# Counteranion effect on the RAFT polymerization of methacrylate-g-pentaarginine and its UCST-type thermoresponsive behavior

Amélie Augé, Tetiana Dorosh, Ayoub Rachdi, Quentin Bievelot, Alain Chaumont, Fouzia Boulmedais, Delphine Chan-Seng

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## ► To cite this version:

Amélie Augé, Tetiana Dorosh, Ayoub Rachdi, Quentin Bievelot, Alain Chaumont, et al., Counteranion effect on the RAFT polymerization of methacrylate-g-pentaarginine and its UCST-type thermoresponsive behavior. European Polymer Journal, 2025, 238, pp.114202. 10.1016/j.eurpolymj.2025.114202. hal-05218688

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Counteranion effect on the RAFT polymerization of methacrylate-g-pentaarginine and its UCST-type thermoresponsive behavior

Amélie Augé,\* Tetiana Dorosh,\textsuperscript{b} Ayoub Rachdi,\textsuperscript{a} Quentin Bievelot,\textsuperscript{a} Alain Chaumont,\textsuperscript{b} Fouzia Boulmedais,\textsuperscript{a}
Delphine Chan-Seng\textsuperscript{a\*}

ABSTRACT

Poly(methacrylate-g-pentaarginine) prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization exhibits an upper critical solution temperature (UCST) behavior with a sharp transition in pure water that can be affected by the presence of salts. In this work after optimizing the conditions for the synthesis of methacrylate-g-pentaarginine (MA-hR5) and its RAFT polymerization, the effect of kosmotropic anions on the polymerization and the thermoresponsive behavior of these polymers in aqueous solutions was investigated experimentally and computationally. The use of chloride, phosphate, and sulfate anions lowered the phase transition temperature, while citrate ones increased it. The latter phenomenon was attributed to the ability of citrate anions to form bridges with the guanidinium groups present on the polymers according the protonation state of the citrate anions.

KEYWORDS

Thermoresponsive polymer, UCST behavior, Hofmeister series, RAFT polymerization

<footnote>\textsuperscript{a} Université de Strasbourg, CNRS, Institut Charles Sadron UPR22, F-67000 Strasbourg, France</footnote>

<footnote>\textsuperscript{b} Université de Strasbourg, CNRS, Laboratoire de chimie de la matière complexe UMR7140, F-67000 Strasbourg, France</footnote>

<footnote>\* corresponding authors : augeameli@gmail.com (A. Augé), delphine.chan-seng@ics-cnrs.unistra.fr (D. Chan-Seng)</footnote>
# 1. INTRODUCTION

Peptide-based polymers offer an attractive platform for the development of new materials for biomedical applications.¹⁻³ Among these macromolecules, guanidinium-rich polymers have attracted a great attention in recent years due to their multiple properties. These macromolecules have been used for their antimicrobial activity as drugs⁴ or coatings on surfaces,⁵,⁶ but also as cell-penetrating agents⁷ enhancing the efficiency of cell internalization⁸ for the delivery of nucleic acids notably.⁹,¹⁰ Some guanidinium-bearing polymers have been reported to exhibit stimuli-responsive properties including response to CO₂,¹¹ salts or specific counterions,¹²⁻¹⁴ and temperature. Multi-L-arginyl-poly-L-aspartic acid produced from recombinant *Escherichia coli* is a thermoresponsive polymer in aqueous solution exhibiting both upper and lower critical solution temperature (UCST and LCST respectively) behaviors.¹⁵ This polymer is composed of a polyaspartate backbone with one arginine or lysine residue on the side chains. The higher the content of lysine in the polymer, the higher the solubility in water allowing the modulation of the UCST phase transition.¹⁶ Our research group has described the synthesis of a regular comb polymethacrylate bearing pentaarginine pendent grafts by reversible addition-fragmentation chain transfer (RAFT) polymerization.¹⁷ These comb polymers exhibit a thermoresponsive behavior in water with an UCST behavior. This behavior was explained by the reversible stacking of the guanidinium groups below the phase transition temperature through hydrophobic interactions that was supported by molecular dynamics (MD) simulations. More recently, the group of Choi has reported the thermoresponsive behavior of a polyether with one guanidinium group per side chain obtained by anionic ring-opening polymerization of allyl glycidyl ether and post-polymerization modification by thiol-ene reaction with 2-aminoethanethiol followed by guanylation.¹⁸,¹⁹ Liquid-liquid phase separation in water was triggered by adding monovalent salts such as NaCl to the polymer solution exhibiting an UCST behavior.

While reports on thermoresponsive polymers with guanidinium groups are relatively scarce, the properties of the guanidinium group and arginine-rich peptide sequences have been more extensively investigated. The guanidinium cation ($Gdm^+$) is composed of three nitrogen atoms bound to a central carbon atom forming a planar Y-shaped conjugated structure.²⁰ Due to its high pKa value (13.8) in water the guanidinium group of arginine is predominantly charged even in a strong basic environment.²¹⁻²⁴ Its hydration occurs mostly in the molecular plane while its planar faces are water-deficient leading to the stacking of $Gdm^+$ groups defying the electrostatic repulsion expected between cationic molecules. Usually cations have less effect than anions in the Hofmeister series; however, $Gdm^+$ cation is a better denaturing ion than Cl⁻ anion due to its capacity to interact both with the hydrophilic parts of the protein through hydrogen bonds and with the hydrophobic ones. Schneider *et al*. have investigated the role of anions on the ability of $Gdm^+$ to stabilize or denature the protein α-chymotrypsinogen A classifying them into two categories.²⁵ The first ones (acetate and chloride ions) have lower interactions with guanidinium groups forming weaker anion-cation hydrogen bonds and having a lower capacity to form anion-arginine-anion bridges leading to a more random distribution of molecules. However, phosphate, sulfate, and citrate anions are able to interact strongly with the guanidinium group notably through hydrogen bonds. Due to the large number of hydrogen bond donors and acceptors these anions are able to form anion-arginine-anion bridges that can lead to the formation of clusters stabilizing the native form of proteins. Arginine-rich peptides have been reported to exhibit UCST-mediated coacervation in the presence of multivalent
anions. Wang *et al.* have reported the coacervation of the mussel foot protein mfp-3b at pH 3.0 in the presence of citrate.²⁶ In the absence of citrate no coacervation is observed. The formation of coacervate below the UCST is attributed to the decrease of hydrogen bonding between the hydrophilic residues and the increase of citrate deprotonation. Kim *et al.* have investigated the coacervation of protamine in aqueous solution.²⁷ When protamine is mixed with either sodium citrate or sodium tripolyphosphate coacervates are observed with a reversible UCST behavior, which is not the case when using sodium chloride. The authors have suggested that the multivalent anions behave as crosslinkers through electrostatic interactions with the positively charged protamine.

The effect of anions on protein solubility in aqueous solutions known as “salting in” and “salting out” according to the type of anions (i.e. chaotropic and kosmotropic respectively)²⁸, ²⁹ has been a guide when investigating the effect of salts on the phase transition temperature of thermoresponsive polymers in solution. Some reports have highlighted that the use of kosmotropic anions lowers the phase transition temperature of polymers with a LCST behavior in aqueous solution,³⁰, ³¹ while the phase transition temperature of polymers with an UCST behavior tends to increase with the addition of kosmotropic anions.³², ³³ This difference in tendency could be attributed to the type of interactions involved in the phase transition phenomenon. While the LCST behavior is mostly entropy-driven involving hydrophobic interactions, the UCST one is enthalpy-driven usually associated to intra- and intermolecular hydrogen bonding or ionic interactions.³⁴ However, the UCST behavior of the comb polymer with pentaarginine pendent grafts, P(MA-hR5), we previously reported, is induced by the stacking of the guanidinium groups through hydrophobic interactions.¹⁷ Herein we propose to investigate the use of different anions for the polymerization of methacrylate-g-pentaarginine (MA-hR5). After optimizing the synthesis of MA-hR5 and its polymerization in water, the polymerizations were conducted in aqueous solution with NaCl, a weakly chaotropic salt, and various kosmotropic salts including sodium acetate (NaOAc), sodium phosphate dibasic (Na₂HPO₄), sodium sulfate (Na₂SO₄), and sodium citrate (NaCit). The effect of these salts on the polymerization and the phase transition of these polymers in aqueous solution are discussed based on experiments and simulations.

## 2. RESULTS AND DISCUSSION

### 2.1. Optimization of the synthesis of MA-hR5

The improvement in the elution conditions for the LC-MS analysis revealed the presence of a large number of deleted sequences (not detected by MALDI-ToF mass spectrometry) when using the precedent synthesis conditions,¹⁷ while irreproducibility from batch to batch in the ratio between the different species composing the product obtained was observed. The coupling agent, *N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU)*, and the racemization inhibitor, 1-hydroxybenzotriazole (HOBt), were replaced by (1-cyano-2-ethoxy-2-oxoethylidenaminoxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU) and ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) respectively that are reported as more efficient and stable. This change permitted to reduce the reaction time for the coupling reactions and the number of repetitions for the addition of each arginine unit. However, due to their similarities in structure and the tendency of arginine-rich peptides to have low retention on C18 column when attempting purification,
the protocol was modified to add a step of *N*-capping (step 2 on Scheme 1) for each repeating cycle of arginine addition. This step aims at preventing further reactions at unreacted sites and could help in the isolation of the targeted peptide from the deleted sequences. The most common strategy of *N*-capping considers the use of acetic anhydride (Ac₂O) leading to the acetylation of the unreacted amino groups,³⁵, ³⁶ which we first considered for the synthesis of R5 that was monitored by LC-MS (Figure 1). A single peak leaving the column in the dead volume (0.51 min) was observed on the total ion chromatogram (TIC) for the synthesis of R5 without *N*-capping, while a second peak at 0.82 min was also detected when conducting the *N*-capping with Ac₂O. The mass spectrum resulting from the integration of this second peak on the TIC showed the m/z values corresponding to the capped R4 resulting from the acetylation: 172.35, 229.47 and 343.71 (Figure 1e). Ball *et al*. have proposed the use of *N*-(2-chlorobenzyloxycarbonyloxy)succinimide (2(ZCl)-OSu) as capping agent.³⁷ Using this *N*-capping strategy, two peaks were observed on the TIC. The first one at 0.51 min corresponds to R5, while the second one at 9.01 min was associated to R4 capped with 2(ZCl)-OSu (m/z at 203.75, 271.33 and 406.50). The following steps to obtain MA-hR5 were conducted as previously reported. MA-hR5 was characterized by LC-MS showing a peak at a retention time between 8.5 and 9.5 min, which is close to the one of R4 capped using 2(ZCl)-OSu. The most suitable capping agent for the synthesis MA-hR5 was Ac₂O allowing its purification by reverse-phase liquid chromatography on C18 affording MA-hR5 in 52 % yield with a purity higher than 95 % (ESI, Figure S1).

<figure><img src="image_3.png" /><figcaption>**Scheme 1.** Optimization of the synthesis of MA-hR5 by adding the *N*-capping step for each repeating cycle done for the addition of arginine residues.</figcaption></figure>
<figure><img src="image_4.png" /></figure>

<table><caption>Figure 1. Effect of the N-capping on the synthesis of R5: total ion chromatograms (a) of R5 synthesized with and without N-capping along with MA-hR5 (R5 highlighted in green, the N-capped species in blue and MA-hR5 in red), from which are extracted the mass spectra of the peaks present on the chromatograms for the synthesis of R5 conducted without N-capping (b), using Ac<sub>2</sub>O (c) or 2-(ZCl)-OSu (d) as capping agent. The table (e) is provided to facilitate the identification of the expected and deleted species.</caption><thead><tr><th rowspan="2"></th><th colspan="3">no N-capping</th><th colspan="3">Ac<sub>2</sub>O</th><th colspan="3">2(ZCl)-OSu</th></tr><tr><th>R5</th><th>R4</th><th>R3</th><th>R4-C1</th><th>R3-C1</th><th>R4-C2</th><th>R3-C2</th><th>R5</th><th>R4</th><th>R3</th></tr></thead><tbody><tr><td>Theoretical molar mass</td><td>798.51</td><td>642.41</td><td>486.31</td><td>685.41</td><td>529.31</td><td>810.99</td><td>654.89</td><td>799.51</td><td>643.41</td><td>487.31</td></tr><tr><td>m/z</td><td>+1</td><td>+2</td><td>+3</td><td>+4</td><td>-</td><td>-</td><td>-</td><td>400.26</td><td>322.21</td><td>267.17</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>200.63</td><td>161.60</td><td>200.63</td></tr></tbody></table>
## 2.2. P(MA-hR5) prepared by RAFT polymerization in water

The polymerizations of MA-hR5 were previously conducted in a mixture of water and MeOH at pH 3 due to the low solubility of the commercially available chain transfer agent used, i.e. 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, in aqueous solution.¹⁷ However, the solubility of MA-hR5 and the corresponding polymers are poor in alcoholic solutions requiring first the optimization of the polymerization of MA-hR5 in water. 4-Cyano-4-(((ethylthio)carbonothiol)thiol)pentanoic acid (CTA) was selected due to its better solubility in water and its suitability for the polymerization of methacrylate under RAFT conditions.³⁸, ³⁹ 4,4'-Azobis(4-cyanovaleric acid) (V-501) and 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044), two azo initiators suitable for polymerization in water, were considered to evaluate the impact of their half-life at 70 °C, 600 and 20 min respectively, on the polymerization while keeping the CTA/I molar ratio as 1/0.5. MA-hR5 was polymerized in water at 70 °C using a MA-hR5/CTA/I molar ratio of 20/1/0.5 for 4 h. The polymers were purified by dialysis against water at 65 °C and lyophilized before analyses by NMR spectroscopy and size-exclusion chromatography (SEC). The polymerizations using VA-044 and V-501 led to similar results reaching a monomer conversion of 77% and 74% respectively, which corresponds to a degree of polymerization of 15 and a theoretical molar mass of 16 193 g·mol⁻¹ that are in agreement with the experimental SEC results (Mn = 16 400 g·mol⁻¹ and D = 1.11 for the polymerization conducted with VA-044; Mn = 15 700 g·mol⁻¹ and D = 1.08 for the polymerization conducted with V-501) indicating in both cases an experimental degree of polymerization of 15. These results along with the low dispersity of the polymers (ESI, Figure S2) is indicating a good control of the polymerization. The absence of termination reactions during the polymerization was investigated by running polymerization kinetics using V-501 as initiator and drawing the first-order kinetics plot. As depicted on Figure 2 after an induction period of 26 min the plot of ln([M]₀/[M]) evolved linearly as a function of the polymerization time. The polymerization using V-501 as initiator was also attempted for other targeted degrees of polymerization (i.e. 10 and 50). The same trend was observed on the first-order kinetics plots. However, phase separation was observed during the polymerization after 90 min when targeting a degree of polymerization of 50. This was attributed to the high concentration in guanidinium groups in the reaction medium.
<figure><img src="image_5.png" /><figcaption>**Figure 2.** First-order kinetics plots for the polymerization of MA-hR5 using 4-cyano-4 (((ethylthio)carbonothiol)thiol)pentanoic acid as CTA and V-501 as initiator in pure water at 70 °C targeting different degree of polymerization: Xn, target = 10 (●), 20 (■), and 50 (▲).</figcaption></figure>

The thermoresponsive behavior of P(MA-hR5) obtained using a targeted degree of polymerization of 20 was investigated at different concentrations in water by turbidimetry at 500 nm (Figure 3a). The phase transition temperature was determined from these curves by extracting the maximum of the first derivative of transmittance vs. temperature (dT%/dT) and reported in Figure 3b. The cloud point (TCL), i.e. phase transition temperature observed upon cooling, increased from 14 to 42 °C when increasing the polymer concentration from 1 to 5 mg·mL⁻¹.
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

Figure 3. UCST behavior of P(MA-hR5)₁₅ in water at various polymer concentrations: (a) transmittance vs. temperature curves upon heating (open symbols) and cooling (filled symbols) at a polymer concentration of 1 (●), 2 (▲), 3 (◆), 4 (■), and 5 (▼) mg·mL⁻¹ and (b) evolution of the cloud point as a function of the polymer concentration

## 2.3. Polymerization of MA-hR5 in the presence of salts

The phase separation during the polymerization in water limits the access to polymers of higher degree of polymerization. As salts of the Hofmeister series influence the solubility of polymers in aqueous solution⁴⁰⁻⁴² the effect of salts (Table 1) was investigated on the polymerization of MA-hR5 targeting a degree of polymerization of 50 keeping the other polymerization parameters as fixed as previously, i.e. 4-cyano-4-(((ethylthio)carbonothiol)thiol)pentanoic acid as chain transfer agent and V-501 as initiator, CTA/I = 1/0.5, and [MA-hR5]₀ = 10 mg·mL⁻¹ at 70 °C for 4 h. The first salt considered was trifluoroacetate ions as during the synthesis of MA-hR5 the removal of the Pbf protecting groups of the guanidinium groups on the macromonomer was done with a solution of trifluoroacetic acid (TFA). The addition of an excess of TFA (0.1 v% in water) to the polymerization solution prevented the phase separation to occur during the polymerization. The degree of polymerization (theoretical as revealed by the monomer conversion and experimental from the SEC results) and the value of T<sub>CL</sub> determined by turbidimetry at a polymer concentration of 5 mg·mL⁻¹ in water were similar to those observed in pure water, i.e. around 50 °C. In the peptide field, trifluoroacetate counterions are commonly replaced by chloride ones by counterion exchange.⁴³ NaCl was added at two concentrations, i.e. 0.15 and 0.5 M, and led to higher
conversion of MA-hR5 and thus polymers of higher molar mass with an increase of the dispersity. However, T<sub>CL</sub> was similar to the one obtained for the polymerization in pure water when conducted at 0.15 M NaCl and significantly decreased when the polymerization was conducted at 0.5 M NaCl. These results suggested that replacing TFA counterions by chloride ones affected the stacking of the guanidinium groups leading to a shift of T<sub>CL</sub> to lower temperature upon increasing the concentration in NaCl, which is in agreement with our previous results when NaCl was added to a solution of P(MA-hR5) in water.<sup>17</sup>

<table><caption>**Table 1.** Polymerization of MA-hR5 in aqueous solution at 70 °C for 4 h using 4-cyano-4-((ethylthio)carbonothiol)thiol)pentanoic acid as chain transfer agent and V-501 as initiator ([MA-hR5]<sub>0</sub> = 10 mg·mL<sup>-1</sup>, MA-hR5/CTA/I = 50/1/0.5)</caption><thead><tr><th>Salt</th><th>[salt]<br>(M)</th><th>Phase<br>separation<sup>a</sup></th><th>Conversion<sup>b</sup><br>(%)</th><th>X<sub>n,th</sub><sup>c</sup></th><th>M<sub>n</sub><sup>d</sup><br>(g·mol<sup>-1</sup>)</th><th>Đ<sup>d</sup></th><th>X<sub>n,SEC</sub><sup>e</sup></th><th>T<sub>CL</sub><sup>f</sup><br>(°C)</th></tr></thead><tbody><tr><td>none</td><td>0</td><td>yes</td><td>51</td><td>26</td><td>19 500</td><td>1.40</td><td>18</td><td>51</td></tr><tr><td>TFA</td><td>-g</td><td>no</td><td>55</td><td>28</td><td>20 300</td><td>1.44</td><td>19</td><td>55</td></tr><tr><td>NaCl</td><td>0.15</td><td>no</td><td>63</td><td>32</td><td>33 600</td><td>1.61</td><td>31</td><td>50</td></tr><tr><td>NaCl</td><td>0.5</td><td>no</td><td>89</td><td>45</td><td>47 600</td><td>1.61</td><td>45</td><td>&lt;10</td></tr><tr><td>NaOAc</td><td>0.15</td><td>no</td><td>62</td><td>31</td><td>33 300</td><td>1.61</td><td>31</td><td>48</td></tr><tr><td>Na<sub>2</sub>HPO<sub>4</sub></td><td>0.15</td><td>no</td><td>77</td><td>39</td><td>16 300</td><td>1.33</td><td>15</td><td>16</td></tr><tr><td>Na<sub>2</sub>SO<sub>4</sub></td><td>0.15</td><td>yes</td><td>30</td><td>15</td><td>41 300</td><td>1.57</td><td>39</td><td>50</td></tr><tr><td>Na<sub>3</sub>Cit</td><td>0.15</td><td>yes</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Citrate-phosphate<br>buffer</td><td>-</td><td>no</td><td>72</td><td>36</td><td>28 600</td><td>1.35</td><td>27</td><td>&gt;80</td></tr></tbody></table>

Further insights into the structure and dynamics of these systems were obtained from molecular dynamics (MD) simulations. Comparison between two systems containing ten MA-hR5 molecules and either forty TFA⁻ or Cl⁻ anions has been done. We observed large differences between both systems after 1 µs of dynamics (Figure 4). For the system containing TFA⁻ counterions an aggregate formed of seven MA-hR5 molecules was observed leaving three free monomers in solution, while for the system containing Cl⁻ anions all ten MA-hR5 remained as free monomers in solution. This behavior was further reflected by the C<sub>ARG</sub>-C<sub>ARG</sub> radial distribution function (RDF), C<sub>ARG</sub> being the central C atom of a guanidinium group (Gdm⁺), where a more pronounced first peak was observed when TFA⁻ anions were present in solution as compared to the Cl⁻ ones (ESI, Figure S3). The presence of this peak is an indication of π-π interaction between Gdm⁺ moieties in these systems. Integration of this first peak up to 6 Å shows that there were about twice as much π-π stacking interactions when TFA⁻ anions (0.26) were present in solution compared to when Cl⁻ anions were present (0.12). TFA⁻ anions have a stronger tendency to interact with the Gdm⁺ groups than Cl⁻ anions, hence leading to a stronger stabilization of the π-π interaction between Gdm⁺ groups. A clear indication for this can be derived from the RDFs of TFA⁻ anions around C<sub>ARG</sub> atoms in comparison to the RDFs of Cl⁻ anions around the same atoms (ESI, Figure S4). Integrating these RDFs up to the first minimum indicates that, on average, 0.12 TFA⁻ was found around a Gdm⁺ group, whereas in the case of Cl⁻ only 0.03 was found around a Gdm⁺ group.

<footnote><sup>a</sup> phase separation observed during the polymerization; <sup>b</sup> determined by <sup>1</sup>H NMR spectroscopy; <sup>c</sup> theoretical degree of polymerization determined from monomer conversion; <sup>d</sup> number-average molar mass (M<sub>n</sub>) and dispersity (D) determined by SEC measurements 20/80 acetonitrile/water with 0.1 M NaCl and 0.1% TFA on hydrophilic silica-based SEC columns; <sup>e</sup> determined from the number-average molar mass obtained by SEC; <sup>f</sup> cloud point determined by turbidimetry using a polymer concentration of 5 mg·mL<sup>-1</sup> in MilliQ water; <sup>g</sup> corresponds to 0.1 v% TFA in water</footnote>
<figure><img src="image_8.png" /><figcaption>**Figure 4.** Snapshot after 1 µs of the [10 MA-hR5, 40 X] systems with X = TFA (top line) or X = Cl (bottom line). Each MA-hR5 is represented by a different color, TFA⁻ in tan, Cl⁻ in green, water molecules not shown for clarity.</figcaption></figure>

The effect of the anion while keeping sodium as cation and the concentration in salt fixed to 0.15 M was investigated considering sodium acetate (NaOAc), sodium phosphate dibasic (Na₂HPO₄), sodium sulfate (Na₂SO₄), and sodium citrate (NaCit) towards more kosmotropic ions from the Hofmeister series (salting out effect). As for NaCl no phase separation during the polymerization was observed when NaOAc and Na₂HPO₄ were present in the polymerization solution contrary to the ones conducted with Na₂SO₄ and NaCit. The results for the polymerization in NaCl and NaOAc were identical in terms of conversion, molar mass, dispersity, and TCL, as both anions are considered as moderate stabilizers in the Hofmeister series. Citrate and sulfate anions, the most strongly hydrated anions of the Hofmeister series and thus prone to salting out, led to phase separation. For the polymerization conducted in the presence of NaCit phase separation was observed at the early stage of the polymerization (i.e. within the first few minutes). This phenomenon was attributed to the increased formation of hydrogen bonding due to the tricarboxylic acid nature of citrate anions. No characterization by NMR spectroscopy and SEC could be run for this experiment due to the too low recovery in product (the formation of species of low molar masses that were eliminated by dialysis was suspected). The polymerization in aqueous solution with 0.15 M Na₂SO₄ led to a polymer of high molar mass and showed discrepancies between the theoretical and experimental degree of polymerization that could be explained by a loss of the polymerization control, but prevented from determining the effect of the sulfate counterion on TCL. Our results follow the same trends as the ones described by Schneider et al.<sup>25</sup> with a tendency of the polymer to phase separate when strong interactions between the polymer and the anions existed except for the phosphate anion. The
polymerization in 0.15 M Na₂HPO₄ aqueous solution did not show any phase separation as it could have been expected and exhibited high conversion of MA-hR5, but unexpectedly a polymer of low molar mass and dispersity.

As the presence of citrate ions led to phase separation but not the phosphate ones, the possibility of ion exchange was suspected. The polymerization was carried out in the citrate-phosphate buffer solution and did not show any phase separation. The polymers prepared under this condition had relatively high molar mass. Polymerization kinetics was carried out in citrate-phosphate buffer solution using MA-hR5/CTA/V-501 molar ratios of 10/1/0.5, 20/1/0.5, and 50/1/0.5. After an induction period of approximately 50 min the plot of ln([M]₀/[M]) evolved first linearly as a function of the polymerization time (ESI, Figure S5) demonstrating the absence of termination reactions during the polymerization for all the conditions investigated. However, a deviation from the linearity was observed after a certain time (i.e. 325, 270, and 180 min when targeting a degree of polymerization of 10, 20, and 50 respectively with a monomer conversion of 47% (Xn,th = 5), 37% (Xn,th = 9), and 23% (Xn,th = 12)). This behavior could be associated to the fact that when the polymerization was conducted aiming at higher degree of conversion polymers higher molar masses were reached at lower conversion due to earlier phase separation. The polymer prepared when targeting a degree of polymerization of 50 was dissolved in water at a same concentration as previously (5 mg·mL⁻¹) exhibited a cloud point above 80 °C (measurements above this temperature could not be recorded to avoid damaging the spectrophotometer due to water vapor forming when approaching the boiling point of water), which was drastically higher than the values of TCL measured for any of the polymers synthesized in the presence of salt or not. The cloud point could be tuned by reducing the polymer concentration in water (ESI, Figure S6) or by dissolving the polymer in a 0.15 M NaCl aqueous solution (ESI, Figure S7). Figure 5 shows the increase of the cloud point with the polymer concentration and its decrease with the concentration of NaCl in the aqueous solution. To elucidate the effect of each ion of the buffer solution when conducting ion exchange, the cloud point of P(MA-hR5) synthesized in water was first measured at a concentration of 1 mg·mL⁻¹ in water. The same polymer was brought into contact with a NaCit, Na₂HPO₄, or citrate-phosphate buffer solution for 3 h at 70 °C. After dialysis against water to remove the excess of salts and lyophilization, the cloud point was measured in water at the same polymer concentration. When conducting the ion exchange with Na₂HPO₄ the cloud point decreased from 14 °C to below 8 °C (measurements below this temperature could not be recorded due to water condensation affecting the assessment of the transmittance), while TCL raised to 33 °C for NaCit and 29 °C for the citrate-phosphate buffer solution (Figure 6). The polymer solution treated with the citrate-phosphate buffer solution was analyzed by ¹H and ³¹P NMR spectroscopy (ESI, Figure S8). The citrate was observed on the ¹H NMR spectrum as a quadruplet at 2.80 ppm, while no peak was detected on the ³¹P NMR spectrum confirming the occurring of the ion exchange from trifluoroacetate ions into citrate ones that contributed in the thermoresponsive behavior of the polymer. Citric acid being a tricarboxylic acid with three pKa values (3.1, 4.8, and 6.4),⁴⁴ the citrate counterion associated to the polymer could have different protonation states. The effect of the pH on the thermoresponsive behavior was investigated on P(MA-hR5) post-polymerization treated with the citrate-phosphate buffer solution (Figure 7). A small variation in the pH from 4.5 to 5.3 led to a significant shift of TCL raising from 26 to 55 °C, while at lower pH, i.e. pH = 3.4, TCL was below 10 °C.
<figure><img src="image_9.png" /><figcaption>**Figure 5.** Evolution of the cloud point of P(MA-hR5) synthesized in citrate-phosphate buffer solution as a function of (a) the polymer concentration in water (●) and in 0.15 M NaCl (▲), and (b) the concentration of NaCl in the aqueous solution at a concentration of 2.5 mg·mL⁻¹.</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>**Figure 6.** Turbidimetry measurements in water at a polymer concentration of 1 mg·mL⁻¹ using a P(MA-hR5) obtained by polymerization in water followed by ion exchange with a NaCit (■), Na₂HPO₄ (▲) or citrate-phosphate buffer (●) solution. The curve of the P(MA-hR5) solution before treatment in water (▼) is added for reference.</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>Figure 7. Turbidimetry measurements in water of P(MA-hR5) after treatment with a citrate-phosphate buffer solution upon heating (open symbols) and cooling (filled symbols) at various pH: 3.45 (♦), 4.50 (▲), and 5.30 (●).</figcaption></figure>

In order to better understand the influence of the various citric acid protonation states (Cit⁰, Cit⁻, Cit²⁻, and Cit³⁻) on the interaction between MA-hR5 molecules we have performed four MD simulations each containing ten MA-hR5 molecules and citric acid molecules in each of its four protonation states (the main characteristics of the simulated systems are indicated in Table S1). Final snapshots after 1 µs of simulation of the final configurations of these four systems are depicted in Figure 8. From these snapshots two situations are observed. In the presence of Cit²⁻ and Cit³⁻ anions, a large aggregate was formed by all ten MA-hR5 molecules in solution. In the presence of Cit⁻ and Cit⁰ a smaller aggregate was formed by seven MA-hR5 molecules leaving three free MA-hR5 molecules in solution, similar to what was previously observed for TFA⁻ only containing solutions; hence the situation observed in the latter case may be due to the presence of TFA⁻ and not so much of Cit⁻ or Cit⁰. The C<sub>ARG</sub>-C<sub>ARG</sub> radial distribution function (RDF) clearly reflected again the aggregation degree of MA-hR5 (ESI, Figure S9). The first peak in these RDFs clearly decreased with the charge of the Cit anion, excepted when shifting from Cit⁻ to Cit⁰ for which the increase in the first peak can be related to the presence of TFA⁻ anions in solution enhancing the π-π stacking. This was further corroborated by the integration of this first peak up to 6 Å which decreased from 1.16 (Cit³⁻) to 0.89 (Cit²⁻) to 0.224 (Cit⁻) and 0.38 (Cit⁰), respectively. At the same time the RDF of the central C atom of Cit around C<sub>ARG</sub> decreased (ESI, Figure S10). Integration of the first peak of these RDFs indicates that the number of Cit moieties around a Gdm⁺ group decreased from 0.71 (Cit³⁻) to 0.62 (Cit²⁻) to 0.14 (Cit⁻) and 0.03 (Cit⁰), respectively. This suggests that interactions between citrate and MA-hR5 decreased with the charge on the citrate counterion.
<figure><img src="image_12.png" /><figcaption>**Figure 8.** Snapshot after 1 µs of the [10 MA-hR5, 40 TFA⁻, 15 Cit⁰] (1st line), [10 MA-hR5, 25 TFA⁻, 15 Cit⁻¹] (2nd line), [10 MA-hR5, 10 TFA⁻, 15 Cit⁻²] (3rd line), and [10 MA-hR5, 5 Na⁺, 15 Cit⁻³] (4th line) systems. Each MA-hR5 is represented by a different color, TFA⁻ in tan, CA in ochre, while water molecules are not shown for clarity.</figcaption></figure>
### 3. CONCLUSION

The purity of the macromonomer MA-hR5 and thus the composition of the corresponding polymer has an effect on the thermoresponsive behavior of the polymer and its phase transition temperature. Ensuring the absence or at least reducing to the minimum the content in deleted sequences should offer a more reproducible and reliable way to control the polymerization and the properties of this polymer. Due to its thermoresponsive behavior phase separation occurred during the polymerization in pure water affecting the chain propagation; however, the presence of anions of different natures in the aqueous media could prevent it. The counterion exchange from trifluoroacetate ions to more kosmotropic ones (i.e. chloride, sulfate, phosphate) from the Hofmeister series led as expected to a decrease in the phase transition temperature. However, the exchange with citrate ions exhibited a significant increase of the phase transition temperature that was explained by the ability of citrate ions to form bridges with guanidinium groups depending on its protonation state. Ion exchange post-polymerization seemed a more straightforward approach to investigate the effect of anions of the Hofmeister series as compared to the polymerization of MA-hR5 in the presence of the anion of interest.

### 4. EXPERIMENTAL SECTION

#### 4.1. Materials

2-Chlorotrityl chloride resin (CTC, 100-200 mesh, 1,6 mmol·g⁻¹) was purchased from Iris Biotech. Fmoc-Arg(Pbf)-OH (98 %), (1-cyano-2-ethoxy-2-oxoethylidenaminoxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU, 98 %), ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma, 95 %) were purchased from BLDpharm. N,N-Diisopropylethylamine (DIPEA, for synthesis, > 99.0 %), piperidine (99 %), propargyl methacrylate (98 %, stabilized with 250 ppm of 4-methoxyphenol) and trifluoroacetic acid (TFA, 99 %) were purchased from Alfa Aesar. Anhydrous N,N-dimethylformamide (anhydrous DMF, 99.8 %), anhydrous dichloromethane (anhydrous CH₂Cl₂, ≥ 99.8 %, stabilized with 40-150 ppm amylene), copper(I) bromide (Cu(I)Br, 98 %), triisopropylsilane (TIPS, 98 %), N-(2-chlorobenzyloxycarbonyloxy)succinimide (Z(2-Cl)-OSu), acetic anhydride (ReagentPlus, ≥99 %), C18 reversed-phase matrix (Discovery® DSC-18 SPE bulk packing), 4,4'-azobis(4-cyanovaleric acid) (V-501, 98%), citric acid (anhydrous), sodium phosphate dibasic heptahydrate (Na₂HPO₄·7H₂O, ACS reagent), and trifluoroacetic acid-d (TFA-d) were purchased from Sigma-Aldrich. N,N,N',N'',N'''-Pentamethyldiethylenetriamine (PMDETA, 98 %), 2,2,2-trifluoroethanol (TFE, > 99 %), and 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044) were purchased from TCI. Cuprisorb™ was purchased from Seachem. Methanol (MeOH, 99.8 %) and N,N-dimethylformamide (DMF, 99.8 %) were purchased from VWR Chemicals. Dichloromethane (CH₂Cl₂, 99.8 % ) and acetone (99.8 %) were purchased from Carlo Erba. Sodium chloride (NaCl, >99.5%), sodium citrate dihydrate (>99%), sodium sulfate anhydrous (Na₂SO₄, 99,6%), sodium acetate, acetic acid (99.8 %), and formic acid (99 %, quality LC/MS Optima™) was purchased from Fisher Chemical. All the reagents and solvent were used as received except if noted otherwise. Aqueous solutions were prepared using Milli-Q water having a resistivity of ~ 18 MΩ·cm. Copper(I) bromide was purified by stirring in glacial acetic acid overnight, washing with absolute ethyl alcohol followed by anhydrous diethyl ether, and drying under vacuum at room temperature. The purified CuBr was stored under argon at room temperature. V-501 was purified by recrystallization in methanol. All the syntheses on solid support were performed in solid phase extraction (SPE) tubes (60 mL polypropylene SPE tubes with polyethylene frits, 20 µm porosity purchased from SUPELCO®). 4-Cyano-4-(((ethylthio)carbonothiol)thiol)pentanoic acid (CTA)⁴⁵, ⁴⁶ and 6-azidohexanoic acid⁴⁷ were synthesized as previously reported.
## 4.2. Characterizations

**Nuclear magnetic resonance (NMR) spectroscopy.** ¹H, ¹³C, and ³¹P NMR spectra were recorded in deuterium oxide (D₂O, 99.96%, Eurisotop) on a 400 MHz Bruker Avance III HD spectrometer at 25 °C.

**Size exclusion chromatography (SEC).** SEC line is composed of a HPLC part (1260 Infinity II, Agilent, Santa Clara, USA) an Optilab™ NEON refractometer and a DAWN™ NEON multi-angle light scattering detector (MALS, Wyatt technology, Dernbach, Germany). The fractionation was carried out through three Bio SEC-5 columns (100 Å, 150 Å and 300 Å) from Agilent (Santa Clara, USA). The eluent was deionized H₂O (resistivity ~ 18 MΩ·cm) with 0.1 % in volume of trifluoroacetic acid and the presence of 0.1 M NaCl. Measurements were performed at room temperature using a constant flow rate of 0.5 mL·min⁻¹. Solutions were prepared at concentration between 1 and 2 mg·mL⁻¹ and filtered on a 0.45 µm hydrophilic PTFE membrane (Millex-LCR, Millipore, Burlington, USA) prior injection. The light scattering detector allows determining the molecular weight of each fraction. By considering a refractive index increment value dn/dc = 0.183 mL·g⁻¹ and a Zimm model of zero order, the analysis of the chromatograms gives the weight average molecular weight, the number average molecular weight and the dispersity.

**Liquid chromatography coupled to mass spectrometry (LC-MS).** Analyses were conducted on a HPLC 1260 Infinity II system coupled to a single quadrupole mass spectrometer from Agilent Technologies. The system was controlled by the ChemStation software. 1 µL of MA-hR5 was injected and eluted on Zorbax SB-C18 column (2.1 x 50 mm, 1.8 µm, Agilent Technologies) at 35 °C. The mobile phase was composed of two solvents: MilliQ water (A) and acetonitrile (B, HiPerSolv CHROMANORM®, ≥ 99.9 %, VWR), both containing 0.1 v% formic acid (LC/MS Optima™ quality, 99.0+ %, Fisher Chemical). MA-hR5 eluting from the column was analyzed by UV-absorption at 210 nm followed by MS detection. The electrospray ionization source operated in positive mode at 3000 V. The fragmentor value was 115 V and full scan was recorded from 100 m/z to 1100 m/z. The mass detector was calibrated using Tuning mix (Agilent Technologies).

**Fourier transformed infrared (FT-IR) spectroscopy.** Spectra were recorded with a resolution of 2 cm⁻¹ on a Bruker Vertex 70 spectrometer using the attenuated total reflectance (ATR) technique for wavenumber between 600 and 4000 cm⁻¹.

**Turbidimetry.** Turbidimetry measurements were carried out on UV-Vis-NIR Cary 5000 spectrophotometer (Agilent) equipped with a thermostated multicell holders. The polymer solution was heated above the phase transition temperature before starting the measurements. The transmittance was recorded at 500 nm every 1 °C during a cooling-heating cycle using a rate of 1 °C·min⁻¹.

## 4.3. Synthesis of macromonomer MA-hR5

**Loading of the resin.** CTC resin (1.50 g, 2.40 mmol of functional groups) was weighted in a solid phase extraction (SPE) tube, swollen in CH₂Cl₂ for 10 min, and washed six times with CH₂Cl₂. Fmoc-Arg(Pbf)-OH (1.93 g, 1.80 mmol) was added to the vessel, which was degassed by performing three vacuum/argon cycles. 15 mL of anhydrous CH₂Cl₂ was added to the vessel followed by 0.6 mL (3.50 mmol) of DIPEA. The vessel was agitated on an orbital shaker at 280 rpm for 2 h at room temperature. The solution was then filtered and the resin was washed six times with 15 mL of DMF (addition of DMF, shaking for 1 min, and filtration), then six times with 15 mL of CH₂Cl₂. 15 mL of CH₂Cl₂/MeOH/DIPEA (80/15/5 v%) was added to the SPE tube, agitated for 10 min at room temperature, and filtered (twice). The resin was then washed six times with 15 mL of DMF. The Fmoc-protecting groups were removed by adding a 20 v% piperidine solution in DMF (15 mL) and the SPE tube that was agitated for 20 min. After filtration, this step was
repeated with an agitation of 20 min. The resin was washed six times with DMF (15 mL), six times with CH₂Cl₂ (15 mL), and six times with MeOH (15 mL). The resin was dried under vacuum for 48 h. The loading density of the resin was determined by gravimetry as 1.22 mmol of Pbf-protected arginine per gram of resin (1.44 mmol, 60%).

**Addition of the other Pbf-protected arginine units to the loaded resin.** Fmoc-Arg(Pbf)-OH (2.80 g, 4.32 mmol), COMU (1.85 g, 4.32 mmol), and Oxyma (0.61 g, 4.32 mmol) were added in an Erlenmeyer flask and solubilized in 15 mL of anhydrous DMF. DIPEA (1.51 mL, 8.64 mmol) was added to the solution and after rapid homogenization, the mixture was transferred to the SPE tube containing the resin. The reaction mixture was agitated on an orbital shaker for 1 h at room temperature. The resin was washed six times with DMF (15 mL) and then six times with CH₂Cl₂ (15 mL). The Kaiser test was performed: i) if the solution was blue, the coupling was repeated or ii) if the solution was yellow, the Fmoc removal reaction was performed as previously using a solution of piperidine in DMF. Once the Kaiser is negative (no primary amines present), the growing peptide sequence was N-capped either with acetic anhydride or N-(2-chlorobenzyloxycarbonyloxy)succinimide (Z(2-Cl)-OSu) before adding the next arginine residue.

**N-Capping using acetic anhydride.** For a CTC resin functionalized with 0.9 mmol of Arg(Pbf)-OH, the N-capping step after addition of the arginine residue was conducted by adding 4 equivalents of capping agents. Briefly, 10 mL of DMF was added to the SPE tube containing the resin followed by 0.29 mL of pyridine (3.6 mmol) and 0.34 mL of acetic anhydride (3.6 mmol). The reaction mixture was agitated on an orbital shaker for 30 min at room temperature. The solution was filtered and the resin washed six times with DMF and six times with CH₂Cl₂.

**N-Capping using 2-(ZCl)-OSu.** For a CTC resin functionalized with 0.9 mmol of Arg(Pbf)-OH, the N-capping step after addition of the arginine residue was conducted by adding 4 equivalents of capping agents. Briefly, 1.02 g of 2-(ZCl)-OSu (3.6 mmol) was dissolved in a 1/1 DMF/CH₂Cl₂ solution. 1.2 mL of DIPEA (6.9 mmol) and 3.5 mL of DMF was added to this solution before adding it to the SPE tube containing the resin. The reaction mixture was agitated on an orbital shaker for 10 min at room temperature. The solution was filtered and the resin washed six times with DMF and six times with CH₂Cl₂.

**Addition of 6-azidohexanoic acid to the pentaarginine grafted on the resin.** 6-Azidohexanoic acid (0.68 g, 4.32 mmol) was added to the SPE tube containing the resin. COMU (1.85 g, 4.32 mmol) and Oxyma (0.61 g, 4.32 mmol) were dissolved in 15 mL of anhydrous DMF. DIPEA (1.51 mL, 8.64 mmol) was added to the solution that was hand-shaken and then added to the SPE tube containing the resin. The reaction mixture was agitated on an orbital shaker for 2 h at room temperature. The solution was filtered, and the resin was rinsed six times with DMF (15 mL), and six times with DCM (15 mL). The Kaiser test was performed: i) if blue, the coupling was repeated or ii) if yellow, FT-IR spectroscopy was performed to confirm the presence of the azide stretching band around 2120 cm⁻¹. The R5 sequence functionalized with azide termination will be denoted hR5

**Copper-assisted alkyne-azide cycloaddition (CuAAC) with propargyl methacrylate.** As the CuAAC was performed in solution, hR5 was cleaved first from the resin. The resin was transferred in round bottom flask and 100 mL of TFE/CH₂Cl₂ (1/4) mixture was added. The mixture was stirred on an orbital shaker at 280 rpm for 45 min at room temperature. The resin was removed by filtration and the organic solvent was evaporated using rotary evaporator.

Cu(I)Br (103 mg, 0.72 mmol) was added to the flask followed by three vacuum/argon cycles. 15 mL of anhydrous CH₂Cl₂ was then added followed by PMDETA (0.30 mL, 1.44 mmol) and propargyl methacrylate (0.54 mL, 4.32 mmol). The reaction mixture was shaken under magnetic stirring. The completion of the reaction was monitored by the disappearance of the characteristic stretching band of azide groups at 2120 cm⁻¹ by FT-IR spectroscopy (after about 1 h). The organic solvent was evaporated by rotary
evaporator. Copper was removed using Cuprisorb® beads. A large quantity of trapping beads was washed with water, then methanol and dichloromethane. MA-hR5 was dissolved in dichloromethane and the washed Cuprisorb® was added to the macromonomer solution. The mixture was placed on orbital stirrer at 280 rpm and room temperature until the solution become yellow. The monomer was filtered, and the dichloromethane was evaporated by rotary evaporator.

**Removal of the Pbf protecting groups and purification.** The Pbf protecting groups were removed using a mixture of TFA/TIPS/H₂O (95.0/2.5/2.5 v%). The monomer was dissolved in this mixture and stirred overnight at room temperature and the solvent was evaporated by rotary evaporator. 50 mL of Milli-Q water was added and the aqueous phase was washed three times with diethyl ether. The aqueous solution was lyophilized. Finally, the monomer was purified by chromatography using reversed phase C18 as stationary phase (Discovery® DSC-18 SPE Bulk Packing from Sigma-Aldrich) and water/acetonitrile mixture as eluent. A gradient was performed to improve the separation of the product from the impurities starting from 100 v% to 85 v% water. The collected fractions containing the product were lyophilized. The product was obtained as a light-yellow powder and stored at -20 °C.

MA-hR5: 618 mg (23 % yield). ¹H NMR (400 MHz, D₂O (0.5 mL with 5 µL of TFA)) δ: 8.06 (s, 1H), 6.12 (s, 1H), 5.71 (s, 1H), 5.30 (s, 2H), 4.42 (t, J = 6.8 Hz, 2H), 4.31 (dd, 9H), 4.25-4.15 (m, 9H), 3.31-3.05 (m, 20H), 2.25 (t, 2H), 1.97-1.47 (m, 47H), 1.23 (q, J = 7.3 Hz, 2H) ppm. ¹³C NMR (126 MHz, D₂O + TFA) δ: 215.37 (acetone), 176.87, 175.07, 174.01, 173.53, 169.06, 156.69, 135.49, 127.26, 125.37, 57.59, 53.46, 52.39, 50.28, 40.49, 34.96, 30.19, 28.96, 27.99, 27.56, 24.95, 24.40, 17.30 ppm. MALDI-ToF (m/z) [M+H]⁺ calculated from C₇₃H₁₄₀N₄₃O₁₄, 1843.156; found 1844.303.

## 4.4. RAFT Polymerization of MA-hR5

<figure><img src="image_13.png" /><figcaption>Scheme 2. RAFT polymerization of MA-hR5 in aqueous solution.</figcaption></figure>

All polymerizations were conducted with the same quantity of MA-hR5 (50 mg, 47 µmol) and aqueous solution (5 mL) to reach a monomer concentration of 10 mg·mL⁻¹ (Scheme 2). The concentration in initiator (I, either VA-044 or V-501) and 4-cyano-4-(((ethylthio)carbonothiol)thiol)pentanoic acid used as chain transfer agent (CTA) was adjusted to the targeted degree of polymerization (i.e. Xn = 10, 20 or 50) by adding the needed volume of the I and CTA solutions prepared in MeOH at a concentration of 10 and 5 mg·mL⁻¹ respectively while maintaining the CTA/I ratio to 1/0.5. After bubbling the reaction mixture with argon for 30 min, the flask was placed in a pre-heated oil bath thermostated at 70 °C. The reaction was stopped by placing the reaction flask in an ice bath. The polymer was purified by dialysis using a regenerated cellulose dialysis membrane (Spectrapor® 6, MWCO = 2000 g·mol⁻¹) against deionized water at 65 °C for 24 h followed by lyophilization and storage at -20 °C. When running the polymerization kinetics experiments, aliquots of 500 µL of the polymerization mixture were withdrawn periodically, lyophilized, and analyzed by ¹H NMR spectroscopy in D₂O containing 1 v% TFA-d.
## 4.5. Molecular dynamics simulations

Classical molecular dynamics “MD” were performed using the AMBER.20 GPU software suite⁴⁸ in which the potential energy U of the system is empirically described by a sum of bond, angle and dihedral deformation energies and a pair wise additive 1-6-12 potential (electrostatic + van der Waals) between non-bonded atoms (Eq 1).

$$ U = \sum_{bonds} k_b (r - r_0)^2 + \sum_{angles} k_{\theta} (\theta - \theta_0)^2 + \sum_{dihedrals} \sum_{n} V_n (1 + \cos(n\varphi - \gamma)) + \sum_{i<j} \left[ \frac{q_i q_j}{R_{ij}} - 2\epsilon_{ij} \left(\frac{R_{ij}^*}{R_{ij}}\right)^6 + \epsilon_{ij} \left(\frac{R_{ij}^*}{R_{ij}}\right)^{12} \right] $$

Eq 1

The different simulated system consisted of 10 MA-hR5 molecules immersed in a cubic solvent box of about 118.0 Å side length. Furthermore, for each system different anions (Cl⁻, TFA⁻ or Cit⁻ (n = 0 to 3)) were introduced to neutralize the system. Additionally, each system was also composed of about 54000 H₂O molecules. A summary of the main characteristics of the different simulated system is given in Table SI-1. Force field parameters for the arginine amino acids were taken from the AMBER ff14SB force field,⁴⁹ while those for the remaining fragments of the MA-hR5 unit as well as those of the Cit⁻ (n = 0 to 3) entities were taken from the GAFF Force Field,⁵⁰ while those for Cl⁻ stem from the work of Cheatham et al.⁵¹ Water molecules were represented using the OPC3 model.⁵² Atomic charges for the non-arginine fragments were obtained using the RESP procedure.⁵³ Cross terms in van der Waals (vdW) interactions were constructed using the Lorentz-Berthelot rules. 1-4 van der Waals and 1-4 electrostatic interactions were scaled by a factor of 2 and 1.2, respectively. Simulations were performed using 3D periodic conditions. An atom-based cutoff of 12 Å for electrostatic and vdW interactions was applied, while long-range electrostatic interactions were calculated using the particle mesh Ewald method. After equilibration an MD simulation of a total of 1.0 µs in the NVT ensemble was performed. The temperature of the system was maintained at 333.15 K using a Berendsen thermostat⁵⁴ with a relaxation time of 1.0 ps. A time step of 1 fs was used to integrate the equations of motion via the Verlet leapfrog algorithm. Snapshots along the trajectory were taken using the VMD software.⁵⁵

## CREDIT AUTHOR CONTRIBUTION STATEMENT

Amélie Augé: conceptualization, methodology, investigation, formal analysis, writing, visualization.
Tetiana Dorosh: investigation. Ayoub Rachdi: investigation. Quentin Bievelot: investigation. Alain Chaumont: supervision, writing, visualization. Fouzia Boulmedais: funding acquisition, supervision.
Delphine Chan-Seng: conceptualization, funding acquisition, supervision, writing, visualization.

## CONFLICTS OF INTEREST

There are no conflicts to declare.
DATA AVAILABILITY

Data will be available upon request.

ACKNOWLEDGMENTS

This research was supported by the Frontier Research in Chemistry Foundation (project THEOS),
the région Grand Est (Fonds Régional de la Coopération pour la Recherche - project ERMES - and
contrat doctoral call - project MARS), and the French National Research Agency (project ARHES,
ANR-22-CE06-0011). This work of the Interdisciplinary Institute HiFunMat, as part of the ITI 2021-
2028 program of the University of Strasbourg, CNRS and Inserm, was supported by IdEx Unistra
(ANR-10-IDEX-0002) and SFRI (STRAT'US project, ANR-20-SFRI-0012) under the framework of the
French Investments for the Future Program. The postdoctoral position of AA is supported by the
Frontier Research in Chemistry Foundation and the doctoral position of QB by the ANR and the
region Grand Est. T.D. acknowledges the financial support from "Agence Nationale de la
Recherche" (CASH, ANR-21-CE06-0033) for PhD funding. The authors are thankful to M. Legros for
SEC measurements. The authors thank the polymer characterization facilities at the Institut
Charles Sadron and the Cronenbourg NMR core facilities.

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