Porphyrin tripod as a monomeric building block for guest-induced reversible supramolecular polymerisation

Reversible supramolecular polymerisation and depolymerisation of biomacromolecules are common and fundamental phenomena in biological systems, which can be controlled by the selective modication of biomacromolecules through molecular recognition. Herein, a porphyrin tripod (DP Zn T) connected through a triazole bridge was prepared as a monomeric building block for guest-induced supramolecular polymerisation. Although the lone pair electrons in triazolic nitrogen potentially bind to the zinc porphyrin units through axial ligation, the intrinsic steric hindrance suppressed the coordination of the triazole bridge to the porphyrin unit in DP Zn T. Therefore, DP Zn T formed spherical nanoparticles through π-π interactions. The addition of 1,3,5-tris(pyridine-4-yl)benzene (Py 3 B) caused the guest-induced brous supramolecular polymerisation of DP Zn T by forming a 1:1 host-guest complex, which was further assembled into a brous polymer. Furthermore, addition of Cl − to DP Zn T induced the transformation of spherical nanoparticles to brous supramolecular polymers. The brous supramolecular polymers of DP Zn T obtained by adding Py 3 B or Cl − were depolymerised to their original spherical particles after adding Cu(ClO 4 ) 2 or AgNO 3 , respectively.


Introduction
Reversible supramolecular polymerisation and depolymerisation, controlled by the selective modi cation of biomacromolecules through molecular recognition processes, are common and fundamental phenomena in biological systems [1][2][3][4][5][6][7] . These guest-induced supramolecular polymerisations are of great interest because of their important roles in the biological systems. The growth of microtubules and actin laments is a representative example of guest-induced brous supramolecular polymerisation 3,5,7 . The growth and degradation of microtubules are initiated by the binding of GTP and hydrolysis of phosphate, respectively. Similarly, the binding of ATP to G-actin initiates the polymerisation of actin to form micro laments, which are F-actin. Supramolecular polymerisations with signi cantly more complicated structural transformations compared with brous assemblies are also often found in nature. For example, clathrin with a triskelion structure is polymerised into a networked structure by recognising adaptor proteins to form clathrin-coated membranes. 8 Although several examples of controlled supramolecular assembly growth and guest-induced supramolecular polymerisation have been reported, [9][10][11][12][13][14][15][16][17][18][19] the reversible transition of self-assembled structures during supramolecular polymerisation has not yet been actively studied. Therefore, it is still challenging to develop arti cial self-assembled structures that mimic both multivalency and morphological transitions observed in natural systems. From this perspective, we demonstrate the guest-induced supramolecular polymerisation and depolymerisation accompanied by morphological changes from spherical particles to brous nanostructured polymers. Morphological changes were realised through various noncovalent interactions such as π-π interaction, metal-ligand coordination, and hydrogen bonding. In this study, we prepared a porphyrin tripod (DP Zn T; Figure 1) that formed spherical nanoparticles owing to intrinsic structural restriction. The triazole group in DP Zn T did not form an axial coordination complex with the zinc porphyrin units in the neighbouring DP Zn T. The spherical nanoparticles of DP Zn T transformed into brous or networked supramolecular polymers upon adding guest molecules, depending on the guest species. Furthermore, the supramolecular polymers dissociated into spherical nanoparticles after removing the guest species. This rationally designed porphyrin tripod enabled us to mimic the supramolecular polymerisation and depolymerisation occurring in natural biomolecules.

Results And Discussion
Spherical nanoparticle formation of DP Zn T The synthesis of porphyrin tripods was conducted by a copper-catalysed click reaction between 1,3,5tris(azidometyl)benzene and ethynyl-bearing porphyrin derivatives. As control compounds for DP Zn T, the freebase and copper-coordinated forms of porphyrin tripods (DP FB T and DP Cu T, respectively; Fig. 1) were prepared to investigate the effect of axial coordination. Several structural fragments of DP Zn T, DPP Zn , TB, m-TB, and TC were also prepared for control experiments. The details of the synthetic procedures and characterisation of porphyrin tripods and compounds used in this study are summarised in the Supplementary Information (Supplementary Scheme 1).
The Lewis acidic zinc porphyrins generally show a strong binding a nity to the lone pair electrons in triazolic nitrogen to form axial coordination complexes. 20 The binding of axial ligands causes a bathochromic absorption shift of zinc porphyrins; however, the Soret absorption band of DP Zn T appeared at approximately 412 nm, which is consistent with that of DPP Zn ( Fig. 2A). Therefore, the UV/vis absorption spectroscopic observation of DP Zn T indicated the absence of axial coordination interactions between the triazole and porphyrin units. UV/vis titrations of TB and m-TB to DPP Zn in toluene were performed to elucidate the absence of coordination interactions between the triazole and zinc porphyrin units in DP Zn T. When TB was added to DPP Zn , the absorption did not change even after the addition of 13,000 equivalents of TB ( Supplementary Fig. 1A). In contrast, upon the successive addition of m-TB (0-9,600 eq.), the Soret absorption band of DPP Zn exhibited a signi cant bathochromic shift with the existence of clear isosbestic points at 414.5, 543.5, and 575.0 nm (Supplementary Fig. 1B-C). The computer aided modeling of TB showed coplanar geometry between the triazole and the neighbouring phenyl group (Fig. 2B). These observations indicate that the triazole moiety with the neighbouring phenyl group cannot be coordinated to zinc porphyrin because of steric hindrance. Although DP Zn T did not show a bathochromic absorption shift, a relatively large shoulder at approximately 400 nm was observed owing to the π-π stacking of the porphyrin units. 21,22 However, the intensity of the shoulder near 400 nm increased when a small amount of n-hexane (15%) was added to the solution of DP Zn T in toluene, which could be attributed to the enhanced molecular interaction in DP Zn T ( Fig. 2A). The solution of DP Zn T in toluene was spin-coated onto a freshly cleaved mica surface, and atomic force microscopy (AFM) was performed to observe the morphological aspects of DP Zn T. The AFM image showed spherical nanoparticles with a height of approximately 3 nm (Fig. 2C). The formation of nanoparticles was also observed when DP Zn T was spin-coated onto highly oriented pyrolytic graphite (HOPG) ( Supplementary   Fig. 2). However, the average height of the nanoparticles was slightly shorter than that observed in the AFM specimen prepared on the mica substrate. The attening of nanoparticles can explain the decreased average height of the nanoparticles on HOPG because of the high a nity of the alkyl chains to HOPG. 23,24 Grazing-incidence wide-angle X-ray scattering (GIWAXS) was conducted on the drop-cast lms of DP Zn T. Thus, the in-plane and out-of-plane diffraction of DP Zn T overlapped well (Fig. 2D), indicating the absence of angular dependency in the wide-angle region owing to the formation of spherical nanoparticles.
Because DP Zn T formed spherical nanoparticles, the morphological aspects of DP FB T and DP Cu T, noncoordinatable derivatives of DP Zn T, were again measured by AFM. The AFM images of both DP FB T and DP Cu T also showed the formation of spherical nanoparticles ( Supplementary Fig. 3), indicating that the coordination interaction does not contribute to the formation of spherical nanoparticles. Similar to DP Zn T, the UV/vis absorption spectra of both DP FB T and DP Cu T exhibited a shoulder in the blue-shifted region of the Soret absorption of DPP FB and DPP Cu , respectively, indicating that the driving force for the spherical particle formation of porphyrin tripods is π-π interactions among the porphyrin units. Guest-induced supramolecular polymerisation of DP Zn T The guest-induced supramolecular polymerisation of DP Zn T was investigated (Fig. 3A). Considering the three zinc porphyrin units in DP Zn T, we envisaged that DP Zn T could also form a host-guest complex with 1,3,5-tris(4-pyridylbenzene) (Py 3 B; Fig. 1) through axial coordination interactions. [25][26][27] The UV/vis titration of DP Zn T on the addition of Py 3 B showed distinct spectral shifts with isosbestic points at 412, 541, and 575 nm (Fig. 3B). The binding isotherm recorded at 417 nm showed that the absorption reached saturation upon adding 1 equiv. of Py 3 B, indicating a strong binding a nity of Py 3 B toward DP Zn T (Fig.   3C). Although it is di cult to estimate the binding constant between Py 3 B and DP Zn T because pristine DP Zn T forms spherical nanoparticles, the apparent binding constant exceeds the upper limit (K > 10 8 ), which can be measured by the absorption changes. 28 Analysis using the continuous variation method (modi ed Job's plot analysis) suggested the formation of a 1:1 host-guest complex between DP Zn T and Py 3 B (DP Zn T•Py 3 B) ( Supplementary Fig. 4). [29][30][31] Energy-minimised molecular modelling indicates that DP Zn T•Py 3 B adopts a cone-shaped geometry, wherein the porphyrin units of DP Zn T are exposed to the outer environment. Therefore, the host-guest complex could undergo further aggregation to avoid unfavourable exposure to the exterior solvent molecules, similar to our previous study. 32 Transmission electron microscopy (TEM) images showed the formation of brous supramolecular polymers of ca. 4.0 nm width (Fig. 4D). In addition, AFM images of DP Zn T with 1 equiv. of Py 3 B demonstrated brous supramolecular polymer formation ( Supplementary Fig. 5). DP Zn T, noncoordinatable DP FB T, and DP Cu T exhibited no absorption spectral changes upon adding Py 3 B ( Supplementary Fig. 6). Therefore, the binding of Py 3 B to DP Zn T plays a critical role in the formation of brous supramolecular polymers. The formation of supramolecular polymers of DP Zn T•Py 3 B was further con rmed by 1 H diffusion-ordered spectroscopy ( 1 H DOSY) NMR experiments. DP Zn T•Py 3 B showed concentration-dependent changes in the diffusion coe cient (D) value ( Supplementary Fig. 7A), indicating the concentration-dependent elongation of the supramolecular polymers.
In contrast, DP Zn T formed networked supramolecular polymers on adding Cl -. DP Zn T has two types of anion-binding sites. The rst type is the triazole groups generated by the click reaction, and the second type are Lewis acidic zinc atoms in the porphyrin wings. Anionic species can bind to triazolic C-H and zinc atoms in porphyrin wings through C-H•••Xhydrogen bonding and axial ligation, respectively. 33,34 The binding of anionic species to triazolic C-H and zinc porphyrin can be monitored by 1 H NMR spectroscopy. However, the 1 H NMR measurements of DP Zn T are not eligible because DP Zn T forms spherical nanoparticles. Therefore, the 1 H NMR spectral change of TC, a structural fragment of DP Zn T, was monitored upon adding Clto con rm the binding of halide ions to the triazole C-H group ( Supplementary Fig. 8). Thus, the triazole C-H peak (H a ) of TC was down eld shifted from 7.23 to 7.38 ppm when Clwas added in the form of tetrabuthylammonium salt, indicating the binding of Clto TC through the C-H•••Clhydrogen bonding. The UV/vis absorption of DPP Zn also showed a bathochromic shift upon adding Clthrough axial coordination complex formation ( Supplementary Fig.  9). The absorption spectra exhibited a similar bathochromic shift with clear isosbestic points at 417.5, 547.0, and 580.0 nm when Clwas added to DP Zn T (Fig. 3E). The binding isotherm obtained from the absorption changes at 413 nm suggested that the absorption reached saturation after adding 20 equiv.
of Cl - (Fig. 3F). A toluene solution of DP Zn T with 10 equiv. of Cl -(DP Zn T•Cl -) was subjected to AFM after spin-coating onto a freshly cleaved mica surface to observe the morphological aspects. Therefore, we observed the formation of a brous network structure (Fig. 3G). Unlike pristine DP Zn T, the in-plane and out-of-plane diffractions of the GIWAXS signals did not overlap with each other (Supplementary Fig.  10). The angular dependency on WAXS indicates that the packing structure of the mixture system of DP Zn T with Cldiffers from that of the spherical particles of pristine DP Zn T. The D values obtained from 1 H DOSY NMR of DP Zn T with 10 equiv. of Cl-in toluene-d 8 con rmed the concentration-dependent elongation of the network structure. The D values gradually decreased with increasing concentration, indicating the elongation of the supramolecular polymer and formation of a brous network ( Supplementary Fig. 7B). 35 The importance of Clcoordination on zinc porphyrin in DP Zn T was supported by control experiments using DP FB T and DP Cu T. Unlike DP Zn T, DP FB T and DP Cu T did not exhibit a bathochromic shift in the absorption spectra ( Supplementary Fig. 11), indicating the absence of axial coordination of Clto porphyrin units in both DP FB T and DP Cu T. However, 1 H NMR spectral studies showed that triazole units in DP FB T interact with Clthrough C-H•••Clhydrogen bonds; the triazolic C-H (H a ) signal was down eld shifted from 8.17 to 8.33 (Δd = -0.16 ppm) (Supplementary Fig. 12). The morphological aspects of DP FB T and DP Cu T were observed by AFM after adding 10 equiv. of Cl -. AFM images showed that the shape of the spherical particles did not change; however, the size of the particles signi cantly increased. Therefore, we concluded that both C-H•••Clhydrogen bonding and axial coordination interactions simultaneously contribute to the formation of networked supramolecular polymers.

Reversible supramolecular depolymerisation
Because the binding of Py 3 B or Clto DP Zn T induced supramolecular polymer formation, the removal of Py 3 B or Clcould lead to the dissociation of the supramolecular polymers. First, we attempted to remove Py 3 B from the brous supramolecular polymers. Pyridyl group could be removed by adding copper ions as they form stable metal-coordination complexes with pyridyl ligands. 36 As aforementioned, DP Zn T showed a bathochromic absorption shift during the formation of a 1:1 host-guest complex with Py 3 B. The absorption spectrum almost recovered to that of pristine DP Zn T (Fig. 4A) when Cu(ClO 4 ) 2 was added to DP Zn T•Py 3 B,. After the solution was ltered to remove the insoluble precipitates, morphological aspects were observed by AFM. The AFM results indicated the formation of spherical nanoparticles in pristine DP Zn T (Fig. 4B). For the networked supramolecular polymers, Clwas removed by AgNO 3 treatment. 37 With the addition of AgNO 3 to DP Zn T•Clsolution in toluene, the Soret absorption band at 423.5 nm completely recovered to the absorption of the pristine DP Zn T (Fig. 4C). After adding AgNO 3 , the insoluble salt was removed by ltration, and the ltrate solution was spin-coated onto the mica surface for AFM measurements. The AFM results also indicated the recovery of the original spherical nanoparticles formed by pristine DP Zn T (Fig. 4D). The UV/vis absorption spectral changes were monitored upon the addition of Cland AgNO 3 (Fig. 4E) observe the reversibility of this process. The absorption changes at both 413 and 423.5 nm upon the successive treatment of Cland AgNO 3 supported the reversible changes in supramolecular polymerisation and depolymerisation (Fig. 4F).
In summary, we prepared a triazole-bearing tripodal porphyrin, DP Zn T, that formed spherical nanoparticles. UV/Vis titration with structural fragments of DP Zn T and molecular modelling revealed that axial coordination of the triazole groups to the zinc porphyrin units was prevented owing to steric hindrance. The addition of Py 3 B resulted in the formation of a 1:1 host-guest complex between Py 3 B and DP Zn T, and this host-guest complex was further aggregated to form a linear brous supramolecular polymer. The removal of Py 3 B from the host-guest complex resulted in the formation of spherical nanoparticles by the reversible depolymerisation of linear brous supramolecular polymers. In contrast, the spherical nanoparticles of DP Zn T were transformed into networked supramolecular polymers through the binding of Cl -. The original spherical nanoparticles of DP Zn T were recovered by the reversible depolymerisation of networked supramolecular polymers when AgNO 3 was added to remove Cl -. Because DP Zn T has successfully undergone supramolecular polymerisation and depolymerisation upon the treatment with Py 3 B/Cland Cu 2+ /Ag + , respectively, the results provide insight into a better understanding of molecular-level association processes in natural systems that exhibit structural transformation.

Reagents and synthesis
All commercially available chemicals were of reagent grade and used without further puri cation. Dichloromethane (CH 2 Cl 2 ), n-hexane, acetonitrile, tetrahydrofuran (THF), and toluene were distilled before use. The 1 H NMR spectra were recorded at 25°C on a Bruker Avance DPX 250 and DPX 400 spectrometer.
The 13 C NMR spectra were recorded at 25°C using a Bruker DPX 400 spectrometer. NMR data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet), and integration. 2D-DOSY NMR spectra were recorded at 25°C using a Bruker Avance 600 spectrometer. Matrix-assisted laser desorption ionisation-time-of-ight mass spectrometry (MALDI-TOF-MS) was performed on a Bruker Daltonics LRF 20 mass spectrometer with dithranol (1,8,9-trihydroxyanthracene) as the matrix. Recycling size exclusion chromatography was performed on a JAI LC-9201 chromatograph equipped with JAIGEL-1H, JAIGEL-2H, and JAIGEL-3H columns using THF (DUCKSAN Pure Chemicals, Republic of Korea) as the eluent. Measurements and sample preparation UV/Vis absorption spectra were recorded on a JASCO V-760 spectrometer equipped with a thermostatic cell holder coupled with a controller (ETCS-761, JASCO, Japan). The absorption spectral measurements were performed using a quartz cuvette with a path length of 1 cm. AFM images were obtained using a Park Systems NX10 instrument. For the AFM measurements, a drop of each solution was spin-coated onto freshly cleaved mica or HOPG substrates (3000 rpm, 60 s). TEM images were obtained using a JEM-1400 instrument operating at 120 kV (JEOL, Japan). For the TEM measurements, a drop of each sample in the solvent was placed on a carbon-coated copper grid and allowed to evaporate under ambient conditions. The sample was then allowed to rest for at least 1 min, after which the excess solution was wicked off using lter paper. The thin lms for WAXS measurements were cast from each solution in toluene on a silicon substrate. WAXS measurements were performed on the PLS-II 9A beamline in the Pohang Accelerator (PAL, Pohang, Republic of Korea). The X-rays generated from the in-vacuum undulator were monochromated by Si(111) double crystals and focused on the detector position using the K B -type mirror system. X-rays with a wavelength of 11.065 Å were used.

Modelling studies
The structural optimisation of the ground state of TB was obtained using the semi-empirical PM6 method in the Gaussian 09 software. A molecular modelling study for the complex of DP Zn T with Py 3 B was conducted using Accelrys Materials Studio 7.0.

Data availability
All data needed to evaluate the conclusions of this study are available in the main text or Supplementary Information. The data that support the ndings of this study are available from the corresponding  Spherical nanoparticle formation of DP Zn T.
A. Normalised absorption spectra of DP Zn T (20 mM) in toluene (blue) and 15% n-hexane-containing toluene (red), and DPP Zn (20 mM) in toluene (black), with TB (pink) and m-TB (green). B. Computer-aided molecular modelling TB with a schematic illustration of coordination and steric repulsion between TB Reversible transformation from supramolecular polymer to nanoparticles of DP Zn T.
A. Absorption spectral change of DP Zn T by adding Py 3 B and successive treatment of Cu(ClO 4 ) 2 . B. AFM height image of DP Zn T after removal of Py 3 B using Cu(ClO 4 ) 2 treatment. C. Absorption spectral changes of DP Zn T by adding Cland successive treatment of AgNO 3 . D. AFM height image of DP Zn T after