Communication Chemistry—A European Journal doi.org/10.1002/chem.202101660 Unveiling Polymerization Mechanism in pH-regulated Supramolecular Fibers in Aqueous Media Nicolás M. Casellas,[a, b] Lorenzo Albertazzi,[c, d] Sílvia Pujals,*[c, e] Tomás Torres,*[a, b] and Miguel García-Iglesias*[a, b, f] technologies.[3] It is, therefore, of paramount importance to find Abstract: An amine functionalized C3-symmetric benzotri- a balance between the different supramolecular interactions[4] thiophene (BTT) monomer has been designed and synthe- to control the polymerization mechanism and the final proper- tized in order to form pH responsive one-dimensional ties of the aggregates.[5] supramolecular polymers in aqueous media. While most of Several pH-responsive supramolecular monomers have the reported studies looked at the effect of pH on the size been described in the last decade, where their supramolecular of the aggregates, herein, a detailed mechanistic study is polymerization can be tuned via electrostatic repulsive forces, reported, carried out upon modifying the pH to trigger the varying pH,[6] leading to highly promising materials for formation of positively charged ammonium groups. A biomedical applications, such as pH-triggered drug delivery.[7] dramatic and reversible change in the polymerization Additionally, the frustrated growth induced by the presence mechanism and size of the supramolecular fibers is of Coulombic repulsive forces of charged monomeric species observed and ascribed to the combination of Coulombic have been recently employed to control the shape and size of repulsive forces and higher monomer solubility. Further- the resulting supramolecular aggregates with high precision.[8] more, the induced frustrated growth of the fibers is further In this framework, the pioneering work of Besenius et al., employed to finely control the one-dimensional unveiled the importance of the polymerization mechanism supramolecular polymerisation and copolymerization proc- under the influence of repulsive interactions, and more esses. specifically, the cooperativity as a key property to rule the physical properties of the supramolecular polymers.[9] However, most of the studies reported examples in this area are focused on the size control of the supramolecular polymers Self-assembly is a powerful tool to access a great variety of or copolymers, while the influence of the Coulombic repulsion structures, where the reversibility of the supramolecular inter- forces on the polymerization mechanism and therefore, the actions endows such materials with the possibility to respond structure-mechanism relations,[10] is generally overlooked. towards different external stimuli, a key feature for a plethora of Herein, we present the design, synthesis and self-assembly applications.[1] study of a C3-symmetric benzotrithiophene (BTT)[11] monomeric In this context, the rational design of responsive unit where peripheral amine groups have been incorporated in supramolecular 1-dimensional polymers in water[2] presenting a order to introduce electrostatic repulsive forces triggered by pH precise control over shape, size, and stability of the aggregates changes. The detailed experimental study presented unravels is of special interest for applications in biomedical key mechanistic aspects of the supramolecular polymerization [a] Dr. N. M. Casellas, Prof. T. Torres, Dr. M. García-Iglesias [e] Dr. S. Pujals Department of Organic Chemistry Department of Electronics and Biomedical Engineering Universidad Autónoma de Madrid (UAM) Faculty of Physics Institute for Advanced Research in Chemical Sciences (IAdChem) Universitat de Barcelona Calle Francisco Tomás y Valiente, 7, 28049 Madrid (Spain) Av. Diagonal 647, 08028, Barcelona (Spain) E-mail: tomas.torres@uam.es [f] Dr. M. García-Iglesias [b] Dr. N. M. Casellas, Prof. T. Torres, Dr. M. García-Iglesias QUIPRE Department IMDEA Nanociencia University of Cantabria c/ Faraday 9, Cantoblanco, 28049 (Spain) Avd. de Los Castros, 46, 39005 Santander (Spain) [c] Dr. L. Albertazzi, Dr. S. Pujals E-mail: giglesiasm@unican.es Nanoscopy for Nanomedicine group Supporting information for this article is available on the WWW under Institute for Bioengineering of Catalonia (IBEC) https://doi.org/10.1002/chem.202101660 The Barcelona Institute of Science and Technology (BIST) © 2021 The Authors. Published by Wiley-VCH GmbH. This is an open access Carrer Baldiri Reixac 15–21, 08024 Barcelona, (Spain) article under the terms of the Creative Commons Attribution License, which E-mail: spujals@ibecbarcelona.eu permits use, distribution and reproduction in any medium, provided the [d] Dr. L. Albertazzi original work is properly cited. Department of Biomedical Engineering and ICMS Eindhoven University of Technology 5600 MB Eindhoven (The Netherlands) Chem. Eur. J. 2021, 27, 11056 – 11060 11056 © 2021 The Authors. Published by Wiley-VCH GmbH Communication Chemistry—A European Journal doi.org/10.1002/chem.202101660 Figure 1. Chemical structures of BTT derivatives. (a). Changes of BTT-F-NH2 ammonium formate (c = 5 × 10 5 M) in water at room temperature in (b) absorption and (c) CD intensity as a function of increasing pH by addition of NaOH from 6.8 (red line) to 13.4 (blue line), as a result of the acid-base equilibrium of terminal amine groups. (d) Normalized CD signal intensity Irel = CDx-CDmin/CDmax-CDmin at λ = 287 nm (red dots) and λ = 271 nm (black dots) of BTT-F-NH2 fitted with the non-protonated/protonated ratio using the Henderson-Hasselbalch equation (line) as a function of pH. in the presence of Coulombic interactions. Moreover, this study perfect agreement with the protonation states and pKa values provides a rationale for the designed control over the size of of the terminal amine groups[14] and can be nicely fitted by the the supramolecular polymers in aqueous media upon modify- Hendersen-Hasselbalch equation. This fact points out that along ing the pH. the pH-dependent experiments, BTT-F-NH2 self-assembly is The molecular design of the self-assembling unit BTT-F-NH2, triggered by the disappearance of ionic repulsion between the has been planned based on a monomeric structure recently positively charged ammonium groups and the decrease in the reported that directs the supramolecular polymerization into monomer solubility. However, the absence of proximity effects strictly one-dimensional aggregates in a highly cooperative lowering the pKa values indicates large distances between fashion in water (BTT-F-OH), providing a good balance between ammonium moieties.[15] This fact might indicate the participa- directional hydrogen-bonding and non-directional hydrophobic tion of cation-π interactions between ammonium cations and interactions.[12] Terminal amine groups have been incorporated the electron-rich aromatic core in the equilibrium, protecting in such structure to the water solubilizing octaethyleneglycol against stacking and deprotonation.[16] side-chains, located next to the L-phenylalanine, in order to The spectroscopic observations were corroborated by TEM confer to the resulting fibrillar structures pH-responsiveness negative staining, observing the formation of fibrillar assemblies (see Figure 1a) (see Supporting Information for synthetic with a diameter of 5 nm in water at elevated pH, while non- characterization). appreciable objects could be observed at pH 6.5 (see Figure 2). Changes in structure of BTT-F-NH2 were monitored as a In summary, we have demonstrated that the self-assembly function of pH by different techniques such as UV-vis, circular of BTT-F-NH2 into one-dimensional supramolecular polymers in dichroism (CD) spectroscopy and TEM. water can be switched ‘on’ and ‘off’ as a function of pH when The UV spectrum of BTT-F-NH2 ammonium formate at introducing Coulombic forces. Moreover, this process is fully pH 6.8 shows an absorption maximum at 301 nm (Figure 1b, reversible (Figure S3). red line), indicating that monomers are molecularly dissolved as Once determined the spectroscopic properties and the a consequence of the high electrostatic repulsion forces morphology of this pH-switchable system, we aim to investigate between the three-fold ammonium moieties.[12] In contrast, the interactions driving the self-assembly under pH influence. when increasing pH by stepwise addition of NaOH to the Therefore, in order to study the polymerization mechanism of aqueous solution up to pH = 13.4, the compound spectrum this system, temperature dependent experiments in water were evolves to an absorption band less intense and hypsochromi- carried out from 280 to 356 K, monitoring changes to the CD, or cally shifted, located at 284 nm. This fact indicates the UV spectra at pH = 13 and pH = 7 at different monomer formation of H-aggregates of BTT cores when the amine groups concentrations. are in their neutral state (see Figure 1b, blue line). Similar hints were perceived by CD spectroscopy during the pH-dependent experiments in water, where at pH 6.8 the CD signal is minimal, indicating non-aggregation between molecules, while a strong bisignated Cotton effect in CD spectra turns up when raising pH (see Figure 1c). This ratifies the formation of helical columnar aggregates with a preferred handedness at elevated pH when the monomers are in their non-protonated state.). These results are in good agreement with the data previously reported by us showing the characteristic features of BTT derivatives either molecularly dissolved or aggregated in fibrillar structures by UV and CD spectroscopy.[12,13] Moreover, the normalized CD (Figures. 1d and S1) and UV Figure 2. TEM images of BTT-F-NH2 in water (c = 4 × 10 5 M) (a) one-dimen- (Figure S2) signals during the pH-dependent experiments are in sional fibers at pH 13 and (b) non-appreciable objects at pH 6.5. Chem. Eur. J. 2021, 27, 11056 – 11060 www.chemeurj.org 11057 © 2021 The Authors. Published by Wiley-VCH GmbH Communication Chemistry—A European Journal doi.org/10.1002/chem.202101660 According to the temperature dependent experiments at observed, indicating the formation of small aggregates with pH 13, a complete disruption of the BTT-F-NH2 supramolecular little Cotton effect amplification (see Figure S6). polymers was observed when raising the temperature (see A detailed inspection of the UV spectra against temperature Figure S4). The different melting curves obtained by CD at revealed sigmoidal temperature-dependent profiles at pH 7, in different concentrations showed a non-sigmoidal behavior, strong contrast with the previous case (Figures 3b and S7–8). suggesting a nucleation-elongation polymerization process. The resulting data was precisely fitted with an an isodesmic or This fact was confirmed by successfully fitting the data with the Equal-K polymerization model (Table 2) in which a clear cooperative model developed by Eikelder, Markvoort, Meijer decrease in the degree of polymerization was observed, leading and co-workers[17] (see Figure 3a and S5). Thermodynamic to the formation of discrete objects constituted by few parameters derived from the fitting disclosed a high degree of monomers (see Figure S9), in good agreement with CD cooperativity and thermodynamic parameters similar than spectroscopy and TEM images. those previously reported by us for similar structures termi- In order to rule out a major influence of the ionic strength nated by -OH instead -NH2 forming highly ordered one-dimen- in the self-assembly of BTT-F-NH2 with respect to the experi- sional supramolecular polymers 12 (see Table 1). ments carried out at elevated pH, temperature dependent Analogous experiments were performed at pH 7, where the experiments at pH 7 in the absence and presence of 1 M conc presence of aggregated species was only observed when using of NaCl were performed. In those experiments non-appreciable 102-fold higher concentrations than the previous experiments changes in the UV spectra and temperature dependent plots carried out at elevated pH, revealing lower stability of the were appreciated (Figure S10). system either by inferior stability of the supramolecular polymer Summarizing, there is a striking difference in the polymer- or more favourable interactions between monomer and solvent. ization mechanism at the two pH values examined. The non- At those concentrations, the monomer stacking was nicely protonated three amino groups exhibit a highly cooperative detected by UV spectroscopy by the formation of a band behaviour at pH 13, which is typically known to lead longer and located at 284 nm, as previously observed at elevated pH, monodisperse assemblies.[18] However, when the terminal indicating plane to plane aggregation. However, by CD charged primary ammonium are formed at lower pH, the spectroscopy a bisignated signal with very low intensity was energy associated with the Coulombic interactions represents a thermodynamically unfavourable component in the overall Figure 3. Temperature-dependent aggregation behavior of. BTT-F-NH2. Plots of the CD intensity recorded at 287 nm as a function of temperature at different concentrations at pH = 13 (a). Plot of the variation of the absorbance of BTT-F-NH2 ammonium formate at λ = 300 nm versus temperature at different concentrations (b). The cooling rate for both plots was 2 K min 1. The red lines in (a) and (b) correspond to the fit using Eikelder-Markvoort-Meijer model.[17] Table 1. Thermodynamic parameters obtained from temperature-depend- ent CD experiments of BTT-F-NH2 in water at pH 13 at different concentrations on the basis of the ten Eikelder-Markvoort-Meijer model17. λ = 287 nm. Table 2. Thermodynamic parameters obtained from the temperature- ΔHELO[a] ΔS[b] ΔHNP[c] Ke[d]* Kn[e]* σ[f]* dependent UV/Vis, experiments of BTT-F-NH2 ammonium formate in water [kJ mol 1] [Jmol 1K 1] [kJ mol 1] [106 M 1] [M 1] [10 5] at pH 7 at different concentrations using an isodesmic model. λ = 300 nm. 65 85 26 9.1 214 2.3 Ka[a]* ΔH[b] ΔS[c] ΔG[d] [a] Elongation enthalpy. [b] Entropy of elongation. [c] Nucleation penalty. [104 M 1] [kJ mol 1] [Jmol-1 K 1] [kJ mol 1] [d] Elongation constant. [e] Nucleation constant. [f] Degree of coopera- 2.9 80 183 25.4 tivity of cooperativity associated with the polymerization process, calculated as σ = Kn/Ke. *Ke, Kn and σ were calculated at 298 K. The cooling [a] Association constant. [b] Enthalpy. [c] Entropy. [d] Gibbs free energy. * rate was 2 K min 1. Ka was calculated at 298 K. The cooling and heating rate were 2 K min 1. Chem. Eur. J. 2021, 27, 11056 – 11060 www.chemeurj.org 11058 © 2021 The Authors. Published by Wiley-VCH GmbH Communication Chemistry—A European Journal doi.org/10.1002/chem.202101660 Figure 4. TEM images of mixtures (c = 4 × 10 5 M) at pH = 7 of BTT-F-OH : BTT-F-NH2 ammonium formate at different ratios, a) 100 : 0, b) 75 : 25, c) 50 : 50, d) 25 : 75, e) 0 : 100 respectively, showing one-dimensional fibers in water at room temperature. In yellow, average length for the fibers. Scale bar 200 nm. process, being responsible for a drastic change in the polymer- Acknowledgements ization mechanism, and leading to a isodesmic behavior, that can also be attributed as in previous examples to an Financial support from MINECO (CTQ2017-85393-P) and ERA- anticooperative effect due to the frustrated growth of the NET/European Commission/MINECO (EuroNanoMed2017-191/ aggregates. PCIN-2017-042) is acknowledged by T.T. IMDEA Nanociencia After investigating the pH-dependent self-assembly of BTT- acknowledges support from the “Severo Ochoa” Programme for F-NH2, we turned our attention to the copolymerization study Centres of Excellence in R&D (MINECO, Grant SEV2016-0686). of mixtures of BTT-F-OH and BTT-F-NH2 monomeric species M.G.I thanks Santander Talent Atraction Research (STAR2) for (Figure 1a) at pH = 7. Different studies of pH-regulated finantial support. This work was also financially supported by supramolecular co-polymerizations have been reported by Spanish Ministry of Science and Innovation (PID2019-109450RB- Besenius et al., by mixing complementary charged monomeric I00/AEI/10.13039/501100011033), European Research Council/ species.[19] Horizon2020 (ERC-StG-757397), la Caixa Foundation (ID In this case, the copolymerization will allow us to combine 100010434) and by the Generalitat de Catalunya (2017 SGR the fine control over structure and dispersity in a cooperative 01536) to S.P. and L.A. supramolecular polymer together with the frustrated growth by Coulombic repulsive forces to control the formation of colum- nar assemblies of defined length. Conflict of Interest Therefore, mixtures of different ratio between co-monomers were studied by CD and UV spectroscopy at pH = 7, observing a The authors declare no conflict of interest. clear reduction in the fraction of aggregated molecules when increasing the ratio of the charged monomeric species of Keywords: pH responsivity · polymerization mechanism · self- BTT F NH2 vs. BTT F OH (see Figure S11). assembly · supramolecular chemistry · supramolecular polymers The same trend was seen by negative staining TEM (see Figure 4), being able to gain a fine control over the length. We could go gradually from long fibers at 100 % of BTT-F-OH to [1] a) A. H. Gelebart, D. JanMulder, M. Varga, A. Konya, G. Vantomme, E. W. very short ones in a mixture 25 : 75 of BTT-F-OH : BTT-F-NH2. Meijer, R. L. B. Selinger, D. J. Broer, Nature 2017, 546, 632–636; b) A. Feula, X. Tang, I. Giannakopoulos, A. M. Chippindale, I. W. Hamley, F. Shortening of the fibers was supported by Zeta potential Greco, C. P. Buckley, C. R. Siviour, W. Hayes, Chem. Sci. 2016, 7, 4291– measurements (see Figure S11), going from a negative mean 4300; c) M. Sofos, J. Goldberger, D. A. Stone, J. E. Allen, Q. Ma, D. J. value for 100 % BTT-F-OH and reaching a positive maximum Herman, W. W. Tsai, L. J. Lauhon, S. I. Stupp, Nat. Mater. 2009, 8, 68–75. [2] a) X. Ma, H. Tian, Acc. Chem. Res. 2014, 47, 1971–1981; b) N. M. value at BTT-F-OH:BTT-F-NH2 50 : 50. Matsumoto, R. P. M. Lafleur, X. Lou, K. C. Shih, S. P. W. Wijnands, C. In summary, the study we present unveils the delicate Guibert, J. W. A. M. van Rosendaal, I. K. Voets, A. R. A. Palmans, Y. Lin, interplay between the forces interacting in supramolecular E. W. Meijer, J. Am. Chem. Soc. 2018, 140, 13308–13316. [3] a) E. Krieg, B. Rybtchinski, Chem. Eur. J. 2011, 17, 9016–9026; b) S. I. systems, including key mechanistic aspects of the frustrated Stupp, Nano Lett. 2010, 10, 4783–4786; c) D. Chandler, M. J. Webber, polymerization growth induced by a combination of Coulombic E. A. Appel, E. W. Meijer, R. Langer, Nat. Mater. 2016, 15, 13–26; d) Y. Li, repulsive forces and a higher monomer solubility in pH- Y. Wang, G. Huang, J. Gao, Chem. Rev. 2018, 118, 5359–5391. [4] a) C. M. A. Leenders, L. Albertazzi, T. Mes, M. M. E. Koenigs, A. R. A. responsive processes. This mechanistic study paves the way for Palmans, E. W. Meijer, Chem. Commun. 2013, 49, 1963–1965; b) M. B. the preparation of size-controlled one-dimensional stimuli- Baker, L. Albertazzi, I. K. Voets, C. M. A. Leenders, A. R. A. Palmans, G. M. responsive supramolecular polymers and in aqueous media. Pavan, E. W. Meijer, Nat. Commun. 2015, 6, 6234; c) D. Bochicchio, M. Salvalaglio, G. M. Pavan, Nat. Commun. 2017, 8, 147. [5] E. Krieg, M. M. C. Bastings, P. Besenius, B. Rybtchinski, Chem. Rev. 2016, 116, 2414–2477. [6] a) K. J. C. Van Bommel, C. Van Der Pol, I. Muizebelt, A. Friggeri, A. Heeres, A. Meetsma, B. L. Feringa, J. Van Esch, Angew. Chem. Int. Ed. 2004, 43, 1663–1667; Angew. Chem. 2004, 116, 1695–1699; b) D. Spitzer, V. Marichez, G. J. M. Formon, P. Besenius, T. M. Hermans, Angew. Chem. Int. Chem. Eur. J. 2021, 27, 11056 – 11060 www.chemeurj.org 11059 © 2021 The Authors. Published by Wiley-VCH GmbH Communication Chemistry—A European Journal doi.org/10.1002/chem.202101660 Ed. 2018, 57, 11349–11353; Angew. Chem. 2018, 130, 11519–11523; c) R. [12] N. M. Casellas, S. Pujals, D. Bochicchio, G. M. Pavan, T. Torres, L. Otter, K. Klinker, D. Spitzer, M. Schinnerer, M. Barz, P. Besenius, Chem. Albertazzi, M. García-Iglesias, Chem. Commun. 2018, 54, 4112–4115. Commun. 2018, 54, 401–404; d) S. M. Darnall, C. Li, M. Dunbar, M. Alsina, [13] N. M. Casellas, I. Urbanaviciute, T. D. Cornelissen, J. A. Berrocal, T. Torres, S. Keten, B. A. Helms, T. Xu, J. Am. Chem. Soc. 2019, 141, 10953–10957; M. Kemerink, M. García-Iglesias, Chem. Commun. 2019, 55, 8828–8831. e) R. Appel, S. Tacke, J. Klingauf, P. Besenius, Org. Biomol. Chem. 2015, [14] X. Xia, M. Yang, Y. Wang, Y. Zheng, Q. Li, J. Chen, Y. Xia, ACS Nano 2012, 13, 1030–1039; f) B. J. Cafferty, R. R. Avirah, G. B. Schuster, N. V. Hud, 6, 512–522. Chem. Sci. 2014, 5, 4681–4686. [15] B. Baumeister, A. Som, G. Das, N. Sakai, F. Vilbois, D. Gerard, S. P. Shahi, [7] a) A. Ghosh, M. Haverick, K. Stump, X. Yang, M. F. Tweedle, J. E. S. Matile, Helv. Chim. Acta 2002, 85, 2740–2753. Goldberger, J. Am. Chem. Soc. 2012, 134, 3647–3650; b) C. C. Lee, J. A. [16] a) D. A. Dougherty, Acc. Chem. Res. 2013, 46, 885–893; b) K. Fujisawa, M. MacKay, J. M. J. Frechet, F. C. Szoka, Nat. Biotechnol. 2005, 23, 1517– Humbert-Droz, R. Letrun, E. Vauthey, T. A. Wesolowski, N. Sakai, S. 1526. Matile, J. Am. Chem. Soc. 2015, 137,11047–11056. [8] a) D. González-Rodríguez, J. L. J. van Dongen, M. Lutz, A. L. Spek, [17] A. J. Markvoort, H. M. M. ten Eikelder, P. A. J. Hilbers, T. F. A. de Greef, A. P. H. J. Schenning, E. W. Meijer, Nat. Chem. 2009, 1, 151–155; b) Y.-L. E. W. Meijer, Nat. Commun. 2011, 2, 509. Wu, K. E. Brown, D. M. Gardner, S. M. Dyar, M. R. Wasielewski, J. Am. [18] a) C. Rest, R. Kandanelli, G. Fernández, Chem. Soc. Rev. 2015, 44, 2543– Chem. Soc. 2015, 137, 3981–3990; c) M. García-Iglesias, T. Torres, D. 2572; b) C. Kulkarni, E. W. Meijer, A. R. A. Palmans, Acc. Chem. Res. 2017, González-Rodríguez, Chem. Commun. 2016, 52, 9446–9449. 50, 1928–1936. [9] a) P. Besenius, G. Portale, P. H. H. Bomans, H. M. Janssen, A. R. A. [19] a) H. Frisch, Y. Nie, S. Raunser, P. Besenius, Chem. Eur. J. 2015, 21, 3304– Palmans, E. W. Meijer, Proc. Natl. Acad. Sci. USA 2010, 107, 17888–17893. 3309; b) P. Ahlers, H. Frisch, P. Besenius, Polym. Chem. 2015, 6, 7245– b) K. K. Kartha, F. Wendler, T. Rudolph, P. Biehl, G. Fernández, F. H. 7250. Schacher, Chem. Eur. J. 2020, 26, 606–610. [10] T. F. A. de Greef, M. M. J. Smulders, M. Wolffs, A. P. H. J. Schenning, R. P. Sijbesma, E. W. Meijer, Chem. Rev. 2009, 109, 5687–5754. [11] a) A. Demenev, S. H. Eichhorn, Chem. Mater. 2010, 22, 1420–1428; b) X. Manuscript received: May 10, 2021 Guo, S. Wang, V. Enkelmann, M. Baumgarten, K. Müllen, Org. Lett. 2011, Accepted manuscript online: June 7, 2021 13, 6062–6065. Version of record online: June 29, 2021 Chem. Eur. J. 2021, 27, 11056 – 11060 www.chemeurj.org 11060 © 2021 The Authors. Published by Wiley-VCH GmbH