# Surfmer versus Polymer Effect toward Solubilization and Stabilization of Membrane Proteins

Floriane Mangin, Vinay Chauhan, Pierre Guillet, Marjorie Damian, Marine

Soulié, Jean-Louis Banères, Grégory Durand

► To cite this version:

Floriane Mangin, Vinay Chauhan, Pierre Guillet, Marjorie Damian, Marine Soulié, et al. Surfmer versus Polymer Effect toward Solubilization and Stabilization of Membrane Proteins. ACS Applied Polymer Materials, 2024, 6 (6), pp.3139-3149. 10.1021/acsapm.3c02858. hal-04560472

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Distributed under a Creative Commons Attribution 4.0 International License
Surfmer versus Polymer Effect Towards Solubilization and
Stabilization of Membrane Proteins

Floriane Mangin,ª Vinay Chauhan,ª† Pierre Guillet,ª,c

Marjorie Damian,ᵇ Marine Soulié,ª,c Jean-Louis Banères,ᵇ Grégory Durand*ª,c

ªInstitut des Biomolécules Max Mousseron (UMR 5247 UM-CNRS-ENSCM) & Avignon Université,
301 rue Baruch de Spinoza – 84916 AVIGNON cedex 9 (France) ;

ᵇInstitut des Biomolécules Max Mousseron (UMR 5247 UM-CNRS-ENSCM), 1919 route de Mende, 34293 Montpellier Cedex 5 (France) ;

ᶜEquipe Synthèse et Systèmes Colloïdaux Bio-organiques, Unité Propre de Recherche et d'Innovation, Avignon Université, 301 rue Baruch de Spinoza – 84916 AVIGNON cedex 9 (France).

To whom correspondence should be addressed: [gregory.durand@univ-avignon.fr](mailto:gregory.durand@univ-avignon.fr)
**Abstract.**

We have synthesized four acrylamide-based monomers whose polar head group consists of one or two glucose or one maltose moieties and whose alkyl chain is made of a *n*-decyl or a *n*-octyl hydrophobic chain. These surface active monomers, called surfmers, were polymerized by free-radical telomerization in presence of a thiol-based transfer agent leading to four non-ionic polymers that belong to the class of amphipols. A very good reproducibility and control of the polymerization was noted leading to average polymerization degrees in perfect agreement with the initial ratio R₀ and to quite narrow polydispersity index after purification. Surface tension (SFT) measurements indicate that the four surfmers self-assemble at concentration ranging from ~0.5 to 4 mM. Dynamic light scattering (DLS) experiments suggest the formation of rather small homogenous aggregates of 5 nm diameter for the monomers while the size of the aggregates ranges from 5 to 16 nm diameter for the polymers. The ghrelin receptor GHSR, a typical rhodopsin-like G protein-coupled receptor (GPCR) was used as a challenging model because of its conformational instability in vitro. The surfmers solubilized the receptor from membrane fractions more efficiently than their polymeric counterparts. In contrast, the polymers were significantly more efficient at stabilizing the functional fold of the isolated protein in solution. This demonstrates the opposite effect of surfmers and their polymer counterparts towards the solubilization and stabilization of fragile membrane proteins.

**Keywords:** Surfmers, non-ionic amphipols, micelles, membrane proteins, solubilization, stabilization.
## Introduction.

Membrane proteins (MPs) are involved in numerous biological processes such as transport of ions, molecules and electrons as well as signal transduction, enzymatic reactions and intercellular communication.<sup>1-2</sup> Therefore, they are essential for the steady-state homeostasis of cells and their reaction to environmental changes which makes them of primary importance in drug development as they are the targets for more than half of all drugs.<sup>3</sup>

In order to study MPs, it is necessary to extract them from their complex environment and keep them stabilized in their native form. Detergents are traditionally used for the solubilization of membrane and the subsequent extraction and purification of MPs.<sup>4-5</sup> However, due to their delipidating nature and the non-native environment they offer to MPs, detergents may induce denaturation of the proteins. As a result, MPs and their activities are altered.<sup>6</sup>

To overcome the dissociating and inactivating effects of detergents towards MPs, chemical compounds with original architectures have been designed. There are currently several approaches aiming at the development of milder compounds able to keep MPs stable and water-soluble: homogeneous systems made of chemically-defined amphiphiles as well as non-detergent heterogeneous systems such as polymers and nanodiscs.<sup>7-9</sup>

Amphipols (APols) are amphipathic polymers containing hydrophilic backbones with hydrophobic side chains. They have been used to stabilise MPs by surrounding their transmembrane domains while retaining their functions.<sup>10-11</sup> So far, A8-35 has been the most extensively used amphipol. A8-35 is made of a polyacrylate chain of ~35 acrylate residues onto which octylamine or isopropylamine have been randomly grafted. The alkyl chains of A8-35
adsorb onto the hydrophobic transmembrane surface of MPs through multiple contacts in a
noncovalent but quasi-irreversible manner and the resulting complex remains water-soluble
through the hydrophilic free carboxylic groups.<sup>11-12</sup>

Other carboxylic acid amphipols have been developed,<sup>13</sup> as well as zwitterionic,<sup>14</sup> phosphorylcholine,<sup>15</sup> sulfonated<sup>16</sup> and more recently cyclic amphipols.<sup>17</sup>

Totally non-ionic APols called NAPols have also appeared appealing as, at variance with A8-
35, they are pH- and calcium-insensitive and they offer milder environment to the proteins.
First NAPols were developped using the hydroxyl moieties of Tris groups to confer
hydrophilicity,<sup>18</sup> but the solubility of the resulting polymers was too low. More water soluble
glycosylated non-ionic APols have then emerged,<sup>19-20</sup> the latest version of which are
homopolymers prepared from the polymerization of an amphiphilic monomer.<sup>21</sup> This most
advanced series of NAPols was successfully used to reconstitute and stabilize model proteins
as well as pharmacologically relevant GPCR and were found compatible with cell-free
synthesis of MPs, as well as NMR studies.<sup>22</sup> The application of NAPols for the cryo-EM
structure determination of MPs was also demonstrated in 2019.<sup>23</sup> Successful detergent-free
extraction and stabilization of GPCR have been achieved using SMA,<sup>24-25</sup> DIBMA,<sup>24</sup> and
PMA<sup>26</sup> copolymers. The two main advantages of such polymer-based extraction and
stabilization lies in the direct extraction of GPCR from membranes and the formation of
nanoscale polymers-lipids particles maintaining a native environement during purification.<sup>24-26</sup>
However these three polymers have the disadvantage of being sensitive to pH and to the
presence of high concentration of Ca<sup>2+</sup> although DIBMA and PMA are more tolerant than
SMA.

The structure of the most advanced NAPols is based on a repeating amphiphilic unit that
belongs to the class of surfmers Surfmers are elaborated monomers that combine the
functionalities of surface-active agents with the reactivity of classical monomers.<sup>27-30</sup> We
report herein the synthesis of four surfmers made of an acrylamide as a polymerizable function. The four detergent-like monomers were polymerized by free radical telomerization in the presence of a transfer agent to lead to four NAPols. The hydroxyl groups of tris(hydroxymethyl) acrylamidomethane (THAM) were used to graft both hydrophilic and lipophilic groups and different chemical modifications were considered to modulate the self-aggregation and biochemical properties of the monomers and of their resulting polymers: the nature of the sugar-based polar head *i.e.* glucose or maltose; the number of glucose *i.e.* one or two ; and the length of the alkyl chain *i.e.* n-octyl or n-decyl.

The micellar properties of the four surfmers were evaluated by means of surface tension (SFT) measurements and their self-assemblies and those of their resulting polymers were studied by dynamic light scattering (DLS) experiments. We then evaluated the ability of these compounds for extracting a model membrane protein, the ghrelin receptor GHSR, from its lipid bilayer environment and then stabilizing its native fold in solution. To the best of our knowledge this is the first example of the comparison of solubilizing and stabilizing potencies between a detergent molecular unit and its polymeric form.

**Experimental section.**

*Materials.* All starting materials were commercially available and were used without further purification. All solvents were of reagent grade and used as received unless otherwise indicated. Anhydrous solvents were dried by simple storage over activated 4 Å molecular sieves for at least 24 h. Molecular sieves were activated by heating *in vacuo* for 24 h. The 2-((benzoyloxy)methyl)-2-(3-mercaptopropanamido)propane-1,3-diyl dibenzoate (10)²⁰ was synthesized according to earlier reported procedure while N-(3-hydroxy-2,2-bis(hydroxymethyl)propyl)acrylamide (1)³¹ was synthesized by a different procedure. The progress of the reactions was monitored by thin-layer chromatography. The compounds were detected either by exposure to ultraviolet light (254 nm) or by spraying with sulfuric acid (5%
ethanol) and/or ninhydrin (5% ethanol), followed by heating at ca. 150 °C. Weakly acidic ion-exchange resin Amberlite™ CG-50 was used as received from Sigma-Aldrich to neutralize the pH of methanolic solution after deprotection of acetate groups under Zemplén conditions. Flash chromatography purifications were carried out on silica gel (pore size 60 Å, 230–400 mesh particle size, 40–63 µm particle size). Size exclusion chromatography purifications were carried out on Sephadex LH-20 resin. ¹H and ¹³C NMR experiments were performed at 400 and 100 MHz, respectively. Chemical shifts are given in ppm relative to the solvent residual peak as a heteronuclear reference for ¹H and ¹³C. Abbreviations used for signal patterns are: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublet; dt, doublet of triplet. High-resolution mass spectra were determined on a Synapt G2-S (Waters) mass spectrometer with a TOF mass analyzer in a positive ionization mode. Milli-Q water (resistivity of 18.2 MΩ cm, surface tension of 71.45 mN/m at 25 °C) was employed for all physicochemical experiments. HPLC experiments were performed on a Waters XTerra RP18 (100 × 2.1 mm, 5 µm) column using MeCN + 0.1% TFA/H₂O + 0.1% TFA as mobile phase starting from 10% to 95% MeCN on 35 min. Individual synthetic protocols as well as complete chemical characterization of monomers and polymers are reported in the supporting information.

*General procedure for carbamate synthesis.* Acrylamide derivative (2 or 3 eq.) was dissolved in dry DMF (15 mL), under argon, then trimethylamine (1.5 eq.) was added and the resulting solution was heated to 80 °C before adding isocyanate (1 eq.) dropwise. The reaction mixture was stirred at 80 °C for 2 h, then the solvent was evaporated under reduced pressure. The crude mixture was taken up in EtOAc/CH₂Cl₂ (60 mL), filtered and the solvent was evaporated under reduced pressure. The crude product was then purified by flash chromatography (Cyclohexane/EtOAc 1:1 to 3:7 v/v).
*General procedure for glycosylation.* Carbamate derivative (1 eq.) and peracetylated sugar (5 or 9 eq.) were dissolved in dry CH₂Cl₂ (73 mL) under argon and stirred for 10 min at room temperature. BF₃.Et₂O (1 eq./sugar) was then added dropwise and the reaction mixture was stirred at room temperature for 18 h. CH₂Cl₂ (60 mL) was added and the crude mixture was washed with saturated aqueous solution of NaHCO₃ (121 mL) and brine (120 mL). The organic layer was dried over Na₂SO₄ and evaporated under reduced pressure. The residue was taken up in CH₂Cl₂ (73 mL), then Ac₂O (1.3 eq./sugar) and triethylamine (1.5 eq./sugar) were added and the reaction mixture was stirred at room temperature for 16 h. CH₂Cl₂ (60 mL) was added and the crude mixture was washed with saturated aqueous solution of NaHCO₃ (121 mL) and brine (120 mL). The organic layer was dried over Na₂SO₄, evaporated under reduced pressure and the crude product was purified by flash chromatography (Cyclohexane/EtOAc 6:4 to 4:6 v/v).

*General procedure for deprotection of monomers.* To a solution of protected monomer (1 eq.) in MeOH (40 mL) was added a catalytic amount of NaOMe. The resulting solution was stirred at room temperature until total consumption of the reagent. Ion-exchange resin Amberlite™ CG-50 was then added and the reaction mixture was gently stirred until pH = 6-7. The resin was filtered and the solvent was evaporated under reduced pressure to afford the deprotected monomer as a white solid.

*General procedure for polymerization and treatment.* The monomer (1.0 g, 15 eq.), transfer agent **10** (1.0 eq.) and AIBN (0.5 eq.) were dissolved in THF (1.0 mL) in a Schlenk tube. This solution was degassed by 5 Freeze-Pump-Thaw cycles followed by inerting the atmosphere with argon and sealing the tube. The polymerization was conducted at 65 °C for 4 h. Consumption of the monomer was evaluated by ¹H NMR after the polymerization was stopped. Monitoring the disappearance of the specific signals of the acrylamide double bond witnessed that in most of the case, the percentage of remaining monomer was lower than 5%,
otherwise the polymer was discarded. After analysis, the solvent was evaporated under reduced pressure and the crude was purified by gravity size-exclusion chromatography to exclude potential traces of monomer, the smallest telomers and impurities from AIBN decomposition. In addition, fractionation was also used as a way to collect together polymers with closer distribution chain length (based on their elution on TLC) and discriminate the longest and the shortest polymers.

To purify 1g of crude polymer, the column dimensions were set to 5.5 cm of diameter and 40 cm of height filled with Sephadex™ LH-20 resin. The binary mixture of solvent CH₂Cl₂/MeOH 1:1 v/v was used as mobile phase. About 250 mL of eluant was passed through the column before collecting the polymer as 3 to 6 fractions of 10 mL each. All the fractions collected were analyzed by TLC (eluant: CH₂Cl₂/MeOH 9:1 v/v) and the composition of the different fractions collected is described in Figure S1. For the selected fractions, the solvent was removed under reduced pressure to give the protected NAPol as a white powder.

D̅Pₙ determination by NMR. The average degree of polymerization of the protected polymers was obtained by ¹H NMR calibration, by comparing the integral area of aromatic protons from the phenyl groups at 7.4–8.1 ppm with those of the methyl from the chain of each protected monomer constituting the polymer backbone (Figure 1). The number of monomers in the polymer chain was taken as the D̅Pₙ and the transfer agent was not taken in the calculation of the D̅Pₙ but was included in the average molar mass (M̄w) calculation.

*Polymer analysis by size exclusion chromatography.* Size exclusion chromatography (SEC) analysis were performed on a Shimadzu system, equipped with two detectors: a Shimadzu RID-20A RI detector and a Shimadzu UV detector SPD-40. Separation is done using a PSS guard column GRAM 10 µm (8×50 mm) and two PSS column GRAM 100 Å 10 µm (8×300 mm). The mobile phase was dimethylformamide (DMF), at a flow rate of 0.5 mL/min at 40
°C. The calibration was established using 8 standards of poly(methyl methacrylate) (PMMA, 540-46900 Da) from Agilent Technologies and led to average molar mass in number (M̄n) and in weight (M̄w) of the protected polymers.

*General procedure for deprotection of polymers.* To a solution of protected NAPol (500 mg to 1g) in MeOH (10 mL) was added a catalytic amount of NaOMe. The resulting solution was stirred at room temperature for a couple of hours. Ion-exchange Amberlite™ CG-50 resin was then added until pH = 6-7 and the reaction mixture was gently stirred for a few minutes. The resin was filtered off, the solvent was concentrated under reduced pressure and the crude product was precipitated in diethyl ether and filtered to give the deprotected NAPol as a white solid in quantitative yield.

*Dialysis.* An aqueous solution of polymer (10 g/L) was dialyzed through a 1 kDa membrane against approximatively 2 L of deionized water under continuous stirring. The exterior water was changed three times every 4 h. The resulting dialyzed solution was frozen, then lyophilized to give pure polymer as a white foam.

*Surface Tension Measurements.* The surface activity of detergents in solution at the air/water interface was determined using a K100 tensiometer (Kruss, Hamburg, Germany). Surface tensions were determined at different concentrations by dilution of a stock solution (ca. 3 x CMC for **3** and **5b**, 9 x CMC for **5a** and 4 x CMC for **8**) using the Wilhelmy plate technique. In a typical experiment, surface tensions were measured for 12 concentrations, with a maximum of 30 min between each measurement. All measurements were performed at 25.0 ± 0.5 °C.

The surface excess, Γmax, and an estimate of the area per molecule, Amin, at the interface were derived from the Gibbs adsorption isotherm with:

$$Γ_{max} = -(1/RT)(dγ/d(ln C))$$
where Γmax is the surface excess (moles per unit area), R is the universal gas constant, T is the absolute temperature, γ is the surface tension, and C is the surfactant concentration; and with:

$$A_{\min} = 1/(NA \Gamma_{\max})$$

where NA is the Avogadro number.

The standard molar Gibbs free energy change accompanying micelle formation takes the form:

$$\Delta G_{S}^{m/aq, \circ} = -RT \ln K_{S}^{m/aq} = RT \ln \frac{CMC}{55.5 \text{ M}}$$

*Dynamic Light Scattering.* Hydrodynamic particle size distributions were determined on a Nano Zetasizer ZS90 (Malvern Instruments, UK) equipped with a He–Ne laser (λ = 633 nm). Samples were centrifugated at 10 000 rpm for 5 minutes then filtered on 0.45 µm cellulose acetate syringe filter. All measurements were performed at (25 ± 0.5) °C in pure water at different concentrations: from 2 x CMC to 10 x CMC for monomers and from 1 g/L to 25 g/L for polymers **12a** and **12d** and from 1 g/L to 10 g/L for **12c**.

*Receptor expression, membrane isolation and solubilization.* Sf9 insect cells membranes containing the ghrelin receptor GHSR were obtained as previously described.³² These membranes were resuspendend at 1 mg/mL in a 25 mM Tris-HCl pH 8, 200 mM NaCl, 5% glycerol buffer and stock solutions of β-DDM (Anatrace) or of the different compounds were diluted to achieve the desired concentrations, i.e. 10 times the CMC for the monomeric compounds and the detergent or 0.5% (w/w) for the polymers. After 2 h incubation at 4°C, the suspension was ultracentrifuged at 100,000 xg for 1 h at 4 °C and the supernatant recovered.

*Ligand binding assay.* The ligand binding properties of the isolated receptor in the supernatant after ultracentrifugation were assessed using a fluorescence polarization-based assay with the GHSR inverse agonist LEAP2(1-17) labeled with dy647.³³ In this assay, GHSR was incubated for 1 h with the fluorescent compound (samples prepared in triplicates).
Polarization was then measured using a Fluoromax 4 (Horiba) equipped with a plate reader, with an excitation wavelength of 650 nm (bandwidth 5 nm) and an emission wavelength of 670 nm (bandwidth 5 nm). Non-specific binding was assessed using control solutions obtained after solubilization of GHSR-free *sf9* membranes.

**Results and Discussion.**

*Synthesis of Surfmers.* The 2-amino-2-methylpropane-1,3-diol was used as starting material for the synthesis of three surfmers (Scheme 1). The two hydroxyl groups allow the connection of both the hydrophobic tail and the hydrophilic part. In first place, *N*--(1,3-dihydroxy-2-methylpropan-2-yl)acrylamide (1) was obtained by condensation of commercially available prop-2-enoyl chloride, commonly called acryloyl chloride, onto 2-amino-2-methyl-1,3-propandiol in methanolic potassium hydroxyde, in satisfactory yield (56%). Then the alkyl hydrophobic chain was introduced on one hydroxyl group *via* the formation of a carbamate bond, with an alkylisocyanate *i.e.*, *n*-decylisocyante or *n*-octylisocyanate, in the presence of triethylamine in DMF, leading to compounds **2a-b** with good yields (88% and 76%, respectively).

Finally, the anomeric glycosylation of the last free hydroxyl functions was carried out. There have been numerous methodologies described for anomeric glycosylation.<sup>34-35</sup> One can cite three conditions that are often used: 1) a Lewis acid catalyst (BF<sub>3</sub>.Et<sub>2</sub>O, TMSOTf...) with glycosyl esters or pre-activated compounds, 2) the Koenigs-Knorr-like procedures using halogen as leaving groups activated by silver or mercury salts or Lewis acids, and 3) through a radical reaction starting from thioglycosides and catalyzed in acidic conditions by NBS or NIS.

Compound **3** was obtained by *O*-alkylation of the peracetylated *β-D*-glucose in presence of BF<sub>3</sub>.Et<sub>2</sub>O followed by direct reacetylation using acetic anhydride and pyridine in 46% yield
(Scheme 1). The reacetylation step was necessary due to the use of a large excess of glucose and Lewis acid (4.5 eq.) that leads to the deprotection of some hydroxyls.

For the synthesis of monomers **6a** and **6b**, three methodologies of maltose introduction were attempted. We first used octaacetylated β-D-maltose in the presence of BF₃.Et₂O, which allowed us to obtain the targeted compounds however with two main issues. On the one hand, this requires the use of a large amount of octaacetylated β-D-maltose which makes the purification complex. On the other hand, the formation of a side-product was observed (Figure S18). But despite the difference in their retention times, 14.99 min for the side-product vs 16.69 min for compound **5b**, their separation was tricky whatever the chromatography phase employed, normal or reversed phase. With this method, we obtained the desired maltosylated compound in a moderate yield of 34%.

<figure><img src="image_2.png" /><figcaption>**Scheme 1.** Synthesis of **4**, **6a** and **6b**<sup>a</sup></figcaption></figure>

<footnote><sup>a</sup>Reagents and conditions: (a) Acryloyl chloride, KOH/MeOH 3N, MeOH, RT, 24h; (b) alkylisocyanate (0.3 eq.), NEt₃ (0.5 eq.), DMF, 80 °C, 5h; (c) sugar (4.5 eq./OH), BF₃.Et₂O (4.5 eq.),</footnote>
$CH_2Cl_2$, RT, 16h, then $Ac_2O$ (1.3 eq./sugar), Pyridine (1.5 eq./sugar), $CH_2Cl_2$, RT, 16h; (d) $MeONa$ (cat.), $MeOH$, RT, 16h.

We next investigated the use of acetobromo-α-D-maltose, prepared according to a standard procedure by treatment of peracetylated maltose with hydrobromic acid in glacial acetic acid,³⁶ and introduced in slight excess (2.4 eq.), in the presence of a silver salt (AgOTf) and 2,4,6-collidine.³⁷ Unfortunately, this method did not allow us to obtain the desired monomers.

Finally, the last route carried out was to use the heptaactetyl-β-D-maltose trichloroacetimidate (TAC) with TMSOTf as promoter. This method requires prior preparation of the TAC derivative, carried out in two steps from octaacetylated β-D-maltose. The anomeric position of octaacetyl-β-D-maltose was selectively deprotected with ammonium acetate in DMF.³⁸ Then the alcohol obtained was converted into trichloroacetimidate with $K_2CO_3$ in dichloromethane.³⁹ With this method, which has the advantage of not using a large excess of the glycosyl donor, we observed the formation of another side-product during the reaction between **2b** and octaacetylated β-D-maltose. However, we were not able to isolate the desired compound and we obtained a mixture containing 80% of the desired compound in 65% yield as estimated by HPLC. Therefore, we relied on the first method for the introduction of maltose polar head group.

<figure><img src="image_3.png" /><figcaption>**Scheme 2.** Synthesis of 9.ª</figcaption></figure>

<footnote>ªReagents and conditions: (a) decylisocyanate (0.3 eq.), $NEt_3$ (0.5 eq.), DMF, 80 °C, 5h; (b) Peracetylated D-glucose (9 eq.), $BF_3.Et_2O$ (4.5 eq.), $CH_2Cl_2$, RT, 16h, then $Ac_2O$ (2.6 eq.), Pyridine (3.0 eq.), $CH_2Cl_2$, RT, 16h; (c) $MeONa$ (cat.), $MeOH$, RT, 16h.</footnote>
A fourth surfmer was synthesized in three steps from THAM (Scheme 2). The alkyl chain was first introduced by the selective alkylation of one hydroxyl group by the commercially available *n*-decylisocyanate in the presence of triethylamine in DMF. Compound **7** was obtained in 62% yield. The introduction of the glucose moieties on the two hydroxyl groups was next carried out by *O*-alkylation of the peracetylated D-glucose in presence of BF₃.Et₂O followed by direct acetylation using acetic anhydride and pyridine. This acetylation was necessary due to the use of a large excess of glucose and Lewis acid (4.5 eq. per hydroxyl group), that led to partial removal of acetyl protecting groups during the glycosyl bond formation. Compound **8** was obtained in 47% yield.

In order to determine the colloidal properties of those monomers, deprotection of the acetyl groups was carried out under mild basic conditions using a catalytic amount of sodium methoxide in methanol (Scheme 1 and 2). The deprotected monomers **4**, **6a-b** and **9** were obtained as white powders which were solubilized in water then freeze-dried before being further investigated.

A common nomenclature was used to name the deprotected surfmers: M, MG and DG, indicate the nature of the polar head *i.e.* maltose, monoglucose and diglucose, respectively. C# indicates the length of the alkyl chain, 10 or 8 carbon atoms. Therefore, compounds **6a** and **6b** are respectively called MC10 and MC8 and compounds **4** and **9** are respectively called MGC8 and DGC10.

*Synthesis of polymers.* The polymers were synthesized by free-radical telomerization of the monomers in the presence of the transfer agent **10** and AIBN as a radical initiator, in a sealed Schlenk tube under inert conditions, in THF (Scheme 3).<sup>20-21</sup> Telomerization reactions, in contrast to polymerization reactions, are generally regarded as reactions which lead to oligomers having very low molar masses. Telomerization describes a kinetic situation where
rates of chain growth and chain transfer are in near balance.⁴⁰ This balance is quantitatively stated in terms of chain transfer constants (C_T). Thiol-based transfer agents also called telogens have C_T close to one which allows good correlation between the initial ratio of monomer to transfer agent (R₀) in the feed and the obtained degree of polymerization.⁴⁰⁻⁴¹ The thiol-based transfer agent **10** was first obtained following a previously described procedure, starting from mercaptopropionic acid.²¹ Briefly, the thiol function was first protected with a trityl group, then tris-(hydroxymethyl)-amidomethane (Tris) was condensed onto the carboxylic acid moiety. The three free hydroxyl functions were next protected with benzoyl groups followed by the deprotection of the thiol to lead, after purification, to the transfer agent in about 40% overall yield in four steps. The presence of the benzoyl groups on the transfer agent **10** allows NMR end-chain calibration of the polymers as previously reported.²⁰

<figure><img src="image_4.png" /><figcaption>**Scheme 3.** Synthesis of nonionic polymers.ᵃ</figcaption></figure>

ᵃReagents and conditions: (a) AIBN (0.5 eq.), THF, 65 °C, 4.5 h; (b) MeONa (cat.), MeOH, RT, 16 h.

The number-average degree of polymerization was set by the initial ratio of monomer to transfer agent (R₀), for all our reactions, we set it to 15. The determination of the apparent degree of polymerization and the molecular mass of the final polymer were achieved by ¹H NMR measurements, by comparison between signals of aromatic groups grafted onto Tris and signal corresponding to the terminal CH₃ of the alkyl chain of monomer units (Figure 1).
Finally, all hydroxyl groups of polymers were deprotected under mild basic conditions using a catalytic amount of sodium methoxide in methanol before being dialyzed.

<figure><img src="image_5.png" /><figcaption>**Figure 1.** ¹H NMR spectrum of (A) Protected MC10 surfmer (5a) in CDCl₃; (B) protected polymer pMC10 (11c) in CDCl₃, and (C) deprotected polymer MC10NA10 (12c) in MeOD-d₄.</figcaption></figure>

To name the protected polymers, the letter p, which indicate the polymeric protected form, was placed before the name of the monomer: pMGC8 and pDGC10 for the two glucose-based polymers and pMC10 and pMC8 for the two maltose-based polymers. The final deprotected polymers were named by adding after the name of the monomer, NA for non-ionic amphipols followed by the average molar mass in kDa of the polymer chain.

*Characterization of protected polymers by size exclusion chromatography and NMR spectrometry.* Before being characterized, all the protected polymers were purified by gravity exclusion chromatography onto Sephadex™ LH20 resin (Figure S1). Telomerization can indeed lead to the formation of small adducts containing a couple of monomers (1:1 monoadduct, 1:2 dimer, 1:3 trimer...) as well as small organic side products that can be removed by gravity exclusion chromatography (Figure S1). Of note, only, for one
polymerization of pMC8 monomer, we did a fractionation of the collected fraction as described in Figure S1C.

Polymerization degree (DP̅n), average molar mass (in number, M̅n, and in weight, M̅w) and polydispersity of all polymers were determined by coupling two techniques, namely ¹H NMR and SEC chromatography (Table 1).

<table><caption>**Table 1.** Characterization of protected polymers.</caption><thead><tr><th rowspan="2">Protected polymers</th><th colspan="2">NMR<sup>a</sup></th><th colspan="3">SEC<sup>b</sup></th></tr><tr><th>DP̅n</th><th>M̅n (g.mol<sup>-1</sup>)</th><th>M̅n (g.mol<sup>-1</sup>)</th><th>M̅w (g.mol<sup>-1</sup>)</th><th>M̅w/M̅n</th></tr></thead><tbody><tr><td>pMGC8</td><td>18</td><td>12100</td><td>5900</td><td>7800</td><td>1.31</td></tr><tr><td rowspan="5">pDGC10</td><td>15</td><td>15900</td><td>5100</td><td>5600</td><td>1.10</td></tr><tr><td>15</td><td>16100</td><td>5200</td><td>5700</td><td>1.09</td></tr><tr><td>15</td><td>16200</td><td>5100</td><td>5600</td><td>1.10</td></tr><tr><td>17</td><td>18200</td><td>5200</td><td>5600</td><td>1.09</td></tr><tr><td>16</td><td>15500</td><td>6900</td><td>8100</td><td>1.18</td></tr><tr><td rowspan="3">pMC8</td><td>11<sup>c</sup></td><td>10300</td><td>5400</td><td>6700</td><td>1.23</td></tr><tr><td>19<sup>c</sup></td><td>18300</td><td>6800</td><td>7800</td><td>1.15</td></tr><tr><td>27<sup>c</sup></td><td>26000</td><td>8500</td><td>9300</td><td>1.10</td></tr><tr><td rowspan="4">pMC10</td><td>11</td><td>11100</td><td>6000</td><td>6800</td><td>1.14</td></tr><tr><td>12</td><td>12100</td><td>6000</td><td>7100</td><td>1.16</td></tr><tr><td>13</td><td>13000</td><td>5900</td><td>6800</td><td>1.15</td></tr><tr><td>14</td><td>14000</td><td>5900</td><td>6900</td><td>1.16</td></tr></tbody></table>

The initial ratio of monomer to transfer agent (R₀) was set to 15, M̅n is the average molar mass in number, M̅w is the average molar mass in weight and DP̅n is the average polymerization degree. <sup>a</sup>Determined by ¹H NMR. <sup>b</sup>Determined by SEC experiments. <sup>c</sup>The polymer was fractioned into three samples by gravity size exclusion chromatography onto Sephadex LH-20 resin (according to Figure S1).

A very good reproducibility and control of the polymerization was noted. Indeed, polymerization of the diglucose surfmer DGC10 led after purification by size exclusion chromatography on Sephadex LH-20 to average polymerization degrees of 15 to 17 in perfect agreement with the initial ratio R₀. A quite narrow polydispersity index was also noted indicating the efficiency of the purification. Polymerization of the two protected maltose MC10 and MC8 led to average polymerization degrees of 11 to 16 in good agreement with the initial ratio R₀ and confirm the rather good control of the polymerization. This suggests
that the chain transfer constants ($C_T$) could be close to one as previously observed for other thiol-based transfer agents.<sup>40-41</sup> In previous work, we used the same transfer agent for NAPol synthesis and the $C_T$ was estimated close to one although we did not measure it.<sup>21</sup> Of note, fractionation of one batch by size exclusion chromatography onto Sephadex LH-20 gravity column led to three polymer distribution of 11, 19 and 27 degrees of polymerization.

Average molar masses determined by SEC indicate significantly shorter polymers than expected. Since we used PMMA as standards for the SEC calibration which differ largely from the chemical structures of our polymers, the SEC data should be taken with caution regarding molar masses determination. By contrast, <sup>1</sup>H NMR determination based on the integration of characteristic signals of aromatic protons of the transfer agent and those of the terminal CH<sub>3</sub> of the alkyl chains provided more coherent number average molar masses. Of note, in our <sup>1</sup>H NMR determination of DPn we made approximation that there was no irreversible termination of the growing chain and that one telogen is present on each polymer chain.

Despite the variations of the two methods, a rather good correlation is obtained when plotting the number average molar masses obtained by the two techniques (Figure S#).

## Colloidal characterization.

*Water solubility.* At room temperature, all the surf polymers, under their deprotected form, were freely soluble in water at concentrations varying from 4 g/L to 12 g/L (details are given in Table 2). Those concentrations, giving transparent clear solutions, were the maximum concentrations used for physicochemical studies but they do not represent the limit solubility of the compounds.

The polymers, under their deprotected form, were soluble in water: at 25 g/L for MC10NA12 and MC8NA10; at 100 g/L for DGC11NA14 and DGC10NA12. Their solutions remained
transparent and fluid for both diglucose derivatives, and very viscous for both maltose derivatives. By contrast, MGC8NA9 was very poorly soluble in water (<5 g/L) and therefore could not be further studied (Table 2).

*Surface tension measurement.* Micellization processes of the amphiphilic monomers were characterized by surface tension measurements using the Wilhelmy plate technique from which we derived micellar parameters (Figure 3, Table 2).

<figure><img src="image_6.png" /><figcaption>**Figure 3.** Surface tension of the four monomers.</figcaption></figure>

The branched diglucose monomer DGC10 exhibits a CMC of 1.37 mM which is 2.7-fold higher than that of DGC11 (0.51 mM).²⁹ For the two maltose monomers, MC8 and MC10, the CMC was found to be 3.74 mM and 0.47 mM, respectively. The drop in the CMC observed when adding one methyle is in quite good agreement with the Traube's rule that is 3 times. For a given C10 chain, the contribution of the diglucose polar head to the polarity is more pronounced than that of the maltose polar head as the CMC of DGC10 is 3 times higher than that of MC10. The branched arrangement of the two glucose moieties is likely the origin of this increased polarity and solubility compared to the linear maltose. When looking at the two C8 derivatives, the polar contribution of a single glucose group in MGC8 is obviously lower
than that of the maltose group of MC8 with the CMC of MGC8 (2.47 mM) being 1.5 times lower than that of MC8.

Despite the differences in their chemical structures, no significant changes were observed in the surface tension at the CMC of the four monomers whose value is comprised between 34 and 37 mN/M in full agreement with that of hydrogenated sugar-based detergents.

Surface excess concentration ($Γ_{max}$) and surface area per molecule ($A_{min}$) were determined using the Gibbs equation. MGC8 leads to the highest $Γ_{max}$ value and the lowest $A_{min}$ which indicates a better packing at the interface due to the small single glucose polar head. The addition of a second glucose in the polar head increases by ~20% the $A_{min}$ when comparing MGC8 and DGC10. For the given maltose polar head, changing the alkyl chain length has no effect on the packing properties as MC8 and MC10 exhibit similar $A_{min}$ and $Γ_{max}$ values.

<table><caption>**Table 2.** Micellar and colloidal properties of monomers and polymers.</caption><thead><tr><th></th><th>MONOMERS</th><th>MGC8 (4)</th><th>MC10 (6a)</th><th>MC8 (6b)</th><th>DGC10 (9)</th></tr></thead><tbody><tr><td></td><td>Molar mass (g/mol)</td><td>476.3</td><td>666.4</td><td>638.3</td><td>682.4</td></tr><tr><td></td><td>Minimal solubility in water (g/L)</td><td>&gt; 12</td><td>&gt; 4</td><td>&gt; 10</td><td>&gt; 7</td></tr><tr><td rowspan="5">SFT<sup>a</sup></td><td>CMC (mM)<sup>b</sup></td><td>2.49 ± 0.05</td><td>0.47 ± 0.06</td><td>3.74 ± 0.11</td><td>1.37 ± 0.08</td></tr><tr><td>γ<sub>cmc</sub> (mN/m)<sup>c</sup></td><td>33.7 ± 0.3</td><td>36.2 ± 2.7</td><td>35.5 ± 0.9</td><td>37.2 ± 1.1</td></tr><tr><td>Γ<sub>max</sub> x 10<sup>-12</sup> (mol/mm<sup>2</sup>)<sup>d</sup></td><td>3.96 ± 0.07</td><td>2.87<sup>f</sup></td><td>3.03 ± 0.23</td><td>3.32 ± 0.19</td></tr><tr><td>A<sub>min</sub> (A<sup>2</sup>)<sup>d</sup></td><td>42.0 ± 0.7</td><td>57.8<sup>f</sup></td><td>55.0 ± 4.1</td><td>50.2 ± 2.9</td></tr><tr><td>ΔG<sup>mic</sup> (kJ/mol)<sup>e</sup></td><td>-24.8 ± 0.1</td><td>-29.0 ± 0.3</td><td>-23.8 ± 0.1</td><td>-26.3 ± 0.2</td></tr><tr><td rowspan="3">DLS</td><td>D<sub>h</sub> (nm) 6×CMC<sup>g</sup></td><td>5.3 ± 1.1</td><td>5.8 ± 1.5</td><td>n.a<sup>i</sup></td><td>4.8 ± 1.2</td></tr><tr><td>D<sub>h</sub> (nm) 4×CMC<sup>g</sup></td><td>4.9 ± 1.1</td><td>5.7 ± 1.7</td><td>n.a<sup>i</sup></td><td>4.7 ± 1.3</td></tr><tr><td>D<sub>0</sub> (nm)<sup>h</sup></td><td>5.4</td><td>5.8</td><td>n.a<sup>i</sup></td><td>4.6</td></tr><tr><td></td><th>POLYMERS</th><th>MGC8NA9</th><th>MC10NA12</th><th>MC8NA10</th><th>DGC10NA12</th></tr><tr><td></td><td>Molar mass (KDa)</td><td>8.79</td><td>11.5</td><td>10.4</td><td>11.8</td></tr><tr><td></td><td>DP<sub>n</sub><sup>j</sup></td><td>18</td><td>17</td><td>16</td><td>17</td></tr><tr><td></td><td>Minimal solubility in water (g/L)</td><td>&lt; 5</td><td>25</td><td>25</td><td>100</td></tr><tr><td rowspan="3">DLS</td><td>D<sub>h</sub> (nm) 5 g/L</td><td>n.d.<sup>k</sup></td><td>18.2<sup>l</sup></td><td>8.7<sup>l</sup></td><td>5.7 ± 1.5</td></tr><tr><td>D<sub>h</sub> (nm) 1 g/L</td><td>n.d.<sup>k</sup></td><td>18.2<sup>l</sup></td><td>8.5 ± 4.1</td><td>5.6 ± 1.5</td></tr><tr><td>D<sub>0</sub> (nm)<sup>h</sup></td><td>n.d.<sup>k</sup></td><td>15.6</td><td>8.2</td><td>5.5</td></tr></tbody></table>

<footnote><sup>a</sup>Data are averages of at least two experiments. ± indicates standard errors from the two or three experiments.</footnote>

<footnote><sup>b</sup>Critical micellar concentration (CMC).</footnote>
cSurface tension attained at the CMC.

dThe surface excess ($\Gamma_{max}$) and the surface area per molecule ($A_{min}$) were estimated from the slope of the surface tension curve.

eGibbs free energy of micellization.

fCalculated from one experiment only.

gHydrodynamic diameter measured in water at 4 and 6 × CMC.

hHydrodynamic diameter estimated at the infinite dilution of the monomer or polymer.

iNon applicable.

jDetermined by ¹H NMR.

kNon determined.

lDetermined with one measurement.

*Dynamic light scattering.* We next determined the hydrodynamic diameters of the aggregates formed in water by DLS at different concentrations (Table 2). Due to the non-ionic nature of both monomers and polymers we did not used saline solution nor buffer as control experiments. Whatever the monomer studied, we observed unimodal volume-weighted (Figure 4A) and number-weighted particle size distribution (data not shown) which indicates the formation of aggregates whose hydrodynamic diameter is comprised between 4.8 and 5.8 nm, except for MC8. For this latter, we noted the formation of large and poorly defined aggregates as suggested by both volume-weighted size distributions (>70 nm) and number-weighted distribution (around 30 and 50 nm) in addition to a second population of bigger aggregates of 100 to 1000 nm diameter (Figure S55). Concerning the intensity-weighted size distribution, we observed for the other three derivatives multimodal patterns, a first peak around 6 nm diameter corresponding to the main population of particles and other larger peaks bigger than 60 nm diameter (Figure S56A). Finally, upon dilution no significant difference in the volume-weighted distributions was observed for all those three monomers (Table 2, Figure 4B).
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**Figure 4.** Volume-weighted particle size distributions (A) and volume-weighted hydrodynamic diameter vs monomers concentration (B). Volume-weighted particle size distributions (C) and volume-weighted hydrodynamic diameter vs polymers concentration (D).</figcaption></figure>

Because of the low water solubility of the mono- glucose polymer MGC8NA9, it was not included in the DLS study. The other three polymers form relatively well-defined particles comprised between 5 nm and 16 nm (Table 2). Indeed, we observed unimodal volume-weighted distributions for the three compounds, centered, at 5 g/L, around 5.7 nm for DGC10NA12, 8.7 nm for MC8NA10 and 18.2 nm for MC10NA12. While self-aggregation of DCG10NA12 led particles with narrow size distribution, those obtained with maltose-based polymers appeared broader and more heterogeneous (Figure 4C). This was confirmed when the data were looked in intensity-weighted distribution (Figure S56B) and indicates that modification in the polar head induce significant changes in the size of the aggregates at the macromolecular scale. By contrast, we have previously shown that the $\overline{DP}_n$ of the polymer has only a slight influence on the particles sizes.²¹ Upon dilution no change in the volume-weighted distribution was observed for DGC10NA12 while for the two maltose derivatives a slight decrease in the particle size was observed. (Figure 4D).
For the two maltose derivatives, the increase of the alkyl chain length by two CH₂ causes a significant increase in the hydrodynamic diameter (5.5 nm and 8.2 nm for MC10NA12 and MC8NA10, respectively at infinite dilution). Comparing DGC10NA12 and MC10NA12 the difference is even bigger, the former forming aggregates 3 times smaller than the second. This contrasts with the similar hydrodynamic diameter of the aggregates formed by the monomers.

**Biochemical evaluation.**

We then evaluated the ability of the different compounds to extract a membrane protein from its lipid environment and then stabilize its native, functional fold in solution. We used here as a model the ghrelin receptor GHSR, which is a typical G protein-coupled receptor (GPCR). As most of the members of the GPCR family, GHSR is a most challenging membrane protein to manipulate *in vitro* because of its conformational instability. For the sake of comparison, Surfmer DGC11 and its polymeric counterpart DGC11NA13, the only non-ionic amphipol commercially available, were included in the evaluation. The different compounds were first compared for their ability for membrane solubilization. To this end, GHSR-containing insect cells membrane fractions were prepared as described in Materials and Methods. These membranes were then incubated with the different compounds at a final concentration corresponding to 10 times their CMC for the monomeric compounds or to 0.5% (w/w) for the polymers. After ultracentrifugation to remove insoluble material, the amount of functional receptor was assessed using a fluorescence-based assay and compared to the amount of ligand-competent receptor solubilized with the reference detergent, β-DDM. To be noted, this assay only takes into account the amount of functional receptor, which is the fraction of interest for further studies, regardless of the total amount of protein solubilized. As shown in Figure 5A, all monomers solubilized the ghrelin receptor in a ligand-competent state with yields in the 50-60% range, compared to β-DDM. The two diglucose surfmers were slightly
more efficient than the two maltose derivatives and no effect of the chain length was noted.
Of note, β-DDM is the most potent detergent for the solubilization of GHSR and therefore the
50-60% yield of solubilization afforded by the four surfmers indicates efficient solubilization.
In contrast, the polymeric compounds were far less efficient at solubilizing the receptor from
membrane fractions, with yields in the 10% range compared to β-DDM, indicative of a poor
detergency property of these polymers. This confirms that amphipols are in general poor
solubilizing agents.

We next analyzed the stability of the ghrelin receptor after solubilization in each of the
compounds under the conditions of Figure 5A. To be noted, although the solubilization of
GHSR with the polymers was low, sufficient amounts of functional protein were nevertheless
obtained for the assay to be carried out. In the case of the monomers, the higher
solubilization yields were fully compatible with the assay. To assess the stability of the
ghrelin receptor, we monitored the fraction of fluorescent tracer bound to the isolated receptor
as a function of time. As shown in Figure 5B, a fast and significant decrease in the amount of
ligand-competent receptor was observed when GHSR was solubilized in any of monomeric
compounds. In contrast, the polymers preserved the functional properties of the ghrelin
receptor, as almost 80% of ligand-competent GHSR was still observed 24 hours after
exchange of β-DDM with the polymer. Hence, although poor detergent, the polymeric species
display good stabilizing properties, consistent with previous observations with A8-35 and
NAPols. However, one cannot exclude at the present stage of the analysis that the lower
stability observed in the assays would be due, at least to some extent, to the more efficient
solubilization properties of the monomers that would extract from the membrane alternate
receptor populations of lower stability. Assessing this point on an experimental basis will
require a further detailed pharmacological analysis of the different GHSR populations
obtained after membrane solubilization. The commercially available NAPol DGC11NA13 is
slightly more stabilizing than its C10 analog, DGC10NA8 and the two maltose polymers MC10NA9 and MC8NA10 but the four polymers outperform β-DDM. As such, these three non-ionic amphipols could be an important addition to the toolbox used for stabilizing membrane proteins in the context of structural and functional analyzes.

<figure><img src="image_11.png" /></figure>

**Figure 5.** (A) Solubilizing and stabilizing properties of the monomers and polymers. (A) Amount of soluble receptor able to bind the fluorescent inverse agonist LEAP2(1-17) after incubation of the membrane fractions with the different compounds. Data are normalized to the amount of ligand-competent receptor obtained with β-DDM. (B) Amount of ligand-competent receptor as a function of time. Data are normalized to the amount of functional receptor initially present. In all cases, data are presented as mean ± SD of three experiments.
**Conclusion.**

We have synthesized four monomers bearing a sugar-based polar head and an alkyl chain as hydrophobic moiety. These non-ionic monomers behave like a classical detergent exhibiting strong surface activity and forming small micelles of ~ 5nm in diameter with CMC in the millimolar range of concentration and therefore can be classified as surfmers. They were next successfully polymerized by classical free radical polymerization in the presence of a transfer agent and led to water soluble non-ionic amphiphilic polymers of about 15 units. These polymers form rather small aggregates of 5 to 16 nm diameter. When comparing the biochemical properties of the surfmers to their polymer counterparts, we found out that the monomeric species solubilized efficiently the fragile ghrelin receptor in a ligand-competent state. By contrast, the polymers proved much more efficient at maintaining the native fold of the ghrelin receptor out of the membrane. This could provide an original strategy that would combine the respective properties of the compounds, where the monomers would be used to solubilize the target protein from the membrane and then exchanged for their polymeric counterpart for subsequent stabilization out of the membrane environment. In addition we demonstrate that the three non-ionic amphipols could be an important addition to the toolbox used for stabilizing membrane proteins in the context of structural and functional analyzes.

**Supporting information.** Synthesis of compounds **1, 7, 2a-b, 8, 3, 5a-b, 9, 4, 6a-6b, 11a-d, 12a-d**, schematic description of the purification process of the polymers, ¹H and ¹³C NMR spectra for compounds **1, 2a-b, 3-4, 5a-b, 6a-b, 7-9**; ¹H spectra for polymers **11a-d** and **12a-d**; HPLC chromatogram and HR-MS of compounds **3-4, 5a-b, 6a-b, 8, and 9**; SEC chromatogram of polymers **11a-d**; Contin distribution of compound **6b** and intensity-weighted distributions of monomers and polymers.

**Corresponding author.**
**Notes.**

The authors declare the following competing financial interest(s): Grégory Durand is co-inventor on a patent that covers NAPols. The NAPol DGC11NA13 used in this study is commercially distributed by ANATRACE (USA).

**Acknowledgements.**

This work received support from the Agence Nationale de la Recherche (ANR) through grants no. ANR-17-CE18-0022 and ANR-20-CE92-0028 and the Fondation Pour la Recherche Médicale (EQU202103012736). We acknowledge the financial support of the European Regional Development Fund, the French Government, the “Région Provence Alpes Côte d'Azur”, the “Département de Vaucluse” and the “Communauté d'agglomération Grand Avignon” for access to the NMR platform (CPER 3A). We thank Dr Marco Antônio G.B. Gomes for technical assistance with some SEC experiments.

**Present address:** †V.C.: Shoolini University, School of Advanced Chemical Sciences, Solan, 173212, India

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