent coatings, fluorescent adhesives, bio-fluorescence, and multi-sensing. This study not only provides a paradigm for the molecular design of NTLPs but also lays the foundation for developing a multifunctional material platform integrating optics, mechanics, and intelligent responses, which is of great significance for the development of next-generation biointegrated devices and adaptive optoelectronics.

Methods
Chemical and materials
A list of all chemicals and materials can be found in the supplementary information section.

Synthesis and sample preparation
The synthesis procedure of UPy precursor was shown in Supplementary Fig. 1. A mixture of 2-acetylbutyrolactone (5.12 g, 40 mmol) and guanidine carbonate (3.6 g, 40 mmol) was refluxed in absolute ethanol (40 mL) in the presence of Et3N (11.0 mL, 80 mmol) for 1 h. Then the mixture became clear, and the reaction continued for 3 h. The final obtained product was precipitated and filtered to yield a light-yellow solid, which was further washed with ethanol for three times and dried in vacuum to obtain a white powder solid, i.e., 5-(2-hydroxyethyl)-6-methyl-2-aminouracil. The chemical structure was confirmed by 1H-NMR (Supplementary Fig. 2).

The synthesis procedure of UPy-DHDI was also shown in Fig. 1. The 5-(2-hydroxyethyl)-6-methyl-2-aminouracil (0.85 g, 5.0 mmol) was dissolved in a mixture of 10 mL of 1,6-diisocyanatohexane (HDI) and 1 mL of pyridine under a nitrogen atmosphere. The mixture solution was stirred at 100 °C for 1.5 h followed by precipitation into tenfold excess of petroleum ether. The resulting solid was then washed with pentane and dried in vacuum to yield a white powder. The chemical structure of UPy-DHDI was confirmed by 1H NMR spectra as shown in Supplementary Fig. 3.

The synthesis procedure of PU-UPy was also shown in Supplementary Fig. 4. PTMG-2000 (2.0 g, 1 mmol) firstly reacted with UPy-DHDI (1.010 g, 2 mmol) in anhydrous N,N-Dimethylformamide (DMF, 15 ml) at 70 °C under nitrogen atmosphere for 1 h, yielding a prepolymer. Subsequently, BDO (1,4-butanediol, 0.09 g, 1 mmol) and the catalyst dibutyltindilaurate (0.5 mol% for isocyanate or alcohol units) in anhydrous N,N-Dimethylformamide (DMF, 2 ml) were added into the mixture and further stirred for another 12 h at 70 °C. The solution was poured into a Teflon casting dish dried under 70 °C overnight followed by 70 °C for 48 h under vacuum. After drying, the resulting polymer sheet was peeled off from the Teflon mold and used for further measurements. The chemical structure was confirmed by 1H-NMR (Supplementary Fig. 5) and FTIR (Supplementary Fig. 6). Blank sample PU-b was synthesized using the same synthesis method (UPy-DHDI was replaced with HDI).

Characterization
Solution NMR experiments were performed on a Bruker AVANCE III NMR spectrometer with a proton resonance frequency of 400.13 MHz. The samples were dissolved in deuterated chloroform or DMSO-d6 or DMF-d with a small amount of TMS as the internal reference standard. Solid-state NMR experiments were performed on a Bruker AVANCE NEO wide-bore (89 mm) NMR spectrometer operating at a proton frequency of 400.18 MHz. A conventional 1.3 mm double resonance MAS DVT 400 W1 probe was used, the 1H 90° pulse length was 1.5 µs and the recycle delay was set as 6 s. The fast magic angle spinning (MAS) was automatically controlled at 60 kHz within ±1 Hz with a MAS speed controller. For 1H two-dimensional (2D) double-quantum/single-quantum (DQ/SQ) NMR experiments, the BABA-xy16 pulse sequence48 was used for DQ coherence excitation and reconversion. The spectral width on t1 and t2 dimension was set as 60 kHz and 25 kHz, respectively.

The FT-IR spectra were obtained using a TENSOR II (Bruker, Germany). The infrared spectra were recorded with a resolution of 8 cm−1 and 16 scans per sample.

UV–vis absorption of solutions was recorded on the UV–vis spectrophotometer (Specord plus 210). UV–vis absorption of solid were recorded on the UV–vis spectrophotometer equipped with integrating spheres (Hitachi U-4100).

The steady-state excitation, emission spectra, and quantum yield of solid and solution samples were measured by steady-state transient fluorescence spectroscopy (Edinburgh FS5).

The fs-TAS spectra were obtained with a self-built femtosecond transient absorption spectroscopy. The light source we used is a Pharos laser with a center wavelength of 1030 nm, an average power of 20 W, a pulse adjustable range of <290 fs-10 ps, and a repetition rate of 10 kHz–200 kHz. The optical parametric amplifier is used to generate pump pulses with specific wavelengths, and the optical pulse width of the compressed signal is <100 fs. We adjust the arrival time of pump-probe light through optical delay line, in which the stepping accuracy of optical delay line is 0.1 μm, corresponding to the time of light passing time is 0.33 fs, and can provide a time window of 8 ns. The specific optical path was built with reference to the previous work49. The transient absorption optical path diagram is shown in Supplementary Figs. 12 and 13.

DSC measurements were performed on a Mettler-Toledo DSC1 differential scanning calorimeter with a heating rate of 10 K/min under nitrogen atmosphere. About 10 mg samples were encapsulated in 40 μL aluminum pans before measurements.

XRD measurement was carried out on a Bruker Model D8 FOCUS X-ray diffractometer with Cu Kα radiation (λ = 1.5406 Å) at a generator voltage of 40 kV and a current of 40 mA.

The SAXS experiments were performed at room temperature using a Xenocs Xeuss 3.0 SAXS system operated at 50 kV and 30 mA. The wavelength of the incident X-ray beam from Cu Kα radiation was 0.154 nm. The long period d is inversely related to the wave vector at the scattering peak, d = 2π/q.

TGA experiments were conducted on a NETZSCH TG 209 instrument at a linear heating rate of 10 °C min−1 from 25 to 600 °C under nitrogen atmosphere.

Mechanical properties of the samples were tested using a UTM6103 microcomputer-controlled desktop electronic universal testing machine. Micrographs of the self-healing experiments were captured using an Olympus BX53 microscope equipped with a hot stage.

TD-DFT calculations were performed using the Gaussian 16 software. Structural optimization was conducted at the B3LYP/6-31G* level of theory, utilizing the Gaussian 16 and Multiwfn software packages40,41. To obtain the equilibrium conformations of UPy-DHDI dimers and tetramers, we performed molecular dynamics (MD) simulations on UPy-DHDI in the poor solvent hexane. Atomic types and force field parameters for UPy-DHDI and hexane were assigned using the General Amber Force Field50 (GAFF). The atomic partial charges of each atom in UPy-DHDI were generated by the restrained electrostatic potential (RESP) fit method based on the calculated electrostatic potential at the M06-2X/6-31G** level by the Gaussian 16 package51–53. The initial configurations of the UPy-DHDI dimers and tetramers were generated through randomly placing one UPy-DHDI dimer (two molecules) and two parallel UPy-DHDI dimers (four molecules) into a 6 nm cubic box solvated by hexane, respectively. For each system, the energy minimization was first performed using the steepest descent algorithm. Subsequently, 2 ns pre-equilibrated MD simulations were performed under the NVT (T = 300 K) and NPT ensembles, respectively. Finally, a 100 ns production MD simulation was carried out for each system under the NPT ensemble (T = 300 K, P = 1 bar) to obtain equilibrated dimers and tetramers. During the equilibrium simulation, temperature and pressure were controlled by the velocity rescaling thermostat54 and Berendsen barostat55, respectively. The classical Newtonian equations of motion were performed by leapfrog algorithm with an integral step of 2 fs. For all systems, the conformations of the production MD simulations were stored every 4 ps for analysis. Electrostatic interactions were treated using the particle mesh Ewald (PME) method.56,57 All MD simulations were performed using the GROMACS package (version 5.1.5)58. Trajectory analysis was performed with the help of the utility tools included in the GROMACS package58 and VMD software59.

The biocompatibility of PU-UPy was evaluated using L929 mouse fibroblasts. A material extract was prepared at 0.1 g/mL in serum-free DMEM and incubated at 37 °C for 24 h. The extract was diluted with complete medium to various concentrations. L929 cells were seeded in 24-well plates and, after 24 h, exposed to the diluted extracts, complete medium (control), or blank medium. After 1, 3, and 5 days, cell viability was measured with a CCK-8 assay. The absorbance at 450 nm was recorded, and relative viability was calculated. All groups were tested in sextuplicate. The error bars in the figures represent the standard deviation derived from three independent replicate measurements for each data point. They were calculated as the positive square root of the sample variance for each set of triplicates and are displayed as ±1 SD (More details about the cell experiments are listed in the supplementary information).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This paper presents the advancement of non-traditional luminescent polymers (NTLPs) by developing a multifunctional\nfluorescent supramolecular polyurethane (PU-UPy) inspired by green fluorescent protein (GFP). The authors achieve a high\nphotoluminescence quantum yield (PLQY) of up to 52% through the modulation of ureidopyrimidinone (UPy) units,\nleveraging excited-state proton transfer (ESPT) and quadruple hydrogen bonding. The study investigates the luminescence\nmechanism and validate its multifunctionality. However, according to a high standard for Nature Communications, several\nshortcomings and issues need to be addressed.\n1.Authors claim “Non-traditional luminescent polymers (NTLPs) exhibit remarkable advantages in bio-diagnostics and\nintelligent materials”, but in this paper any information about bio-diagnostics and intelligent materials did not emerge. Some\nbio-diagnostics experiments should be supplemented to improve the quality of the paper and self-consistent their claim.\n2.The authors claim that their work presents the \"first proposal of its ESPT luminescence mechanism\" for UPy motifs. While\nthis may be true for ESPT, the paper does mention a prior related reference for \"isomerization behavior of UPy\". More\nprecise explanations how they differ from previously reported photophysical behaviors of UPy would be necessary.\nEspecially the references about the luminescence works using UPy are missed.\n3.The paper proposes that the UPy dimer undergoes \"cooperative multi-proton transfer and dynamic hydrogen bond\nreorganization\" upon excitation. While the fs-TA spectroscopy provides compelling evidence for the keto → enol\ntautomerization, the paper does not provide detailed evidence that the transfer is \"cooperative\" or \"multi-proton\". While this is\na reasonable assumption given the quadruple hydrogen bonds, a insightful analysis or discussion of this specific kinetic\npathway, perhaps with computational modeling of the transition state, would strengthen this claim.\n3. Authors demonstrate that the PLQY remains stable after five cycles of dissolution and remolding. However, a key proplem\nis the lack of a statement on the reproducibility of the initial 52% PLQY across multiple synthesis batches. Providing data on\nbatch-to-batch consistency would add confidence in this key claim and support the robustness of the synthesis.\n4.Authors claim that the fluorescence quenching by FeCl₃ to excited-state intermolecular charge transfer. While this is a\nvalid hypothesis, the authors also state that FeCl₃ treatment did not disrupt the multiple hydrogen-bonding structure. The\nwork does not provide a detailed mechanism for how the Fe³⁺ ion interacts with the UPy motif or other parts of the polymer to\nfacilitate charge transfer without affecting the crucial hydrogen bond network. A more detailed exploration of this interaction\nwould be added.\n5.The work describes UPy-DHI as having \"extremely weak fluorescence emission\" with a quantum yield \"below 10%\".\nHowever, the weak fluorescence is then attributed to the \"residual UPy dimer with quadruple hydrogen bonds\", which is a\npotential contradiction. The authors should clarify whether the weak emission is from the bulk crystalline material or a minor\namorphous component. Quantifying the amount of this \"residual UPy dimer\" and its contribution to the overall fluorescence\nwould provide a more precise and academically rigorous explanation.\n6.The authors claim that the large interplanar spacing in UPy-DHDI indicates a \"non-π-π close packing arrangement,\"\nfor\nluminescence. The term itself is somewhat vague, and a clearer explanation of how this specific type of packing, as opposed\nto close packing in the crystalline UPy-DHI, prevents non-radiative decay pathways (such as aggregation-caused\nquenching, ACQ) and facilitates efficient ESPT would improve the clarity and rigor of the argument.\n7.The disordered amorphous structure of UPy-DHDI is responsible for the high luminescence, while the crystalline structure\nof the control sample UPy-DHI leads to weak emission. The authors attribute this to the amorphous structure suppressing\nexciton annihilation. However, the paper does not provide a detailed mechanistic explanation of why the amorphous state is\nmore favorable for the ESPT process itself. A insightful evidence of how the dynamics of the hydrogen bond network or the\nconformational freedom in the amorphous state specifically facilitating proton transfer would be offered.\n8.The paper posits the existence of a second enol form (Enol₂) to explain the long-wave emission peak observed in CHCl₃\nsolution. While this is supported by theoretical calculations that show a good match between the predicted and experimental\nwavelengths, this evidence is primarily computational. A significant flaw is the absence of any direct experimental\nverification of the Enol₂ form's existence, which would substantially strengthen the proposed ESPT mechanism. Further\nexperimental techniques, such as time-resolved spectroscopy specifically designed to probe for this second tautomer, could\nbe necessary.", "reviewer_2": "The manuscript by Zuo et al deals with unexpected fluorescence properties induced by a\nwell know moiety (ureidopyrimidinone, UPy) within a polymer. A combination of\nexperimental (synthesis, spectroscopies, various applications) and theoretical (TD-DFT,\nMD) approaches has been convincingly provided to present, rationalize and prove the\neHiciency and the potential of such systems. Very interestingly, they propose a\nmechanism involving tautomerization and excited state proton transfer to explain the\nboost in fluorescence eHiciency while the restriction of motions is often argued to explain\nit. Please find below some questions and comments I had after reading the manuscript.\nSome of them should be taken into account to clarify or precise some points within the\nmain text.\nComments & questions:\n- As no details about the TD-DFT calculations are provided (oscillator strength,\nnature and description in terms of orbitals/involved states), does the maximum\nabsorption wavelength corresponds to S state, as only the S was optimized to\n1 1\nretrieve the emission wavelength? Are HOMO and LUMO the only orbitals involved\nwithin the transition?\n- How was the curve of figure 9b in SI obtained? Was it a simple scan (relaxed or\nnot?) with potential energies reported or is it a “real” energy barrier with frequency\ncalculations involved? This should be clarified within the manuscript and SI.\n- Does the tautomerization process take place at the ground state (GS), towards the\nenol form, or only in the excited state (ES)? Is there a competition between the\ntautomerizaton at the GS and the ES? In the excited state, following figure 9b in SI,\nthe keto form seems to be more stable than the enol form, is it compatible with the\nfollowing steps?\n- Are the 4 H-bonds always eHective during MD simulations? What are the extra H-\nbonds observed? Do they correspond to “peripheric” H-bonds? It should be stated\nthat the keto form was considered for MD simulation. Was the MD simulation of\nthe keto form done with the GS or ES structure (if the structures/charges/density\nrepartition are diHerent between GS and ES)? Why the enol1 and enol2 were not\nconsidered during MD simulations?\n- Why are there such harsh thresholds on the emission spectra of UPy-DHI (figure\n7b in SI)? Were the spectra recorded over the entire wavelength range?\n- Why was hexane used for MD simulations and not DMF or chloroform?\n- On figure 2a, the second ESPT appears as reversible (double arrow) but not the first\none (simple arrow). Why? Is it due to energetic considerations? This can be added\nto the figure and/or the text if the authors have the energy barriers or an\napproximation of it.\n- Are the Enol2 dimers also stable as there are only 2 intermolecular H-Bonds,\ncompared to 4 H-Bonds for the keto/enol1 dimers?\nOverall, the manuscript is dense in results, the explanations are convincing, and it\nprovides new results about a multifunctional fluorescent polymer, and I recommend its\npublication after minor corrections/precisions (see above).\nTypos:\nFigure 5: (e) Plots of PL intensity versus temperature (?) for PU-UPy under 365 nm UV light\nirradiation.\nSpectral characterization revealed that UPy-DHI exhibited a quantum yield below 10%\nand displayed excitation wavelength-dependent fluorescence emission (Supplementary\nFigs. 10b, d). → There is no Fig 10b, d in SI.\nFigure 12 in SI. Graph b does not represent the emission spectra at diHerent wavelength\nbut at diHerent concentration.\nFigure 9b in SI. Unit of the y axis should be kcal mol-1"}, "author_response": "Response Letter to Reviewers\nReviewer #1 (Remarks to the Author):\nThis paper presents the advancement of non-traditional luminescent polymers (NTLPs) by\ndeveloping a multifunctional fluorescent supramolecular polyurethane (PU-UPy) inspired by green\nfluorescent protein (GFP). The authors achieve a high photoluminescence quantum yield (PLQY)\nof up to 52% through the modulation of ureidopyrimidinone (UPy) units, leveraging excited-state\nproton transfer (ESPT) and quadruple hydrogen bonding. The study investigates the luminescence\nmechanism and validates its multifunctionality. However, according to a high standard for Nature\nCommunications, several shortcomings and issues need to be addressed.\nReply: We sincerely appreciate the reviewer's comments and insightful suggestions. In response,\nwe have carefully revised the manuscript (with changes highlighted in yellow) and provide a point-\nby-point response to all comments below.\n1. Authors claim “Non-traditional luminescent polymers (NTLPs) exhibit remarkable advantages in\nbio-diagnostics and intelligent materials”, but in this paper any information about bio-diagnostics\nand intelligent materials did not emerge. Some bio-diagnostics experiments should be supplemented\nto improve the quality of the paper and self-consistent their claim.\nReply: We thank the reviewer for pointing this out. In the introduction, we have outlined the\napplications of various NTLPs, and included the statement “Non-traditional luminescent polymers\n(NTLPs) exhibit remarkable advantages in bio-diagnostics and intelligent materials”. However,\ngiven that each material possesses certain advantages and limitations, the descriptions provided are\nnot intended to imply that every NTLP is suitable for biomedical diagnostics or/and smart materials.\nThe core objective of this paper is to elucidate a novel luminescence mechanism and discovery of a\nnew luminescent polymer with breakthrough in the versatile integration of functionalities within\nNTLP materials. The reported material demonstrates greater suitability for macro-scale smart\nmaterials, as exemplified by its applications in temperature response and ion detection, among\nothers. Nevertheless, we fully acknowledge the reviewer’s perspective that further exploration and\nexpansion of the material’s potential in biomedical diagnostics are warranted. To investigate the\nbiocompatibility of the material, we co-cultured the extract of PU-UPy with L929 cells and assessed\nits biocompatibility by measuring cell viability. The corresponding data have been added in the\nrevised Supplementary Fig. 37. The extracts at various dilution ratios-maintained cell viability\nabove 90% over culture periods of 1, 3, and 5 days, showing no significant difference compared to\nthe negative control group. The results demonstrate that PU-UPy exhibits excellent biocompatibility\nand holds potential for applications in bioimaging and diagnostics. However, limited by the\ndeficiencies in our experimental conditions for biosensing and other aspects, further relevant\nexperiments may be the direction of our future research.\nWe have added a discussion of the biocompatibility results to the revised manuscript, as\nhighlighted: “We cultured L929 cells using an extract of PU-UPy and assessed its biocompatibility\nby measuring cell viability over a 1–5 days period. The results, presented in Supplementary Figure.\n37, demonstrate that the extracts at various dilution ratios showed no cytotoxicity compared to the\nnegative control, confirming the material’s potential for applications in the biomedical field” .\nFigure R1. Cell viability of different dilution factors of the experimental materials group\n(compared with the negative control group).\n2. The authors claim that their work presents the \"first proposal of its ESPT luminescence\nmechanism\" for UPy motifs. While this may be true for ESPT, the paper does mention a prior related\nreference for \"isomerization behavior of UPy\". More precise explanations how they differ from\npreviously reported photophysical behaviors of UPy would be necessary. Especially the references\nabout the luminescence works using UPy are missed.\nReply: We sincerely appreciate the reviewer's valuable insight and suggestion. The \"UPy\nisomerization\" mentioned in the cited reference refers to a ground-state conformational change\nachieved through chemical modification or physical treatment, which does not involve\nphotophysical processes. This fundamentally differs from the photoinduced excited-state proton\ntransfer (ESPT) process reported in our work. In accordance with the reviewer's suggestion, we have\nprovided a more detailed explanation in the revised manuscript.\nIn the introduction of this paper, we present a comprehensive discussion on the luminescence\nresearch related to UPy, particularly highlighting “UPy has been employed as an auxiliary unit to\nenhance traditional fluorophores29, or as an energy transfer medium to improve phosphorescence\nefficiency”. In these previous studies, UPy was not utilized as a luminophore but rather served as\nan auxiliary unit to construct supramolecular assemblies. To the best of our knowledge, no literature\nhas reported the intrinsic fluorescence behavior of UPy. This work represents the first report of this\nphenomenon, which also constitutes the key innovation of our study. We have expressed the\ndiscussion on the photoinduced isomerization behavior of UPy more clearly in the revised\nmanuscript, as highlighted: “However, its intrinsic luminescent properties and photoinduced keto–\nenol tautomerism have remained undiscovered until now ”, and “It has been reported in the\nliterature that the existence of the inherent tautomerization of heterocyclic ring-based system such\nas UPy derivatives38. And the structure control of intramolecular proton tautomerism has been an\ninspired and fruitful research area in supramolecular chemistry30. In fact, the equilibrium of proton\ntautomer of UPy is solvent and temperature dependent40, referring to a permanent conformational\nchange in the ground state. Based on the experimental results presented above as well as the\nexistence of the inherent tautomerization of UPy, we hypothesize that UPy undergo photoinduced\ntautomerism in the excited state for the first time”.\n3.The paper proposes that the UPy dimer undergoes \"cooperative multi-proton transfer and dynamic\nhydrogen bond reorganization\" upon excitation. While the fs-TA spectroscopy provides compelling\nevidence for the keto → enol tautomerization, the paper does not provide detailed evidence that the\ntransfer is \"cooperative\" or \"multi-proton\". While this is a reasonable assumption given the\nquadruple hydrogen bonds, a insightful analysis or discussion of this specific kinetic pathway,\nperhaps with computational modeling of the transition state, would strengthen this claim.\nReply: We sincerely thank the reviewer for raising this critical question, which prompted us to\nperform more detailed calculations and thereby refine our mechanistic hypothesis. To describe the\nproton transfer process more clearly, we carried out energy calculations for the intramolecular\nproton transfer and intermolecular proton transfer processes step by step, the computational results\nwere shown in Fig. R2. It was found that directly completing the intermolecular proton transfer\n(Proton from N3–C4) from the keto configuration to form the enol structure requires overcoming\na relatively high energy barrier (2.97 eV), which is unlikely to occur under conventional\nphotoexcitation. However, we observed that the intramolecular ESPT (N1–C2) process only\nrequires an energy barrier of 1.12 eV, suggesting that the intramolecular ESPT is likely to occur\npreferentially. Furthermore, after the proton transfers from N1 to C2, the electronic distribution of\nthe molecule changes, leading to an increased charge density around the N3 atom, which facilitates\nproton transfer between N3 and C4. Based on these two-step calculation results, we propose that\ndue to the lower energy barrier, intramolecular proton transfer occurs prior to intermolecular proton\ntransfer. The intramolecular proton transfer provides a favorable configuration for the subsequent\nintermolecular proton transfer, reducing the energy barrier and thereby promoting the overall\nprocess. These additional computational results provide supporting evidence for the hypothesized\nconcerted proton transfer mechanism. We have expanded the discussion regarding these new\nfindings as highlighted: “To more accurately describe the proton transfer process, we performed\npotential energy surface scans for both the intramolecular and intermolecular proton transfer\npathways (Supplementary Fig.13). The results suggested that direct intermolecular proton transfer\nfrom the keto configuration faces a high energy barrier (2.97 eV), making it unlikely under\nconventional photoexcitation. In contrast, the intramolecular pathway has a much lower barrier\n(1.12 eV), suggesting it occurs preferentially. Once the intramolecular transfer is complete, the\nresulting electronic rearrangement significantly reduces the barrier for subsequent intermolecular\ntransfer, facilitating a concerted mechanism. Therefore, the ESPT of UPy dimers should be a\nintramolecular-intermolecular cooperative multi-proton transfer process” in the revised\nmanuscript.\nFigure R2. Potential energy surface scanning for intramolecular (a) intermolecular (b) ESPT.\n4. Authors demonstrate that the PLQY remains stable after five cycles of dissolution and remolding.\nHowever, a key problem is the lack of a statement on the reproducibility of the initial 52% PLQY\nacross multiple synthesis batches. Providing data on batch-to-batch consistency would add\nconfidence in this key claim and support the robustness of the synthesis.\nReply: We sincerely appreciate the reviewer's valuable suggestion. As requested, we tested the\nphotoluminescence quantum yield (PLQY) of three different batches of the material, with each batch\nmeasured at three different points and averaged. The average PLQY values for the three batches\nwere 51.96%, 50.90%, and 52.57%, respectively. The result calculated based on standard deviation\nis 51.81 ± 0.7. These results confirm the reproducible high quantum yield and the stability of the\nmaterial synthesis. We have presented the PLQY spectra of three different batches in Fig. R3. We\nhave emphasized this point in the revised manuscript as highlighted: “The PLQY data from different\nbatches also serves as evidence for the stability of the synthesis (Supplementary Fig. 22)”.\nFigure R3. PLQY testing of three different batches of PU-UPy (a-c).\n5. Authors claim that the fluorescence quenching by FeCl₃ to excited-state intermolecular charge\ntransfer. While this is a valid hypothesis, the authors also state that FeCl₃ treatment did not disrupt\nthe multiple hydrogen-bonding structure. The work does not provide a detailed mechanism for how\nthe Fe³⁺ ion interacts with the UPy motif or other parts of the polymer to facilitate charge transfer\nwithout affecting the crucial hydrogen bond network. A more detailed exploration of this interaction\nwould be added.\nReply: We thank the reviewer for this suggestion. To further investigate the interaction mechanism\nbetween Fe³⁺ and the material, we conducted additional UV-vis absorption (Fig. R4) and ¹³C\nCPMAS solid-state nuclear magnetic resonance (SSNMR) experiments (Fig. R5). The relevant\nresults have been included in Supplementary Figs. 33-34. The UV-vis absorption spectra revealed\nthat after binding with FeCl₃, a new absorption band emerged in the 400–600 nm range for PU-UPy,\nwhich differs from the intrinsic n-π* absorption band of UPy or the d-d transition band of Fe³⁺. This\nprovides direct evidence for the formation of a charge-transfer complex. Fig. R5 shows the\nexpanded UPy region of the ¹³C CPMAS NMR spectra of both PU-UPy and PU-UPy–Fe samples.\nUpon the addition of Fe³⁺, significant line broadening (measured at 25% peak height) was observed\nfor the C=O peak at 172.0 ppm and the peak at 114 ppm in UPy. Moreover, all UPy peaks shifted\ntoward lower frequency (increased shielding), indicating an interaction between Fe³⁺ and the\ncarbonyl groups of UPy. These results further clarify the site where charge transfer occurs. In\nSupplementary Fig. 30 (Fig. R6) of the original manuscript, we presented the ¹H MAS spectrum of\nthe Fe³⁺-treated sample. The peaks associated with multiple hydrogen bonding remained unchanged\nin position and intensity, suggesting that the hydrogen-bonding configuration remained relatively\nstable. Additionally, Raman spectroscopy results indicated no significant formation of new\ncoordination bonds, implying that the interaction between Fe³⁺ and UPy is not a strong chemical\nbinding but rather results from spatial proximity and electron cloud overlap. Collectively, these\nfindings elucidate the specific interaction mode between Fe³⁺ and UPy and the underlying quenching\nmechanism. This point has been emphasized in the revised manuscript. The added text (highlighted\nfor review) states: “The formation of a new absorption band in the 400–600 nm range in the UV-\nvis spectrum (Supplementary Fig. 33), along with the broadening of the carbonyl peak in the solid-\nstate ¹³C NMR spectrum (Supplementary Fig. 34), indicates the presence of excited-state charge\ntransfer between Fe³⁺ and the carbonyl groups of UPy due to spatial proximity”.\nFigure R4. Normalized UV-vis absorption spectra of PU-UPy, PU-UPy+FeCl and FeCl in\n3 3\nDMF.\nFigure R5. Solid-state 13C CPMAS NMR spectrum of PU-UPy and PU-UPy-Fe.\nFigure R6. 1H-Solid state NMR spectra of PU-UPy, PU-UPy-TFA and PU-UPy-Fe.\n6. The work describes UPy-DHI as having \"extremely weak fluorescence emission\" with a quantum\nyield \"below 10%\". However, the weak fluorescence is then attributed to the \"residual UPy dimer\nwith quadruple hydrogen bonds\", which is a potential contradiction. The authors should clarify\nwhether the weak emission is from the bulk crystalline material or a minor amorphous component.\nQuantifying the amount of this\" residual UPy dimer\" and its contribution to the overall fluorescence\nwould provide a more precise and academically rigorous explanation.\nReply: We thank the reviewer for their valuable comments and insightful suggestion. To investigate\nthe effect of amorphous regions on the weak fluorescence emission, we designed the following\nexperiment: A chloroform solution of UPy-DHI was quenched in liquid nitrogen and then rapidly\nfreeze-dried to remove residual chloroform, thereby obtaining a UPy-DHI sample with poor\ncrystallinity. XRD results (Fig. R7) confirmed that this quenching treatment significantly reduced\nthe crystallinity and substantially increased the amorphous content of the sample. Quantum yield\ntesting (Fig. R8) was employed to evaluate the changes in the fluorescence properties of the samples\nbefore and after the quenching process. 1H DQ/SQ solid-state NMR experiments (Fig. R9) were\nconducted to examine changes in the hydrogen-bonding configurations before and after the\nquenching process. We observed that although the amorphous content increased notably, no\nsignificant change in the quantum yield (6.4%–6.8%) for quenched UPy-DHI sample. This\nindicates that the fluorescence does not come from the amorphous components in the samples. In\naddition, the ¹H DQ/SQ NMR results indicate that the hydrogen-bonded structures of the UPy-DHI\nsamples remain largely unchanged after liquid nitrogen treatment, with a significant portion of UPy\ngroups not forming quadruple hydrogen bonds. This suggests that the quadruple hydrogen-bond\nnetwork in UPy-DHI is disrupted compared to that in UPy-DHDI, thereby preventing the\nestablishment of an effective ESPT pathway. However, owing to the limited resolution of the ¹H\nsolid-state NMR spectra, a quantitative analysis of the hydrogen-bonded UPy groups is unfeasible.\nNonetheless, the reviewer's question is undoubtedly valuable and will be pursued in our future\nstudies. Based on these new results, we have provided a more detailed explanation of this issue in\nthe revised manuscript. The added text as highlighted states: “Subjecting the UPy-DHI sample to\nliquid nitrogen quenching produced an amorphous material (Supplementary Fig. 14) that still\nexhibited weak fluorescence (Supplementary Fig. 15), confirming that crystallization is not the\norigin of the fluorescence differences”, and “Notably, the ¹H NMR spectrum of UPy-DHI—both\ncrystalline and amorphous—shows a distinct signal for the isolated proton Hc′, which is not\ninvolved in UPy hydrogen bonding (Fig. 3e and Supplementary Fig. 18). This directly demonstrates\nthe disruption of the quadruple hydrogen-bonding network in UPy-DHI, which we propose\nsuppresses the intermolecular proton transfer process, leading to its markedly weaker fluorescence”.\nFigure R7. XRD patterns of UPy-DHI before and after quenching treatment.\nFigure R8. PLQY testing of UPy-DHI before and after quenching treatment\nFigure R9. Solid-state NMR two-dimensional DQ/SQ correlation spectra of UPy-DHI sample\nbefore and after quenched in liquid nitrogen.\n7. The authors claim that the large interplanar spacing in UPy-DHDI indicates a \"non-π-π close\npacking arrangement,\" for luminescence. The term itself is somewhat vague, and a clearer\nexplanation of how this specific type of packing, as opposed to close packing in the crystalline UPy-\nDHI, prevents non-radiative decay pathways (such as aggregation-caused quenching, ACQ) and\nfacilitates efficient ESPT would improve the clarity and rigor of the argument.\nReply: We sincerely appreciate the reviewer's suggestion. For clarity, the term “non-compact\npacking arrangement' has now been changed and more specifically described as 'larger molecular\nspacing and more disordered molecular orientation.” We have accordingly revised the description\nof how this packing type influences the luminescence mechanism. The “larger molecular spacing\nand more disordered molecular orientation” directly reduce intermolecular π-π stacking. This\npromotes efficient fluorescence generation through two mechanisms. On one hand, π-π stacking\nstructures can act as quenching sites for excitons, and this specific packing configuration reduces\nsuch quenching sites, ensuring the efficient occurrence of the ESPT process. On the other hand, the\nweakening of π-π stacking suppresses the ACQ (aggregation-caused quenching) effect, preventing\nthe fluorescence generated via the ESPT process from dissipating through non-radiative transitions.\nWe thank the reviewer for this valuable suggestion, which guided us in more clearly describing the\ninfluence of the aggregated state structure on the fluorescence mechanism. We have modified the\nrelevant descriptions in the revised manuscript as highlighted: “The increased molecular spacing\nand more disordered molecular orientation directly weaken intermolecular π–π stacking. This\nstructural change enhances fluorescence efficiency through two distinct mechanisms. First, by\nreducing the number of π–π stacking structures that can act as exciton quenching sites, it ensures\nthe efficient progression of the Excited-State Proton Transfer (ESPT) process. Second, the\nsuppression of π–π stacking mitigates aggregation-caused quenching (ACQ), thereby preventing the\ndissipation of ESPT-generated fluorescence via non-radiative pathways”.\n8. The disordered amorphous structure of UPy-DHDI is responsible for the high luminescence,\nwhile the crystalline structure of the control sample UPy-DHI leads to weak emission. The authors\nattribute this to the amorphous structure suppressing exciton annihilation. However, the paper does\nnot provide a detailed mechanistic explanation of why the amorphous state is more favorable for\nthe ESPT process itself. A insightful evidence of how the dynamics of the hydrogen bond network\nor the conformational freedom in the amorphous state specifically facilitating proton transfer would\nbe offered.\nReply: We thank the reviewer for their valuable comments and insightful suggestion. We have\nprovided further explanation regarding the reason why the amorphous state facilitates the ESPT\nprocess. The amorphous state offers a degree of conformational freedom, allowing dynamic\nreorganization of the hydrogen-bonding network, which is crucial for the efficient occurrence of\nESPT. InSupplementary Figure 14 of the original manuscript, we included solid-state NMR results\nrelated to molecular mobility that effectively support this conclusion. The table below lists the ¹³C\nT₁ relaxation time measurements, where α, β, and δ represent carbon atoms involved in forming\nmultiple hydrogen-bonding structures. Among the two samples, UPy-DHDI exhibits much shorter\n¹³C T₁ relaxation times, indicating faster molecular motion and higher conformational freedom,\nwhich facilitates the proton transfer process and enables highly efficient luminescence. In contrast,\nUPy-DHI shows significantly longer ¹³C T₁ times, suggesting restricted molecular mobility that\nhinders the subtle conformational adjustments required for ESPT. We have discussed these findings\nin the revised manuscript as highlighted: “Furthermore, these results provide an explanation for the\nhighly efficient fluorescence of UPy-DHDI from the perspective of conformational freedom. The\nrelatively amorphous structure offers greater conformational flexibility, thereby allowing dynamic\nrearrangement of the hydrogen-bonding network, which is crucial for the ESPT process”.\nTable R1. T relaxation time of 13C nucleus in UPy core\n1\n9. The paper posits the existence of a second enol form (Enol₂) to explain the long-wave emission\npeak observed in CHCl₃ solution. While this is supported by theoretical calculations that show a\ngood match between the predicted and experimental wavelengths, this evidence is primarily\ncomputational. A significant flaw is the absence of any direct experimental verification of the Enol₂\nform's existence, which would substantially strengthen the proposed ESPT mechanism. Further\nexperimental techniques, such as time-resolved spectroscopy specifically designed to probe for this\nsecond tautomer, could be necessary.\nReply: We sincerely appreciate the reviewer's comment and insightful suggestion. The direct\nexperimental verification of the Enol₂ form's existence, can indeed enhance credibility of the ESPT\nmechanism we proposed here. Following the reviewer's suggestion, time-resolved emission\nspectroscopy (TRES) was further employed to detect the emission peak from Enol2 configuration,\nas shown in Figure R10. In the time-resolved emission spectra excited at 405 nm, three distinct\nemission peaks (1-3) are observed at different decay time, which should be attributed to the keto\nform and two enol forms. Among them, Peak 3 appears gradually during the decay process, and its\nrelative intensity increases over time, strongly suggesting the existence of the Enol2 configuration.\nAs the delay time increases, the intensities of peaks 1 and 2 gradually weaken, while that of peak 3\ngradually strengthens. We hypothesize that the Enol2 forms at a later stage and exists in relatively\nlow abundance, making it challenging to capture its signal in both transient absorption and steady-\nstate fluorescence spectra. Herein, TRES based on time-correlated single photon counting (TCSPC),\ncan capture the weak emission signal from Enol2 with a long delay time once peak 1 and peak 2\nhave weakened. This supplementary experimental data is crucial for supporting the mechanism we\nhave proposed. Therefore, the relevant discussions have been incorporated into the revised\nmanuscript, as highlighted: “Time-resolved fluorescence spectroscopy based on time-correlated\ntime-correlated single photon counting (Supplementary Fig. 12), was employed to capture the weak emission signal\nfrom Enol2 with a long delay time. The evolution of the emission spectra with delay time\ndemonstrates the existence of the ESPT process and the existence of the Enol2 form”.\nFigure R10. Time-resolved emission spectrum of PU-UPy.\nReviewer #2 (Remarks to the Author):\nThe manuscript by Zuo et al deals with unexpected fluorescence properties induced by a well know\nmoiety (ureidopyrimidinone, UPy) within a polymer. A combination of experimental (synthesis,\nspectroscopies, various applications) and theoretical (TD-DFT, MD) approaches has been\nconvincingly provided to present, rationalize and prove the efficiency and the potential of such\nsystems. Very interestingly, they propose a mechanism involving tautomerization and excited state\nproton transfer to explain the boost in fluorescence efficiency while the restriction of motions is\noften argued to explain it. Please find below some questions and comments I had after reading the\nmanuscript. Some of them should be taken into account to clarify or precise some points within the\nmain text.\nReply: We sincerely appreciate the reviewer's positive comments and the constructive suggestions.\nA response to all concerns has been systematically address the concerns addressed point-by-point\nas follows:\n1. As no details about the TD-DFT calculations are provided (oscillator strength, nature and\ndescription in terms of orbitals/involved states), does the maximum absorption wavelength\ncorresponds to S1 state, as only the S1 was optimized to retrieve the emission wavelength? Are\nHOMO and LUMO the only orbitals involved within the transition?\nReply: We thank the reviewer for pointing this out. The maximum absorption wavelength indeed\ncorresponds to the S₁ state, and the HOMO and LUMO are the sole molecular orbitals involved in\nthis transition.\n2.How was the curve of figure 9b in SI obtained? Was it a simple scan (relaxed or not?) with\npotential energies reported or is it a “real” energy barrier with frequency calculations involved? This\nshould be clarified within the manuscript and SI.\nReply: The curve in Figure SI-9b was obtained through a simple scan (relaxed) of potential energies,\nand this has been explicitly stated in both the main text and the supplementary materials of the\nrevised manuscript.\n3.Does the tautomerization process take place at the ground state (GS), towards the enol form, or\nonly in the excited state (ES)? Is there a competition between the tautomerizaton at the GS and the\nES? In the excited state, following figure 9b in SI, the keto form seems to be more stable than the\nenol form, is it compatible with the following steps?\nReply: We thank the reviewer for pointing this out. The tautomerization process occurs exclusively\nin the excited state (ES), as the energy barrier between the keto and enol configurations in the ground\nstate is too high to overcome. We simulated the infrared spectra of three configurations of UPy-\nDHDI, and the simulated spectrum of the keto form showed excellent agreement with the\nexperimental results, confirming that the ground-state configuration is the stable keto structure (Fig.\nR11).\nFigure R11. (a) FTIR spectra of UPy-DHDI. (b) Simulated FTIR spectra of three configurations of\nUPy-DHDI: keto, enol1, enol2.\nUpon careful re-examination of the results in SI 9b, we identified an error in the original labeling\nof the x-axis: it should represent the N–H distance rather than the O–H distance. This labeling error\nhas been corrected in the revised manuscript. With this correction, the computational results are\nconsistent with the subsequent processes. We sincerely appreciate the reviewer’s thoroughness and\nrigor, which helped us rectify this detail and prevent potential misunderstandings.\nFigure S9. (a) Optimized structure of UPy-DHDI. (b) The potential energy curve of ESPT\nproduct.\n4. Are the 4 H-bonds always effective during MD simulations? What are the extra H-bonds observed?\nDo they correspond to “peripheric” H-bonds? It should be stated that the keto form was considered\nfor MD simulation. Was the MD simulation of the keto form done with the GS or ES structure (if\nthe structures/charges/density repartition are different between GS and ES)? Why the enol1 and\nenol2 were not considered during MD simulations?\nReply: We sincerely thank the reviewer for these insightful questions. As follows, we will respond\nto the reviewers point by point:\n(1) The quadruple hydrogen bonds (H-bonds) between UPy dimers remained stable and effective\nthroughout the MD simulations. This is supported by the consistently maintained number of H-\nbonds within the dimer and tetramer systems. Specifically, the dimeric system exhibited between 4\nand 6 H-bonds, while the tetrameric system maintained between 8 and 10 H-bonds during the\nsimulation period.\n(2) The additional H-bonds beyond the core quadruple set primarily involve intramolecular\ninteractions, specifically between the hydrogen atom in the amino group and the oxygen atom in the\ncarbonyl group of the UPy units (Fig. R12). These can indeed be classified as \"peripheric\" H-bonds,\nwhich contribute additional stability to the supramolecular assembly without disrupting the core\nquadruple H-bonding network.\n(3) The MD simulations were conducted using the ground-state (GS) keto form of the UPy dimer.\nThis form represents the thermodynamically stable configuration underground state conditions, as\nfurther corroborated by the results in Figure R11.\n(4) The enol forms (Enol1 and Enol2) are photogenerated species that predominantly exist in the\nexcited state (ES) following excited-state proton transfer (ESPT). Their lifetimes are on the order\nof picoseconds, as evidenced by our transient absorption spectroscopy data. Conventional MD\nsimulations, which operate on nanosecond to microsecond timescales under thermal equilibrium\nconditions, are not suitable for capturing these short-lived excited-state tautomer.\nWe have revised the manuscript and explicitly stating the use of the keto form in the MD\nsimulations, as follows: “MD simulations based on keto form of UPy-DHDI were used to study the\naggregated structure of dimers and tetramers, revealing the influence of aggregated structure of\nchromophores for fluorescent properties.”\nFigure R12. One structure of UPy-DHDI dimers extracted from the MD simulation, with the red\ndotted line representing the H-bonds.\n5.Why are there such harsh thresholds on the emission spectra of UPy-DHI (figure 7b in SI)? Were\nthe spectra recorded over the entire wavelength range?\nReply: Since the tail of the excitation light may interfere with the fluorescence signal of the sample,\na strict threshold cutoff was applied to the detection wavelength to minimize this effect and enable\nnormal detection of the sample's emission signal. However, due to the fact that the emission signal\nfrom the UPy-DHI sample is weaker than the tail signal of the excitation light, the aforementioned\n\"harsh thresholds\" were employed. The spectrum covers the wavelength range where sample\nemission is present.\n6.Why was hexane used for MD simulations and not DMF or chloroform?\nReply: In molecular dynamics (MD) simulations, the use of a poor solvent is necessary to simulate\naggregated states such as dimers or tetramers. In experiments, n-hexane acts as a poor solvent for\nthe system, while DMF and chloroform are good solvents. Therefore, n-hexane was selected as the\nsolvent for the simulations.\n7.On figure 2a, the second ESPT appears as reversible (double arrow) but not the first one (simple\narrow). Why? Is it due to energetic considerations? This can be added to the figure and/or the text\nif the authors have the energy barriers or an approximation of it.\nReply: Since our previous ultrafast spectroscopic experiments did not detect signals corresponding\nto the enol1→enol2 transition, we initially hypothesized that this process might involve ultrafast\nrelaxation and thus represented it with a reversible arrow in the schematic diagram. In the revised\nmanuscript, following reviewer1's suggestion, we successfully detected the enol1-enol2 transition\nsignal using transient fluorescence spectroscopy (Fig. R10), confirming that the process is\nirreversible. Accordingly, we have replaced the reversible arrow with an irreversible one in the\nrevised schematic.\n8.Are the Enol2 dimers also stable as there are only 2 intermolecular H-Bonds, compared to 4 H-\nBonds for the keto/enol1 dimers?\nReply: The enol2 configuration is indeed less stable than enol1 in the excited state, representing an\nunstable structure, which is consistent with our experimental observations. Although it possesses\nfewer hydrogen bonds compared to the keto and enol1 configurations, its higher degree of\nconjugation provides the driving force for the enol1→enol2 transition.\nTypos:\nFigure 5: (e) Plots of PL intensity versus temperature (?) for PU-UPy under 365 nm UV light\nirradiation.\nSpectral characterization revealed that UPy-DHI exhibited a quantum yield below 10% and\ndisplayed excitation wavelength-dependent fluorescence emission (Supplementary Figs. 10b, d). →\nThere is no Fig 10b, d in SI.\nFigure 12 in SI. Graph b does not represent the emission spectra at diHerent wavelength but at\ndiHerent concentration.\nFigure 9b in SI. Unit of the y axis should be kcal mol-1\nReply: We thank the reviewer for the thorough review of our manuscript. The diligent work has\nbrought to our attention several minor errors and oversights in the original version, which greatly\ncontributed to improving the quality of the paper. In the revised manuscript, we have carefully\nreviewed and corrected the issues mentioned above."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authers have resovled my concenrd issues, I think this work can be pulished", "reviewer_2": "Zuo and co-authors did a great work considering the comments and questions of both reviewers.\nEspecially, they completed the experimental part going upon the test of biocompatibility of their systems, demonstrating they\nare non cytotoxic and that they can be used in biomedical applications but they also added time-resolved emission\nspectroscopy to prove the existence of a particular enol form.\nThe information they added, for example about the energy barriers for ESPT, support the affirmations within the manuscript.\nThe clarifications about some technical details regarding the calculations will also help the future readers and finally all the\nquestions were adressed and the missing information added to the manuscript.\n\n======================================================================\nATTACHED REVIEWER REPORT — IMPORTANT\nThe text below is a complete reviewer report submitted as a PDF\nattachment. It appears after the Creative Commons license block.\nAssign it to whichever reviewer wrote 'see pdf' or had no inline text.\n======================================================================\n\nThe manuscript by Zuo et al deals with unexpected fluorescence properties induced by a\nwell know moiety (ureidopyrimidinone, UPy) within a polymer. A combination of\nexperimental (synthesis, spectroscopies, various applications) and theoretical (TD-DFT,\nMD) approaches has been convincingly provided to present, rationalize and prove the\neHiciency and the potential of such systems. Very interestingly, they propose a\nmechanism involving tautomerization and excited state proton transfer to explain the\nboost in fluorescence eHiciency while the restriction of motions is often argued to explain\nit. Please find below some questions and comments I had after reading the manuscript.\nSome of them should be taken into account to clarify or precise some points within the\nmain text.\nComments & questions:\n- As no details about the TD-DFT calculations are provided (oscillator strength,\nnature and description in terms of orbitals/involved states), does the maximum\nabsorption wavelength corresponds to S state, as only the S was optimized to\n1 1\nretrieve the emission wavelength? Are HOMO and LUMO the only orbitals involved\nwithin the transition?\n- How was the curve of figure 9b in SI obtained? Was it a simple scan (relaxed or\nnot?) with potential energies reported or is it a “real” energy barrier with frequency\ncalculations involved? This should be clarified within the manuscript and SI.\n- Does the tautomerization process take place at the ground state (GS), towards the\nenol form, or only in the excited state (ES)? Is there a competition between the\ntautomerizaton at the GS and the ES? In the excited state, following figure 9b in SI,\nthe keto form seems to be more stable than the enol form, is it compatible with the\nfollowing steps?\n- Are the 4 H-bonds always eHective during MD simulations? What are the extra H-\nbonds observed? Do they correspond to “peripheric” H-bonds? It should be stated\nthat the keto form was considered for MD simulation. Was the MD simulation of\nthe keto form done with the GS or ES structure (if the structures/charges/density\nrepartition are diHerent between GS and ES)? Why the enol1 and enol2 were not\nconsidered during MD simulations?\n- Why are there such harsh thresholds on the emission spectra of UPy-DHI (figure\n7b in SI)? Were the spectra recorded over the entire wavelength range?\n- Why was hexane used for MD simulations and not DMF or chloroform?\n- On figure 2a, the second ESPT appears as reversible (double arrow) but not the first\none (simple arrow). Why? Is it due to energetic considerations? This can be added\nto the figure and/or the text if the authors have the energy barriers or an\napproximation of it.\n- Are the Enol2 dimers also stable as there are only 2 intermolecular H-Bonds,\ncompared to 4 H-Bonds for the keto/enol1 dimers?\nOverall, the manuscript is dense in results, the explanations are convincing, and it\nprovides new results about a multifunctional fluorescent polymer, and I recommend its\npublication after minor corrections/precisions (see above).\nTypos:\nFigure 5: (e) Plots of PL intensity versus temperature (?) for PU-UPy under 365 nm UV light\nirradiation.\nSpectral characterization revealed that UPy-DHI exhibited a quantum yield below 10%\nand displayed excitation wavelength-dependent fluorescence emission (Supplementary\nFigs. 10b, d). → There is no Fig 10b, d in SI.\nFigure 12 in SI. Graph b does not represent the emission spectra at diHerent wavelength\nbut at diHerent concentration.\nFigure 9b in SI. Unit of the y axis should be kcal mol-1"}, "author_response": ""}]

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