Hamid Ghorbani Shiraz
email: hamid.ghorbani.shiraz@liu.semikhailvagin-mikhail.vagin@liu.se

Zia Ullah Khan

Daniel Péré

Xianjie Liu

Yannick Coppel

Mats Fahlman

Magnus Berggren

Radoslaw Chmielowski

Myrtil L Kahn

Mikhail Vagin

Xavier Crispin

Hamid Gh

Shiraz

3R-TaS 2 as an Intercalation-Dependent Electrified Interface for Hydrogen Reduction and Oxidation Reactions

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nanosheets. XRD and XPS data reveals an increase of the interlayer distance by 22%. The HER and HOR were characterized in a Pt group metal-free electrochemical system. The pristine sample shows a low overpotential of -0.016 V at onset. The intercalated sample demonstrates a large shift on its performance for HER. It is revealed that the intercalation is a potential key strategy for tuning the performance of this family of catalysts. The inhibition of HER by intercalation is considered as the increase of the operational window of water-based electrolyte on negative electrode, which is relevant to technologies of electrochemical energy storage.

Introduction. For many decades, hydrogen has been identified as a key energy carrier for a sustainable future society, as it offers a high energy density and principally no negative direct impact on the environment, with respect to CO2 emissions. Hydrogen generation, as a first step towards a hydrogen economy, has been a matter of debate among scientists. Although many production methods, like photochemical or cracking reformation, or gasification of biomass, are being operated all over the world, there are several problems with current methods such as low yield or major environmental impacts.

One of the most efficient and clean processes to produce hydrogen is via electrochemical hydrogen evolution reaction (HER). There are many electrocatalysts for HER that possess high efficiencies, among which Pt, Rh, and Pd 1, 2 are commonly considered and explored. Although numerous candidates like hybrid structures (core-shell, alloys, composites, etc.) [START_REF] Bai | Surface Polarization Matters: Enhancing the Hydrogen-Evolution Reaction by Shrinking Pt Shells in Pt-Pd-Graphene Stack Structures[END_REF][START_REF] Yan | Three-Dimensional Crystalline/Amorphous Co/Co3o4 Core/Shell Nanosheets as Efficient Electrocatalysts for the Hydrogen Evolution Reaction[END_REF] , doped atom systems [START_REF] Luo | One-Dimensional Porous Hybrid Structure of Mo2c-Cop Encapsulated in N-Doped Carbon Derived from Mof: An Efficient Electrocatalyst for Hydrogen Evolution Reaction over the Entire Ph Range[END_REF] , metal organic frameworks [START_REF] Lin | Ru Doped Bimetallic Phosphide Derived from 2d Metal Organic Framework as Active and Robust Electrocatalyst for Water Splitting[END_REF] , metal oxide arrays [START_REF] Zhu | Metal Oxide-Based Materials as an Emerging Family of Hydrogen Evolution Electrocatalysts[END_REF] , etc. have shown proper performance characteristics, several major problems still lie ahead. Several studies are being conducted in the search for a cost-effective (especially targeting Platinum group metal-free catalysts), efficient, and scalable electrocatalyst. In that context, transition metal dichalcogenides (TMDs) constitute another class of two-dimensional materials where the elemental periodic unit is composed of an atomic layer of a transition metal (Ti, Ta, Mo, W, etc.) in the center with a chalcogen atomic layer (S, Se) on either side. The geometry of chalcogen atoms with respect to transition metal results in various crystal structures (1T, 2H and 3R, etc.) where T, H and R represent overall structures (trigonal, hexagonal, and rhombohedral) and symmetry repetition is given with the number (1, 2, 3) of layers [START_REF] Dunnill | Superconducting Tantalum Disulfide Nanotapes; Growth, Structure and Stoichiometry[END_REF] . TMDs like WS2 display significant activity for HER [START_REF] Geng | Engineering Sulfur Vacancies in Basal Plane of Mos2 for Enhanced Hydrogen Evolution Reaction[END_REF] . Also, MoS2 is one of the most investigated TMDs towards HER [START_REF] Voiry | Conducting Mos2 Nanosheets as Catalysts for Hydrogen Evolution Reaction[END_REF][START_REF] Li | Mos2 Nanoparticles Grown on Graphene: An Advanced Catalyst for the Hydrogen Evolution Reaction[END_REF][START_REF] Yu | Layer-Dependent Electrocatalysis of Mos2 for Hydrogen Evolution[END_REF] . Among TMDs, tantalum disulfide (TaS2) exhibits the highest temperature-dependent electronic conductivity [START_REF] Bulaevskiĭ | Structural Transitions with Formation of Charge-Density Waves in Layer Compounds[END_REF][START_REF] Gamble | Intercalation Complexes of Lewis Bases and Layered Sulfides: A Large Class of New Superconductors[END_REF][START_REF] Gamble | Superconductivity in Layered Structure Organometallic Crystals[END_REF] , which motivates the investigation of HER electrocatalytic activity of this 2D material for the use as (see Fig. 1a andb). It is demonstrated that the majority of 2D structures are catalytically, several times more active at the edge sites, as compared to the basal plane [START_REF] Zhang | A Facile Preparation of Ws2 Nanosheets as a Highly Effective Her Catalyst[END_REF] . To improve the (electro)catalytic activity, several modifications with the aim of activation of TaS2 sheets are studied, such as plasma treatment [START_REF] Li | Atomic-Sized Pores Enhanced Electrocatalysis of Tas2 Nanosheets for Hydrogen Evolution[END_REF] , chemical exfoliation [START_REF] Wu | Exfoliated 2d Transition Metal Disulfides for Enhanced Electrocatalysis of Oxygen Evolution Reaction in Acidic Medium[END_REF] , atom-scale dispersion [START_REF] Wang | In Atom-Scale Dispersed Palladium in a Conductivepd0. 1tas2 Lattice with a Unique Electronic Structurefor Efficient Hydrogen Evolution[END_REF][START_REF] Yu | Tuning the Hydrogen Evolution Performance of Metallic 2d Tantalum Disulfide by Interfacial Engineering[END_REF] , interfacial engineering, etc. Yu et al.

have been using theoretical calculations that shows that cracked eight-awn star TaS2 nanostructure possesses significant electrocatalytic properties [START_REF] Feng | Cracked Eight-Awn Star Tas 2 with Fractal Structures Used as an Efficient Electrocatalyst for the Hydrogen Evolution Reaction[END_REF] . They revealed that both basal and edge sites contribute to HER. Also, Hanlin et al. demonstrated that the electro-exfoliation of bulk TaS2 into nanosheets can significantly enhance the electrocatalytic activity [START_REF] Chen | Highly Effective Electrochemical Exfoliation of Ultrathin Tantalum Disulfide Nanosheets for Energy-Efficient Hydrogen Evolution Electrocatalysis[END_REF] ; leading to low overpotential and a favorable Tafel slope of 197 mV (@10 mA.cm -2 ) and 100 mV.dec - 1 , respectively, while compared to those of the parent material (overpotential and Tafel slope of 547 mV and 216 mV.dec -1 , respectively). This demonstrates that the exfoliation of bulk TaS2 can improve the performance, which may be ascribed to the increase of the interlayer space.

Hence, nano-organization of the material is crucial, as it impacts the diffusion of protons via accommodation of more protons at the edge sites where the hot reaction sites are located.

Interestingly, TaS2 can be exfoliated into nano-flakes, which can host a variety of linear and ring-shaped organic molecules in the inter-layer gaps (Fig. 1c). To the best of our knowledge, we have not found any prior study reporting the role of intercalated organic molecule on the catalytic efficiency of the HER. In this work, we studied the effect of the intercalation of hexylamine (HA) in TaS2 nanosheets on the HER performance. The exfoliated TaS2 nanoflakes were studied with scanning electron microscopy (SEM) and their intercalation with the hexylamine was studied by X-ray diffraction (XRD). The surface compositions were analyzed with X-ray and UV-photoelectron spectroscopies (XPS, UPS). Additional evidence of the structure of the interaction between the organic molecules and exfoliated-TaS2 were obtained by solid-state NMR spectroscopy and, finally, HER was measured in a standard electrochemical cell. Ta and S were mixed in stoichiometric ratio in an agate mortar and then transferred to quartz ampoule through a funnel. The ampoule was evacuated and flame-sealed. Later, it was placed in a vertical furnace for 48 hours at 900˚C and then quenched in water at room temperature.

The ampoule was opened inside a N2 glovebox, and the powder was passed through a 125 µm mesh. The powder was heated to 650 ˚C under vacuum to produce the 3R-TaS2 crystal structure with an electrical conductivity (σ) of 500 S/cm. HA and TaS2 were then mixed in 20:1 molar ratio inside a sealed glass bottle and stirred at 50 ˚C for 5 days. To suppress the agglomerated particles, the inks of the electrocatalysts were subjected to ultrasonication just before the casting of the films.

Structural Characterization. The X-ray diffraction patterns (XRD) were collected at room temperature using a D8 Bruker diffractometer equipped with a copper anode and a point detector. The scanning electron microscopy (SEM) investigations were carried out with a Hitachi S-4700 equipped with a field emission gun. For both XRD (Bruker D8 Advance / copper / theta-2theta mode / 5° -67° / step 0.04° / time : 10s per step) and SEM (5.00 kV and 12.5*2.50k SE(M) and 10.0 kV and 13.2*5.00k SE(M) for top view and cross section images, respectively) analyses, the TaS2 powders were drop-casted on glass substrates which were heated at 100˚C for 30 minutes inside glovebox to evaporate ethanol used as a solvent. XPS/UPS (X-ray, ultraviolet photoemission spectroscopy) measurements have been performed in Scienta ESCA 200 system under the base pressure of 2E-10 mbar with SES 200 electron analyzer, a monochromatic Al Ka x-ray source (h=1486.6 eV) and a helium discharge lamp (h=21.22 eV) for XPS and UPS, respectively. All spectra were collected at normal emission and at room temperature. The spectrometer was calibrated by a sputter-cleaned Au film with the Fermi level at 0 eV and Au 4f7/2 peak at 84.0 eV with its full width at half maximum being 0.65 eV for XPS. The total energy resolution of UPS is about 0.08 eV estimated from the width of the Fermi edge of a clean Au film. The work function of sample was extracted from the edge of the secondary electron cutoff in UPS spectrum with a bias of -3V applied in sample.

NMR experiments were recorded on Bruker Avance 400 III HD spectrometer operating at magnetic fields of 9.4 T. Samples were packed into 2.5 mm zirconia rotors under argon inside a glove box. The rotors were spun between 10 and 15 kHz at 295 K. [START_REF] Jin | Mechanochemical-Assisted Synthesis of Ternary Ru-Ni-S Pyrite Analogue for Enhanced Hydrogen Evolution Performance[END_REF] H MAS were performed with the DEPTH pulse sequence and a recycle delay of 3 s. [START_REF] Bulaevskiĭ | Structural Transitions with Formation of Charge-Density Waves in Layer Compounds[END_REF] C MAS and CP-MAS were recorded with recycle delays of 5 s and 1.5 s, respectively and with a contact time of 2 ms for CP-MAS. Chemical shifts were externally referenced to liquid TMS. The NMR experiments had to be realized in small rotors (2.5 mm) because of the unfavorable electromagnetic properties of the samples. Electrochemical Characterization. Electrochemical measurements were carried out using a three-electrode setup (BioLogic SP200 potentiostat) in 0.5 M H2SO4 solution. A graphite felt was used as a counter electrode to provide the highest possible surface area, and to avoid any metallic contamination [START_REF] Gu | Is Base-Inhibited Vapor Phase Polymerized Pedot an Electrocatalyst for the Hydrogen Evolution Reaction? Exploring Substrate Effects, Including Pt Contaminated Au[END_REF] . The structure and performance of carbon felt characterized and proved during measurement. An Ag/AgCl and a rotating glassy carbon disk electrodes were used as a reference and working electrodes, respectively. A 5 wt% Nafion was used as a protonconductive binder. The measurements were done in the electrolyte where the working electrode was rotating at 800 rpm and the system was purging using pure hydrogen gas. For analysis, all the potentials are converted to reversible hydrogen electrode using Nernst equation; in 0.5 M H2SO4 (pH=0) the equation is simplified as 𝐸(𝑅𝐻𝐸) = 𝐸(𝑆𝐶𝐸) + 0.242.

Linear sweep scan rate (LSV) curves were measured at the scan rate of 5 mV.sec -1 , in the range of 0.2 to -1 V versus RHE, all reported values are IR-compensated. The electrochemical active surface area measurements were recorded at scan rates 10 to 500 mV.sec -1 in a potential window of 0.14-0.24 (Vs. RHE).

Results.

From the synthesis of TaS2, we succeeded to obtain a powder of the 3R-TaS2 crystal structure, which was supported by XRD studies. It was reported [START_REF] Feng | 3r Tas2 Surpasses the Corresponding 1t and 2h Phases for the Hydrogen Evolution Reaction[END_REF] that this structurallycontrolled electrocatalyst showed a highest HER activity in comparison with the other TaS2 crystal structures illustrated by the lowest overpotential for HER (0.2 V, compared to 0.47 V for 2H-TaS2 and 0.55 V for 1T-TaS2 and 0.02 V for Pt) and the lowest Tafel slope of 85 mV/dec (compared to 100 mV/dec for 2H-TaS2, 155 mV/dec for 1T-TaS2, and 30 mV/dec for Pt/C).

The SEM image of the powder shows platelets of TaS2 of various sizes (Fig. 2a). A compressed layer of this powder displays an electrical conductivity of 500 S/cm suggesting that it has a metallic character. To intercalate HA into the crystal strucutre, the following simple recipe was used: HA and the TaS2 powder were mixed at a 20:1 molar ratio inside a sealed glass bottle and stirred at 50 ˚C for 5 days. That step results in the full intercalation of HA into partially exfoliated Ta S2 nano-flakes.

After exfoliation and intercalation, the suspension was casted on top of a glass substrate to form a thin layer. SEM cross-section images, given in Fig. 2b To better understand the intercalation phenomenon of HA in 3R-TaS2, the samples were analyzed by X-ray photoelectron spectroscopy (XPS). To emphasize the change on the valency and different chemical environments, all XPS data were plotted with the binding energy relative to the vacuum level. The Ta 4f spectrum of the pristine 3R-TaS2 clearly indicated that there is more than one type of Ta valency (left panel in Fig. 3a). The doublet around 27 and 29

eV are from 3R-TaS2, where the doublet around 30.5 and 32.5 eV can be attributed a surface oxide (TaOx) [START_REF] Chamlagain | Thermally Oxidized 2d Tas2 as a High-Κ Gate Dielectric for Mos2 Field-Effect Transistors[END_REF] due to the exposure of 3R-TaS2 to ambient air. Upon HA intercalation, the Ta4f doublet from the surface oxide TaO5x can be significantly decreased, which indicates that HA participates to the removal of the oxide. Also meanwhile, it can be clearly seen that the doublet from 3R-TaS2 has a slight shift to the high binding energy, around 0. We pursuit the surface analysis by measuring the UV photoelectron spectra of the samples. A first observation is that the secondary electron cutoff of the UPS spectra (Fig. 3c) demonstrates that the work function of 3R-TaS2 decreases from 4.3 eV, for the pristine airexposed TaS2 layer, to 3.9 eV after the intercalation of HA. The drop in work function by 0.4 eV indicates a significant change in the interfacial energetics. The most significant change of the UPS spectra upon HA intercalation is the appearance of the valence features of TaS2 at 0.5 eV, 1.1 eV and 2.6 eV relative to the Fermi level (HA has a high valence band feature). On the contrary, there is no visible feature in the pristine 3R-TaS2, and the spectral intensity is very low and there is almost featureless close to the Fermi level, which is mostly due to the presence of the insulating oxide surface layer with a large energy gap. The surface oxide TaOx has the dominant contribution to UPS spectrum of the 3R-TaS2 surface. Indeed, by zooming in on the energy region close to the Fermi level (inset of Fig. 3d), the Fermi edge of the metallic TaS2 is visible together with the first band at 0.5 eV on the pristine TaS2 sample. The removal of the surface oxide by the HA treatment leads to an enhancement of about 50 times of the valence bands in addition to clear valance band features.

Further insight on the structure of HA interacting with exfoliated TaS2 were obtained from solid-state NMR spectroscopy. Similar results were obtained regardless of the HAfunctionalized TaS2 batch suggesting that the synthesis is reliable and reproducible. [START_REF] Jin | Mechanochemical-Assisted Synthesis of Ternary Ru-Ni-S Pyrite Analogue for Enhanced Hydrogen Evolution Performance[END_REF] H MAS spectrum of TaS2 alone displays a weak and broad signal centered at 3 ppm that can be tentatively assigned to isolated hydroxyl groups and physisorbed water molecules (Fig 4a bottom). The 1 H MAS spectrum of HA-intercalated TaS2 (Fig. 4a top) shows stronger signals with a characteristic alkyl chain peak at 1.24 ppm and a shoulder at 7.24 ppm attributable to ammonium. This assignment is confirmed by signal deconvolution (Fig. S2), with a ratio between the 7.24 and 1.24 signals equal to 0.18 (±0.03), that corresponds to the expected value of 0.2 (3/15, (NH3 + )/C6H15) for the ammonium species. The 13 C CPMAS and MAS spectra (Fig. 4b) show broad peak characteristic of disordered structures. The alkylamines adopt several different conformations with varying coordination modes. The 13 C CPMAS and MAS spectra are very similar with a good sensitivity of the CP MAS experiment indicating that the amines are rigid in the material [START_REF] Spataro | Prominence of the Instability of a Stabilizing Agent in the Changes in Physical State of a Hybrid Nanomaterial[END_REF] . The position of the central CH2 groups of the alkyl chain around 30 ppm indicates that the aliphatic chain of the amines is in the form of rigid pellets. Finally, we investigate the electrocatalytic performance of the TaS2 with and without HA intercalation for both hydrogen evolution reaction (HER) and hydrogen oxidation reaction (HOR) in acidic medium. The electrode is prepared in the following way: the TaS2 powder or the HA-TaS2 nanoflakes are dispersed in an ethanol-water solution and mixed with 5wt% of Nafion as a binder, followed by a final deposition on a glassy carbon substrate. We have chosen the Nafion as the binder as it is not soluble in water and is also stable in acids. Moreover, it promotes the transport for protons inside the TaS2 electrode layer, at humid/aqueous conditions. The linear sweep voltammetry visualizes a strong HER activity on the pristine 3R-TaS2 (Fig. 5a). The potentials are referred to the zero-voltage corresponding to the thermodynamically dictated electronic energy threshold (reversible potential) of the reaction:

𝐻 + + 𝑒 -↔ 𝐻 0 (1)
b

The zero voltage demarcates the potentials of HER and HOR manifested in reversible hydrogen electrode (RHE). The increase of electronic energy, which corresponds to the application of negative electric potential, beyond a threshold (so called overpotential)

prioritizes the reaction (1) from left to right, i.e. the HER. The overpotential for our 3R-TaS2/Nafion electrode is as low as 0.016 V at the onset. We speculate that the negatively charged Nafion might lead to small interfacial potential drop with respect to the electrolyte.

So, the overpotential is very small and the HER-driven current rise is remarkable all through -70 mA cm -2 , at -0.08 mV, illustrating a diffusion-free behavior typical for fast proton transport.

This impressive performance shows that the 3R-TaS2/Nafion electrode is competitive with other state-of-the-art electrocatalysts for HER. In contrast, the HA-TaS2/Nafion electrode with HA intercalation leads to a significantly higher overpotential to observe the onset of HER and a current level of -7 mA cm -2 at -0.34 V, which is significantly smaller than with the TaS2/Nafion electrode. Note that the blank current collector (glassy carbon) shows an overpotential of ca. 0.74 V, thus demonstrating that it has no impact on the current observed for the TaS2-based electrodes deposited on glassy carbon.

The application-relevant steady-state measurements (Fig. 5b and5c) confirm the increase HER rate on the TaS2 electrode in comparison with HA intercalated electrode.

Importantly, the steady-state measurements allow for the observation of HOR currents corresponding to the proceeding of the reaction (1) running from right to left. The data presentation in Tafel coordinates (Fig. 5c) enables us to investigate in detail the electrocatalytic phenomena of HER and HOR. In contrast to smooth glassy carbon electrode, both TaS2-based electrodes showed non-monotonous dependence of current density on the overpotential of HER. Specifically, the region of current densities of 0.27 mA cm -2 (ln(J) ~ -1.3, Fig. 5c) is characterized with the change of the slope. This might illustrate the effect of kinetic limitation by the mass transport on the highly porous electrocatalyst. The low current densities enable the full involvement of the electrocatalyst surface distributed within nanoscale pores, while the higher current densities are characterized by a limitation of reagent or product transport inside the pores. All electrode systems showed a Tafel slope of ca. 120 mV dec -1 . This implies that the rate-determining step in HER on all electrodes of this study is a Volmer step [START_REF] Shinagawa | Insight on Tafel Slopes from a Microkinetic Analysis of Aqueous Electrocatalysis for Energy Conversion[END_REF] :

𝐻 3 𝑂 + + 𝑒 -+ 𝑀 ↔ 𝑀 ⋯ 𝐻 0 + 𝐻 2 𝑂 (2).
where M denotes the surface empty site.

Coherently with linear sweep voltammetry measurements, HER showed a higher rate on pristine TaS2 in comparison with the intercalated TaS2. Interestingly, HOR current characteristics reveals an insensitivity to the effect of intercalation of HA in the TaS2 electrodes. Note, however, that the HOR current is much higher than the vanishingly small current level of the glassy carbon collector. Both pristine and intercalated TaS2-modified electrodes show a change of Tafel slopes from 40 mV dec -1 to 120 mV dec -1 as current densities increase. This corresponds to Volmer step as a rate-determining in HOR (reaction (2) proceeds from right to left) [START_REF] Shinagawa | Insight on Tafel Slopes from a Microkinetic Analysis of Aqueous Electrocatalysis for Energy Conversion[END_REF] .

The extrapolation of Tafel slopes (120 mV dec -1 ) for both HOR and HER yields the interception point corresponding to the exchange current density of HOR/HERoverpotentialfree rate of the process, where the rates of both HOR and HER are equal. In order to take into account the effect of the nanoscale roughness of porous electrocatalysts [START_REF] Voiry | Best Practices for Reporting Electrocatalytic Performance of Nanomaterials[END_REF] , we normalized the estimated exchange current densities to the capacitive current densities, which are assumed to represent the electrochemically available surface area (EASA) of the electrode. The HA intercalated TaS2 electrode shows up to 20 times higher capacitive currents as compared to the pristine material of the same mass load. We identified this difference as due to the exfoliation process taking place upon HA treatment while producing the nanoflakes (Fig. 2b). The exchange current densities estimated from the regions of small overpotentials were 0.041 mA cm -2 (ln(J0) ~ -3.18) and 0.033 mA cm -2 (ln(J0) ~ -3.39) for pristine and intercalated TaS2. The exchange current densities estimated from the high overpotentials, featured with porous mass transport limitations, were 0.025 mA cm -2 (ln(J0) ~ -3.68) and 0.021 mA cm -2 (ln(J0) ~ -3.87)

for pristine and intercalated TaS2, respectively. The exchange current density for blank glassy carbon is significantly smaller (0.0015 mA cm -2 (ln(J0) ~ -6.44)). The kinetic characteristics obtained for TaS2 are comparable with the rates of HER/HOR on gold (0.027 mA cm -2 (ln(J0) ~ -3.75)) and significantly smaller than on platinum (0.78 mA cm -2 (ln(J0) ~ -0.23)). The normalized exchanged current densities show a significant poisoning effect of the hexylamine intercalant (Table 1). The rate of HOR/HER on intercalated TaS2 was more than 20 times smaller in comparison with pristine samples. [START_REF] Meffre | Use of Long Chain Amine as a Reducing Agent for the Synthesis of High Quality Monodisperse Iron (0) Nanoparticles[END_REF] , hence we believe that HA reduces TaS2 to functionalize its surface (Fig. 6a) but also to remove the surface oxide of TaS2. Both XPS and UPS results are in agreement with this interpretation. Now, the XRD demonstrates the full intercalation of the HA in the TaS2, hence HA does not only remove the oxide but truly intercalates in the Van der walls gaps of the TaS2. Understanding the nature of the surface and edge of the TaS2 nanoflakes are important to explore the electrocatalytic reactions. The UPS reveals that the metallic electronic structure of TaS2 (high density of states at the Fermi level) is enhanced after HA treatment as the surface oxide is removed. Thus, one could imagine that the electrocatalytic activity would be boosted thanks to available metallic levels on the surface. However, the electrochemical data tells us the opposite. Indeed, a larger overpotential and lower current densities are obtained for the HER upon HA intercalation. It is thus reasonable to think that the HA molecules are also forming sub-monolayer on the surface of the TaS2 nano-flakes and that its interaction with Ta atoms blocks electrocatalytic reactions.

For instance, it is known that HA is forming a monolayer on various 2D materials by a chemical reaction involving hydroxyl groups contaminating their surface in presence of air and humidity [START_REF] Su | Waterproof Molecular Monolayers Stabilize 2d Materials[END_REF] . The mechanism proposed involves the basic character of the amine with a proton transfer from the surface hydroxide groups: Ta-OH + H2N-R → (Ta-O -) ( + NH3-R) (See Fig. 6b). NMR spectroscopic measurements agree with this assumption. It is proved that the resulting formed monolayer can remove water molecules from the Van der walls gaps by expressing its hydrophobic character, which in turn suppresses the nanosheet catalytic activity and ion transport towards the HER. The structure of the aliphatic chain of HA in the form of rigid pellets also agreed with the hydrophobic character of the organic layer. The moderate waterblocking property is also demonstrated when similar architectures of graphene oxide is treated with different alkylamine [START_REF] Abdullahi | Facile Fabrication of Hydrophobic Alkylamine Intercalated Graphene Oxide as Absorbent for Highly Effective Oil-Water Separation[END_REF] . Moreover, the contact angle of HA-treated TaS2 nanoflakes is twice of those measured on non-surface modified systems and further increases for longer alkyl chains of the alkylamine [START_REF] Huang | Tuning the Surface Charge Density of Exfoliated Thin Molybdenum Disulfide Sheets Via Non-Covalent Functionalization for Promoting Hydrogen Evolution Reaction[END_REF] .

Apart from that, surface charges will remain at the surface of the structure, which gives that the zeta potential of the intercalated TaS2 likely decreases. This decrement in surface charges has a detrimental effect on the electron transfer between the surface and the protons [START_REF] Huang | Tuning the Surface Charge Density of Exfoliated Thin Molybdenum Disulfide Sheets Via Non-Covalent Functionalization for Promoting Hydrogen Evolution Reaction[END_REF][START_REF] Shiraz | Transition Metal Sulfides for Electrochemical Hydrogen Evolution[END_REF] ; thus, negatively impress the HER activity of the electrocatalyst.

Moreover, the intercalation with HA increases the band gap, meaning that the higher overpotential is needed to drive the reaction of hydrogen reduction [START_REF] Hunt | Band Gap Engineering of Graphene Oxide by Chemical Modification[END_REF] . In principle, the HA intercalation will increase the interlayer gap between nanosheets. However, the effect of impurity was not inevitable in this study, as the impurity is detected by XRD spectrum (Fig. 2). We assume that we have some impurity during the synthesis of electrocatalyst, since the materials were transferred to ampoule and later passed through a mesh. These introduces impurities and possible defects to the film, which may impact the performance of electrocatalysts [START_REF] Toh | Ultrapure Molybdenum Disulfide Shows Enhanced Catalysis for Hydrogen Evolution over Impurities-Doped Counterpart[END_REF][START_REF] Strange | Electrodeposited Transition Metal Dichalcogenides for Use in Hydrogen Evolution Electrocatalysts[END_REF] . The optimized effect of defects might enhance the performance of electrocatalyst, though, as it is already reported in case of transitional metal dichalcogenide HER application [START_REF] Wang | The Mechanism of Hydrogen Adsorption on Transition Metal Dichalcogenides as Hydrogen Evolution Reaction Catalyst[END_REF] . The reason is that the optimized level of defects can offer local higher charge density [START_REF] Jia | A Heterostructure Coupling of Exfoliated Ni-Fe Hydroxide Nanosheet and Defective Graphene as a Bifunctional Electrocatalyst for Overall Water Splitting[END_REF] . Although there are some reports regarding the one-spot electrodeposition directly on electrode [START_REF] Strange | Electrodeposited Transition Metal Dichalcogenides for Use in Hydrogen Evolution Electrocatalysts[END_REF] , the side deposition of species is not inevitable.

Conclusions. In summary, 3R-TaS2 nanosheets and the hexylamine-treated derivative were investigated for hydrogen evolution. Aliphatic intercalant can conduct a high electron donor process of intercalation. XPS and XRD spectroscopy measurements shows that the interlayer distance in nanosheets has been increased as a result of hexylamine intercalation. Polarization curves show us an excellent performance of the pristine 3R-TaS2 sample, while the intercalated sample reveals a deteriorated performance. The suppressing effect of HA-TaS2 could be arisen from the i) decrease of the surface charges associated to a decrease of their mobility, ii) water molecule-blocking, and iii) bandgap increment, as post-intercalation effects. Apart from that, the hydrogen oxidation measurements express that both catalysts are quite active for the reverse reaction when hydrogen is oxidized, converted to electricity. While the current density for catalyst are exponentially increases as potential increases, glassy carbon doesn't show any potential dependency and stays at current density of 0 V. Tafel slopes also show the Volmer reaction as the rate determining step for both evolution and oxidation of hydrogen. This work demonstrates TaS2 nanosheets as versatile electrified interface, which can be used either as efficient HER/HOR electrocatalyst or as an inert current collector.

Fig. 1 :c

 1 Fig. 1: (a) configuration of sulfurs (yellow) and Metal (dark blue) for different crystal phases,

Fig. 2 :

 2 Fig. 2: SEM images of the TaS2 (a) top view of the bulk powder; (b) cross-section of the film

  c

Fig. 3 :

 3 Fig. 3: Comparison of XPS spectra pristine and HA-intercalated samples; (a) Ta 4f and (b) S

Fig. 4 :

 4 Fig. 4: a) 1 H MAS spectrum of HA-intercalated TaS2 and pristine TaS2; a closer view clearly

Fig. 5 .

 5 Fig. 5. HER/HOR electrocatalysis on TaS2. Linear sweep (a, 5 mV s -1 ) and steady-state (b,

Fig. 6 .

 6 Fig. 6. (a) reductant character of the hexylamine leading to electron transfer and chemical

Table 1 . Kinetic parameters of HER/HOR on TaS2.

 1 To improve reaction rates, a critical phenomenon of interest is the removal/reduction/dissolution of the Ta2O5 surface oxide by the reaction with HA. Alkyl amines are known to serve as reducing agents for metal oxide nanoparticles

	Interface	J0, mA cm -2	J0 / Jcapacitive
		Small	High	Small	High
		overpotentials	overpotentials	overpotentials	overpotentials
	Pristine	0.041	0.025	0.041	0.025
	TaS2
	Intercalated	0.033	0.021	0.0016	0.0010
	Glassy carbon
		0.0015
	(Current collector)
	Gold 31	0.027
	Platinum 32	0.78

Acknowledgments

This work was financially supported by the Swedish Research Council (VR 2016-05990), the Knut and Alice Wallenberg Foundation (KAW 2019.0604; 2021.0195), Karl Erik Önnesjös Foundation, and the Swedish Government Strategic Research Area in Materials Science on Advanced Functional Materials at Linköping University (Faculty Grant SFO-Mat-LiU No. 2009-00971).

Supporting Information

Cyclic Voltammograms, 1 H MAS


Pedro Salas-Ambrosio

Antoine Tronnet

Mostafa Badreldin

Luzangel Reyes

Marc Since

Sandra Bourgeade-Delmas

Bruno Dupuy

Pierre Verhaeghe

Colin Bonduelle

Star-like poly(peptoid)s with selective antibacterial activity

come

Star-like poly(peptoid)s with selective antibacterial activity Pedro Salas-Ambrosio,a Antoine Tronnet,b,c, Mostafa Badreldin,a Luzangel Reyes,a Marc Since,d Sandra Bourgeade-Delmas,e Bruno Dupuy,c Pierre Verhaeghe,b,f Colin Bonduelle*a

We developed new macromolecular engineering approaches enabling the preparation of star-like polypeptoids by ring-opening polymerization. Parallely to the evaluation of their cytotoxicity of the HepG2 human cell line, their screening toward a wide variety of Grampositive and Gram-negative bacteria higlighted several compounds showing not only good but also selective antimicrobial activity.

Introduction, Antimicrobial peptides (or AMPs) are specific macromolecules made of amino acids that are mainly produced by microorganisms to fight against other competing microorganisms. 1-3 As selective biocides, AMPs are potentially active against all types of microorganisms, thanks to their ability to destabilize cell membranes. [START_REF] Wimley | Describing the Mechanism of Antimicrobial Peptide Action with the Interfacial Activity Model[END_REF][START_REF] Reddy | Antimicrobial Peptides: Premises and Promises[END_REF][START_REF] Schmidt | Criterion for Amino Acid Composition of Defensins and Antimicrobial Peptides Based on Geometry of Membrane Destabilization[END_REF][START_REF] Arora | Antibacterial Properties of Human Beta Defensin-3 Derivative: CHRG01[END_REF] This particular mechanism of action is due to various physicochemical properties including their amphiphilic character.6,8 So far, the pharmaceutical development of AMPs remains limited for various reasons including elevated cost of production, scalability issues and their sensitivity to proteases, preventing for instance their oral administration.9-12 This limitation is a tremendous drawback to treat several infections such as the ones caused by Clostridioides difficile, a very important nosocomial pathogen infecting the intestinal tract (an environment full of proteases).13,14 Synthetic macromolecules are certainly the best candidates to overcome the limitations posed by the production and use of AMPs. [START_REF] Salas-Ambrosio | Synthetic Polypeptide Polymers as Simplified Analogues of Antimicrobial Peptides[END_REF][START_REF] Ergene | Biomimetic Antimicrobial Polymers: Recent Advances in Molecular Design[END_REF] In this direction, access to large amounts of material is an important asset of using polymer chemistry. Like AMPs, polymers are macromolecules, but unlike AMPs, they are polydisperse in size and constituted of monomer units whose sequence is not controlled.17,18 Among all polymeric analogs, those made of amino acids called polypeptide polymers are showing great potential by providing one of the best biomimetic and bioactive structures for further biomaterials science applications. [START_REF] Salas-Ambrosio | Synthetic Polypeptide Polymers as Simplified Analogues of Antimicrobial Peptides[END_REF] Although polypeptide polymers demonstrated very efficient antimicrobial activity (including against drug resistance bacteria), they are susceptible to protease degradation. In this context, "polypeptoids", also called poly(N-substituted glycines) are N-alkylated analogs of synthetic polypeptide that can provide enhanced resistance to proteolysis. [START_REF] Miller | Comparison of the Proteolytic Susceptibilities of Homologous L-Amino Acid, D-Amino Acid and N-Substituted Glycine Peptide and Peptoid Oligomers[END_REF][START_REF] Salas-Ambrosio | Cyclic Poly(α-Peptoid)s by Lithium Bis(Trimethylsilyl)Amide (LiHMDS)-Mediated Ring-Expansion Polymerization: Simple Access to Bioactive Backbones[END_REF] The simple preparation of polypeptoids involves the use of building blocks called N-alkylated-Ncarboxyanhydrides or NNCAs that are polymerized through the ring-opening polymerization (ROP) using nucleophiles as initiators. [START_REF] Rasines Mazo | Ring Opening Polymerization of α-Amino Acids: Advances in Synthesis, Architecture and Applications of Polypeptides and Their Hybrids[END_REF][START_REF] Sun | High Antibacterial Activity and Selectivity of the Versatile Polysulfoniums That Combat Drug Resistance[END_REF] This facile route of synthesis enables the preparation of well define copolymers whose chemical composition can easily be adjusted. [START_REF] Deming | Facile Synthesis of Block Copolypeptides of Defined Architecture[END_REF] A very interesting point is that ring-opening polymerization reaction paves the way to various topologies (cyclic, branched, conjugates, etc.) that would be difficult to implement using other preparation methods (biotechnology, solid-phase synthesis, etc.). [START_REF] Salas-Ambrosio | Synthetic Polypeptide Polymers as Simplified Analogues of Antimicrobial Peptides[END_REF] For instance, star polymers are macromolecules with elongated polymeric arms using a core constituted of more than 3 arms and they are known to have the lowest entanglement as compared to cyclic and linear polymers. [START_REF] Wu | Star Polymers: Advances in Biomedical Applications[END_REF][START_REF] Fox | Soluble Polymer Carriers for the Treatment of Cancer: The Importance of Molecular Architecture[END_REF] In infectiology, star-like polypeptides have shown impressive antimicrobial activities. They greatly enhanced antimicrobial property as compared to linear polymers, meanwhile they decreased cell toxicity. [START_REF] Lam | Combating Multidrug-Resistant Gram-Negative Bacteria with Structurally Nanoengineered Antimicrobial Peptide Polymers[END_REF][START_REF] Chen | Star-Shaped Polypeptides Exhibit Potent Antibacterial Activities[END_REF][START_REF] Shirbin | Architectural Effects of Star-Shaped "Structurally Nanoengineered Antimicrobial Peptide Polymers" (SNAPPs) on Their Biological Activity[END_REF][START_REF] Pan | Unnatural Amino-Acid-Based Star-Shaped Poly( L -Ornithine)s as Emerging Long-Term and Biofilm-Disrupting Antimicrobial Peptides to Treat Pseudomonas Aeruginosa -Infected Burn Wounds[END_REF] Scheme 1. Preparation of star-like polypeptoids from NNCAs by ring-opening polymerization. Herein, the goal was to extend the use of polypeptoids copolymers obtained by ring-opening polymerization to these new star-like architectures. We based our design on copolypeptoids merging cationic (ZLys-NNCA) and hydrophobic (Phe-NNCA) monomers and used dendritic macroinitiators (poly(amidoamine) (PAMAM). The goal of this study was to evaluate if this macromolecular engineering approach enhanced or modulated the antimicrobial properties of their linear copolymer analogues (Scheme 1).

Results and discussion

Synthesis and characterization of star-like poly(sarcosine). Several methodologies are well established to design star-like polymers such as atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain-transfer (RAFT) polymerization, living anionic polymerization, ring-opening metathesis polymerization and ring-opening polymerization (ROP).24 Usually, a dendritic core with "n" functional groups is used to promote polymerization growth.24 For instance, star-like polypeptides (from NCAs)26 were successfully developed with PAMAM dendrimers that are displaying primary amines as terminal end groups. With N-alkylated NCA, an early study already reported the preparation of polysarcosine from poly(trimethyleneimine) dendrimers with 64 primary amine groups.30 Here, we decided to employ PAMAM dendrimers macroinitiators to perform various copolymerizations of NNCAs monomers. In a first step, we optimized the ROP from PAMAM-(NH2)16 with Sar-NCA (as a model NNCA) targeting a DP = 30 (for each -NH2 terminal group). According to already published procedures, we set up three different methodologies (table 1): Method A) Direct addition of initiator: we prepared a 0.4 M Sar-NCA (480 equiv.) solution in anhydrous DMF and we added an aliquot of PAMAM-(NH2)16 (1 equiv., solution in DMF at 0.01 M), Method B) Direct addition of monomer:26 we prepared a solution of Sar-NCA (480 equiv.) that we added to a solution with PAMAM-(NH2)16 (1 equiv.) targeting a final concentration of 0.37 M in DMF (the volume ratio of DMF was 16:1 for monomer:initiator), Method C) Reduced concentration: we prepared a solution of Sar-NCA (480 equiv.) that we added to a solution with PAMAM-(NH2)16 (1 equiv.) targeting a final concentration of 0.1 M in DMF (the volume ratio of DMF was 1:1 for monomer:initiator). The polymerizations were followed by FTIR monitoring the NNCA peak at 1850 cm-1. Upon full conversion, the s-(P(Sar)30)16 were precipitated in Et2O, dried under vacuum and isolated in good yields 78-87%. Then, we characterized the polymers obtained from each method by SEC and 1H-NMR (see table 1 and figure 1). From the SEC analyses in DMF, we characterized polymers with numbered-molar masses (Mn) similar to the theoretical values (M/I theoretical, table 1). Careful considerations of the MW values and of dispersity made us conclude that the best methodology was the use of a lower concentration providing a dispersity value ÐM = 1.11 and also the highest yield (87%). With this method, the ROP from different PAMAM (PAMAM with 4, 8, 16 -NH2 terminal groups) further showed that conversions were not significantly influenced by the number of arms and that the efficiency of the polymerization of each arm was approximately the same (data not shown). We then characterized the s-(P(Sar)30)16 by 1H-NMR in DMSO-d6. We observed the characteristic peaks corresponding to poly(sarcosine) signals a and b (3.80-4.42 and 2.50-3.10 ppm) but we were not able to determine the DPNMR because i) the signals of the dendrimer initiator were overlapped by the signals of poly(sarcosine)31 and i) the signals of the dendrimer initiator were very weak due to the low proportion of the PAMAM protons, as compared to the protons belonging to the copolymer arms (1:480). In addition to NMR, SEC analyses provided clear evidence that we successfully prepared star-like polypeptoids in quantitative yields (see esi figure S1). Synthesis and characterization of star-like poly(peptoid)s copolymers. In infectiology, early studies involving branched copolymers constituted of methylmethacrylate and dimetyhyl-aminoethyl-methacrylate demonstrated strong inhibition growth of Escherichia coli and Staphylococcus aureus in diffusion agar plates.32 Since then, similar works were developed to apply this macromolecular engineering approach with other polymers including antimicrobial polypeptides.32-35 Overall, star-like polypeptide were shown to enhance the antimicrobial efficacy: to access such star-shaped copolymers means preparing macromolecules incorporating of cationic and hydrophobic side chains. So far, no one has extended the use of this design to antibacterial polypeptoids. Using methodologies previously developed for cyclic polypeptoids,20 we prepared copolymers by mixing N-protected-lysine-like NNCA (ZLys-NNCA) and phenylalanine-like NNCA (Phe-NNCA) using PAMAM dendrimers initiators. Monomers were prepared through an optimized three-step synthesis method (see esi). We then performed the ROP from PAMAM with different numbers of arms (PAMAM with 4, 8, 16 -NH2 terminal groups) at M/I = 30. We mixed ZLys-NNCA and Phe-NNCA in equal stoichiometry (to target of 50% of hydrophobic content) in DMF and under argon atmosphere (0.1 M at RT, step 1, Scheme 2) Scheme 2. Synthesis of star-like copolypeptoids from ZLys NNCA and Phe-NNCA varying the number of arms.

We monitored the NNCA disappearance of the N-COO signal at 1850 cm-1 by FTIR. Then we isolated the star-protected copolymers S-P(ZNLys-NPhe) 1-3 by precipitation, drying under high vacuum in 40-45% yields and characterized them by SEC and NMR analyses (table 2, figure 2 and figure S2,S3). ‡ First, we characterized the copolymers 1-3 by 1H-NMR in DMSO-d6 (figure 2A and figure S3-S7). We found signals attributable to the polymer backbone (CH2, peak a,a'), signals attributable to the N-Cbz-aminobutyl-glycine (ZNLys) moiety (peaks b-h) and signals attributable to N-benzyl-glycine (NPhe) moiety (peaks i,j). At 20°C, the determination of the hydrophobic content from the integrations of these signals gave us values far from what we expected (figures 2A, S4A, S5A). An 1H NMR analysis performed at higher temperatures further showed that this deviation comes from a poor solubility of the hydrophobic monomer units in the organic solvent (peak j+h and i, figure 2B). On another hand, we could not determine the DPNMR due to i) the high monomer/initiator ratio (120:1 for PAMAM with 4 arms and even higher for PAMAM with 16 arms) and ii) to the PAMAM signals overlapped with the copolymer (peaks b, e, a, a'). Second, we characterized the copolymers 1-3 by SEC in DMF. These analyses allowed to calculate a 2-fold lower weightedmolar mass (Mw) for the different synthesized star copolymers. This result was not surprising as star polymers are known to elute at lower volumes, compared to their linear counterparts, this being due to a difference on the hydrodynamic radius. 36 As other factors such as solvation can also give rise to differences, the decrease in hydrodynamic radius was better evidenced by plotting the double logarithm of mean square radius (RMS radius) versus molar masses (MW) between linear and star-like polymers (figure 2D).37 Indeed, comparing linear copolymer P(ZNLys-Nphe) initiated with allylamine at M/I = 30 with the star-like copolypeptoid S-P(ZNLys-NPhe) 1, the difference in slope values was in agreement with the difference expected for branched polymers (values of slope < 0.5).38 This SEC characterizations also evidenced that we prepared star-shaped copolymers with 2 and 3 (see figure S3). It is to note that we obtained a polymer dispersity that increased with the number of arms (ÐM = 1.18 for 4 arms and up to 1.40 for 16 arms), a feature already reported with star-like polypeptides.28 Following a methodology we previously developed,20 we then performed the deprotection of the Z-Lys side chains in TFA by adding 2 equivalents of HBr (relative to cationic side chains in TFA, RT). After 3 h, the copolymers (S-P(NLys-NPhe)) were isolated by precipitation in diethyl ether, dialyzed (MWCO = 10 kDa), and lyophilized (yields 21-62%, table 2, figure 2C). ‡ To verify the chemical composition in monomer units, we analyzed the samples by 1H-NMR in D2O (figure 2C). We found signals attributable to the polymer backbone (CH2, peak a,a'), signals attributable to the N-aminobutyl-glycine (NLys) moiety (peaks b-e) and signals attributable to NPhe moiety (peaks i,j). We calculated the hydrophobic content by carefully comparing the integration of NLys (peaks c,d) and NPhe (peak j). In marked contrast to hydrophobic contents before deprotection, these contents were in agreement with the theoretical ratio given by the stoichiometry of NNCA monomers (48-54%, table 2). Once again, we could not determine the DPNMR due to the high monomer/initiator ratio (120:1 for PAMAM with 4 arms and even higher for PAMAM with 16 arms) and due to the PAMAM signals overlapped with the copolymer (peaks b, e, a, a').

Antimicrobial potency: number of arms

A key work reported in 2016 by G. Qiao et al. was that star-like polypeptides showed good in vitro and in vivo activities against multidrug-resistance bacteria.26 Composed of several polymeric arms (either 16 or 32 with a polymerization degree of 30), containing lysine and valine (hydrophobic content 30%), these star-like polypeptides were active in killing Gramnegative and Gram-positive bacteria, including multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii. The antibacterial structure-activity relationship studies revealed that increasing arm numbers [START_REF] Ergene | Biomimetic Antimicrobial Polymers: Recent Advances in Molecular Design[END_REF] and length [START_REF] Aoi | Synthesis of a Novel Star-Shaped Dendrimer by Radial-Growth Polymerization of Sarcosine N-Carboxyanhydride Initiated with Poly(Trimethyleneimine) Dendrimer[END_REF] enhanced antimicrobial activity but this increase was associated with an increase in cytotoxicity.28 More recently, I. Kim et al. prepared copolymers made of TFA-lysine and Cbz-glutamate monomer units by grafting from tris-(2-aminoethyl)amine initiators, using ring-opening polymerization. Copolymers bearing 3 arms, having a polymerization degree of 30 and containing 20% of hydrophobic content were shown to be very good antimicrobial activity meanwhile keeping low hemolytic effects.39 Even more recently, J. Cai et al. reported the preparation of a star polymer with 8 arms attached to polyethyleneimine dendrimers and constituted of mixed cationic side chains (lysine or ornithine with diaminoheptylic acid). [START_REF] Pan | Unnatural Amino-Acid-Based Star-Shaped Poly( L -Ornithine)s as Emerging Long-Term and Biofilm-Disrupting Antimicrobial Peptides to Treat Pseudomonas Aeruginosa -Infected Burn Wounds[END_REF] The best properties were found with ornithine at M/I = 20 with improved activity against Gram-positive methicillin-resistant S. aureus and S. epidermidis, and P. aeruginosa. In this work, copolymers 1-3 were used to evaluate the antimicrobial potency towards representative pathogens: Gram-positive (C. difficile, Methicillin-sensitive-and -resistant S. aureus, Enterococcus faecalis, Listeria monocytogenes and Streptococcus pneumoniae), Gram-negative bacteria (E. coli, P. aeruginosa, A. baumanii), and anaerobic or microaerophilic bacteria (Bacteroides fragilis and Helicobacter pylori). The results are presented in figure 3 (and table S1): we determined the MIC from the microdilution method bacteria (see experimental section, approximately 105 CFU/mL) for 24-48 h with increasing concentrations of polypeptoids. We found that S-P(Nlys-Nphe) 1 showed interesting activity against most of Gram-positive and Gram-negative bacteria: the MIC was found below 62.5 µg/mL except for S. pneumoniae (MIC above 250 µg/mL). The best antimicrobial activities were obtained against B. fragilis (anaerobic Gram-negative bacteria) with a MIC value of 7.8 µg/mL and against L. monocytogenes (Gram-positive bacteria) with a MIC value of 15.6 µg/mL in comparison with the positive control amoxicillin that was less active: 15.6 µg/mL for B. fragilis and 250-500 µg/mL for L. monocytogenes (see ESI). S-P(Nlys-Nphe) 2 (figure 3 and table S1) was comparatively less active against the different Gram-positive and Gram-negative bacteria. However, as compared to copolymer 1, we observed a similar activity against L. monocytogenes (MIC value of 15.6-31.25 µg/mL) and an enhanced activity against B. fragilis (MIC value of 7.8 µg/mL). Finally, the antimicrobial potency of S-P(Nlys-Nphe) 3 was very similar to the one of 2: the copolymer was moderately active against most of the different Gram-positive and Gram-negative bacteria tested with several MIC values found below 250 µg/mL (figure 3). Copolymer 3 was more selective with an optimized antimicrobial efficacy found against L. monocytogenes and B. fragilis. Interestingly, 3 showed decreased anti-B. fragilis activity reflected in 4-fold increase of the inhibitory concentration (MIC = 31.25 µg/mL) value as compared to 2. In line with previous work on polypeptides,26-29 antimicrobial activities evaluated on a single arm (linear copolymer, Figure S8 and Table S4 in esi) showed that the star-like architecture optimized the anti-infective potency, especially for S. aureus, E. faecalis, P. aeruginosa and B. fragilis. Overall, these first studies established that star-like polypeptoids can promote antibacterial properties with a significantly broad spectrum of action although some antibacterial activities were more selective. Also in agreement with the literature, the number of arms was an important parameter to tune the activity:27,28 MIC values against B. fragilis were for instance optimized with the star copolymer 2 bearing 8 arms.

Antimicrobial potency: varying the hydrophobic content

To better understand the structure/antimicrobial activity relationship, we also modified the cationic/hydrophobic ratio of each arm of the star-like topology to see how this parameter could also influence the antimicrobial efficacy. Indeed, the hydrophobic content is an important parameter when the antibacterial activity involves membrane destabilization. [START_REF] Deming | Facile Synthesis of Block Copolypeptides of Defined Architecture[END_REF][START_REF] Wyrsta | A Parallel Synthetic Approach for the Analysis of Membrane Interactive Copolypeptides[END_REF][START_REF] Lee | What Can Machine Learning Do for Antimicrobial Peptides, and What Can Antimicrobial Peptides Do for Machine Learning?[END_REF] In this new study, we prepared a small series of star-P(ZNlys-Nphe) using a PANAM dendrimer having 16 (-NH2) terminal functions because this topology was expected to present the lowest cytotoxicity. [START_REF] Lam | Combating Multidrug-Resistant Gram-Negative Bacteria with Structurally Nanoengineered Antimicrobial Peptide Polymers[END_REF] We performed the polymerization using ZLys-NNCA (1, 1.4 and 1.8 equiv.; 50, 70, 90 hydrophobic content) and Phe-NNCA (1, 0.6, 0.2 equiv.; 50, 30, 10% hydrophobic content) using PAMAM initiator at M/I = 30 (4.16x10-3 equiv.) in DMF 0.1 M at RT under argon atmosphere (scheme 3). We followed the conversion of the ROP by monitoring FTIR until reaction completion (NNCA stretching at 1850 cm-1). Purification of the copolymers was achieved by precipitation in Et2O and drying under high vacuum. Star like copolymers were then isolated in 39-45% yields and characterized by 1H-NMR in DMSO-d6 (figure S5-S7). We found signals attributable to the polymer backbone (CH2, peak a,a'), signals attributable to the ZNlys moiety (peaks b-h) and signals attributable to Nphe moiety (peaks i,j). Once again, i) the determination of the hydrophobic content from the integrations of these signals gave us values far from what we expected (figures S4-S7); ii) we could not determine the DPNMR due to the high monomer/initiator ratio (120:1 for PAMAM with 4 arms and even higher for PAMAM with 16 arms) and due to the PAMAM signals overlapped with the copolymer (peaks b, e, a, a'). Scheme 3. Synthesis of star-like copolypeptoids from ZLys NNCA and Phe-NNCA varying the hydrophobic content.

We also characterized the copolymers by SEC in DMF (see esi figure S2,S3). These analyses allowed calculating a 2-fold lower weighted-molar mass (Mw) for the different star copolymers synthesized 3-5, similarly to polymers 1 and 2. Moreover, we observed a decrease in hydrodynamic radius by plotting the double logarithm of mean square radius (RMS radius) versus molar masses (MW) between linear and star-like polymers (figure S3)37 demonstrating that we successfully prepared star-shaped copolymers (dispersities ÐM = 1.3-1.4). Then, we performed Z-Lys side chains deprotection in TFA by adding 2 equivalents of HBr (relative to cationic side chains in TFA, RT). After 3 h, the copolymers were purified by precipitation in diethyl ether, dialyzed, and lyophilized (yields 42-62%, see esi). To determine the DPNMR and the chemical composition in monomer units, we analyzed the samples by 1H-NMR in D2O (see figure S5-S7). We calculated the hydrophobic content by carefully comparing the integration of signals belonging to NLys side chains (peak c,d) and NPhe side chains (peak j) and we found values that were in agreement with the expected ones (table 2). Moreover, NMR characterization confirmed that the copolymers did not suffer from chemical modifications during the deprotection step and that cationic copolymers containing hydrophobic units were prepared. This new series of copolymers were then studied in biology to evaluate the antimicrobial activity. The results are presented in table S2. First, we observed that copolymer 4 with a hydrophobic content of 30%, was very active against L. monocytogenes with a MIC value of 15.6 µg/mL and B. fragilis with a MIC value of 31.25 µg/mL. However, other MIC values obtained with other Gram-negative and Gram-positive bacteria tests were found higher (MIC = 62.5 µg/mL and above) as compared to the copolymer 3 composed of higher amount of hydrophobic units. With even lower hydrophobic content, star-like copolymer 5 provided a good MIC value of 15.6-31.25 µg/mL against L. monocytogenes. Surprisingly, this activity was more selective than for copolymer 4 because other MIC values were found above 62.5 µg/mL. Overall, we observed that star-like copolymer 4 with a 30% of hydrophobic content presented a broader and better antimicrobial activity (MIC < 62.5 µg/mL) as compared to copolymers with 10 and 50% or linear counterpart (figure S8 and table S4). This study demonstrated that modulating hydrophobic content in star-like polypeptoids modify the activity against bacteria as we also previously observed for cyclic polypeptoids.20 Later, we also assessed the cytotoxicity of the copolymers (1-5) using the human hepatic CaCO-2 cell line (table 3). We observed moderated cytotoxicity for all the star-like copolypeptoids and contrary to star-like polypeptides, no significant change in cytotoxicity values was observed neither by modifying the amount of arms nor by changing the hydrophobic content.2 Thus, the values of the selectivity index indicated that some star-like copolypeptoids could be interesting starting points for the conception of further antimicrobial compounds against B. fragilis or against L. monocytogenes (table 3).

Membrane disruption and protease resistance.

It is expected that the antimicrobial activity of our polypeptoid structures (made of cationic and hydrophilic side chains) was explained by bacterial membrane destabilization. We verified in vitro this possible mechanism by using two different liposome models containing carboxyfluorescein (CF): i) neutral liposome membranes constituted of soybean phosphatidylcholine and cholesterol (SPC:Chol, 8:2) with a zeta potential of ζ = -3.9±0.9 mV and ii) negatively charged liposome membranes constituted of 1,2-dioleoyl-sn-glycero-3phosphoglycerol (PG) and cholesterol (PG:Chol, 8:2) with a zeta potential of ζ = -33.3±2.2 mV. The liposomes were first loaded with the CF fluorophore. Then, the star-like copolymers P(NLys-NPhe) 1-5 were prepared at a concentration of 90 µg/mL and mixed with the liposomes. Upon incubation (1.5 h), the concentration of released of carboxyfluorescein was measured by fluorimetry (λex = 485 nm and λem = 528 nm) and reported in percentage of release (%CF release). As depicted in figure 4, we clearly observed that copolymers having a hydrophobic content of 50% were better in destabilizing both negatively and neutral charged liposome models (80-90% leakage, figure 4). This result was verified, whatever the number of arms. In marked contrast, changing the hydrophobic content was strongly influencing the membrane destabilization of the liposomes: the lower the hydrophobic content in the 16-arm polypeptoid was the lower ability to destabilize the liposomes we observed (5 afforded 0% and 3% of CF release). The results obtained with this model of membrane destabilization was particularly in agreement with the MIC results obtained against B. fragilis and could explain why 5 had the lowest MIC value (250 µg/mL) against this pathogen. However, the antibacterial tests presented in table S2 also showed that copolymer 4 (hydrophobic content of 30%) exhibited a broader spectrum of action and some higher antibacterial activities than copolymers 3 and 5 (with hydrophobic content of 10 and 50%). We attributed this difference to the fact that bacterial membranes are much more complex than a simple mixture of phospholipids. Overall, by using liposomes, we verified that the star-like polypeptoids could destabilize the bacterial cell membrane with a significant influence of the hydrophobic content on this destabilization. Finally, peptoids are known to resist proteolytic degradation mediated by proteases. [START_REF] Miller | Comparison of the Proteolytic Susceptibilities of Homologous L-Amino Acid, D-Amino Acid and N-Substituted Glycine Peptide and Peptoid Oligomers[END_REF][START_REF] Salas-Ambrosio | Cyclic Poly(α-Peptoid)s by Lithium Bis(Trimethylsilyl)Amide (LiHMDS)-Mediated Ring-Expansion Polymerization: Simple Access to Bioactive Backbones[END_REF] This characteristic made them as an attractive backbone to develop therapeutic agents that could be administered orally. However, proteolysis of cationic polypeptoids is scarcely documented. [START_REF] Salas-Ambrosio | Cyclic Poly(α-Peptoid)s by Lithium Bis(Trimethylsilyl)Amide (LiHMDS)-Mediated Ring-Expansion Polymerization: Simple Access to Bioactive Backbones[END_REF] In this work, we carried out a proteolysis experiment using trypsin (one of the main proteases presented in the digestive tube), we measured the proteolytic activity implementing a methodology developed for small peptoids, that consisted in reacting 4-Fluoro-7-nitrobenzofurazan (NBDF) with secondary amines (SNAr reaction) resulting from the proteolysis of the amide bonds upon digestion of the polymer (0.1 mM in NaHCO3 buffer 50 mM pH = 7.8) at 37 °C (see esi). The proteolysis was performed for all the star-like copolymers P(NLys-NPhe) (1-5) and compared with a linear polypeptide (poly(lysinephenylalanine) M/I = 200 having 10% hydrophobic content (see esi). We followed the progression of the reaction by taking aliquots at a certain time and upon NBDF treatment we measured the fluorescence (λem = 535 nm, figure S9). These NBDF experiments clearly indicated that all the star-like copolymers were not degraded by trypsin within 50 min, while the linear polypeptide was rapidly degraded in the first 20 min. In agreement with a previous study,20 these results indicated that star-like copolypeptoids could be an interesting approach for further oral administration development. Conclusions In summary, by using grafting-from dendrimers, ring-opening copolymerization of NNCAs was shown to afford antimicrobial structures. This methodology allowed accessing star-like topologies with fine-tuning over the hydrophobic content and the number of arms. The starshaped polypeptoids were containing cationic side chains and a preliminary antibacterial structure-activity relationship study allowed establishing that these copolymers were having significant activity. In general, star-like polypeptoids were better at killing Gram-positive germs, particularly L. monocytogenes with MIC values of 15.6-32.5 µg/mL and star-like copolypeptoids with 8 arms enhanced the antibacterial activity against the anaerobic Gramnegative B. fragilis (MIC = 7.8 µg/mL). All these antimicrobial structures were able to destabilize bacterial membrane at a high hydrophobic content of 50% and they were able to resist trypsin proteolysis. We believe that these dendritic polypeptoids could bring interesting solutions in health applications that merge materials science and infectiology. Experimental Materials. All the chemicals and solvents in this work were purchased from Sigma Aldrich, Fluorochem, Acros, TCI, Strem and, unless otherwise described, were used without any purification. Dimethylformamide (DMF), tetrahydrofuran (THF) were obtained from a solvent system purificator (PureSolv, Innovative Technology), kept under argon atmosphere and freshly used. MilliQ water was obtained from a (Purelab Prima, ELGA). The monomers synthesized were stored at 4 °C under argon atmosphere and weighted in the glove box Jacomex GP13 No. 2675 Methods. The IR spectra were recorded using the FTIR spectrometer (Vertex 70, Bruker), and the samples were measured with the ATR (GladiATR, Pike Technologies) from Fisher technologies performing 32 scans at the LCPO (Bordeaux, France). The raw data were obtained with the Opus7.5 software and processed using the Originlab 2016 software. The NMR spectra were recorded using a Bruker Avance III 400 spectrometer (LCPO, Bordeaux, France). The spectra were analyzed at 295 K and data was analyzed using Mestrenova 14.1.2 software. The chemical shifts of the signals are given in ppm. The spectra obtained were calibrated using the residual solvent signals (CHCl3 7.26 ppm, H2O 4.79 ppm, DMSO-d6 2.50 ppm). The signals were categorized as follows: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m) and broad (br). Polymer molar masses were determined by Size Exclusion Chromatography (SEC) using: a) Dimethylformamide (DMF + lithium bromide LiBr 1g/L) as the eluent (LCPO, Bordeaux, France). Measurements in DMF were performed on an Ultimate 3000 system from Thermoscientific equipped with diode array detector DAD. The system also included a multi-angle light scattering detector MALS and differential refractive index detector dRI from Wyatt technology. Polymers were separated on two Shodex Asahipack gel columns GF310 and GF510 (300 x 7.5 mm) (exclusion limits from 500 Da to 300 000 Da) at a flow rate of 0.5 mL/min. Columns temperature were held at 50°C. The chromatograms were recorded with the Chromeleon 7.2 software and Astra 7.1.0 software and analyzed using the Originlab 2016 software. The dn/dc were determined experimentally. b) Hexafluoro-2propanol (HFIP+ 0,05% KTFA) as the eluent (LCPO, Bordeaux, France). Measurements in HFIP were performed on an Ultimate 3000 system from Thermoscientific equipped with diode array detector DAD. The system also included a multi-angle light scattering detector MALS and differential refractive index detector dRI from Wyatt technology. Polymers were separated on PL HFIP gel column (300 x 7.5 mm) (exclusion limit from 100 Da to 150 000 Da) at a flow rate of 0.8 mL/min. Column temperature were held at 40°C. The chromatograms were recorded with the Chromeleon 7.2 software and Astra 7.1.0 software and analyzed using the Originlab 2016 software. Either the molar mass was calculated using a calibration curve or the dn/dc value. The calibration curve was performed with polystyrene standards with molar masses in the range 0.9-364 kg/mol. The dn/dc were determined experimentally. MALDI-MS spectra were performed at CESAMO facility (Bordeaux, France) on an Autoflex maX TOF mass spectrometer equipped with a frequency tripled Nd:YAG laser emitting at 355 nm. Spectra were recorded in the positive-ion mode using the reflectron and with an accelerating voltage of 19 kV. For MALDI-MS analyses, polypeptoids deposits were prepared according to the two following recipes: 1) Polypeptoids and the cationic agent (NaI) were dissolved in methanol at 10 mg/mL. The α-CHCA matrix (α-cyano-4-hydroxycinnamic acid) solution was prepared by dissolving 10 mg in 1 mL of methanol and the solutions were combined in a 10:1:1 volume ratio (matrix: sample: salt). One microliter of this solution was deposited onto the grid and vacuum-dried before analysis. 2) the polymer and the cationic agent (NaI) were dissolved in methanol at 10 mg/mL. The dithranol matrix solution was prepared by dissolving 10 mg in 1 mL of dichloromethane and the solutions were combined in a 10:1:1 volume ratio (matrix: sample: salt). One microliter of this solution was deposited onto the grid and vacuum-dried before analysis. Synthesis of monomers and aminoacid precursors. The preparation is described in ESI.

Synthesis of star-poly(N-methyl)glycine S-P(Sar). N-Methyl-NCA monomer (Sar-NCA, 0.1 g, 8.7x10-4 mol, 480 equiv.) was weighed in a glovebox under pure argon, introduced in a flame-dried Schlenck vessel, and dissolved with 4 mL of anhydrous DMF. In another Schlenck vessel PAMAM-(NH2)16 29.5 mg (20% w/t in MeOH, 1.81x10-6 mol, 1 equiv.) was dried and dissolved with 4.7 mL of anhydrous DMF. The monomer solution was added to the initiator solution with vigorous stirring avoiding cloudiness during the addition. The mixture was stirred at room temperature under argon until completion (20 days) and confirmed by FTIR. The polymer was then recovered by precipitation in diethyl ether and dried under high vacuum. Yield: 87% (0.059 g, white solid). Molar mass (SEC in DMF, dn/dc = 0.0942): Mn=3.50x104 g/mol ÐM = 1.11. 1H-NMR DMSO-d6 400MHz δ (ppm): 2.50-3.10 (m, 3H, CH3), 3.80-4.42 (m, 2H, CH2). Star-poly[(N-aminobutylglycine)-(N-benzylglycine)] [S-P(Nlys-Nphe)] 4 arms . N-(Cbz-(2aminobutyl))-NCA (ZLys-NNCA, 0.2 g, 6.53x10-4 mol, 60 equiv.) and N-benzyl-NCA (Phe-NNCA, 124.8 mg, 6.53x10-4 mol, 60 equiv.) were weighed in a glovebox under pure argon, introduced in a flame-dried Schlenck vessel, and dissolved with 6 mL of anhydrous DMF. In another Schlenck vessel PAMAM-(NH2)4 28.1 mg (20% w/w in MeOH, 1.09x10-5 mol, 1 equiv.) was dried and dissolved with 7 mL of anhydrous DMF. The monomer solution was added to the initiator solution with vigorous stirring avoiding cloudiness during the addition. The mixture was stirred at room temperature under argon until completion and confirmed by FTIR. The polymer was then recovered by precipitation in diethyl ether and dried under high vacuum. Yield: 45% (0.122 g, yellowish solid). SEC (DMF, dn/dc = 0.0816): 12000 g/mol, ÐM = 1.18 1H-NMR DMSO-d6 400MHz δ (ppm): 1.12-1.65 (m, 4H, 2CH2), 2.86-3.02 (m, 2H, CH2), 3.07-3.27 (m, 2H, CH2), 3.75-4.48 (m, 2H, CH2), 4.87-5.09 (m, 2H, CH2), 6.94-7.47 (m, 5.84H, Ar+NHCO). We could not observe the CH2 of the benzyl group approx. 4.5 ppm. Deprotection method to prepare Star-poly[(N-aminobutylglycine)-(N-benzylglycine)] Polym 1. In a test tube, 100 mg of S-P(Nlys-Nphe)] 4 arms (1.9x10-4 mol, MW = 262.32 g/mol monomer unit and taking into account as 50% mol) were completely dissolved with 1 mL of trifluoroacetic acid. To the polymer solution, 68.5 µL of HBr 33% (3.8x10-4 mol) was added and the solution was stirred at 20 °C for 3 hours. Then, the polymer was precipitated on diethyl ether, centrifuged at 4000 rpm the supernatant was removed, the solid was resuspended in 1 mL of water and the pH was adjusted to pH = 7 with a NaHCO3 saturated solution. The solutions were dialyzed using regenerated cellulose membranes (MWCO 10 kDa), the first time with Milli-Q water, followed by phosphates buffer pH = 7 (0.05 M) and two replacements with Milli-Q water. The solutions were freeze-dried (BenchTop Pro, SP Scientific). Upon freeze-drying, the copolymer was recovered as a yellowish powder in 21% yield (0.0185 g). Calculated hydrophobic content by comparison of 2CH2 (1.37-1.92 ppm) and Ar (6.93-7. Antimicrobial assays: To test the activity against bacteria the methodology was as follows: mother solutions of the polypeptoids under assay were prepared by adding 1 mL of sterile distilled water (SDW) directly in the Eppendorf containing the product, the solution was transferred in a sterile tube. Eppendorf was rinsed 3 times (SDW) and the resulting solutions were added to the tube. The final volume was adjusted to a concentration of 500 µg/mL with SDW. Shaking (4 times/h) during 1 h. Mother solutions were homogenized and appear clear expected. Storage at -20°C. Culture maintaining: S. aureus, E. faecalis, L. monocytogenes, E. coli, A. baumanii, P. aeruginosa: Muller Hinton agar, incubation 24 h at 36 ± 1°C under aerobiosis. S. pneumoniae: Muller Hinton agar, incubation 24 h at 36 ± 1°C under anaerobiosis. B. fragilis and H. pylori: Columbia agar + 5% sheep red cells, incubation 24 -48 h at 36 ± 1 °C under anaerobiosis for B. fragilis (and under microaerophilic conditions for H. pylori). Assays: S. aureus, E. faecalis, L. monocytogenes, E. coli, A. baumanii, P. aeruginosa: Muller Hinton broth, and Muller Hinton agar, incubation 24 h at 36 ± 1°C under aerobiosis. S. pneumoniae: Muller Hinton broth, supplemented with 10% SVF and Muller Hinton agar, incubation 24 h at 36 ± 1°C under anaerobiosis. B. fragilis: Muller Hinton broth and agar, incubation 24 h at 36 ± 1°C under anaerobiosis. H. pylori: Muller Hinton broth supplemented with 10% SVF and Muller Hinton agar supplemented with 10% horse blood, incubation 24 h at 36 ± 1°C under microaerophilic conditions. MIC (Minimal Inhibitory Concentration): Strains were maintained on agar specified above. A fresh suspension was prepared just before each assay in Tryptone salt and adjusted to 108 CFU/mL. Each well of a 96-well microtiter plate was filled with 100 µL of specify broth. Then, 100 µL of a mother solution were added to the first well of two lines of the microtiter plate and 2 fold dilutions were performed from well 1 to well 10. All the wells (except column 11: sterility control) were inoculated with 1 to 3 µL of the tested suspension (multi inoculator Denley). Microplates were then incubated under conditions described above. MIC was determined as the lowest concentration without any visible growth versus positive control. C. difficile strain was cultured in an anaerobic chamber with an atmosphere of 5% H2, 5% CO2, 90% N2. Cells were cultured on a Brain-Hearth Infusion (BHI) medium at 37 °C. The Minimal Inhibitory Concentration (MIC) of tested polymers was performed by the micro dilution method. A twofold serial dilution of tested polymers and control drugs were prepared in highly purified water to give a final concentration range from 1000 to 8 µg/mL and 20 µL of each concentration was added to a 96-well plate. Then, an overnight bacterial solution of C. difficile having an OD600 between 0.45 and 0.60 (107-108 CFU/mL) was diluted in BHI at 105 CFU/mL, each well was inoculated anaerobically with 180 µL and incubated at 37°C. MIC reported is the minimal drug concentration that totally suppressed the growth of bacteria after 24 h at 37 °C, by visual determination. The experiments were performed by triplicates. The uninoculated medium was used as a negative control to test for contamination of the growth medium. The positive control consisted of a well inoculated with C. difficile but no antimicrobial compound was added.

Membrane destabilization studies:

The analyses were performed at the Centre d'Etudes et de Recherche sur le Médicament de Normandie (CERMN, Caen Normandie, France). The procedure is described as follows. Liposome mixtures of SPC:cholesterol (SPC:Chol) or DOPG:cholesterol (PG:Chol) in a molar ratio of 8:2 were formulated. Liposomes were formulated according to the adapted method of the thin lipid film hydration.58 Lipid solutions in chloroform/methanol (4:1) were evaporated under nitrogen flow and left under vacuum for 2-3 h to form a lipid film. Carboxufluoresceine (CF) was dissolved in phosphate buffer Saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) to reach 70 mM, pH was adjusted with concentrated NaOH to 7,4. This thin lipid film was then hydrated in the CF solution and vortexed 1h. At RT the yielded multilamellar vesicles (MLVs) were then extruded 13 times with a mini-extruder (Avanti Polar Lipids, Inc., Alabaster, Alabama, USA) through polycarbonate membranes with a pore diameter of 100 nm (Avanti Polar Lipids, Inc.). The obtained LUVs were separated from possible unincorporated CF by passage through a Sepharose® CL-4B loaded (Sigma-Aldrich) column, using PBS buffer as eluent. The LUVs size was assessed by DLS (NanoZS®, Malvern Instruments, Worcestershire, UK) after a 1:100 dilutions in the PBS buffer. Liposomes were stable within 4 month: SPC:Chol having a Zaverage 153.1±0.5 nm, PDI=0.095 ±0.02 and Z-potential -3.9±0.9: PG:Chol having a Z-average 138.1±1.7 nm with a PDI=0.093 ±0.04 and Z-potential -33.3±2.2. The entrapment of CF was measured by the dequenching of fluorescence after the addition of 2 µL 20% (v/v) Triton X-100 measured with a Synergy 2 microplate reader (Biotek, Colmar, France) equipped with the appropriate filters (λex = 485/20 nm and λem = 528/20 nm). CF release assay was performed in a final volume of 100 μL, using 10 μM LUVs in PBS buffer. Polypeptoids solubilized in PBS buffer were then added to the solution to reach a final concentration of 90 µg/mL. The fluorescence was recorded immediately (F0) and for 1.5 h at 25 °C. It was compared with that measured at the end of the experiment after the addition of 2 μL of 20% Triton X-100 solution to achieve complete liposome leakage (Fmax). The percentage of CF release was calculated according to the following equation:59 〖% CF leakage〗_((t))=(F_t -F_0)/(F_max-F_0 )×100 where Ft was the fluorescence intensity at time t, F0 the initial fluorescence intensity, and Fmax the final fluorescence intensity after adding Triton X-100. Fluorescences of PBS and polypeptoids alone at the same concentration were measured as negative controls.

Cytotoxicity assays:

The evaluation of the tested peptides cytotoxicity by MTT assay on the Caco-2 cell line (human epithelial cell line) was done according to Mosmann with slight modifications. The experiments were performed at PHARMA-DEV in Toulouse, France. Briefly, cells (1.105 cells/mL) in 100 µL of the complete medium [DMEM High Glucose supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin) and 1X NEAA] were seeded into each well of 96-well plates and incubated at 37 °C in a humidified 5% CO2 with 95% air atmosphere. After a 24 h incubation, various concentrations of peptides and appropriate controls were then added (100 µL) and the plates were incubated for 72 h at 37 °C. Each well was then microscope-examined for detecting possible precipitate formation before aspiration of the medium. MTT solution (0.5 mg/mL in complete DMEM High Glucose, 100 µl) was then added to each well. Cells were incubated for 2 h at 37 °C. The MTT solution was then removed and DMSO (100 µL) was added to dissolve the resulting formazan crystals. Plates were shaken vigorously (300 rpm) for 5 min. The absorbance was measured at 570 nm with a microplate spectrophotometer (Eon Bio Tek). Water was used as blank and doxorubicin (Sigma Aldrich) as a positive control. CC50 were calculated by non-linear regression analysis processed on dose-response curves, using TableCurve 2D V5 software. CC50 values represent the mean value calculated from three independent experiments. Protease assay: The protocol was adapted from a known method for peptoid analysis. [START_REF] Miller | Comparison of the Proteolytic Susceptibilities of Homologous L-Amino Acid, D-Amino Acid and N-Substituted Glycine Peptide and Peptoid Oligomers[END_REF] A polymer solution was prepared (0.1 mM) in 1 mL of NaHCO3 buffer 50 mM pH = 7.8 equilibrated at 37 °C for 10 min and 47 µL of trypsin 42 µM (1 mg/mL) was added. At a certain time, 2.3 µL of the reaction mixture were taken, placed in an Eppendorf tube, quenched with 200 µL acetonitrile and 200 µL sodium borate buffer 0.1 M and kept in an ice bath. 4-Fluoro-7-nitro-benzofurazan (NBDF) (10 µL, acetonitrile solution 10 mM) was added, immediately the solution was incubated at 60 °C for 3 min and 500 rpm in the Eppendorf thermomixer R. The sample was placed on ice and 20 µL of 3 N HCl were added to stabilize the 4-amino-7-nitro-benzofurazan product. The fluorescence was measured in a Spectra max M2 (Molecular devices) with λex = 470 nm and λem = 540 nm. The values were adjusted using the appropriate blank. The raw data was obtained from SoftMaxPro V5 and analyzed in Origin2016 software ‡ The yields with the copolymers are lower than the yields obtained with polysarcosine: we attribute this observation to the difference in solubility that exists between these two structures whereas they were purified by the same process (precipitation in diethyl ether).

Figure 1 .

 1 Figure 1. Star-like polysarcosine. A) The preparation was achieved by ROP of NNCAs from PAMAMs. B) 1H NMR in DMSO-d6 of the s-(P(Sar)30)16 obtained with 3 different methods (A: direct addition of initiator; B: direct addition of monomers; C: reduced concentration).Table 1. Comparison of SEC characterizations and yields obtained by analyzing s-(P(Sar)30)16 from three methodologies studied M/Itheo MWtheo (g/mol) Mn (g/mol)a Mw (g/mol)a ÐM Yield (%) Method A 30 3.7x103 4.69x103 5.29x103 1.13 86 Method B 30 3.7x103 3.68x103 4.12 x103 1.12 78 Method C 30 3.7x103 3.5x103 3.90 x103 1.11 87 a SEC performed in DMF. b Calculated from the 1H-NMR spectrum in DMSO-d6

Figure 2 .

 2 Figure 2. Representative characterizations of star-[P(ZNLys-NPhe)]: copolymer 1 with 4 arms. 1H-NMR spectra A) in DMSO-d6 before deprotection at 20 °C; B) in DMSO-d6 before deprotection at 70 °C; C) in D2O upon deprotection. D) Double logarithm plot of the root means square radius plot versus molar masses of a linear copolypeptoid M/I = 30 and the star-like copolymer bearing 4 arms of P(ZNLys-NPhe) 1 M/I = 30. The slope for linear polymer was 0.8±0.04 and for star polymer slope 0.4±0.04.

Figure 3 .

 3 Figure 3. Minimum inhibitory concentrations (MIC) of star-like polypeptoids (S-P(Nlys-Nphe) 1-3 against representative pathogens. *MRSA = Methicilline resistant S. aureus.

Figure 4 .

 4 Figure 4. Membrane destabilization by active cyclic copolypeptoids using two different liposome models. SPC:Chol (8:2); PG:Chol (8:2).

  39 ppm) according to: H/C%=〖XH〗_Ar/5*50 H/C% = 52% 1H-NMR D2O 400 MHz δ (ppm): 1.34-1.92 (m, 4H, 2CH2), 2.75-3.04 (m, 2H, CH2), 3.09-3.35 (m, 2H, CH2), 3.373-4.63 (m, 4H, 2CH2), 6.93-7.39 (m, 5.2H, Ar). Star-poly[(Z-N-aminobutylglycine)-(N-benzylglycine)] [S-P(ZNlys-Nphe)] 8 arms. The protected polymer was obtained according to the 4 arm copolymer trough ROP of Phe-NNCA and ZLys-NNCA. Yield: 43% (0.118 g, yellowish solid). SEC (DMF, dn/dc = 0.0816): 17500 g/mol, ÐM = 1.36. Hydrophobic content calculation by comparison of Ar (6.94-7.47 ppm) and Calculated hydrophobic content by comparison of Ar (1.37-1.92 ppm) and 2CH2 (6.93-7.39 ppm) = 12%. 1H-NMR D2O 400 MHz δ (ppm): 1.34-1.92 (m, 4H, 2CH2), 2.75-3.04 (m, 2H, CH2), 3.09-3.35 (m, 2H, CH2), 3.373-4.63 (m, 4H, 2CH2), 6.93-7.39 (m, 1.19H, Ar).

Table 1 .

 1

	Comparison of SEC characterizations and yields obtained by analyzing s-(P(Sar)30)16
	from three methodologies studied
	M/Itheo	MWtheo
	(g/mol)	Mn (g/mol)a Mw (g/mol)a ÐM Yield (%)
	Method A	30	3.7x103	4.69x103	5.29x103
	1.13 86
	Method B	30	3.7x103	3.68x103	4.12 x103	1.12 78
	Method C	30	3.7x103	3.5x103	3.90 x103	1.11 87
	a SEC performed in DMF. b Calculated from the 1H-NMR spectrum in DMSO-d6

Table 2 .

 2 Star-[P(Nlys-Nphe)] copolymers: molar masses from SEC, hydrophobic content from 1H-NMR and yields.

	Arms M/I H/C (%)	MWtheo
	(g/mol)	Mn (g/mol)a Mw (g/mol) ÐM Yield before deprotection (%) ‡	H/C
	upon deprotection (%)c	Yield upon deprotection (%) ‡
	S-P(ZNLys-NPhe) 1 4	30	50	2.78x104	1.20x104	1.42x104	1.18
	45	52	21
	S-P(ZNLys-NPhe) 2 8	30	50	5.24x104	1.75x104	2.38x104	1.36
	43	55	41
	S-P(ZNLys-NPhe) 3 16	30	50	1.02x105	3.59x104	5.05x104	1.40
	40	53	62
	S-P(ZNLys-NPhe) 4 16	30	30	1.13x105	3.62x104	5.11x104	1.41
	39	35	55
	S-P(ZNLys-NPhe) 5 16	30	10	1.24x105	4.05x104	5.28x104	1.30
	45	12	42
	a SEC performed in DMF. b Hydrophobic content calculated from the 1H-NMR spectrum in
	DMSO-d6. c Calculated from the 1H-NMR spectrum in D2O

Table 3 .

 3 Cytotoxicity over CaCO-2 cells and selectivity indices (SI) of star-like polypeptoids. Minimum inhibitory concentration (MIC) determined on B. fragilis and L. monocytogenes.

	Polym CaCO-2 CC50 (µg/mL) MIC
	B. fragilis (µg/mL)	SI

  at the Laboratoire de Chimie des Polymères Organique (LCPO, Bordeaux, France). Studies using Clostridioides difficile 630Δerm were performed at LPBA Pasteur Institute, Paris, France; studies using Staphylococcus aureus CIP 4.83, Staphylococcus aureus ATCC 33591 (MRSA or methicilline resistant strain), Enterococcus faecalis CIP 103015T, Streptococcus pneumoniae CIP 104.471, Listeria monocytogenes CIP 82.110T, Escherichia coli CIP 53.126, Acinetobacter baumanii CIP 70.34T, Pseudomonas aeruginosa CIP 82.118, Bacteroides fragilis CIP 77.16 and Helicobacter pylori were performed at FONDEREPHAR, Toulouse, France.

Acknowledgements

The authors acknowledge Amelie Vax and Sylvain Bourasseau for assistance with sizeexclusion chromatography. Christelle Absalon is acknowledged for MALDI TOF analysis. This work has benefited from the facilities and expertise of the CESAMO platform (Bordeaux University). PSA received support from CONACYT (scholar-ship holder No. 548662). This work was supported by the French national agency for research (ANR): grant N° ANR-17-CE07-0039-01. Christine Roques and Catherine Feuillolay (FONDEREPHAR TEAM in TOULOUSE).

Conflicts of interest

"There are no conflicts to declare".