Hachfi R Soussi
email: rima.hachfi-soussi@inrae.fr

Marie-Hélène Famelart

Florence Rousseau

Pascaline Hamon

Said Bouhallab

Lactoferrin/β-lactoglobulin complex coacervates: rheological properties

Keywords: Complex coacervation, Co-assembly, Rheology, β-Lactoglobulin, Lactoferrin

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.


Hachfi R Soussi
email: rima.hachfi-soussi@inrae.fr

Marie-Hélène Famelart

Florence Rousseau

Said Bouhallab. Pascaline Hamon

Said Bouhallab

Rheological properties of lactoferrin/β-lactoglobulin complex coacervates

Keywords: Complex coacervation, Co-assembly, Rheology, β-Lactoglobulin, Lactoferrin. [1]. Tavares

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.


Hachfi R Soussi

Marie-Hélène Famelart

Florence Rousseau

Pascaline Hamon

Said Bouhallab

Said Bouhallab Rheological

Professeur Yannick Outreman

Agrocampus Ouest

Professeur Alain Vian

Rheological properties of lactoferrin /β-lactoglobulin complex coacervates

Keywords: Alimentation, Complex coacervation, Rheology

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.


Dr Florian Daressy

Dr Florian Malard

Line Seguy

Vincent Guérineau

Dr Cécile Apel

Dr Vincent Dumontet

Aude Robert

Dr Anne-Claire C Groo

Dr Marc Litaudon

Dr Jérôme Bignon

Dr Sandy Desrat

Aurélie Malzert-Fréon

Dr Joëlle Wiels

Dr Ewen Lescop

Fanny Roussi
email: fanny.roussi@cnrs.fr

Dr A Robert

Dr ] L Seguy

Drimane derivatives as the first examples of covalent BH3-mimetics that target MCL-1

Drimane sesquiterpenoid dialdehydes are natural compounds with antiproliferative properties. Nevertheless, their mode of action has not yet been discovered. In this publication, we have demonstrated that various drimanes are potent inhibitors of MCL-1 and BCL-xL, two proteins of the BCL-2 family that are overexpressed in various cancers, including lymphoid malignancies. Subtle changes in their structure significantly modified their activity on the target proteins. The two most active compounds are MCL-1 selective and bind in the BH3 binding groove of the protein. Complementary studies, by NMR and mass spectrometry analyses but also synthesis showed that they are covalent inhibitors of MCL-1, via the formation of a pyrrole adduct. In addition, cytotoxic assays revealed that these two compounds show a cytotoxic selectivity for BL2, a MCL-1/BCL-xL dependent cell line and induce apoptosis.

Introduction

Apoptosis is a well-known form of programmed cell death that is essential for tissue homeostasis through the elimination of useless or potentially harmful cells. [START_REF] Strasser | [END_REF] One of the most studied protein families implicated in the intrinsic (mitochondrial pathway) of apoptosis is the BCL-2 family. [2] This protein family is divided into anti-and pro-apoptotic members. The anti-apoptotic proteins such as MCL-1, BCL-xL and BCL-2 are multidomain proteins and they share short conserved sequenced regions (BH1 to BH4 motifs). Pro-apoptotic proteins are of two types and play a distinct role: either multidomain like Bax and Bak with three to four domains of homology (BH1 to BH4) or monodomain (BH3-only) like Bad, Bid or Bim. In normal conditions, the anti-apoptotic proteins sequester the BH3 domain of the pro-apoptotic proteins at a hydrophobic groove formed by their BH1, BH2 and BH3 domains via protein-protein interactions (PPIs), thus preventing apoptosis. [3] Cellular stress signals such as DNA damage lead to the up-regulation of BH3-only proteins that trigger apoptosis. They bind to antiapoptotic proteins, unleashing pro-apoptotic multidomain proteins that oligomerize in the mitochondrial outer membrane leading to its permeabilization, release of cytochrome c into the cytoplasm, activation of caspases and cell death. [4] Numerous studies have shown that overexpression of the anti-apoptotic BCL-2 proteins is involved in the development of many kinds of cancers or confers resistance to apoptosis induced by standard anticancer therapies. [5] For example, proteins of the BCL-2 family play a central role in lymphocytes biology and are very often deregulated in lymphoid malignancies. Indeed, anti-apoptotic members of this family of proteins are overexpressed through translocation, amplification of the gene or upon action of transforming agents (such as oncogenic viruses for example) in haematological malignancies. [6] On the other hand, a recent survey of various solid tumours revealed that, most of the time, MCL-1 is more abundant than BCL-2 or BCL-xL, thereby suggesting that there might be some benefit in specifically targeting MCL-1 in these tumours. [7] Similarly, in haematological cancers, patients with poor prognosis are often associated with an elevated level of MCL-1. [8] These findings are not surprising because MCL-1 is one of the most amplified genes, [9,10] and its overexpression is directly correlated with tumorigenicity. [11] Consequently, targeting this protein family, and particularly MCL-1, is a highly promising strategy for anticancer therapy that has emerged over the last decades. [12] Particularly, the development of small molecules, called BH3-mimetics, able to bind the hydrophobic cleft of the anti-apoptotic proteins, liberating the pro-apoptotic ones and restoring apoptosis has proven its great efficiency. [13] Currently, such BH3-mimetics that target MCL-1 are evaluated in clinical trials as S63845, [14] S64315, [15] AMG-176, [16] and AZD-5991. [17] Over the past years, our team has been involved in the identification of natural compounds that target the anti-apoptotic proteins of the BCL-2 family thanks to a large screening of ICSN extract library by polarization fluorescence assay (FPA). [18] Various original natural molecules active on BCL-xL, BCL-2, and/or MCL-1 have been isolated, [19,20,21] and chemically modified or synthesized. [22,23,24,25,26,27] A few years ago, we reported the isolation of various drimane sesquiterpenoids 1 from barks and leaves of three Zygogynum (Winteraceae) of New Caledonia and their chemical modification. [28,29] Drimane sesquiterpenoids [30] are widespread secondary metabolites, produced by various higher plants (of the Canellaceae, Taxaceae, Polygonaceae and Winteraceae families), but also fungi, sponges, ferns and nudibranch. They are characterized by a common trans-decalin structure of type 2, decorated by a wide range of substituents (Figure 1). This structural diversity goes along with a vast array of biological activities, including cytotoxic, antibacterial, antifungal and antifeedant properties.

A cinnamate ester at C1 confers originality to compounds of type 1 isolated in our group. We have disclosed that most of these molecules are cytotoxic on HCT-116, HL60 and/or KB cell lines if aldehyde functions are present at C8 and C9. We have also shown that their reduction, oxidation or cyclisation is detrimental. [28] These results were confirmed by Fratoni et al., [31] who have pointed out that drimane derivatives with similar structures as 1 isolated from Drimys brasiliensis (Winteraceae) are cytotoxic against various myeloid and lymphoid cell lines. Contrarily, drimanes like polygodial 2 that do not possess a cynnamoyl group at C1 are found much less cytotoxic. [32,33] Albeit their promising biological activity, little is known about the mode of action and biological target of drimanes of type 1. In this paper, we want to disclose that their cytotoxic effect is due to their interaction with anti-apoptotic proteins from BCL-2 family, and more specifically with MCL-1.

Results and Discussion

As part of our continuing search for novel plant-derived anti-cancer agents, the ethyl acetate bark and leave extracts of various Zygogynum (Winteraceae) from New Caledonia were found to significantly disrupt

O H O Polygodial 3 H 2 O R 1 R 2 R 4 R 3 O R 5 1 1 5 6 9 8
When R 1 = R 2 = CHO : cytotoxicity interactions between the anti-apoptotic proteins BCL-xL with pro-apoptotic proteins Bak. This urged us to investigate the pro-apoptotic potency of various pure drimane sesquiterpenoids 4-9 previously isolated in our team from these plants (Figure 2). [28,29] Figure 2. Structure of some drimane sesquiterpenoids isolated in our team from a-Zygogynum pancheri; b-Zygogynum baillonii and evaluated for their activity on BCL-2 family proteins.

The binding affinities of these natural compounds (4-9) for human isoforms of BCL-2, BCL-xL and MCL-1 has been evaluated by competition against fluorescently Labelled BH3 pro-apoptotic proteins, Bim, Bak and Bid, respectively, as described by Qian et al.. [34] They were compared to three known compounds used as references: meiogynin A a natural pan-BCL-2 inhibitor, [19] ABT 737 a dual BCL-xL and BCL-2 inhibitor, [35] and S63845 [14] that is MCL-1 selective (Table 1). We have found that all tested drimanes are MCL-1 and BCL-xL inhibitors with no affinity for BCL-2 (Table 1). In addition, the apparent affinity of all these compounds toward MCL-1 and BCL-xL is increased by a factor of four when they are incubated in the presence of the proteins for a period of 6 h instead of 2. This is a noticeable result as it is not the case for the control compounds.

Table 1. Binding affinities for compounds 4-9 compared to control molecules (meiogynin A, ABT-737, and S63845) on BCL-xL/Bak, MCL-1/Bid and BCL-2/Bim [a] measured by fluorescence polarization.

Compd

Ki [b] (μM) MCL-1/Bid 2h [c] MCL-1/Bid 6h [d] BCL-xL/Bak 2h [c] BCL-xL/Bak 6h [d] Prussof, [36] The values presented (mean ± S.D.) are from two independent experiments performed in triplicate, [c] Binding affinities were determined after 2 h of incubation, [d] Binding affinities were determined after 6 h of incubation, [e] Displacement assays for control molecule are also described in following references: meiogyinin A see [25]; ABT-737 see [35]; S63845 see [14] The structure-activity relationship studies that can be deduced from these experiments match perfectly to those published by us [28] and Madeiros's group [31] following the cytotoxicity studies on similar compounds. Aldehyde functions in position 8 and 9 are required as demonstrated with compound 9 that presents a low affinity for MCL-1 with Ki = 5.26 µM and is not active on BCL-xL. In addition, position of the hydroxyl group greatly influences the affinity when comparing compounds 4, 5 and 6: an OH at position 5 is detrimental for activity on MCL-1. On the other hand, absolute configuration of aldehyde at C9 is decisive as compounds 9 5 is 16

times more potent than 9 -8. On the contrary, functionalization of the cinnamoyl moiety has a low impact (see compounds 5 and 7).

Finally, the most potent and interesting compounds are 5 and 7 that only differ by the presence of a methoxy group on the cinnamoyl moiety. Functionalization in position 7' does not affect the affinity for MCL-1 as both present sub-micromolar activities (with Ki of 0.35 and 0.57 μM respectively) in the same range of order than S63845. However, the presence of a methoxy group in position 7' increases the affinity of compound 7

for BCL-XL (1.27 μM) compared to compound 5 (3.03 μM). Compound 5 is more selective towards MCL-1 than compound 7 (i.e., it is 9 times more active on Mcl-1 than on BCL-xL compared to twice for compound 7).

As selectivity for MCL-1 with regard to BCL-xL is an important criteria in lymphoid malignancies, where MCL-1 is overexpressed but where BCL-xL is constitutively present and should not be totally inhibited to prevent thrombocytopenia, [37] compound 5 seems more promising than 7.

NMR experiments were carried out to confirm the interaction between compounds 5 or 7 and the proteins MCL-1 and BCL-xL in order to determine more precisely their binding mode. Preliminary solubility experiments (Figure S1) indicated that both molecules are soluble enough (≥ 200-400 μM) in presence of organic co-solvent (3.5 % DMSO) for interaction studies with MCL-1 and BCL-xL proteins.

We first titrated a uniformly 15 N-labeled Mcl-1 solution with increasing concentration of compound 7 (Figure 3).

The titration was followed by 2D 1 H-15 N SOFAST-HMQC NMR experiments that permit the observation of 1 H-15 N correlation peaks for backbone amide groups but also for the long living NH/NH2 side chains from tryptophane, arginine, asparagine and glutamine residues. During the titration, ligand 7 induced two types of successive spectral changes of the protein, in a concentration dependent manner (Figures 3A,3B). The first one was characterized upon addition of compound 7 at low concentration (up to 2 eq.), by the progressive decrease in peak intensity, without chemical shift variation, for a consequent (∼ 15) set of 1 H-15 N resonances.

Notably, no new cross peak appeared in the spectra, suggesting extensive line broadening for the 15 abovementioned residues in the formed complex. Disappearing residues were mapped on MCL-1 structure (Figure 4C). These were located within the BH3-binding groove of the protein composed by α-helices α2, α3, α4 and α5. We observed a similar behaviour in α-helix α6 that does not directly interact with the ligand in most crystal structures, as already reported for MCL-1 complex with BH3-derived peptides. [38] The second type of spectral changes was identified at higher ligand concentration (> 2 eq.), where all signals from MCL-1 decreased uniformly and almost no signal could be seen anymore at maximum ligand concentration (300 μM, 6 eq). In conclusion, compound 7 binds to MCL-1 with a strong affinity in the BH3-binding pocket and likely induces the oligomerization of MCL-1 at high concentration (> 200 μM). These experiments also showed that several NMR signals are missing for residues in the ligand binding sites of the formed complex. This is usually indicative of conformational sampling at the intermediate timescale (~ms) and suggests potential conformational dynamics within the MCL-1/compound 7 complex. In order to locate the binding pocket of compound 5 on human MCL-1, 1 H- 15 N SOFAST HMQC spectra of 15 N-labelled MCL-1 were recorded in presence of increasing amounts of ligands 5 up to its solubility limit (Figure 4). For the highest ligand concentration, we incubated the sample overnight at r. t. to reach apparent system equilibrium. Throughout the titration, spectral perturbations were clearly visible by progressive shifts of several resonances, mainly in rapid exchange regime at the chemical shift timescale, although some peaks also underwent some line broadening (Figures 4A and4B). At the ligand maximum concentration (225 μM), the protein (50 μM) was not fully saturated. Residues located in helical regions (α2, α3, α4 and α5) forming the BH3-binding groove of the protein were predominantly affected upon ligand addition as judged from large chemical shift perturbation upon ligand binding (Figure 4C). This indicates that compound 5 and BH3 motifs do share a common binding pocket in the protein. Consistently, helical regions that do not form the BH3binding groove (α1, α6, α7, α8) did not show perturbation above threshold upon binding of compound 5.

Surprisingly, we observed a new set of cross-peaks in the spectra when increasing ligand concentration (Figure 4B), close to cross-peaks that underwent rapid exchange shifts at lower ligand concentration. For the

O O O H OH O MeO
appearing crosspeaks, higher ligand concentration strengthened signal intensity but did not impact on 1 H-15 N chemical shifts. This suggests a second type of events for MCL-1/compound 5. We excluded the possibility of a second binding site since it would be of stronger affinity as indicated from the slow (or nul) exchange between the new peaks and the first binding mode. Since we previously observed a time-dependent inhibition mode for compound 5 in the FPA experiments, we next hypothesized that compound 5 could chemically crossreact with MCL-1 to yield a covalent complex at longer time and large ligand excess. NMR experiments demonstrated that compound 5 and 7 bind human MCL-1 BH3 domain with good affinity. The same experiments performed with BCL-xL showed that compound 5 binds to BCL-xL in the BH3binding groove of the protein with rather low affinity (Figure S2). Although several residues from the BH3binding groove of the protein (α2, α3, α4 and α5) were affected upon ligand addition, the analysis of the bound BCL-xL spectrum reveals modest changes, with most spectral perturbations in rapid exchange regime.

However, for a very limited number of residues (< 5), intermediate and slow exchange regime shifts could be This indicated the apparent lower affinity of compound 5 for BCL-xl when compared to MCL-1.

A last experience was carried out to compare the relative affinity of compound 7 for MCL-1 and BCL-xL. The 1 H- 15 N SOFAST HMQC spectrum of free 15 N-labelled MCL-1 was compared with the 1 H- 15 N spectra of MCL-1 in presence of compound 7 (1 eq.) with or without BCL-xL (1 eq.). About 75 % of MCL-1 was bound to compound 7 in absence of BCL-xL as seen from the partial disappearing of resonances from residues in the BH3 binding groove of the protein (Figure S3). Upon addition of BCL-xL, no significant change was observed in the spectrum of 15 N-labelled MCL-1 in term of both 1 H- 15 N chemical shifts and intensity (Figure S3). This confirmed the selectivity of compound 7 for human MCL-1 compared to BCL-xL. Considering the relative concentration of the two proteins, this suggests that the relative affinity of compound 7 for BCL-xL could be at least 20 times lower compared to MCL-1 in this kind of experiments. This last NMR experiment as well as the 1 H- 15 N HMQC spectrum of MCL-1 in presence of 4.5 eq. of compound 5 and intriguing FPA results, may also account for the ability of compounds 5 and 7 to form a covalent bond with MCL-1 or BCL-xL via a Paal-Knorr condensation (Figure 5). There are, indeed, few examples in the literature, of Paal-Knorr reactions between natural products and their target. [39,40,41,42] Moreover, the activity of 9b-polygodial 3 on the taste sense has been linked, in the literature, to its ability to interact covalently with a lysine of the taste receptors by a modified Paal-Knorr condensation. [43,44] The inactive 9a-isomer 13 was supposed to be unable of such reaction. So far, only [START_REF] Strasser | [END_REF] H NMR spectroscopic monitoring of 3 and 13 with methylamine and, later on, chemical reaction between 3 and 4-nitroaniline [32,33] were able to support this hypothesis (Scheme 1). Indeed, newly formed pyrroles are most of the time unstable and not isolable. Scheme 1. NMR spectroscopic monitoring between 9b-or 9a-polygodial 3 and 13 and methylamine to mimic the covalent interaction with a lysine of the protein of the taste sense. [43,44] Sodano et al. have shown that only 9b-polygodial is able to react via a Paal-Knorr rearrangement, whereas 9a isomer is unreactive.

In our case, this assumption of a covalent interaction between 9 -5 and 9 -7 and MCL-1 and BCL-xL by a Paal-Knorr reaction is substantiated by the fact that both are much more potent on MCL-1 and BCL-xL than 9 -8. The short residue section (231-238) encompassing lysine 234 and lysine 238 is affected upon addition of compound 5 or compound 7 on MCL-1. Thus, these lysine residues probably contribute in ligand binding, which consequently suggests their side-chain may orient toward the ligand. Note that due to fast solvent exchange at neutral pH and at room temperature, the sidechain amino group of these lysines were not observable in the NMR 2D 1H -15N SOFAST-HMQC maps. Upon the formation of the adduct, the pyrrole nitrogen is 15N labelled. However, the poor sensitivity of 15N direct detection precluded further information regarding the binding mode of these compounds. To support this hypothesis, MALDI experiments were carried out on MCL-1 incubated or not with 0.5, 1 and 2 equivalents of compound 5 (Figures 6A andS4). After 2 h of incubation with 2 equivalents of 5, more than 95% of the peak corresponding to native MCL-1 has disappeared in favour of a peak with a higher mass of about 378 Da. This difference in mass corresponds perfectly to the formation of a covalent adduct between compound 5 and a lysine via a Paal-Knorr rearrangement. A similar result was obtained with 2 equivalents of compound 7 (Figure S5A). On the contrary, the same experiment carried out with 2 equivalents of compound 8 did not show the formation of such covalent adduct (Figure 6B). This observation corroborates that of Sodano [44] who has proposed that the ability to perform this rearrangement depends on the configuration of C9, due to the difference in distance between the two carbonyl functions. In our case, this information is crucial as 5 and 7 are 16 times more active on MCL-1 than compound 8. We assume that this difference in affinity is due to the formation of a covalent bond between the two molecules and the protein. Likewise, MALDI experiments carried out on BCL-xL incubated with either 5 or 7

(2 eq.) showed the formation of covalent adducts but in less extent than with MCL-1 (i.e., of the order of 50%), perfectly in accordance with FPA results (Figures 6C andS5B). To go further and characterize unambiguously the adduct thus formed, compound 5 was engaged in a reaction with protected lysine derivative 14 in wet dichloromethane at room temperature for 48 h (Scheme 2).

1 H NMR analysis of the crude mixture revealed the disappearance of protons at 9.35 and 9.75 ppm corresponding to the aldehyde functions, as well as the apparition of two singlets around 6.4 ppm, characteristic of the protons of a pyrrole ring. In the flask, reactions between 5 and lysine took much more time than within MCL-1. This suggests the protein plays a key role in orientating the aldehydes of 5 so that they interact correctly with the lysine in the BH3 groove. Gratifyingly, the Paal-Knorr products 7a-15 and 7b-16 could be isolated. They resulted from a rearrangement between 5 and lysine 14 followed by a subsequent regioselective addition of water on C7. The 1:3 selectivity in favor of compound 7b-16 can be explained by the preferential addition of water on the less hindered side of the molecule i.e., anti to the adjacent alcohol. Both were sensitive to acidic conditions and prone to degradation. As it has been demonstrated that drimane derivatives are cytotoxic against myeloid and lymphoid cell lines, [31] we intended to associate the cytotoxicity of compounds 5 and 7 to their affinity to anti-apoptotic members of BCL-2, and more particularly to MCL-1 as these proteins are known to play a central role in haematological malignancies tumorigenicity. In vitro tests were realized on 2 haematological cells lines whose dependences to anti-apoptotic members of BCL-2 family have been previously characterized. [25] Thus, BL2, a Burkitt lymphoma cell line, is dependent on BCL-xL and MCL-1 for their survival whereas Remb1, a lymphoblastoid cell line (EBV-transformed normal B lymphocytes), is dependent on BCL-xL. First, cells were treated for 24 h with increasing concentrations of compounds 4 to 9 (0-50 μM) to determine the LC50 (lethal concentration 50%). Cell viability was evaluated by MTT assays (Table 2). Results obtained on both cell lines indicated that, apart 9, which is non cytotoxic, only compounds 5 and 7 showed a real cytotoxic selectivity for BL2, the MCL-1/BCL-xL dependent cell line (4.0 and 3.8 μM respectively) over Remb1 (LC50 > 15 μM), the BCL-xL dependent cell line. The same selectivity was observed for S63845, an MCL-1 inhibitor. On the contrary, ABT 737 a BCL-2 and BCL-xL inhibitor was only cytotoxic for Remb1. As expected, the cytotoxicity associated to compounds 5 and 7 seems correlated to MCL-1 dependency.

Compd. BL2 [a] IC50 (µM)

Remb1 [a] IC50 (µM) For a better characterisation of the nature of the cytotoxicity observed, cells were then treated (or not) with 5 μM of each compound for 24 h. Apoptosis was measured by flow cytometry after labelling the cells with Annexin V-FITC and PI (Figure 7). Compounds 5 and 7 were shown to promote a strong induction of apoptosis on BL2 cell line (respectively 86% and 78% of AV + cells), whereas they had a much weaker effect in Remb1 cell line (respectively 35% and 40% of AV + cells).

Conclusion

In summary, we have shown that drimane-type natural compounds 4-8 are inhibitors of proteins of the BCL-2 family. Among them, compounds 5 and 7 are the most potent molecules, with a sub-micromolar affinity for MCL-1. At cellular level, these two compounds are selective of BL2, a MCL-1/BCL-xL dependent cell line and induce apoptosis. A more detailed study regarding their mode of action, based on NMR and MALDI experiments revealed that compounds 5 and 7 are covalent inhibitors of MCL-1 and in a less extent of BCL-xL. This was confirmed by the synthesis of the corresponding Paal-Knorr adducts 15 and 16. Although for many years, there were reluctances to covalent inhibitors of a target due to safety concerns, there has been recent renewed interest for such compounds. [45,46] In fact, these inhibitors often present a strong affinity for their target, a prolonged effect in patients since they are not eliminated by the bloodstream and limit the risk of acquired resistance. The covalent bond between compounds 5 and 7 and MCL-1 is created thanks to a selective Paal-Knorr rearrangement. If Cimino et al. have hypothesised this rearrangement for the first time in 1984; we were able to confirm it indirectly thanks to the synthesis of compounds 15 and 16. These drimane derivatives 5 and 7 are thus the first examples of covalent BH3-mimetics that target MCL-1. [47] These two compounds can be isolated at multi-gram scale and could constitute good candidates for a new therapeutic strategy.

Experimental Section

General experimental details: All reagents and solvents were used as purchased from commercial suppliers.

Purification by column chromatography on silica gel was performed using Merck Silica Gel 60 (70-230 mesh). Extraction and isolation: Extraction, isolation and characterisation of these compounds have already been described: see [29] for compounds 4, 5, 6, 8 and 9; see [28] and [48] for compound 7.

Human MCL-1 production and purification: Plasmid pET15b (AmpR) encoding for the human MCL-1 deleted for the PEST domain and C-terminal transmembrane segment (MCL-1 sequence from 172 to 329) in fusion with an N-terminal histidine tag followed by a Maltose Binding Protein (MBP) segment was generously shared by L. Carlier (LBM, CNRS/Sorbonne-Universités) and incorporated into BL21 Rosetta pLysS (ChloR) using electroporation method. This biomass was made in a 1 L culture at 37 °C, to an optical density of 0.6-0.9 prior to induction. Induction of protein expression was carried out by adding isopropyl β-D-1thiogalactopyranoside (IPTG) and lasted 18 h at 15 °C, and then the culture was finally harvested and the bacterial pellet containing the protein of interest was collected. After the bacterial lysis, a histidine affinity chromatography was performed (5 mL HisTrap FF crude, GE). The elution product from histidine-affinity chromatography was digested by prescission enzyme. The completion of protein digestion was checked through SDS-PAGE experiments. Once completed, the digestion product was concentrated with an adapted centrifugal filter (Amicon) and the His-TAG was removed by a histidine affinity chromatography column (5 mL HisTrap FF crude, GE). Finally, MCL-1 was purified by size-exclusion chromatography (SEC) with S200 column (buffer: 20 mM Tris pH 7, 300 mM NaCl).

BCL-xL, MCL-1 and BCL-2 binding affinity assays:

The binding affinities of compounds for BCL-2, BCL-xL and MCL-1 were evaluated by competition against fluorescently Labelled reference compounds, Bim, Bak and Bid, respectively, as described by Qian et al.. [34] Kenakin rearranged equation, [49] which is adapted from Cheng and Prusoff equation. [36] Unlabelled peptides Bim, Bak and Bid were used as positive controls. The performance of the assays was monitored by use of Z' factors as described by Zhang et al.. [50] The Z' factors for these assays were 0.85 and 0.

Figure 1 .

 1 Figure 1. General structure of drimane sesquiterpenoids.

Figure 3 .

 3 Figure 3. Compound 7 binds to human MCL-1 with strong affinity in the BH3 binding-groove. (A) Overlay of SOFAST 1 H-15 N HMQC spectra from isolated MCL-1 (50 μM) (black) and in presence of increasing amount of compound 7 with 1 hour incubation at r. t. after each increment and prior to measurement. (B) Disappearance of 1 H-15 N resonances in presence of compound 7 (2 eq.) and corresponding residues (blue) mapped on MCL-1 structure (pdb code: 2MHS): L213, V216, G217, G219, V220, G230, M231, L235, K238, E240, S243, R246, V251, S253, T259, W261, R263, V265, S269, F270, G271, A272, E292, T301, K302 and F315. Residues for which no data are available are highlighted (black). Those include prolines and others that could not be assigned to a 1 H-15 N resonances in the spectrum of free MCL-1. Side-chain of K234 residue is oriented toward the binding pocket (black dash and label); (C) Compound 7.

Figure 4 .

 4 Figure 4. Compound 5 binds to human MCL-1 with micromolar affinity in the BH3 binding-groove. (A) Overlay of 1 H-15 N SOFAST HMQC spectra of MCL-1 alone (50 μM) (black) and in presence of compound 5 at 1 eq. (red), 2 eq. (blue) and 4.5 eq (green). We incubated samples during 1 hour (1eq., 2eq.) or overnight (4.5eq.) prior to measurement. The spectral regions where new cross-peaks were observed at the largest ligand concentration are highlighted (dashed black frames) and the closest neighboring residue 1 H-15 N resonance is labelled for each frame; (B) Close-up view of several spectral regions for which new cross-peaks (black arrow) were observed upon increase of ligand concentration; (C) Mapping of combined 1 H-15 N chemical shift perturbations on MCL-1 structure (pdb code: 2MHS): red > 0.1 ppm (M231, L235, V243, M250, V253, F270), 0.1 > orange > 0.05 ppm (V216, G217, K234, S245, I251, G271), grey < 0.005 ppm. Residues for which no data are available are highlighted (black). Those include prolines and others that could not be assigned to a 1 H-15 N resonances in the spectrum of free MCL-1; (D) Compound 5.

  -xL was far (< 50%) from being saturated at the maximum used concentration of ligand (150 μM) since the rapid exchange shifts are linear regarding the concentration of ligand along the whole titration.

Figure 5 .

 5 Figure 5. Suggested covalent interaction between compound 5 and MCL-1 protein via a Paal-Knorr rearrangement.

Figure 6 .

 6 Figure 6. MALDI experiments of MCL-1 and BCL-xL incubated at 22 °C for 2 h. (A) 1-MCL-1 alone; 2-MCL-1 with 2 eq. of 5; (B) 1-MCL-1 alone; 2-MCL-1 with 2 equivalents of compound 8; (C) 1-BCL-xL alone; 2-BCL-xL with 2 eq. of 5

Scheme 2 .

 2 Scheme 2. Formation of Paal-Knorr pyrrole adducts 7a-15 and 7b-16 by reaction between compound 5 and lysine derivative 14.

  Dependencies: MCL-1/BCL-xL (BL2), BCL-xL (Remb1), [b] IC50 measures the drug concentrations required for 50% cell death after 24 h. The values presented (mean ± S.D.) are from three independent experiments.

Figure 7 .

 7 Figure 7. Apoptosis induction by compounds 5 and 7 on haematological cell lines. Cells were treated (or not) with 5 μM of each compound for 24 h and apoptosis was determined by flow cytometry after labelling with annexin V-FITC and PI the proportion of cells in apoptosis was determined by flow cytometry after labelling with annexin V-FITC and PI. Dot plots representative of at least three independent experiments.

1 H and 13 C% 7 :

 1137 NMR spectra were recorded on Bruker ARX500 or Brucker AVANCE III 700 instruments using CDCl3 or CD3CN as internal reference. Chemical shifts (δ values) are given in parts per million (ppm), and multiplicity of signals are reported as follows: s, singlet; bs, broad singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; m, multiplet. HRMS analyses were performed using a Waters LCT Premier instrument by ElectroSpray Ionization (ESI). Protein-ligand NMR experiments were recorded on a Bruker 600 or 800 (Avance III) and 950 MHz (Avance III HD) spectrometers equipped with cryoprobes. A MALDI-TOF/TOF UltrafleXtreme mass spectrometer (Bruker Daltonics, Bremen) was used for all MALDI-TOF experiments. Mass spectra were of AV/IP labeled cells obtained in linear positive ion mode. The laser intensity was set just above the ion generation threshold to obtain peaks with the highest possible signal-to-noise (S/N) ratio without significant peak broadening. All data were processed using the FlexAnalysis software package (Bruker Daltonics). Sinapic acid (SA, used as the matrix for MALDI-TOF experiments, was of the highest grade available and used without further purification) was purchased from Sigma Aldrich.Plant material: For compounds 4, 5, 6, 8 and 9: Trunk barks and leaves of Z. pancheri subsp. pancheri were collected in the rainforest of Nodela (South Province of New Caledonia) by one of us (V. D.). The corresponding voucher specimen (DUM-578) is kept at the Herbarium of New Caledonia (NOU). See ref[29]. For compound Trunk barks of Zygogynum baillonii were collected in the high-altitude scrubland of "Monts Dzumac", South Province of New Caledonia, by one of us (M.L.). A voucher specimen (LIT-1275) is deposited in the Herbarium of New Caledonia (NOU).

Synthetic procedure and description of compounds 15 and 16 :

 16 8 for BCL-xL/Bak (at 2h and 6h respectively); 0.8 for MCL-1/Bid (at 2 and 6 h); 0.7 for BCL-2/Bim indicating the robustness of such assays.NMR experiments: Spectroscopic measurements were performed in the NMR spectroscopy buffer (NaPO4 20 mM pH 7.4, 50 mM NaCl, 2μM EDTA, 2 mM TCEP, 3.5% DMSO, 95% / 5% H2O / D2O, 0.05% pluronic acid 10% + Roche anti-protease EDTA free) at 298 K. Reference experiments with both isolated,15 N-labelled MCL-1 (human) and BCL-xL (human) were carried out by using 50 μM of protein in a 3 mm (200 μL) NMR tube. For each titration point, the content of the NMR tube was added into a lyophilized aliquot of the desired compound. After vigorous resuspension of the ligand, the solution was transferred back in the NMR tube for measurement. Experiments for the assessment of ligand solubility were performed in the same manner, except that the reference experiment was protein-free and buffer-only. Combined 1 H-15 N chemical shift perturbations were calculated as follows: ∆δ $,& = ((∆δ $ )² + 0,14 * (∆δ & )² With: ∆δ 1 = ∆δ 1,34567 -∆δ 1,89::MALDI-TOF mass Spectrometry: Sample for MALDI analysis was prepared at a concentration of 50 μM in a buffer (NaPO4 20 mM pH 7.4, 50 mM NaCl, 2 μM EDTA, 3.5% DMSO, 0.05% pluronic acid 10% + Roche anti-protease EDTA free). The matrix solution was prepared at a concentration of 45 mM in H2O/CH3CN/TFA 1:1:0.1. The sample was prepared by mixing the sample solution with matrix solution at a volume ratio of 1:9. Dialdehyde compound 5 (61 mg, 0.15 mmol), lysP*HCl 14 (92 mg, 0.31 mmol, 2 eq.) and triethylamine (43 µL, 0.31 mmol) were dissolved in wet dichloromethane. The reaction was stirred at room temperature for 48 h then concentrated in vacuo. The crude residue was purified by flash chromatography on silica gel using a gradient of heptane and ethyl acetate to give compound 7a-15 (4 mg, 4%) and 7b-16(11 mg, 12%). Compound 7a-15: 1 H NMR (700 MHz, CDCl3): δ 7.74 (d, J = 16.0 Hz, 1H), 7.68 (m, 2H), 7.43 (m, 3H), 6.65 (d, J = 16.0 Hz, 1H), 6.54 (s, 1H), 6.26 (s, 1H), 5.46 (s, NH), 4.76 (dd, J = 4.1, 11.3 Hz, 1H), 4.53 (t, J = 4.4 Hz, 1H), 4.19 (td, J = 4.4, 9.9 Hz, 1H), 3.98 (m, 1H), 3.68 (t, J = 6.9 Hz, 2H), 3.61 (s, 3H), 2.99 (brs, 1H, OH), 2.75 (d, J = 10.7 Hz, 1H, OH), 1.82 (m, 3H), 1.77 (m, 1H), 1.58 (m, 3H), 1.49 (m, 1H), 1.46 (m, 1H), 1.38 (s, 9H), 1.37 (s, 3H), 1.19 (s, 3H), 1.19 (m, 2H), 1.17

  (s, 3H), ppm;13 C NMR (175 MHz, CDCl3): δ 174.2, 167.4, 145.5, 135.7, 131.5, 131.0, 130.0 (2C), 129.3 (2C),

  Ki is the concentration corresponding to 50% of inhibition of the binding of the labelled reference compound Bak to Bcl-xL and Bid to Mcl-1, and corrected for experimental conditions according to Cheng and

	[a] Compounds 4-9 show no binding affinity for Bcl-2, [b]
		O			O		O
		O		O	O	O	O	O
		1		9	O	O		O
	7'		5	6
			H			H OH		OH
		O	4 a		O	5 a	O	6 a
		O		O	O	O	O	HO	O
					O	O
	MeO		H OH 7 b		H OH 8 a		H 9 a	OH
	4	5.4 ± 1.8	1.5 ± 0.06	>23	7.0 ± 1.8
	5	1.5 ± 0.1	0.3 ± 0.03	9.6 ± 0.9	3.0 ± 0.4
	6	7.5 ± 0.8	2.0 ± 0.2	>23	11.7 ± 0.8
	7	2.9 ± 0.5	0.6± 0.03	3.9 ± 0.3	1.3 ± 0.05
	8	14.8 ± 4.1	5.7 ± 0.2	>23	>23
	9	17.5 ± 0.5	5.3 ± 0.06	> 23	>23
	Meiogynin A [e]	5.4 ± 0.4	4.7 ± 0.5	4.7 ± 1.4	4.1 ± 1.1
	ABT-737 [e]	ND		ND		0.1 ± 0.01	0.03 ± 0.01
	S63845 [e]	0.2 ± 0.02	0.2 ± 0.02	ND	ND

Table 2 .

 2 Cytotoxic activities of compounds 4-9 on haematological cancer cell lines.

  Fluorescence polarization in millipolarization units was measured with a Beckman Coulter Paradigm® using FP cartridge (λex 485 nm, λem 535 nm). The exposure time was 300 ms per channel. All experimental data were collected using the Biomek Software® (Beckman Coulter, Inc, Brea, CA, USA) and analyzed using Microsoft Excel 2010 (Microsoft, Redmond, WA, USA). Results are expressed as binding activity, i.e. percentage of inhibition of the binding of labelled reference compound, converted into Ki, the concentration corresponding to 50% of such inhibition, and corrected for experimental conditions according to

	Human isoform BCL-2 (isoform 2), human 45-84/C37 BCL-xL
	and human 172-327 MCL-1 proteins were recombinantly produced at ICSN. Bim, FITC-Ahx-Bim-OH, Bak,
	FITC-Ahx-Bak-OH, Bid and FITC-Ahx-Bid-OH peptides were synthetized by GenePep Laboratories
	(Montpellier, France). Labelled and unLabelled peptides were diluted in assay buffer, which contained 20 µM
	Na2HPO4 (pH 7.4), 50 μM NaCl, 2 μM EDTA, 0.05% Pluronic F-68 (without pluronic acid for storage at -20 °C).
	Labelled BH3 peptide (15 nM of Bak and Bid, 3 nM of Bim), 100 nM protein, and 100 μM of unlabelled BH3

peptide or compound (first diluted in 10 mM DMSO and then buffer for final concentration from 10 -9 to 10 -4 M) into a final volume of 20 μL were distributed in a 384-well black flat-bottomed microplate (PS, Hibase, Small Volume, Med. Binding, VWR Garnier Bio-One 784076). The microplate was then incubated at room temperature for 2 and 6 h (except for BCL-2 not stable at RT for 6 h) and shaken before fluorescent polarization was measured.

Acknowledgements

The authors are very grateful to North and South Provinces of New Caledonia, which facilitated our field investigation. The authors warmly thank L. Carlier (LBM, Jussieu) for the gift of MCL-1 plasmid and J.-F. Gallard (ICSN) who performed NMR experiments on the 700 MHz apparatus. This work has been supported by University Paris-Saclay through a PhD fellowship to F. D. and the "IDI 2016" project ANR-11-IDEX-0003-02 (PhD fellowship to F.M.). This work has also benefited from an "Investissement d'Avenir" grant managed by Agence Nationale de la Recherche (CEBA, ref ANR-10-LABX-25-01) and from the IR-RMN-THC FR 3050 CNRS.


Guillaume Taing

Vanessa Legros

Françoise Ta

David Da Silva

Cyril Colas

Marylène Vayer

Marie Schuler

Kamel Chougrani

Christophe Sinturel

Arnaud Tatibouët

Arnaud Tatibouet

Solvent-Free Glycidyl Carbamate Oligomerization and Solvent Affinity of Oligomers

Keywords: glycerol carbonate, glycidyl carbamate, oligoglycerol

published or not. The documents may come

Introduction

Polyethers constitute a class of polymers with manyapplications such as in the paper industry, cosmetics, or biomedical applications. They can be obtained for instance by polymerization of epoxides, such as ethylene oxide, to produce poly(ethylene glycols) (PEGs), well-known in pharmaceutical and biomedical applications for their low toxicity and good solubility. However, concerns have been raised recently due to the potential toxicity of PEGs, in particular with low-molecular-weight PEGs (up to 400 g•mol -1 ), or the suspected hypersensitivity induced by their usage. [START_REF] Thomas | Beyond Poly(Ethylene Glycol): Linear Polyglycerol as a Multifunctional Polyether for Biomedical and Pharmaceutical Applications[END_REF] Furthermore, the absence of functional groups within the chain limits their versatility. To access highly functionalized PEG-like polymers, functional polyethers and polyglycerols (or polyglycidols) have gained a lot of attention thanks to the presence of multiple hydroxyl groups along the chain. Glycidol, which is an epoxide-based monomer derived from glycerol, gives access to many hyperbranched and dendrimeric polyglycerols. [START_REF] Sunder | Controlled Synthesis of Hyperbranched Polyglycerols by Ring-Opening Multibranching Polymerization[END_REF][START_REF] Frey | Dendritic Polyglycerol: A New Versatile Biocompatible Material[END_REF] To build well-defined structures of polyglycerol, synthesis of linear polyglycerol was first investigated. [START_REF] Thomas | Beyond Poly(Ethylene Glycol): Linear Polyglycerol as a Multifunctional Polyether for Biomedical and Pharmaceutical Applications[END_REF] Frey et al. have extensively reviewed the synthesis, modifications, and applications of linear polyglycerols. The main synthetic strategy requires the use of protected glycidol, such as ethoxyethyl glycidyl ether (EEGE), [START_REF] Taton | Synthesis of Chiral and Racemic Functional Polymers from Glycidol and Thioglycidol[END_REF] or commercially available allyl glycidyl ether (AGE) and tert-butyl glycidyl ether (tBuGE) for both homoand copoly-merization. [START_REF] Erberich | Polyglycidols with Two Orthogonal Protective Groups: Preparation, Selective Deprotection, and Functionalization[END_REF] Polymerization of such linear functionalized epoxides has been reviewed several times [START_REF] Brocas | Polyether Synthesis: From Activated or Metal-Free Anionic Ring-Opening Polymerization of Epoxides to Functionalization[END_REF][START_REF] Herzberger | Polymerization of Ethylene Oxide, Propylene Oxide, and Other Alkylene Oxides: Synthesis, Novel Polymer Architectures, and Bioconjugation[END_REF] . The deprotection of obtained polymers under acidic conditions releases free hydroxyl moieties that can further react to form functionalized polyglycerols, with pendent ester, urethane, or carbonate groups. Such modified linear polyglycerol structures have been found in the literature through pathways involving polymer-ization of EEGE, its acetal deprotection, and postfunctionalization of the side chain using phenyl chloroformate and methyl-triethylene-glycol amine [START_REF] Ozdemir | Polyglycidol Based Amphiphilic Double-Comb Copolymers and Their Self-Association in Aqueous Solution[END_REF] or isocyanate. [START_REF] Dimitrov | Synthesis and Self-Association in Aqueous Media of Poly(Ethylene Oxide)/Poly(Ethyl Glycidyl Carbamate) Amphiphilic Block Copolymers[END_REF] Also, in the light of interest for isocyanate-free polyurethanes combined with polyethers, main strategies have been mainly focused on the polymer side chains' functionalization where the choice of monomers is dictated by postpolymerization possibilities rather than their potential properties once polymerized. For example, Brocas et al. described the synthesis of the polyurethane network through epichlohydrin or allyl glycidyl ether polymerization to obtained polyethers further modified to obtain polyurethane networks in the presence of diamine. [START_REF] Brocas | Controlled Synthesis of Polyepichlorohydrin with Pendant Cyclic Carbonate Functions for Isocyanate-Free Polyurethane Networks: Polyepichlorohydrin with Pendant Cyclic Carbonates[END_REF] To our knowledge, there is at the moment no investigation on the direct synthesis of well-defined structures of linear poly(glycidyl carbamates) from glycidyl carbamate (GC) monomers.

Hence, the presence of a carbamate moiety on a functional glycidol derivative opens new perspectives in terms of the synthetic pathway and of properties of the resulting polymers (thermoresponsible, amphiphilic, or resinlike behaviors [START_REF] Dimitrov | Synthesis and Self-Association in Aqueous Media of Poly(Ethylene Oxide)/Poly(Ethyl Glycidyl Carbamate) Amphiphilic Block Copolymers[END_REF][START_REF] Jamróz-Piegza | Hydrophobic Modification of High Molar Mass Polyglycidol to Thermosensitive Polymers[END_REF][START_REF] Edwards | Novel Polyurethane Coating Technology through Glycidyl Carbamate Chemistry[END_REF] ). The synthesis of such monomers has previously been described by our group and presents several advantages: they are prepared from glycerol, one of the most important renewable feedstock nowadays, [START_REF] Pagliaro | From Glycerol to Value-Added Products[END_REF] and the urethane moieties are obtained using amines; thus, they could be seen as an alternative to the use of isocyanates and glycidol for their synthesis as well as functionalizing oligo-and polyglycerol.

Polymerization of glycidol-based monomers by anionic ring-opening polymerization is known in the literature using alkali metals as initiators, such as potassium alkoxides, [START_REF] Dworak | Hydrophilic and Amphiphilic Copolymers of 2,3-Epoxypropanol-1[END_REF]15 or through activated epoxide polymerization by the ZnEt /H2O. catalyst, [START_REF] Taton | Synthesis of Chiral and Racemic Functional Polymers from Glycidol and Thioglycidol[END_REF] or a combination of triisobutylaluminum/alkali metalalkoxides or ammonium salt. [START_REF] Gervais | Synthesis of Linear High Molar Mass Glycidol-Based Polymers by Monomer-Activated Anionic Polymerization[END_REF][START_REF] Billouard | Controlled" High-Speed Anionic Polymerization of Propylene Oxide Initiated by Alkali Metal Alkoxide/Trialkylaluminum Systems[END_REF] Regarding metal-free polymerization of epoxides, many examples of organic initiators can be found in the literature, such as N-heterocyclic carbenes, [START_REF] Raynaud | N-Heterocyclic Carbene-Induced Zwitterionic Ring-Opening Polymerization of Ethylene Oxide and Direct Synthesis of α,ω-Difunctionalized Poly(Ethylene Oxide)s and Poly(Ethylene Oxide)-b -Poly(ε-Caprolactone) Block Copolymers[END_REF] phosphazene bases, [START_REF] Boileau | Activation in Anionic Polymerization: Why Phosphazene Bases Are Very Exciting Promoters[END_REF] or pyridine derivatives. [START_REF] Dell'erba | Homopolymerization of Epoxy Monomers Initiated by 4-(Dimethylamino)Pyridine[END_REF] In this work, we focused our interest on the homopolymerization of glycidyl carbamates 1 (Scheme 1) using different bases as nucleophilic initiators. Glycidyl carbamates have been synthetized by a four-step synthetic pathway from glycerol carbonate, leading to one single regioisomer, where the urethane moiety is located at the β-position from the oxirane ring (NMR 1H of the three monomers used in this article are provided in the Supporting Information). Using a metal-free initiating system, the potential of glycidyl carbamate as a new glycerol-based monomer for the preparation of original functionalized polyglycerols will be discussed. Furthermore, the affinity of these oligomers toward solvents will be explored through an experimental approach using poly(glycidyl carbamates) in the form of thin films.

Experimental

Materials.

4-Dimethylaminopyridine (DMAP) (Acros Organics, 99%), 4-pyrrolidinopyridine (PPY) (Acros Organics, 98%), 9-azajulolidine (AJ) (Aldrich, 97%), calcium hydride (CaH2) (Acros Organics, 93%, 10-100 mm and Alfa Aesar, 92%, coarse powder), sodium iodide (NaI) (Acros Organics, 99%), and dithranol (Sigma, 97%) were used as received. Synthesis of glycidyl N,N-carbamates was achieved using previously reported protocols from our group. [START_REF] Legros | Activated Glycerol Carbonates, Versatile Reagents with Aliphatic Amines: Formation and Reactivity of Glycidyl Carbamates and Trialkylamines[END_REF] Glycidyl carbamates are purified by flash chromatography. Prior to use, they are dried for 2 h over CaH2 under a vacuum and then distilled twice under CaH 2 using a Büchi Glass Oven Kugelrorh.

Instrumentation.

1 H and [START_REF] Pagliaro | From Glycerol to Value-Added Products[END_REF] C NMR spectra were recorded in CDCl3 using a 250 MHz Bruker Avance DPX-250 instrument or a 400 MHz Bruker Avance II 400 instrument.

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) spectra were acquired on a Bruker Autoflex mass spectrometer.

Sample preparation and conditions for MALDI-TOF mass spectrometry (MS).

To prepare the samples, polymers, NaI, and dithranol were dissolved in tetrahydrofuran (THF) at a concentration of 10 mg• mL -1 . Then, 20 μL of the dithranol solution was mixed with 5 μL of the polymer sample and 5 μL of NaI into a 0.5 mL Eppendorf tube; then, 0.5 μL of the solution was spotted on a stainless steel MALDI plate. The solvent was evaporated at room temperature before insertion of the plate into the source.

Each sample was analyzed in linear and reflectron positive ionization modes using the following instrumental settings on FlexControl (v. 3.4).

Reflectron method: ion source 1 at 19 kV, ion source 2 at 16.7 kV, lens 8.4 kV, reflector 21.11 kV, and pulsed ion extracted at 150 ns for the positive mode with a laser frequency of 100 Hz (λ = 355 nm).

Linear method: ion source 1 at 19.5 kV, ion source 2 at 18.3 kV, lens 7 kV, and pulsed ion extracted at 400 ns for the positive mode with a laser frequency of 100 Hz. Laser power was adapted to obtain significant signal-to-noise (S/N) ratio and a good resolution of the mass peaks.

Mass spectra were recorded in the range 400-5000 Da in the reflectron mode and 1-15 kDa in the linear mode. To improve the S/ N ratio, each measured spectrum represented the summations of four times five hundred laser shots. Mass calibration between 400 and 10 000 Da was performed externally with poly(ethylene oxide) (PEO, Mn = 1000, 2000, 4000, and 5000 g•mol -1 ) following the same procedure as described for the polymers (except that PEO was dissolved in a mixture of 50:50 water/acetonitrile). The calibrant ions were detected under a sodium adduct form. The calibration was done with a cubic enhanced algorithm and last fit error of about 30 ppm. MALDI peak picking, average masses (Mn ), and degree of polymerization (DP) of the polymers were determined using FlexAnalysis (v. 3.4) and PolyTools (v. 1.3.1) software (Bruker).

Thin-Film Preparation and Swelling Measurements.

Thin films were obtained by depositing a 30 mg•mL -1 solution of the studied polymer in dichloromethane on silicon substrates (1 × 1 cm 2 ) by spin-coating at 3600 rpm for 30 s. For swelling measurement, the thickness of the films was continuously measured by visible reflectometry (F20, Filmetrics) during an exposure to solvent vapors in a closed poly(tetrafluoroethylene) (PTFE) chamber (total volume of 20 mL) equipped with a transparent lid (allowing for thickness measurement) and containing 1.5 mL of the liquid solvent at the bottom (dichloromethane, ethyl acetate, THF, toluene, cyclohexane, water, acetone, methanol, isopropanol, and ethanol). A partial solvent vapor pressure of 0.8 was maintained in the chamber by mixing the liquid solvent (80% mol•mol -1 ) with a nonvolatile solute (20% mol• mol -1 ). The following nonvolatile solutes were used: squalene for dichloromethane, ethyl acetate, THF, toluene, and cyclohexane; glycerol for water, acetone, methanol, isopropanol, and ethanol.

Polymerization of Glycidyl N,N-Carbamates.

This general procedure applies to each of the three different glycidyl carbamates (GCs). The polymerizations of glycidyl carbamate were carried out in screw-cap sealed tubes under an argon atmosphere. Tubes were previously dried in an oven at 110 °C overnight and cooled down in a dry atmosphere. Then, freshly distilled glycidyl carbamate was added under an argon atmosphere (500 mg) through a septum and left under a vacuum for one more hour. Then, the desired amount of initiator was added to the tube and sealed under argon with a screw-cap. The reaction mixture was stirred at 50 °C for the required amount of time. [START_REF] Thomas | Beyond Poly(Ethylene Glycol): Linear Polyglycerol as a Multifunctional Polyether for Biomedical and Pharmaceutical Applications[END_REF] H NMR was performed after 24-96 h to monitor the conversion of the GC.

The reaction was stopped by addition of 2 mL of ethanol and evaporated under reduced pressure at room temperature, giving a dark-brown sticky paste. [START_REF] Dell'erba | Homopolymerization of Epoxy Monomers Initiated by 4-(Dimethylamino)Pyridine[END_REF] Poly(glycidyl N,N-dimethylcarbamate): 1b. [START_REF] Thomas | Beyond Poly(Ethylene Glycol): Linear Polyglycerol as a Multifunctional Polyether for Biomedical and Pharmaceutical Applications[END_REF]

Derivatization of Poly(glycidyl N,N-dimethylcarbamate).

First, 100 mg of poly(glycidyl N,N-dimethylcarbamate) 1b was dissolved in CH2Cl2 at 0.3 M and stirred with a magnetic stirrer. Then, 0.5 equiv of 3-phenylpropionyl chloride regarding the theoretical molar quantity of 1b was added to the mixture and heated at reflux for 1 h. The reaction was then evaporated under reduced pressure.

Scheme 1 : General Strategy Toward Poly(glycidyl carbamates) from Glycidyl Carbamates 1

Results and discussion

From previous results within the group, the synthesis of N,N-glycidyl carbamates 1 (Scheme 2) is a three-step process starting from glycerol carbonate 2, a renewable molecule. [START_REF] Legros | Activated Glycerol Carbonates, Versatile Reagents with Aliphatic Amines: Formation and Reactivity of Glycidyl Carbamates and Trialkylamines[END_REF][START_REF] Rousseau | Tosylated Glycerol Carbonate, a Versatile Bis-Electrophile to Access New Functionalized Glycidol Derivatives[END_REF][START_REF] Vilkauskaite | Use of Tosylated Glycerol Carbonate to Access N-Glycerylated Aza-Aromatic Species[END_REF] It involves the activation of the primary alcohol as a sulfonate using tosyl chloride to form the tosylated glycerol carbonate 3. This cyclic carbonate then reacts with a primary or a secondary amine, leading to the formation of the carbamate function. We have shown that just a short range of disubstituted amines could be used for this opening: only cyclic secondary amines and dimethylamine proved to be sufficiently reactive to regioselectively ring-open the carbonate and form carbamate 4. The last step is the intramolecular nucleophilic substitution of the O-tosyl group by the secondary alcohol under basic conditions to form epoxide 1. We focused our study on three monomers derived from dimethylamine a, piperidine b, and morpholine c. These reactions were optimized on a multigram scale and obtained with good global yields ranging from 58 to 74% over three steps. Scheme 2: Synthesis of N,N-glycidyl carbamates 1a-c from glycerol carbonate 2

Preliminary Polymerization Tests.

In a metal-free approach of functionalized epoxide polymerization, we first explored various bases including potassium-tert-butoxide (t-BuOK), sodium tert-butoxide (t-BuONa), phosphazene bases, and pyridine derivatives as initiators on the model monomer glycidyl N,N-dimethylcarbamate (GDMC, 1a).

Table 1 : Polymerization of N,N-dimethylglycidylcarbamate (GDMC, 1a) using strong bases as initiators.Our first tests using classical strong bases t-BuOK, t-BuONa, and phosphazene showed poor reliability. t-BuOK ([I]/[M] = 0.05) was used in THF as the solvent or under neat conditions (Table 1). signals, was complete in 72 and 24 h, respectively, for entries 1 and 2,and the poly(glycidyl carbamate) oligomer characterization (molecular weight averages (Mn ) and (Mw ), degree of polymerization (DP), polydispersity, mass of repeat unit, and end-group mass structure) could be performed thanks to matrixassisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in linear and reflectron modes, using the dithranol matrix (for the best ionization process in our case). Despite a good conversion, the presence of several distributions in each sample could be noticed by MALDI-TOF MS in the linear mode with a lower DP value in THF than using the neat conditions (of 9 and 11, respectively). Furthermore, the resulting Mn was lower than the theoretical ones. In the case of polymers with high polydispersity (Đ > 1.2), most studies indicate that MALDI data underestimate the high-mass components, resulting in significantly lower average molecular weight values. One of the reasons for this bias for polymers (in particular with a Đ > 1.2) seems to be mainly related to the sampling and acquisition conditions of MALDI. [START_REF] Stolarzewicz | A New Chain Transfer Reaction in the Anionic Polymerization of 2,3-Epoxypropyl Phenyl Ether and Other Oxiranes[END_REF][START_REF] Murayama | Anionic Ring-Opening Polymerization of a Cyclic Carbonate Having a Norbornene Structure with Amine Initiators[END_REF] Being aware of this constraint, we have tried to minimize these effects by selecting the most favorable matrix preparation (dithranol), matrix/analyte ratio, and desorption conditions (described before) for thecharacterization of our calibrant and samples with a satisfactory degree of confidence. In this case, MALDI results provide a preliminary characterization of the oligomer structures sufficient to consider modulations in the synthetic conditions. We then changed the counterion from potassium to sodium and tested t-BuONa. In this case, the complete conversion was observed at [I]/[M] = 0.025 and 0.075, but the MALDI analysis showed only few signals of ionization and low DPs were obtained even after 168 h of reaction (entries 3 and 4). The oligomerization was finally detected using t-BuONa at the ratio [I]/[M] = 0.125 (entry 5). Quite unexpectedly, the observed DP (13) was higher than the theoretical one (8), which could be attributed to an unfavorable kinetic effect on the initiation step in comparison with the propagation one. We also have tested a phosphazene reagent alone (t-BuP4) (Table 1, entry 6) as an organic initiator, which turned out to be too reactive: full conversion (i.e., transformation) of the glycidyl carbamate was observed by 1 H NMR, but no oligomer could be detected by MALDI-TOF such as entries 3 and 4. To better understand the process of polymerization, we analyzed the oligomers using MALDI-TOF in the reflectron mode (Figure 1). In this mode, we were able to determine that the expected oligomers obtained by initiation with t-BuO -correspond to the structures of the minor population b. The major population on the other hand actually corresponds to oligomers a where the effective initiator is generated through an elimination process (Scheme 3).

The conversion, followed by 1 H NMR spectroscopy looking at the disappearance of characteristic epoxide

These results show that the t-BuOK and t-BuONa lead mainly to small chains induced by their strong basicity and the intrinsic reactivity of the monomer. Indeed, when compared toStolarzewitz (glycidyl ether) [START_REF] De Rycke | Increasing the Reactivity of Nitrogen Catalysts[END_REF] or Hans (glycidyl acetal), 15 the glycidyl carbamate proved less prone to polymerizing. This might be due to an easier hydrogen abstraction of the monomer, which decreases the amount of either the available initiator or monomer (Scheme 3). Considering the proposed mechanism, the degradation and decomposition could even be catalytic.

From these preliminary results, we suspected that, with a strong organic base and a very weak nucleophile, the degradation pathway was predominant. We thus turned our explorations toward more nucleophilic, yet less basic, systems.

Pyridine Derivatives as Initiators.

Three nonmetallic initiators exhibiting higher nucleophilicity and mild basicit were then selected.

Among the various possibilities, the following nitrogen-based initiators were thus chosen: 4-N,Ndimethylaminopyridine (DMAP), 4-pyrrolidinopyridine (PPY), and 9-azajulolidine (AJ). These pyridinederived catalysts are classically used in many reactions such as acylation (quite obviously for acetylation of alcohols), as well as alkylation, 20,27 thanks to their enhanced nucleophilicity related to the donating ability of the exocyclic nitrogen atom that promotes the delocalization of electrons throughout the molecule. [START_REF] Hassouna | Phosphazene/Triisobutylaluminum-Promoted Anionic Ring-Opening Polymerization of 1,2-Epoxybutane Initiated by Secondary Carbamates[END_REF] Hence, they have gained interest in ring-opening polymerization of epoxides such as phenyl glycidyl ether or diglycidyl ether of bisphenol A 20 as an alternative route to organometallic catalysts. Even though the initiation step mechanism is not well-established, it is suggested that the active species are obtained by nucleophilic attack of the catalyst on the epoxide, thus generating a zwitterionic specie with a positively charged nitrogen atom and a propagating alkoxide (Scheme 4). Furthermore, the moderate Scheme 3: Proposed mechanism of polymerization of glycidyl carbamate (Table 1, entry 5).

pKa of pyridine derivatives (9.71 for DMAP, 9.90 for PPY), [START_REF] Montaudo | Characterization of Synthetic Polymers by MALDI-MS[END_REF] compared to alkali metal alkoxide such as t-BuOK (>16 for alkoxides) or P4-tbu (≈42.1 in acetonitrile), may prevent carbamate decomposition. 30 The reactions were performed under neat conditions at 50 °C. The conversion was followed by [START_REF] Thomas | Beyond Poly(Ethylene Glycol): Linear Polyglycerol as a Multifunctional Polyether for Biomedical and Pharmaceutical Applications[END_REF] All of the reactions showed the disappearance of the monomer after 24 h at the ratio [I]/[M] = 0.05, as confirmed by [START_REF] Thomas | Beyond Poly(Ethylene Glycol): Linear Polyglycerol as a Multifunctional Polyether for Biomedical and Pharmaceutical Applications[END_REF] H NMR monitoring (Table 2, entries 1-18). However, decreasing the amount of the initiator to 2.5 mol % leads to traces of the monomer still remaining after 24 h. However, after 4 days (96 h), full conversion was achieved. Reactions lead to dark-brown resinous products. [START_REF] Dell'erba | Homopolymerization of Epoxy Monomers Initiated by 4-(Dimethylamino)Pyridine[END_REF] The longer time required to obtain full conversion might be due to a slower diffusion of the remaining monomers induced by the enhanced viscosity of the medium without the solvent.

The MALDI-TOF MS spectra and information (Mn , DP, and Đ) obtained in linear and reflectron modes were used to elucidate the structure of the obtained poly(glycidyl carbamates) (Table 2). Similarly, as with strong base initiators, several populations with lower than expected Mn were also observed with the pyridine-based initiators.This suggests the presence of several chain-transfer reactions, thus limiting the molar masses obtained. To support the liability of MALDI-TOF MS results, SEC analyses were performed on different samples (Table 2, entries 1, 3, 5, 7, and 13). For the poly(glycidyl carbamates)

initiated by DMAP at [I]/[M] 0.05, Đ for three populations obtained from MALDI-TOF MS analysis shows values between 1.06 and 1.20, in the range where molar mass values of polymers are still reliable using MALDI-TOF. [START_REF] Thomas | Beyond Poly(Ethylene Glycol): Linear Polyglycerol as a Multifunctional Polyether for Biomedical and Pharmaceutical Applications[END_REF] The presence of multiple populations of different Mn within the same sample, still having Đ within the same range (Table 2 entries 1, 2, 3, 7, and 13, the three major populations reported), might be the reason for SEC results showing a higher Đ (1.4-1.5) together with a slightly multimodal aspect of the SEC traces. The two techniques are complementary and orthogonal in describing the oligomers, but SEC relies on polymer properties that cannot be taken into consideration with MALDI-TOF. For instance, the major changes in the structure of the monomers GPC 1b and GMC 1c compared to GDMC 1a could induce fundamentally different hydrodynamic volumes and or interactions with the stationary phase for the corresponding oligomers in SEC. This leads to a reduced DP with this technique (value of 7, Table 2, 2, entry 1). The linear mode shows two main distributions, and the difference between each peak on each population is 145.1 u, which corresponds to the mass of the monomer. The expected mass of the oligomer, if we considered a DP5 zwitterionic form cationized with Na + , is supposed to be 870.5 or 848.5 u if cationized with H + . The closest peak is located at 848.3 m/z (distribution A), which is in accordance with the last suggested specie.

Using MALDI-TOF recorded in the reflectron mode, we were able to show that the second population detected is in fact a combination of two distributions that differ by only 3 mass units, which we were not able to detect previously due to the peak width (distributions B and C). A last population was also detected, with a smaller intensity (distribution D). All of the described distributions are shown in Figure 2.

The sensitivity of the carbamate moiety combined with the transfer reactions we observed strongly suggests that nucleophilic attack might occur during the reaction because of the presence of the alcoholate propagating center and carbamates along the polymeric chain. The suspected fragments possibly lost are shown in Scheme 5. We can observe that the mass of distribution B corresponds to the mass of a chain that has lost one carbamate (-71.1 u) in the case of poly(glycidyl N,N-dimethylcarbamate). The simultaneous presence of both alcohol and alkoxide functions on one chain can lead to intramolecular proton exchange, thus creating several potential sites for chain growth. In that regard, branched polymers might be obtained since the propagation can occur on the backbone of the chain or on the newly generated alkoxide at the lateral chain from carbamate hydrolysis and then proton exchange. Such species have lower hydrodynamic radius compared to the linear counter-parts, which is suggested by the bimodal aspect of SEC traces. Furthermore, the transfer of carbamate from one chain to another theoretically leads to the addition of one carbamate moiety in the ω-end, which actually corresponds to the close-by population on the MALDI-TOF spectrum (+71.1 u), distribution C. Indeed, Hassouna et al. [START_REF] Garipov | Reactivity of Cyclocarbonate Groups in Modified Epoxy-Amine Compositions[END_REF] have shown that carbamates in basic conditions may undergo a dissociation reaction leading to the release of an alkoxide.

Distribution D corresponds to the loss of one carbamic acid moiety (-89.1 u), which is the result of commonly described transfer reactions with glycidyl monomers, and leads to the formation of a double bond in the chain 26 by proton abstraction. These observations are further supported by poly(glycidyl Npiperidinylcarbamate, GPC) or poly(glycidyl N-morpholinyl-carbamate, GMC) spectra, which show exactly similar distributions (see the Supporting Information, more specifi-cally Table 2, entry 13 in details). However, these latter two monomers lead to more complex transfer reactions as several other distributions can be observed from the MALDI-TOFexperiments, which are in fact the result of the combinations of aforementioned transfer reactions. Using [START_REF] Pagliaro | From Glycerol to Value-Added Products[END_REF] C NMR, we can find a signal at 72.5 ppm corresponding to a tertiary carbon, which correlates with a proton signal at 4.9 ppm in [START_REF] Thomas | Beyond Poly(Ethylene Glycol): Linear Polyglycerol as a Multifunctional Polyether for Biomedical and Pharmaceutical Applications[END_REF] H NMR (see the heteronuclear single quantum coherence (HSQC) spectrum in the Supporting Information). This proton is deshielded from 3.5 to 4.9 ppm, which can be attributed to the close proximity of the carbamate moiety (Scheme 6, proton k). This observation is in agreement with our previous work where it has been shown that this type of proton is indeed at around 5 ppm. [START_REF] Legros | Activated Glycerol Carbonates, Versatile Reagents with Aliphatic Amines: Formation and Reactivity of Glycidyl Carbamates and Trialkylamines[END_REF] The lower integration of this signal indicates that this transfer reaction is in minority, corresponding roughly between one-fourth and one-third of the oligomerization process. Whether this reaction principally happens intramolecularly or inter-molecularly is still uncertain, but we suggest a mechanism to obtain such intramolecular migration through a five-membered ring (Scheme 6). This mechanism has indeed been described by Garipov et al., where it was shown that a tetrahedral intermediate is formed through the reaction of a secondary amine with a carbonate moiety to release regioisomeric carbamate products. [START_REF] Klein | Aliphatic Polyethers: Classical Polymers for the 21st Century[END_REF] Although the MALDI is a soft ionization technique leading to little in-source fragmentation, we suspect that carbamate cleavage is induced during the MALDI process. To further confirm the structures and invalidate the hypothesis of MALDI-TOF fragmentation, poly(glycidyl N,N-dimethylcar-bamate) (Table 2, entry 2) was derivatized using 3-phenyl-propionyl chloride under basic conditions. We observed the esterification of the free hydroxyl at the ω-end chain in the case of the main polymer distribution (distribution A), from 1718.9 to 1851.1 m/z, in which the gain of a mass of 132.2 u corresponds to the addition of a 3phenylpropionyl (3PP) moiety. In the case of polymer chains with one carbamate less (distribution B), two free hydroxyl groups are present, one at the ω-end chain and another at the lateral chain (Figure 4). In this case, two esters have effectively been incorporated, with a gain of 264.2 u. In the case of carbamate transfer at the ω-end chain (distribution C), no changes were observed as expected, since there is no available hydroxyl group. In summary, regarding the obtention of poly(glycidyl carbamates) using pyridine derivatives as initiators, no polymers with molecular weights higher than 3000 g•mol -1 have been obtained. The use of these initiators has been motivated by their easy handling and constitute a starting point of the study of the polymerization of glycidyl carbamates. However, in the light of obtained results, the molecular weights have to be increased to considerate real-life applications. Furthermore, we have shown that urethane sensitivity in anionic polymerization conditions may be a drawback if we consider potential copolymerization reactions with other monomers. Further studies will be conducted to decrease side-reactions on the urethane moiety by decreasing the alkoxy reactivity, leading to more controlled polymerization reactions and potential copolymerization with other epoxides.

Having in hand these original poly(glycidyl carbamates), we explored preliminary properties of these materials. Their affinity toward different solvents was determined for potential applications notably as film-forming agents.

Poly(glycidyl carbamate) Affinity with Solvents.

To highlight the differences in properties between the three poly(glycidyl carbamates) we previously synthesized (Table 3, entry 1 (p(GDMC)), entry 7 (p(GPC)), and entry 13 (p(GMC)), a study was conducted to determine the differences regarding their affinity toward solvents. The problematics of solvent selection for polymer applications is still a constant preoccupation in many fields such as biomedical or electronic devices and packaging. Indeed, the affinity of solvents toward polymers influences the solubilization, degradation, and stability of these materials in complex environments such as in painting or cosmetic formulas. This behavior of great importance is mostly driven by the interaction strength between these polymers and solvents. Hence, we have first considered Hansen's solubility parameters (HSPs) to approach the affinity between them. Then, the polymer-solvent affinities were experimentally investigated with a polymer thin film.

Polymer Solubility Using Hansen's Solubility Parameters (HSPs) and Flory-Huggins

Parameters χ. The values of δd (the dispersive component), δp (the polar component), and δh (the hydrogen-bonding component), the Hansen's solubility parameters, were theoretically calculated using the Hoftyzer and van Krevelen method. To do so, we determined the contributions of each structural group of the monomer unit using a group contribution method. [START_REF] Van Krevelen | Properties of Polymers: Their Correlation with Chemical Structure ; Their Numerial Estimation and Prediction from Additive Group Contributions[END_REF] For each structural group, values of HSP components are attributed using tabulated values and the contributions of the structural group to the HSP components are estimated by multiplying these values by the number of occurrences of the structural group in the monomer unit. As an example, the detailed calculation of p(GDMC) (Table 3, entry 1) is presented in Supporting Information. The values of HSP of the three considered p(GC) are reported in Table 3. Then, the polymer-solvent affinity is evaluated from the difference between the HSP value of the considered polymer (P) and one of the solvents (S) of interest. This can be done by calculating the Flory-Huggins parameters χP,S (eq 1). When χP,S < 0.5, the considered solvent has a good affinity for the polymer. When χP,S = 0.5, the solubility is intermediate and is the limit of solubility, and for χP,S > 0.5, the considered solvent is a nonsolvent. where δd,S and δd,P are the HSP contributions of the dispersive forces; δp,S and δp,P are the HSP contributions of the polar interactions; and δh,S and δh,P are the HSP contributions of the hydrogen bonds of the polymer (P) and the solvent (S), respectively. For the three considered p(GC), the values of χP,S are reported in Table 4. Apolar solvents such as toluene and cyclohexane are nonsolvent for p(GDMC) and p(GMC). Diethyl ether is a nonsolvent for p(GDMC) and p(GPC). For polar solvents and intermediate polarity ones, all three p(GC) have generally a good affinity with the majority of solvents.

Experimental Investigation of the Polymer-Solvent Affinity under Thin-Film

Form.

To study the affinity between the p(GC) polymers and solvent using the smallest possible amount of product, we used thin films by deposing p(GC) on silicon substrates. In that regard, with only 2 mL of a 30 mg• mL -1 p(GC) solution, we were able to run about 20 tests to apprehend the polymer-solvent affinities.

The polymer-solvent affinity can show specific behaviors that depend on the processing of polymers.

Specifically, under the thin-film form, our previous work showed that polymer solubility with a solvent might deviate from its predicted solubility determined using χP,S. [START_REF] Vayer | New Insights into Polymer-Solvent Affinity in Thin Films[END_REF] This can be attributed to the concentration of polymer in the film, which is generally above 20%, whereas the HSP generally reflects a situation where the concentration of the polymer is low. The solvents are classified using the different criteria between -good solvents (green colored boxes): χP,S < 0.5, total dissolution of the film for dissolution test and swelling ratio > 1.3 for swelling test,intermediate solvents (yellow colored boxes) (χP,S = 0.5, partial dissolution of the film for dissolution test and SR = 1.3 for swelling test) -non-solvents ( red colored boxes) ( χP,S > 0.5, the film remains unchanged during the dissolution test and SR < 1.3 for swelling test. For swelling test, * indicates that the film is quickly deteriorated to the point that the thickness measurement fails.

In that regard, we deposited the three p(GC) on silicon substrates from a solution in dichloromethane as this solvent shows good solubility with the polymers and a fast evaporation rate. Fortunately, all three p(GC) showed good film-forming properties on silicon substrates, which leads to homogenous films (under optical microscopy observation) that persist for more than several weeks without any storage precaution. To study the affinity of these films with solvents, both solubility and swelling tests were performed. In solubility tests, one drop of solvent is deposited on the film, and whether there is film dissolution or not is an indicator of polymer-solvent affinity. If the film is totally dissolved, the solvent is a good solvent of the polymer. If the film remains unchanged, the solvent is a nonsolvent of the polymer. If the film is partially dissolved, the solvent is an intermediate one. In swelling tests, the film is exposed to the solvent at a controlled partial pressure and the thickness of the film is recorded. The swelling ratio (SR) between the thicknesses of the film before and after swelling is in this case the indicator of polymer-solvent affinity. In a previous study, we have shown that the swelling ratio (SR) does not depend on the molar weight of the considered polymer. Furthermore, the partial pressure of 0.8 is a good compromise between a significant swelling of the film without systematic dewetting. As a general guideline, we considered that an SR ratio of 1.3 is the limit between a good solvent (SR> 1.3) and a nonsolvent (SR < 1.3).

Dissolution tests and swelling tests are presented in Table 4 and compared with the theoretical calculations for the three considered p(GC). First, most of the thin films of p(GDMC) are solubilized or at least deteriorated by deposition of polar solvents, and the swelling ratio is always above 1.3. In contrast, in the case of toluene and cyclohexane, which we considered to be nonsolvent according to the values of χ, the swellings of the films are less than 1.3, which confirms that there is very little affinity with these solvents.

These observations are further supported by the fact that the films are weakly solubilized, which is indeed in good accordance with prediction. The results are similar to p(GPC), with the exception of toluene, which is a good solvent with regard to the two experimental tests. χ is however slightly above 0.5 but still close to that value. On the other hand, p(GMC) shows good correspondence for nonpolar solvents when comparing χ calculations, dissolution tests, and solvent swelling ratios but mixed results regarding polar solvents. In particular, solvents containing alcohol moieties show poor affinity during the dissolution and swelling tests, but the prediction using χ is always below 0.5. Overall, we tend to observe that p(GMC) exhibits lower swelling ratios compared to the other two p(GC). The differences between predicted solubility and experimental observations can be attributed to other factors that are related to the thin-film state: variation between diluted solutions (described by χ) and concentrated state of the thin film exposed to solvent vapors may lead to unpredicted results. Furthermore, viscoelastic properties of the studied compound might also influence the behavior of the thin films.

Conclusions

We have shown that glycidyl carbamates can be polymerized in bulk conditions under organocatalysis conditions and without a solvent. The nucleophilicity of the initiator is mandatory, and simple pyridine derivatives were able to perform the oligomerization. However, the strong basicity of initiators mostly leads to undesired transfer reactions because of the presence of the carbamate moiety. The oligomers obtained have a maximum DP of 10-20 with masses up to 2000 g•mol -1 for poly(glycidyl N,Ndimethylcarbamate), 2800 g•mol -1 for poly(glycidyl N-piperidinylcarbamate), and 3700 g•mol -1 for poly(glycidyl N-morpholinylcarbamate). The oligomer mix-tures are indeed complex due to various transfers, migrations, and or elimination of the carbamate moieties, which make such oligomers difficult to analyze. However, the conditions proved compatible with the three glycidyl carbamates used and allowed the preparation under solvent-free conditions of an "oligo-urethane" based on linear oligoglycerol from biosourced monomers. Regarding the affinity of these new compounds with solvents, we have been able to predict their affinity with several solvents using the theory of Hansen's solubility parameters, providing useful information for the manipulation of these materials. The results are globally in good agreement with experimental tests on these p(GC) thin films. Thus, we have shown here that the method we have developed in a previous work and applied in this article is suitable to investigate the affinity of p(GC) with solvents. [START_REF] Vayer | New Insights into Polymer-Solvent Affinity in Thin Films[END_REF] Extension of this work is currently ongoing in our groups, looking at the optimization of the polymerization methods and the character ization of these novel oligomers to determine their properties, and will be reported in due course.

  H NMR (CDCl3, 250 MHz): 8.2 (d, DMAP-aromatic), 6.4 (d, DMAP-aromatic), 5.1-4.7 (m, -CH-OCON-), 3.4-4.3 (m, -CH2CHO-polymer backbone, CH-CH2O-polymer side chain), 3.2 (s, DMAP-(CH3)2), 2.9 (s, -N-(CH3)2). 13 C NMR (CDCl3, 100 MHz): 156.2 (C-carbamate), 72.4 (CH-O-carbamate), 68.8-72.0 (-O-CH2-CH-O-polymer backbone), 64.4 (-CH2-O-carbamate, polymer side chain), 35.8-35.3 (-N-(CH3)2). Poly(glycidyl N-piperidinylcarbamate): 2a. 1H NMR (CDCl3, 250 MHz): 8.2 (d, DMAP-aromatic), 6.4 (d, DMAP-aromatic), 5.1-4.7 (m, -CH-OCON-), 3.4-4.3 (m, -CH2CHO-polymer backbone, -CH-CH2O-polymer side chain), 3.3 (br, (CH2)2-Ncarbamate), 3.0 (s, DMAP-(CH3)2), 1.5 (br, (CH2)3-piperidinyl).

13 C

 13 NMR (CDCl3, 100 MHz): 156.2 (C-carbamate), 72.4 (CH-O-carbamate), 68.8-72.0 (-O-CH2-CH-O-polymer backbone), 64.4 (-CH2-O-carbamate, polymer side chain), 35.8-35.3 (-N-64.0 (-CH2-O-carbamate, polymer side chain), 44.4 ((CH2)2-N-carbamate), 23.9, 25.3 ((CH2)3piperidinyl). Poly(glycidyl N-morpholinylcarbamate): 3a. 1 H NMR (CDCl3, 250 MHz): 8.2 (d, DMAParomatic), 6.4 (d, DMAP-aromatic), 5.1-4.7 (m, -CH-OCON-), 3.9-4.2 (br, -CH-CH2O-polymer side chain), 3.3-3.7 (br -CH2CHO-polymer backbone; (CH2)2-N-carbamate; (CH2)2-Ocarbamate), 3.0 (s, DMAP-(CH3)2) 13 C NMR (CDCl3, 100 MHz): 155.1 (C-carbamate), 72.7 (CH-carbamate), 68.8-71.9 (-O-CH2-CH-O-polymer backbone), 66.5 (-O-(CH2)2 carbamate), 64.5 (-CH2-O-carbamate, polymer side chain), 44.2 ((CH2)2-N).

Table 1 .

 1 Polymerization of Glycidyl N,N-Dimethylcarbamate (GDMC, 1a) Using Strong Bases as Initiators a,b performed at 50°C in THF except entry 2 which was performed without solvent. [I]/[M] correspond to initiator/monomer molar ratios a Determined by 1 H NMR spectroscopy: >99% indicates the total disappearance of epoxide characteristic signals; b Determined by MALDI-ToF, using the software Polytools; c Determined by 1 H NMR spectroscopy, complete disappearance of monomer, no detection of polymers using MALDI-ToF.

Figure 1 :

 1 Figure1: MALDI-ToF spectrum of poly(glycidyl N,N-dimethylcarbamate) initiated with 12.5 mol% of t-BuONa (table 1, entry 5) , corresponding structures and m/z for DP=6 and theoretical mass.

Scheme 4 :

 4 Scheme 4: Proposed mechanism of polymerization of glycidyl carbamate initiated by DMAP; structures of PPY and AJ.

  H NMR spectroscopy looking at the disappearance of character-istic epoxide signals. All three initiators (i.e., DMAP, PPY, and AJ) were fully soluble in the monomers, and the reaction media started to get colored after about 1 h.

entries 7 and 13 compared to the value of 10 for Table 2 ,

 2 entry 1), whereas MALDI-TOF results show homogeneous DP and Đ for the three different oligomers, together with good confirmation of the polymer structure and accurate determination of absolute molecular weights of different populations of oligomers (polydispersity<1.2).

Figure 2 :

 2 Figure 2: MALDI-ToF spectra of poly(glycidyl N,N-dimethylcarbamate), proposed structures for each population A case example of poly(glycidyl carbamate)'s MALDI-TOF spectrum is presented in Figure 2, referring

Scheme 5 :

 5 Scheme 5: Proposed mechanisms for the transfer reactions of carbamate during the polymerization of glycidyl (carbamate), using DMAP as initiator

Figure 3 :Scheme 6 :

 36 Figure 3: 1 H NMR (250 MHz, CDCl3) spectrum of poly(glycidyl dimethylcarbamate) initiated by DMAP, and protons assignment of the different structures determined.

Figure 4 .

 4 Figure 4. MALDI-TOF spectra recorded in the reflectron mode of poly(glycidyl N,Ndimethylcarbamate)a [polymer n = X + H] + . a MALDI-ToF spectra before (blue) and after esterification (green) by 3-phenylpropionyl chloride for distributions A and B.

  𝑑,𝑃 -𝛿 𝑑,𝑆 ) 2 + 0,25(𝛿 𝑝,𝑃 -𝛿 𝑝,𝑆 )2 + 0,25(𝛿 ℎ,𝑃 -𝛿 ℎ,𝑆 )2 ] (1)

Table 2

 2 presents the experimental conditions and results of polymerization of glycidyl carbamates using pyridine derivatives as initiators obtained by MALDI-TOF MS. Only the main populations observed by MALDI-TOF in the samples are reported in this table, except for Table2, entries 1, 2, 3, 5, 7, and 13, where the three main populations' information are reported (more details can be found in the Supporting Information regarding the other distributions).

Table 3 :

 3 HSP of the 3 p(GC) d : HSP dispersive component, p : HSP polar component, h : HSP hydrogen bonding component, t: total HSP value calculated by T 2 = d 2 + p 2 + h 2

		δd	δp	δh	δt
	p(GDMC)	16.3	8.5	11.2	21.5
	p(GMC)	18.2	8.0	11.4	22.9
	p(GPC)	17.8	6.8	10.0	21.5

Table 4 :

 4 Polymer-Solvent affinity classification

			χ P,S	Dissolution test	Swelling test (SR)
		p(GDMC) p(GMC) p(GPC) p(GDMC) p(GMC) p(GPC) p(GDMC) p(GMC) p(GPC)
	Methanol	0.4	0.4	0.5	1.6	1.3	1.4
	Ethanol	0.2	0.3	0.4	2.6	1.4	2.1
	Isopropanol	0.2	0.2	0.3	1.6*	1.2	1.6
	Ethyl acetate	0.2	0.3	0.2	1.9	1.7	1.9
	Tetrahydrofuran 0.1	0.1	0	1.4	1.3	1.5
	Acetone	0.1	0.2	0.2	*	2.2	2.2
	Dichloromethane 0.2	0.1	0.1	1.5*	1.3*	1.4*
	Diethyl ether	0.5	0.8	0.5	1.0	1.0	1.0
	Toluene	1.0	0.9	0.6	1.3	1.2	1.4
	Cyclohexane	0.8	1.3	1.0	1.0	1.0	1.0

Table 2 :

 2 Experimental conditions of polymerization of glycidyl carbamates using pyridine derivatives as initiators

	1 2		DMAP	0.05 0.025	24 72	>99 97	3025 5750	20 40	1670 1870 1600 1850	11 13 11 13	1460 Đ = 1.4
	3 4	GDMC 1a	PPY	0.05 0.025	24 72	>99 >99	3050 5950	20 40	1630 1860 1400 1600	11 13 9 11	1580 Đ = 1.4
	5 6		AJ	0.05 0.025	24 24	>99 >99	3075 5975	20 40	1540 1620 1950 2020	10 11 13 14	2300 Đ = 1.6
	7		DMAP	0.05	24	>99	3825	20	1920 2580	10 14	1400 Đ= 1.4
	8			0.025	-	-	7525	40	-	-	-
	9 10	GPC 1b	PPY	0.05 0.025	24 24	>99 97	3850 7325	20 40	2050 2800 1830 2570	11 15 10 14
	11 12		AJ	0.05 0.025	24 24	>99 97	3875 7350	20 40	2170 2290 1850 2420	11 12 10 13
	13		DMAP	0.05	24	>99	3850	20	2240 2470	12 13	1425
	14			0.025	degradation	7600	40
	15			0.05	24	99	3900	20	2670 3010	14 16
	16	GMC 1c	PPY	0.025	96	>99	7650	40	2830 3660	14 19 c
	17 18		AJ	0.05 0.025	24 96	>99 >99	3925 7675	20 40	1740 1940 2660 2350	9 10 12

Acknowledgments :

This work has been partly supported by the Université d'Orléans, the Centre National de la Recherche Scientifique (CNRS), the Labex SynOrg (ANR-11-LABX-0029). G.T. thanks the programme Cosmetosciences from the Region Centre Val de Loire for the doctoral fellowship.