Non‐uniform Photoinduced Unfolding of Supramolecular Polymers Leading to Topological Block Nanofibers

Abstract Synthesis of one‐dimensional nanofibers with distinct topological (higher‐order structural) domains in the same main chain is one of the challenging topics in modern supramolecular polymer chemistry. Non‐uniform structural transformation of supramolecular polymer chains by external stimuli may enable preparation of such nanofibers. To demonstrate feasibility of this post‐polymerization strategy, we prepared a photoresponsive helically folded supramolecular polymers from a barbiturate monomer containing an azobenzene‐embedded rigid π‐conjugated scaffold. In contrast to previous helically folded supramolecular polymers composed of a more flexible azobenzene monomer, UV‐light induced unfolding of the newly prepared helically folded supramolecular polymers occurred nonuniformly, affording topological block copolymers consisting of folded and unfolded domains. The formation of such blocky copolymers indicates that the photoinduced unfolding of the helically folded structures initiates from relatively flexible parts such as termini or defects. Spontaneous refolding of the unfolded domains was observed after visible‐light irradiation followed by aging to restore fully folded structures.


Introduction
Modern preparation methods of polymer has enabled us to synthesize av ariety of polymers with unique primary and higher order structures. [1] Such well-designed synthetic polymers are useful not only as functional soft materials wherein their collective behaviors are important, but also as more single-chain nanomaterials that can function like biomacromolecules. [2] Especially block copolymerization techniques allow the synthesis of polymers with distinct structural domains (topologies) in am ain chain like proteins,w hich is crucial to develop polymers as discrete nanomaterials. [3] For supramolecular polymers (SPs), an emerging noncovalent counterparts of polymers, [4] the construction of such "topological" block or "topologically" blocky supramolecular copolymers is particularly challenging because of inherently dynamic nature in monomer binding.I no ther words,t he monomers affording different higher-order structures generally have different molecular structures,a nd accordingly it may be difficult to keep them connected in athermodynamically stable state through non-covalent interactions (heterorecognition). In fact, while several groups have recently reported elegant examples of block or blocky SPs based on thermodynamic [5] or kinetic approaches, [6] all of them have one-dimensionally extended structures.A sa ne xceptional example,werecently reported block supramolecular copolymers consisting of helically folded extended domains by kinetically controlled gradient supramolecular copolymerization of the two molecules with similar chemical structures but affording SPs with distinct higher order structures. [7] However,bottom-up design of such topological block SPs remains af ormidable challenge from the viewpoint of the above compatibility between higher-order structures and heterorecognition, which will limit monomer design to those leading to analogous one-dimensional structures.
We envisage that post-polymerization structural transformation, [8] as has already been applied for covalent polymers,w ould be another strategy to prepare topological block SPs.A sabasis that can verify this idea, we invoke the photoresponsive of monomer 1 we have reported in 2017 ( Figure 1a). [9] This barbiturated azobenzene monomer forms helically folded SPs (SP fold )i nn onpolar media through the formation of six-membered hydrogen-bonded rosettes (Figure 1b). Theh elically folded structure is ar esult of continuous generation of intrinsic curvature upon stacking of the rosettes [10] with translational and rotational displacements. [11] One of the unique features of the SP fold of 1 is unfoldability to linearly extended structures by UV-light, which is due to the perturbation of the intrinsic curvature by the generation of sterically demanding cis-azobenzene units. [12] Importantly,our AFM study showed that the photoinduced unfolding proceeded uniformly throughout the entire fiber, providing unfolded SPs with curvature (SP unfo )a si ntermediate structures ( Figure 1c). Theuniform unfolding suggests that the SP fiber of 1 is flexible enough to allow deformation of the curvature by cis-azobenzene units even in tightly folded internal domains.
Based on the above mechanism, we expected that SP fold consisting of am ore rigid SP fiber allows "non-uniform" photoinduced unfolding,and can provide blocky structures as an intermediate state ( Figure 1d). We thus designed and synthesized new monomer 2 in which azobenzene unit was embedded in ar igid p-conjugated backbone ( Figure 1a). As we will show in this paper, SP fold of 2 shows significant resistance to the photoinduced structural deformation at ambient temperature.A th igh temperature,h owever, photoinduced unfolding proceeds non-uniformly,l eading to topological block SPs consisting of folded and unfolded domains as intermediate structures.O ur molecular dynamics simulations demonstrate that photoisomerization of azobenzene units occurred throughout entire main chains,w hile high inner rigidity led the cooperative unfolding.

Results and Discussion
Monomer 2 was synthesized according to the procedure described in the Supporting Information, and characterized by 1 H-and 13 C-NMR spectroscopies,a nd APCI-MS spectrometry. 1 HNMR demonstrated that the azobenzene unit of as-synthesized molecule 2 was > 99.9 % trans-isomer (Figure S4a). Upon cooling ahot MCH solution of monomeric 2 (c = 10 mM) from 373 to 308 Ka nd subsequently heating at ar ate of 1.0 Kmin À1 ,achange of an absorption shoulder at 465 nm was reversibly observed, indicating aggregation at low temperatures ( Figure 2a). When the temperature-dependence of this new band (l = 465 nm) was monitored as a agg , cooperative (nucleation-elongation) supramolecular polymerization [13] was observed for both cooling and heating curves but with different critical temperatures (T e and T e , Figure 2b). Agradual increase of a agg above T e' upon cooling is probably related to pre-nucleation including conformational change of the p-conjugated system such as planarization. [14] Thethermal hysteresis in our system is mainly caused by the formation of diverse hydrogen bonding species during cooling process,a mong which only discrete cyclic species (rosettes) can nucleate to form SPs ( Figure S5). [15] Atomic force microscopy (AFM) visualized densely folded SP fold ( Figure S6). Theaverage radius of curvature (r ave ), measured by manually fitting acircle with radius r along each curve,was 10.4 AE 0.2 nm ( Figure S7a).
When the above SP fold solution was irradiated with UVlight (l = 365 nm;17WLED lamp) at 308 K, the absorption intensity around 391 nm which is attributable to the p-p * transition of the azobenzene unit decreased (Figure 2c), indicating trans!cis photoisomerization of the azobenzene unit. Photostationary state (PSS) was achieved within 10 min, at which the percentage of cis-isomer was estimated to be 19 %f rom ac ontrol experiment using 1 H-NMR ( Figure S8). This ratio indicates that statistically one or two trans-isomers of 2 per rosette isomerized to the cis-isomers,which is similar to 1 under the same condition. For 1,t his low isomerization ratio was enough to unfold spiral structures into linearly extended fibers. [9] However,n om orphological change was observed for SP fold of 2 even after prolonged UV-light irradiation for 60 min at 308 K( Figure 2d). Because our SPs are composed of stacked rosettes,t he rigidity of monomer structures directly affect the internal rigidity (shape-persistency) of curved SPs.A ccordingly,m ore rigid monomer 2 should provide more rigid SPs in comparison with 1,g iving rise to ac lear difference in the persistence of curvature toward the trans-to-cis isomerization of the azobenzene units.
In order to unfold SP fold by light, UV-light irradiation was attempted at ah igher temperature at which unfolding is entropically more favorable process (Figure 3e). Theheating curve in Figure 2b shows that the dissociation does not occur significantly at 323 K. At this temperature,t he absence of thermal unfolding was confirmed by AFM (Figures 3b and  S9). When the SP fold solution was irradiated with UV-light at 323 K, PSS was achieved by an initial 10-min irradiation, affording 21 %o fcis-isomer (blue line in Figure 3a). AFM observation revealed that the majority of the SP fold have been transformed into topological block structures composed of helically folded and unfolded domains although fully folded SP fold and completely unfolded SP unfo were also observed (Figures 3f-r). Figures 3j-o are AFM images of blocky SPs of which folded and unfolded domains were colored with red and blue,respectively (the original AFM images were shown in Figure S10). Thefractions (%) in lengths of each domain in the topological block SPs were also provided in Figure 3p. Non-uniformity of domain fraction by SPs in combination with the coexistence of intact SP fold (Figure 3f,g) and the fully unfolded SP unfo (Figure 3q,r) strongly suggests that the photoinduced unfolding proceeds cooperatively in individual SP fibers.T he results also imply that the SP main chains initially resist unfolding,b ut they surrender themselves to unfolding once photoinduced deformation occur. Such ac ooperative structural transition reflects the internal rigidity of the SP main chain of 2 in comparison with that of 1 showing the uniform unfolding. [9] It is worthy to note that the unfolding of SP fold of 2 does not necessarily occur from termini as unfolded domains could be observed between the helically folded domains.W epostulated that unfolding could occur also from defected sites, [16] which in our SPs correspond to locally misfolded domains. [17] Prolonged UV-light irradiation of the topological block SP solution at 323 Kfor 60 min afforded fully unfolded SP unfo as confirmed by dynamic light scattering (DLS), in situ small angle X-ray scattering (SAXS) and AFM (Figures 3c,d, S11). DLS measurements before and after the extended UV-light irradiation revealed that the polydispersity index (PDI) of SPs became larger from 0.284 to 0.373 ( Figure S11), suggesting that the transformation from compact to dispersed structures.I nt he SAXS measurements,t he nonperiodic oscillatory features of SP fold that is ac haracteristic of the static intrinsic curvature became unobservable after the UVlight irradiation (Figure 3d). Analysis of the SAXS data of the SP fold ,approximating the helically folded fibers as cylindrical objects with three outer shells (1 = alkyl, 2 = rosette core,3= alkyl), and aLorentzian peak function to represent the helical pitch [18a] gave ah ollow central radius r core = 7.4 AE 0.1 nm and thicknesses (d)ofthe three shells as d 1 = 1.4 nm, d 2 = 4.8 nm, and d 3 = 0.3 nm. This is equivalent to r ave = 11.2 AE 0.2 nm, similar to that found by AFM (r ave = 10.4 AE 0.3 nm). Thepeak position at Q = 0.51 nm À1 equates to apitch of 12 nm. TheXray contrast between the alkyl chains of 2 and the solvent is low,s oS AXS cannot easily distinguish the two.T his,a nd solvent penetration, [18b] explains the lower values of d 1 and d 3 than expected for af ully stretched dodecyl chain (1.7 nm). Thevalue for d 2 ,w hich equates to the rosette diameter, is in line with dimensions obtained by SAXS for similarly sized rosette-forming molecules in previous studies. [18b,c] After UVlight irradiation, the characteristic scattering peaks significantly weakened, indicating deterioration of loop structure. in MCH upon cooling from 373 to 308 Katarate of 1.0 Kmin À1 .T he cooling was ceased at 308 Kt oavoid precipitationu pon further cooling to room temperature. b) Cooling (blue) and heating (red) curves of 2 (c = 10 mM) at arate of 1.0 Kmin À1 obtained by plotting degree of aggregation a agg (calculated from absorption change at 465 nm) as afunction of temperature in MCH. c) UV-Vis absorption spectra of SP fold of 2 in MCH before and after UV-light irradiation at 308 Kf or 60 min. d) AFM image of the SP fold of 2 spin-coated onto highly orientedp yrolytic graphite (HOPG) after UV-light irradiation in MCH at 308 Kf or 60 min.
In line with this,AFM visualized that the SP unfo lack any trace of helically folded domains.I mportantly,u nlike to 1, [9] unfolding up to linearly extended fibers lacking intrinsic curvature was not observed for 2 even after further prolonged UV-light irradiation. AFM analysis of the SP unfo revealed the presence of curvature with r ave of 10.6 AE 0.3 nm, which is almost comparable to that of SP fold ( Figure S7b). This result also reflects the rigid molecular structure of 2,b yw hich the curvature of SPs becomes persistent to the internal perturbation induced by the photoisomerization of the azobenzene unit.
It is worthy to note that the above transformation from topological block SPs to SP unfo proceeded while keeping the constant amount of cis-isomer (21 %) as is evident from no absorption change upon UV-light irradiation at 323 K ( Figure 3a).Namely,t he trans!cis photoisomerization of the azobenzene units drives the unfolding of the main chain, but the generated cis-isomers are smoothly reconverted thermally to the trans-isomers.
Thermodynamic parameters of the SP fold and the photogenerated SP unfo were estimated from the thermal dissociation experiments using UV-Vis spectroscopy in order to gain insight of the impact of temperature on the unfolding.T he non-sigmoidal thermal dissociation curves of SP fold and SP unfo composed of 2,i nw hich all azobenzenes were trans-isomers, recorded at several concentrations (c = 10, 15, 20, and 25 mM), could be fitted with an ucleation-elongation model (Figure S12), [19] and the resulting elongation temperature (T e ) Figure 3. a) UV-Vis spectra of aMCH solution of 2 (c = 10 mM) during UV-light irradiation at 323 K. b,c) AFM images of SP fold of 2 before UVlight irradiation (b) and SP unfo of 2 after UV-light irradiation for 60 min at 323 K(c). d) Change of SAXS profiles of a SP fold solution of 2 (c = 50 mM) upon UV-lighti rradiation at 323 K( from red to blue). The black line is af it to the data using acore-multishell cylinder model. e) Schematic representation of procedure on photo-induced unfolding of SP fold of 2 (f-o,q,r) AFM images of SP fold (f,g), topological block SPs (h-o), and SP unfo (q,r) found in asolution of 2 upon UV-light irradiation for 10 min at 323 K. In (j-o), helically folded and unfolded domains were colored with with red and blue, respectively.p )Fractions in length of helically folded and unfolded domains in the topologicalb lock SPs.
were used to make the modified vantH off plot (Figure S13). [20] Thechanges of standard enthalpy (DH8 8), entropy (DS8 8), and Gibbs free energy (DG8 8)o btained from the plot were summarized in Table 1. Both DH8 8 and DS8 8 of SP unfo are significantly smaller than those of SP fold ,which is ascribable to the stabilization of helically folded structures by interloop van der Waals interactions between alkyl chains. [18a, 21] Thed ifference of DG8 8 values (= DG8 8 (SP unfo )ÀDG8 8 (SP fold )) were 10.3 and 2.7 kJ mol À1 at 308 and 323 K, respectively.S uch large change of DG8 8 against temperature is because of entropic effect. As described above,the no unfolding proceeded under UV-light irradiation at 308 K. These results indicate that the entropic effect is also important factor although main driving force of the unfolding is the large structural change of rosettes.I ndeed, the photo-unfolded SP unfo spontaneously refolded into SP fold upon aging at 308 Kf or 18 h, supporting the drastic impact of entropic effect ( Figure S14).
To obtain am olecular-level insight into the observed inhomogeneous photoinduced unfolding of the SP fiber of 2, we conducted high-resolution molecular simulations for polymeric rosette stacks of 1 and 2 in native unperturbed conditions as well as upon transition of the excited azobenzene units.Recently,molecular models of aphoto-responsive supramolecular tubule allowed to observe that the isomerization of the azobenzene units contained in the self-assembling monomers under UV-light irradiation occurs and proceeds at defected sites in the tubule structure. [16a] Less ordered domains and defects,which are in principle unavoidably present in soft self-assembled materials such as SPs, constitute spots where the transitions of excited units are more probable to occur. [16a] Thecascade of isomerization that tends to localize in proximity of such less defined domains in the assemblies may then provoke an on-uniform isomerization of excited groups over time,a nd ac ooperative response by the assembly structure.
Here we used fully atomistic molecular dynamics (MD) simulations to obtain insights on the mechanism by which the azobenzene isomerization occurs in the SPs.F irst, we built atomistic models of SP fibers of 1 (SP1)a nd 2 (SP2), composed of 192 monomers initially arranged in 32 perfectly pre-stacked rosettes.T hese initially extended fiber models have been then equilibrated via 1 mso fM Ds imulation in explicit MCH solvent at 297 K( see Supporting Information for details on the models and simulations). From these MD runs,w eo btained equilibrated structures of SP1 and SP2 (Figures 4a,b). While the two fibers start from the same configuration, SP2 was observed to be slightly longer and more persistent at the MD equilibrium than SP1,c onsistent with ahigher level of internal rigidity of SP2.Weanalyzed the environment that surrounds the azobenzene units in the two ordered-domain fibers represented by our models.W e calculated the distributions of the azobenzene units in the two equilibrated fiber sections in terms of contacts between them (only azobenzene units) and contacts with the entire surrounding monomers in SP1 and SP2 (Figure 4c,d). The average contact values for the azobenzene units in the two fibers are identified by the black lines in the distribution plots. Theslightly lower average values and the broader distribution obtained for SP1 compared to SP2 suggests as lightly tighter and more ordered packing of the azobenzene units in SP2 compared to SP1.T his can be related to the higher atomic density surrounding the azobenzene units,which is related to their shorter radial distance from the center of the fiber in SP2.F or such geometrical reasons,i nSP1,t his provokes as lightly reduced stacking between the azobenzene units compared to SP2.
Previous studies demonstrated that the degrees of molecular crowding in the environment surrounding the azobenzene units can have an important effect on how the excited chromophores isomerize in the stack:Itcan occur randomly if the crowding effect is reduced whereas it can occur in spatially localized manner if the crowding effect is dominant. [16a] To obtain indications on how the isomerization most likely accumulate in the stack, we used previously validated atomistic model of excited azobenzenes. [22] We investigated the isomerization of the azobenzene units that are extreme in the obtained azobenzene crowding distributions-i.e., those azobenzene groups which are more loosely (blue points, slightly lower contacts than the average) or more tightly (green points in the distributions,slightly higher contacts than the average) packed in the assembled fibers.T he MD simulations showed that the azobenzene units having the highest number of contacts,i fe xcited, have ac haracteristic isomerization timescale that is just $5times slower than that of the azobenzene units with the lowest number of contacts (Figures 4e,f). TheP oisson distributions related to the azobenzene units with higher contacts (Figure 4e,f:s olid curves) are translated on the right (slower kinetics) compared to those of the loosely packed azobenzene units (dashed curves). Black lines in Figures 4e,f identify the characteristic transition times calculated by the cumulative Poisson fits of azobenzene transitions occurring in such molecular models of ordered helical sections of the SPs.I na ll cases,t he MD simulations provided characteristic transition timescales for excited azobenzene units below the nanosecond scale.Inthis model of an ordered section of SP1,t he characteristic isomerization timescale for the excited azobenzene units was found to be in the order of picoseconds,v ery similar to that expected for free/disassembled azobenzenes in solution. [22] This suggests that in SP1,t he eventual presence of defects or less ordered domains than the ones present in such ordered models (likely present in the real systems in such soft SP fibers), will have little impact on how the transitions accumulate in the assembly:i.e., in all cases the isomerization would occur randomly along SP1.T his is consistent with the rather uniform reorganization of SP1 upon light excitation seen in the experiments.Onthe other hand, the isomerization in SP2 occurred with as lower characteristic timescale (i.e., they are less probable) within the ordered section model (Figure 4evs.Figure 4f). This suggests that, in SP2,defects or less ordered domains where the azobenzene units are less tightly packed than in these ordered SP sections (and where the isomerization may occur faster), constitute spots along these assemblies where the isomerization are more favored to accumulate.T his is consistent with the cooperative and nonuniform reorganization of SP2 upon light excitation seen in the experiments. Finally,s tarting from ap reequilibrated structures of the SP fibers (Figure 4a,b), we also carried out MD simulations where 20 %( experimentally observed value at PSS) of the trans-azobenzene units in SP1 and SP2 were converted into cis-isomers (Figure 4g,h: cis isomers in green). Such 20 % cis-SP models were then simulated via MD.Analysis of the global SP structures during the simulations demonstrates that, following to the azobenzene isomerization, the fiber length of SP1 changed over time (Figure 4i:c yan vs.b lue curves). Conversely,t he structure of SP2 remained substantially unchanged (Figure 4j:o range vs.r ed curves). This observation reflects very well the higher rigidity of SP2,and provides ar ational explanation for the experimentally observed nonuniform and cooperative UV-induced unfolding of the SP fold of 2,w hich afforded topological block SPs.

Conclusion
We have demonstrated an ovel strategy to synthesize topological block supramolecular polymers consisting of two distinct higher-order structures,helically folded and unfolded domains.O ur strategy is based on post-polymerization photoinduced unfolding of helically folded supramolecular nanofibers consisting of six-membered hydrogen-bonded rosettes of barbiturates with azobenzene photoswitches.T o achieve non-uniform unfolding of helically folded structures upon isomerization of internal azobenzene units,t he previously designed flexible monomer structure was modified to amore robust structure,bywhich supramolecular nanofibers became more curvature-persistent toward azobenzene isomerization. This monomer-based improvement in the robustness of supramolecular polymer chains has been reproduced by MD simulations of fibers consisting of the previous flexible and the current robust monomers upon isomerization of their azobenzene units.T he improved robustness of supramolecular polymer chains realized non-uniform unfolding of helically folded structures into fully unfolded structures through intermediate topological block structures.O wing to the improved curvature-persistency, spontaneous re-folding of the fully unfolded structures has been observed unlike the Figure 4. a,b) Equilibrated MD snapshots of SP1 (a) and SP2 (b) after 1 msofMDsimulation.B arbiturate groups are colored in blue for SP1 and in red for SP2,r espectively,t he (trans)azobenzeneu nits are colored in black. The rest of the monomers is colored in shaded gray. c,d) Distribution of the azobenzene units based on their interactions/contacts with the surrounding environment:t he x axis reports the number of contacts of the azobenzenes with the other the azobenzeneunits in the fibers, while the y axis reports the numbero fcontacts between the azobenzeneunits and the rest of the monomers. The average is indicated by the blue and red points (average contacts values identified by the black lines). Isolines identify those configurations within 0.5 kcal mol À1 (inner isolines) and 1.0 kcal mol À1 (outer isolines) of free energy penalty from the average (most favorable state) for SP1 (c) and SP2 (d). The monomers showing higher number of contacts and lower numberof contacts are shown as green and blue points, respectively (the other monomers closer to the average are shown in orange). (e,f)Poissonian fits of the isomerization times for excited azobenzeneunits with high (solid line) and low contacts (dashed line) in SP1 (e) and SP2 (f). From the Poisson fits it is possible to calculate the characteristic timescale for the isomerization in the cases of low and high contacts for SP1 and SP2 (black vertical lines intercepting the x axis on the characteristic timescales).g ,h) Equilibrated structures of SP1 (g) and SP2 (h) with 20 % cisisomerized monomers (cis azobenzeneingreen) after 1 msofMDsimulation.i ,j) Variation (in %) of the fiber lengths along the MD simulations calculated respect to the average lengths of the non-isomerized pre-equilibrated SP1 (i)a nd SP2 (j).
previous system. Our study thus demonstrates unprecedented nanofabrication of one-dimensional nanomaterials through the post-self-assembly partial transformation of their structures.W eb elieve that the present study will motivate researchers to develop more precise nanofabrication of supramolecular soft materials.