Félix Grosjean

Emeline Cros-Perrial

Abdenour Braka

Jean-Pierre Uttaro

Laurent Chaloin

Lars Petter Jordheim

Suzanne Peyrottes

Christophe Mathé
email: christophe.mathe@umontpellier.fr

Synthesis and studies of potential inhibitors of CD73 based on a triazole scaffold

The ecto-5'-nucleotidase CD73 is involved in the production of immunosuppressive adenosine in the tumoral microenvironment and recently became a validated target in immunooncology. To avoid formation of CD73-produced adenosine, several series of potential inhibitors of the target enzyme based on a triazole scaffold were synthetized and evaluated on recombinant purified hCD73 and in cell-based assays.

Introduction

Human ecto-5′ -nucleotidase or hCD73 (3.1.3.5), a member of the family of 5′-nucleotidases, is an extracellular enzyme attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor. [1] In addition, CD73 exists as a soluble and circulating form with enzymatic activity similar to its membrane anchored form. The crystal structure of hCD73 has been reported. [2] This enzyme works as a homodimer and is composed of two structural domains, the N-terminal domain and the Cterminal domain, respectively. During its catalytic activity, CD73 undergoes intra and inter domain movements involving these terminal domains that allows switching between an open and a closed conformation. [2][3] This conformational change is critical to the enzymatic activity. The active site is present in the closed conformation, and is situated at the interface between the N-and C-terminal domains. CD73 catalyzes the dephosphorylation of extracellular adenosine 5'-monophosphate (AMP) into adenosine and inorganic phosphate, and as a consequence mediates the formation of immunosuppressive and pro-tumoral adenosine in the tumor microenvironment. [4] Then, the abundance of adenosine exerts immunosuppressive activity on various immune cells such as myeloid-derived suppressor cells, macrophages, dendritic cells, natural killer cells and effector T cells through A2a and A2b receptors recognition. [5] Thus, CD73 appears as an interesting target in oncology owing to its blockade leading to the decrease of tumor growth and metastasis [6] via the use of small molecules [7] and monoclonal antibodies. [8] As a part of our ongoing work targeting CD73, we recently reported ribonucleotide derivatives as substrate analogue inhibitors and evaluated their potential activity against CD73. [9] An alternative approach to inhibit CD73 is to develop small molecules targeting allosteric sites. Thus, by using a bioinformatics approach [3] we identified a binding cavity, distinct from the active site and located at the dimer interface. Such potential inhibitors could block the conformational change and consequently lead to the inhibition of the enzyme. Through molecular docking predictions, we detected suitable scaffolds that could potentially interact with this cavity. Notably, several compounds containing an imidazole ring substituted by aromatics were identified as interesting hits. Some of them being commercially available, they could be evaluated by enzymatic tests and turned out to be strong enzymatic inhibitors. Based on the results from this virtual screening targeting the dimerization site of CD73, we focused on a particular active hit bearing three substitutions on a 1,3-diazole scaffold doubly branched with a biphenyl substituent plus one indole group (hit compound RR3, 93±5% inhibition at 5 µM, Ki = 0.53 µM, Figure 1). [3] Moreover, seven commercially available analogues (AB02, AB03, AB04, AB05, AB06, AB07, AB38) were tested after this hit identification and all analogues showed a significant inhibition of hCD73 activity (78%-94% inhibition at 5 µM). However, numerous analogues of RR3 are patented [10] and their synthetic accessibility is less easy than the triazole ring. [11] Consequently, chemical synthesis of new derivatives was envisaged using triazole as more versatile scaffold instead of diazole in order to explore chemical diversity. Herein, we report the synthesis of potential inhibitors of CD73 based on a triazole scaffold (Figure 1) and the evaluation of their effects in vitro.

Results and Discussion

Synthesis

The Cu(I)-catalyzed azide alkyne cycloaddition reaction (CuAAc) developed by Sharpless [12] et al. allows the synthesis of a huge varieties of 1,4-disubstituted 1,2,3-triazoles from commercially available or synthetic azides and alkynes derivatives. Herein, the synthesis of 1,4-disubstituted 1,2,3triazoles bearing different aromatic cycles are presented as well as their transformations into more complex structures. The first series of compounds considered contains an indole framework as RR3. Indeed, the insertion of indoles on heterocyclic compounds have been extensively investigated due to their wide spectrum of pharmacological activity. Numerous natural and synthetic derivatives of indole have shown a wide spectrum of pharmacological properties including antibacterial [13] and anticancer activities. [14] Its preparation was accomplished from common derivatives 11a and 11b, obtained following reported and adapted procedures [15] and various alkynes (procedure A). The aromatic amine must be protected to incorporate the azido group when n = 1. Formation of the triazole ring (Table 1, compounds 1a-h, n = 1 or 2a-h, n = 2) was achieved in a mixture of dichloromethane/water with catalytic amount of copper sulfate pentahydrate reduced in situ to Cu(I) by sodium ascorbate. Most of the time, the click reaction lasted 5 hours to complete. In this series, we obtained heterocycles with a short linker with good yields (Entry 1-8, n = 1, compounds 1a-h, 35%-97%) and analogues containing an expanded linker (Entry 9 to 16, n=2, compounds 2a-h, 68%-100%) in order to probe the influence of the carbon chain's length against our target. Because the elimination of the Boc protective group in standard TFA conditions [16] lead to degradation of the indole moiety, milder acidic conditions of deprotection have been attempted leading to the same results. [17] Thus, compounds 1a-h and 2a-h were directly evaluated for their activity on hCD73 and on tumor cell lines. Then, we envisaged the synthesis of 1,4-disubstituted 1,2,3triazoles bearing a free indole. Compound 12 was obtained following reported procedures [18] and reacted with different alkynes using similar conditions presented in Table 1, to afford the corresponding 1,4-disubstituted 1,2,3-triazoles (Table 2, Entry 1-5, compounds 3a-e, 44%-89%). Despite many attempts, the synthesis of analogue of compound 12, in which the indole moiety is directly linked to the azido group, as previously reported, [19] did not give any result. Owing to the efficiency of the CuAAc reaction, we also applied it upon alkylated indoles containing azide with good yields (Table 3, Entry 1-2, compounds 4a and 4b, 73%-79%). Nevertheless, starting compounds 13a (see supporting information) and 13b [20] were obtained with low yields, due to low reactivity and stability of their respective alkylated intermediates. Thus, another synthetic strategy was considered. Amine alkylation upon free indole was more effective once the triazole moiety was formed. This allowed us to easily prepare new derivatives with N-alkylated indoles starting from previous compounds 3b-e, that are represented in Table 4 (Entry 1-5, compounds 5a-e, 47%-92%). The amine alkylation was achievable with a large excess of sodium hydride and alkylating As we faced many difficulties during the synthesis of azidoderivatives containing an indole moiety (vide supra), we decided to replace it by aromatic residues analogous to AB06 and AB38 derivatives. Thus, we prepared 1,4-disubstituted 1,2,3-triazoles bearing di-or tri-methoxybenzene substituent instead of the indole moiety (Table 5, compounds 6a-m), using similar conditions as presented above (procedure A).

Table 5. Synthesis of 1,4 disubstituted triazole bearing aromatic derivatives.

[a] Yield over 3 steps from the corresponding alcohol precursor Azido derivatives 14a-f were synthesized from their commercially available precursors (hydroxyl or carboxylic acid form) following reported procedures. [21] Briefly, using diphenylphosphoryl azide (DPPA) and DBU, benzyl derivatives 14a-b (n = 1, R=H or OMe) were obtained in one step. Compounds 14c-f, with longer chain (n = 2 or 3, R = H or OMe), were obtained in a two steps procedure: formation of O-tosylated derivative and nucleophilic substitution using NaN3 as incoming nucleophile. Combination between those azides and two commercial alkynes containing aromatic cores, such as 4-biphenyl and 4-phenoxyphenyl, led to the expected derivatives (Table 5). Incorporation of a 4cyanophenyl group was also accomplished (Entry 7, 6g, 71 %) and can be used as starting material for the subsequent synthesis of more complex structures. In this series, we could afford 1,4disubstituted 1,2,3-triazoles with good yields for compounds 6a-i (Table 5, entry 1-9, 64%-96%) and modest yields for compounds 6j-m (Table 5, entry 10-13, 33%-64%, over 3 steps). Indeed, when n = 3, an impurity was recovered with the tosyle intermediate and could not be separated from the azide, and thus finally removed after the triazole formation.

The third series corresponds to more functionalized entities such as 1,4,5-trisubstituted 1,2,3-triazoles based on 6c-d, 2b, 4b, 5d and 5e as starting materials. The modified Heck coupling developed by Ackermann et al. [22] and catalyzed by palladium, allowed us to obtain derivatives presented in Table 6. With an excess of brominated agent, the Heck coupling was achievable upon 1,4-disubstituted 1,2,3-triazoles with a catalytic amount of commercially available palladium acetate reduced in situ by tricyclohexylphosphine. The reaction was carried out in refluxing toluene for several hours under basic conditions (procedure B). To investigate the influence of the 4-biphenyl and 4-phenoxyphenyl moieties toward our target, we accomplished the synthesis of 7a-d from 6c-d with good yields (71%-80%). This [a] reaction was also applied to 1,4-disubstituted 1,2,3-triazoles bearing N-substituted indole and gave rise to compounds 7e and 7f with modest yields (36%-51%). The presence of the indole moiety (2b, 4b, 5d and 5e) gave rise to the formation of side products and purification of compounds 7e-h was tedious.

Unfortunately, synthesis of 1,4,5-trisubstituted 1,2,3-triazoles could not be done using free indole derivative. We did not observe any reactivity, probably due to the irreversible chelation of palladium by the amine function.

Finally, and in order to solve solubility issues encountered during biological tests, we decided to introduce polar moieties such as a tetrazole ring or the sulfonamide group. We firstly optimized the tetrazole synthesis with a large excess of sodium azide and Lproline in DMF at 130° C. L-Proline was initially used in catalytic amounts [23] but the reaction was not complete after 3 days. Adding up to 2 equivalents of amino-acid and 10 equivalents of sodium azide allowed us to obtain a rapid conversion. These conditions were applied to 4a and 5b and provided compound 8a-b with low to modest yields (Table 7, entry 1-2, 27%-60%). As previously observed the presence of the indole moiety leads to incomplete reactions and partial degradation of the starting materials. Incorporation of the tetrazole ring was also achieved upon 1,4,5trisubstituted 1,2,3-triazoles, previously obtained from compound 6g as shown in Table 8 (procedure C). Except for compound 9a (Entry 1, 85%) for which the tetrazole synthesis was directly achieved, the formation of the latter was subsequently done after the Heck coupling (compounds 9b-e, 2 steps, 42%-47%). In spite of the difficulty to form two tetrazole rings instead of one, we could obtain compound 9b (Entry 2, 42% over 2 steps). We have also synthesized compounds 9c-e (Entry 3-5, 47%-77% over two steps) despite the formation of light sensitive intermediates after the Heck coupling. As in the case of compound 9b, the synthesis of the two tetrazole rings of compound 9e (Entry 5) was delicate and lead to incomplete conversion in both cases. To access the sulfonamide form of 1,4,5-trisubstituted 1,2,3triazoles starting from 6c and 6h, the sulfonamide moiety has to be protected by a Boc group. Then, cleavage of the carbamate bond was accomplished with an excess of trifluoroacetic acid in anhydrous dichloromethane to afford compounds 10a-b (Table 9, entry 1-2, 25%-35% over two steps).

Due to low reactivity and solubility of the starting materials in toluene, both reactions were not complete, and led to the formation of the desired compounds as well as their monodeprotected forms that were pooled for the final cleavage step.

Table 9. Synthesis of sulfonamide derivatives containing 1,4,5-trisubstituted triazole.

[a] Yield over 2 steps

Enzymatic inhibition assays

The compounds were evaluated by kinetics inhibition assays using the recombinant purified enzyme. At high concentration (100 µM) many compounds were able to inhibit CD73 activity (23 derivatives inducing more than 80% of enzyme inhibition, see Table in SI) while a more limited number of derivatives was still active at 10 µM. Compounds 4b, 5b, 6a, 7c and 10a were the most efficient inhibitors at this lower concentration with 68 to 87% of enzyme inhibition.

Cell-based assays

Compounds were also evaluated on their capacity to inhibit the membrane-bound CD73, using CD73-expressing breast cancer cells (MDA-MB-231) as earlier reported. [9a] Many compounds precipitated in the experimental conditions and were not evaluated at 100 µM. Some compounds showed decent inhibitory activity (25-48%) at 100 µM (1d-h, 2a-c, 2e-h, 3c-d, 6c-d, 8b and 9d), whereas none reached 25% when evaluated at 10 µM. The three days survival of cancer cells exposed to these compounds was rather low at 100 µM for the majority of the compounds, whereas only low (<35%) for 3c and 5b at 10 µM (see Table in SI).

Molecular modelling and docking

Molecular docking was carried out in order to better understand the structural rules leading to weak or strong activity of newly synthesized compounds. As shown in Fig. 2, the docking pose obtained for original hit RR3 was compared with commercially available structural analogues. The common diazole scaffold was found to be perfectly superimposed for all of them and in most cased the phenyl groups as well. The analysis of the binding mode of compound 5b showed that the indole group was trapped between three tyrosine residues (Tyr-531 from both chains and Tyr-539, Fig. 3B). Biphenyl group is interacting with CD73 by making hydrophobic contacts with Glu-543 (both chains) and one hydrogen bond with Gly-454. Interestingly, two π-cation interactions were observed between Arg-545 and triazole moiety and between His-456 and the second phenyl group. The cyano group contributes to the binding through hydrogen bonding and hydrophobic contact with Tyr-531. A very similar binding mode was observed for derivative 4b (Fig. 3A, highly superimposable to that of 5b) but without the contacts induced by the cyano group. Subtle modifications such as a methyl group introduced on indole (4b) seemed to strengthen the stacking with Tyr-539 when compared to the structure of derivative 3c even if binding modes were found identical (Fig. 4A). Also, the replacement of the biphenyl group by a phenoxyphenyl in series 6 induced a shift in the binding mode in addition to additional contacts. The resulting orientation did not improve the inhibitory activity for derivative 6b in comparison to that of 6a (Fig. 4B). The length of the linker (n = 1 or 2) between triazole and indole or di/tri-methoxyphenyl was found to play a role for enzymatic inhibition. Indeed, for compounds 8a (n = 1) and 8b (n = 2), a stronger inhibition was observed for 8a and these compounds were found to bind the cavity similarly but with a notable shift (Fig. 5A) leading to weaker interactions between biphenyl group and His-456 residues. Another example is given by dimethoxyphenylbased derivatives 6a (n = 1, 68% of inhibition at 10 µM) and 6e (n = 2, 0% of inhibition at 10 µM) therefore not equivalent in terms of inhibition but exhibiting the same positional (Fig.

In the observation was made in series 3 for derivatives 10a and 10b (Fig. 5C). Here again, the impact of the linker length is confirmed by a shift in the binding mode between these two compounds, complemented by the loss of the interaction with Asp-366 (interaction with the sulfonamide group) for 10b. This feature may represent a general rule for series 2 and 3 with a shorter linker being more advantageous for enzymatic inhibition. However, the impact seems to be different according to the series and the substituting groups used as seen by the exception with compounds 4a and 5b (linker n = 2 promoting a stronger inhibition than n = 1).

Conclusion

Three series of 1,4-disubstituted 1,2,3-triazoles bearing different aromatic cycles as well as complex structures were successfully obtained. All synthesized compounds were evaluated for their ability to inhibit 5'-ectonucleotidase CD73. Among them, compounds 4b, 5b, 6a, 7c and 10a were the most potent inhibitors at 10 µM while more than 20 derivatives producing inhibition superior at 80% of hCD73 at 100 µM. However, these compounds were much weaker inhibitor than the original hit RR3. Several factors could be responsible for the weaker activity such as changing imidazole scaffold into triazole one as well as the nature of substituents/length of the linker which could promote orientation more or less favourable for the interactions with the protein target. When tested in the cell based assay, all the compounds showed marginal inhibition of CD73. We assume that these differences may arise from solubility issues in the culture media and/or the use of the soluble form of the protein, whereas in the cell-based assay the protein is anchored to the membrane. In addition, molecular modeling has helped to identify the interactions between the targeted protein and the compounds exhibiting the best inhibition. Considering the literature data related to the design of inhibitors of CD73, these results will certainly contribute to the establishment of a SAR study and further development of novel derivatives.

Supplementary Information

Experimental procedures, compound characterizations, NMR spectra, data for enzymatic inhibition, cell-based assays and molecular docking methods are provided in supporting information.

General procedure A: 1,4-disubstituted 1H-1,2,3-triazole synthesis. To a stirred solution of appropriate azide (0.26 mmol) and alkyne (0.26 mmol) in CH2Cl2 (2 mL) was added 2 mL of an aqueous solution of a mixture of copper sulfate pentahydrate (0.05 mmol) and sodium ascorbate (0.10 mmol). The mixture was stirred under an argon atmosphere at room temperature for the indicated time, then diluted with CH2Cl2 (60 mL) and water (30 mL). The aqueous layer was extracted with CH2Cl2 (3 x 60 mL). The organic layers were combined, washed with 100 mL of an aqueous solution of EDTA (1%) and ammonia (28%) in a 1:1 ratio, brine (100 mL), dried (Na2SO4), filtered and concentrated in vacuo. Purification was achieved by flash chromatography on silica gel to provide the desired product.

General procedure B: 1,4,5-trisubstituted 1H-1,2,3-triazole synthesis. Appropriate 1,4 disubstituted 1H-1,2,3 triazole (0.074 mmol), aryl bromide (0.111 mmol), Pd(OAc)2 (0.003 mmol), PCy3 (0.006 mmol) and K2CO3 (0.149 mmol) were suspended, under an argon atmosphere, in dry toluene (1 mL) then heated at 110 °C for the indicated time. The reaction mixture was diluted with CH2Cl2 (20 mL) and water (10 mL). The aqueous layer was extracted with CH2Cl2 (3 x 20 mL). The organic layers were combined, washed with a saturated solution of NaHCO3 (30 mL), water (30 mL), brine (30 mL), dried (Na2SO4), filtered and concentrated in vacuo. Purification was achieved by flash chromatography on silica gel to provide the desired product.

General procedure C: tetrazole synthesis. Appropriate nitrile (0.156 mmol), NaN3 (1.56 mmol), and L-Proline (0.312 mmol) were suspended, under an argon atmosphere, in dry DMF (2 mL) then heated at 130 °C for the indicated time. Solvent was removed in vacuo. Purification was achieved by reverse-phase column chromatography (H20/MeOH, 0 to 100%) followed by ion exchange on Dowex 50WX2 (Na + form) and freeze-drying to provide the desired product.

Figure 1 .

 1 Figure 1. Previously identified 1,3-diazole compounds (RR3 and AB02-38) and proposed analogues based on a triazole scaffold.

Fig 2 :

 2 Fig 2: Binding pose obtained by docking at dimerization interface of CD73 dimeric enzyme (grey cartoon representation) for original hit RR3 (cyan thick sticks), AB02 (orange), AB03 (blue), AB05 (green) and AB06 (pink).

Fig. 3 :

 3 Fig. 3: Similar binding mode for compounds 4b (panel A, green sticks) and 5b (panel B, orange sticks). CD73 residues are depicted as blue sticks (yellow and white spheres indicate π-cation and π-stacking interactions, respectively).

Fig 4 :

 4 Fig 4: A) Overlay of binding poses obtained by docking for derivative 3c (white sticks) and 4b (yellow sticks). B) Changes in binding orientation between derivatives 6a (blue) and 6b (pink). The additional residues interacting with 6b are shown as thin orange stick).

Fig. 5 :

 5 Fig. 5: Impact of the linker chain on binding and efficiency of derivatives. A) Binding modes obtained for indole-derived compounds 8a (depicted in light green sticks) and 8b (brown sticks) highlighting the positional offset (indicated by the arrow). B) Comparison of derivatives 6a (blue) and 6e (cyan). C) Comparison of docking poses between derivatives 10a (purple) and 10b (dark yellow) and positional shifting with loss of interaction with Asp-366 (pink).

Table 1 .

 1 Synthesis of 1,4-disubstituted triazoles bearing N-protected indole

Table 2 .

 2 Synthesis of 1,4-disubstituted triazoles bearing free indole.

Table 3 .

 3 Synthesis of 1,4-disubstituted triazoles bearing N-alkylated indole.

Table 4 .

 4 Amine alkylation upon free indole-containing 1,4-disubstituted triazole.

	Entry	R/R1	Solvent	Cpd	Time	Yield
					[h]	[%]
	1	4-cyanophenyl/Me	THF	5a	14	47
	2	4-biphenyl/propanenitrile	THF/DMF (5:1)	5b	4	84
	3	4-methoxyphenyl/TEG	THF/DMF (17:1)	5c	16	85
	4	4-biphenyl/TEG	THF/DMF (9:1)	5d	4	92
	5	4-phenoxyphenyl/TEG	THF	5e	22	84

Table 6 .

 6 Synthesis of tetrazole core upon indole scaffold.

	Entry	R	R1	R2	Cpd	Yield
						[%]

Table 7 .

 7 Synthesis of tetrazole core upon indole scaffold.

Table 8 .

 8 Synthesis

	of 1,4-disubstituted and 1,4,5-trisubstituted triazoles bearing
	tetrazole group.

[a] Yield over 2 steps

Acknowledgements

Institutional funds from the Institut National du Cancer (INCa, project no. 2017-151) supported this work. We are thankful for the financial support provided by the University of Montpellier (PhD grant for F.G.).

Conflict of Interest

The authors declare no conflict of interest.

Entry for the Table of Contents

Insert graphic for Table of Contents here.

Several series of substituted 1,4,5-triazole derivatives, as inhibitors of 5'-ectonucleotidase CD73, were synthetized and evaluated for their potential inhibitory effect on purified recombinant hCD73 and in cell-based assays.


Adrien Rubio

Gregoire Desnos

Mona Semsarilar

Nanostructured Membranes from Soft and Hard Nanoparticles Prepared via RAFT mediated PISA

RAFT dispersion polymerization of benzyl methacrylate (BzMA) was used to prepare a series of near mono-dispersed poly(2-(dimethylamino)ethyl methacrylate)-poly(benzyl methacrylate) (PDMA-PBzMA) diblock copolymer nanoparticles in a ethanol/ water binary mixture via polymerization-induced selfassembly (PISA). A relatively long PDMA 88 chain transfer agent was targeted to ensure that only spherical morphologies were obtained. High (>99 %) BzMA monomer conversions could be achieved within 24 h when targeting degrees of polymerisation (DP) of up to 1000. The resulting particles were analyzed using DLS and TEM. Particles with PBzMA DPs

of 500 and 1000 were used as templates for deposition of silica particles. TEM images confirmed that TMOS precursor was successfully converted into silica sols that were deposited on the surface of the spherical polymer nanoparticles. Thin film membranes were then prepared from these silicified spherical particles (hard spheres) as well as the initial spherical particles devoid of silica coating (soft spheres). These membranes were fully characterized using SEM, AFM and water filtration test.

Introduction

Membranes are an essential part of the majority of industrial processes from pharmaceutics and food production, to water filtration/recycling and chemical industry. [1][2][3] An efficient membrane would need to have regular pores with sharp molecular cut off, and should be robust and mechanically stable. Block copolymers are great candidates for making selective membranes with regular pore size and good mechanical stability since they are highly processable. Polymeric membranes have been made from block copolymers using different techniques such as spin-coating, extrusion, and bulk evaporation. 4 The main disadvantage of these methods is the need for post-fabrication modifications to introduce porosity into the films. This extra step is often not favoured industrially since it increases the production cost.

Recently the use of block copolymer particles in the fabrication of porous membranes has been demonstrated. [5][6][7][8][9][10] Membranes prepared from spherical polymer particles can have relatively well-controlled pore size since the particle diameter dictates the surface area of the gap present in the hexagonally packed spherical particles assembly forming the membrane pores. One of the main drawbacks of membranes made from colloidal polymer particles is their relative fragility especially when dry (they tend to crack and peal when completely dry) or under high pressure (high pressures often compress and deform soft polymer particles).

Polymerization induced self-assembly is a unique method that allows the facile synthesis of polymer nanoparticles without need for any post polymerization step. Also this technique allows the fabrication of nanoparticles at high concentration which is beneficial for the fabrication of membranes from colloidal particles. In the past few years, there have been many reports on RAFT dispersion polymerization formulations using functional macro-CTAs in different media (water, alcohol, alkane). [11][12][13] RAFT dispersion polymerization protocol based on the chain extension of a poly (2-(dimethylamino)ethyl methacrylate) (PDMA) macro-CTA using benzyl methacrylate (BzMA) in ethanol or ethanol/water mixtures have been reported previously. [14][15] It was demonstrated that using a 80/20 ethanol-water binary mixture allows reaching high monomer conversion and high DPs of PBzMA within 24h with good control over the molar mass and the dispersity of the diblock copolymer chains. It was shown that the use of a long PDMA macro-CTA limits the morphology of the resulting particles to kinetically-trapped spheres, allowing targeting higher DPs for the core-forming block. The choice of PDMA stabilizing group also allows the introduction of positive charge on the surface of the particles. This positive charge could be used to deposit silica particles (synthesized via an in-situ sol-gel reaction) on the surface of the PDMA-stabilized particles. 16 Such core-shell spherical particles would benefit from the properties of both the soft polymer and the rigid silica particles. Making membranes from colloidal silica coated particles could allow easy preparation of isoporous membranes endowed with higher resistance to dryness, high pressure and aggressive chemicals such as chlorine (often used to clean membranes).

In the present article, a relatively long PDMA macro-CTA (DP= 88) was used to produce a series of PDMA-PBzMA block copolymer spherical particles via polymerization induced self-assembly in an 80/20 ethanol/water binary mixture at 70°C. The long macro-CTA allowed varying the DP of the core forming PBzMA block up to 1000 reaching full conversion of the BzMA. Selected PDMA-PBzMA particles (according to their sizes) were coated with silica sols via a sol-gel reaction using TMOS. Thin film membranes were then prepared from the silica-coated particles and their porosity, permeation and performance were assessed against membranes prepared from uncoated (naked) counterparts. All the particles and membranes prepared were fully characterized using different microscopy methods such as AFM, SEM and TEM.

Experimental

Materials. All reagents were purchased from Sigma-Aldrich France and used as received unless otherwise noted. Either 4,4'-Azobis(4-cyanovaleric acid) (ACVA) or 2,2'-azobisisobutyronitrile (AIBN) was used as an initiator. Benzyl methacrylate was passed through an inhibitor removal column (also purchased from Sigma) prior to use. 4-Cyano-4-(2phenylethanesulfanylthiocarbonyl) sulfanylpentanoic acid (PETTC) was synthesized as reported previously. 17 ). The sealed reaction vessel was purged with nitrogen and placed in a pre-heated oil bath at 70˚C for 8 h. The resulting polymer (monomer conversion = 87 %; Mn = 14,800 g mol -1 , Mw/Mn = 1.09) was purified by precipitation into excess hexane. The mean degree of polymerisation (DP) of this PDMA macro-CTA was calculated to be 88 using 1 In a typical RAFT dispersion polymerisation synthesis conducted at 20 % w/w total solids, BzMA (1.00 g, 8.7 mmol), AIBN (0.90 mg, 0.006 mmol), PDMA 88 macro-CTA (392.6 mg, 0.028 mmol) was dissolved in 5.6 g of ethanol/water mixture (80/20). The reaction mixture was sealed in a round-bottomed flask, purged with nitrogen gas for 10 minutes and then placed in a pre-heated oil bath at 70˚C for 24 h. The final monomer conversion was determined by 1 H NMR analysis in CDCl 3 or CD 2 Cl 2 by integrating the PBzMA peak (CH 2 ) at 4.9 ppm to BzMA monomer vinyl peaks (CH 2 ) at 5.2 and 5.4 ppm.

In further PDMA-PBzMA diblock copolymer syntheses, the mean DP of the PBzMA block was systematically varied (12, 220, 500 and 1000) by adjusting the amount of BzMA monomer.

Silicification of PDMA-PBzMA diblock copolymer nanoparticles. In a typical silicification reaction, 3.0 g of a 20 % w/w dispersion of PDMA-PBzMA diblock copolymer nanoparticles in ethanol was diluted with ethanol (0.524 g) and water (2.567 g). The pH of the resulting 10% w/w dispersion was adjusted to pH 4 using 50 % acetic acid. Tetramethyl orthosilicate (TMOS) was then added (diblock: TMOS mass ratio = 1:1.1) and the reaction solution was stirred at 25˚C overnight. This dispersion was then diluted with ethanol (19 g) to 5 % w/w total solids and pH was adjusted to 2 by addition of concentrated HNO 3 . The ratio of diblock: TMOS was varied to 1/1.25 and 1/5 to target different silica shell thicknesses.

Analysis and characterization of block copolymers

Copolymer molar mass distributions were determined using size exclusion chromatography (SEC) performed with a double detector array from Viscotek (TDA 305, Malvern instruments, Worcestershire, UK). The Viscotek SEC apparatus was equipped with two mixed-columns with common particle size of 5 µm using THF as an eluent (1.0 ml/min). The Viscotek system contains a refractive index detector (RI, concentration detector), and a fourcapillary differential viscometer. OmniSEC software was used for data analysis and acquisition. The number average molar mass (Mn) and dispersity index (Mw/Mn or Ð) were calculated relative to polystyrene standards. 1 H NMR spectra were acquired with Bruker 300 Mhz spectrometer using CDCl 3 solvent. Dynamic light scattering (DLS) measurements were conducted using a Malvern Instruments Zetasizer Nano series instrument equipped with a 4 mW He-Ne laser operating at 633 nm, an avalanche photodiode detector with high quantum efficiency, and an ALV/LSE-5003 multiple tau digital correlator electronics system.

Transmission electron microscopy (TEM) studies were conducted using a JEOL 1200 EXII instrument operating at 120 kV equipped with a numerical camera. To prepare TEM samples, 5.0 μL of a dilute copolymer solution was placed onto a carbon-coated copper grid, stained (only polymeric samples) using an aqueous solution of Ammonium molybdate 99.98%, and then dried under ambient conditions. Thermogravimetric analysis (TGA) was conducted on vacuum-dried samples using a TA Instruments Q500 thermogravimetric analyser. Samples were heated from 25°C to 800 °C in N 2 at a heating rate of 10°C min -1 . This protocol ensured complete pyrolysis of the organic component. The results were treated using Universal Analysis software.

Filtration and membrane characterizations

Thin film membranes were prepared via deposition of the colloidal dispersion of the nanoparticles on a commercial Nylon membrane (average pore size of 0.2 µm) using a SPS Spin 150 spin coater at 1000 rpm for 120 sec with a speed of 100 rpm.s -1 under dry argon atmosphere. Four different membranes were prepared from bare and silica coated particles of PDMA 88 -PBzMA 500 and PDMA 88 -PBzMA 1000 .

SEM analysis was conducted using a Hitachi S-4500 instrument operating at a spatial resolution of 1.5 nm at 15 kV energy. The samples were dried and coated with an ultrathin layer of electrically conducting platinum deposited by high vacuum evaporation. AFM images were obtained with a Pico SPM II provided by Molecular Imaging. The imagery was controlled by the PicoView 1.10 software. The experiments were all carried out in tapping mode. The types of tips used were PPS-FMR purchased from Nanosensors with a frequency resonance between 45-115 kHz and a force constant between 0.5-9.5 N/m. Gwyddion 2.25 software was used to treat the images.

For filtration tests the prepared membrane (d=2.5 cm) was fitted in a 10 mL filtration cell (Amicon 8010 stirred cell). Then filtration cell was connected to a water reservoir and compressed air line. The measurements were then performed at pressures between 0.1 and 4.0 bars. The mass of the water passing through the membrane (permeate) is recorded by the SartoConnect software at regular time intervals. All filtration experiments were performed at room temperature with dust free ultrapure water (filtered through a 400 micron filter).

Results and Discussion

A PDMA macro-CTA was synthesised by RAFT solution polymerisation in THF (Scheme 1) following a method reported previously. 18 However, in this work a higher DP (up to DP= 1000) was targeted to ensure the exclusive formation of spherical particles. After purification, the DP of this PDMA macro-CTA was calculated to be 88 by 1 H NMR. This PDMA 88 macro-CTA was then chain-extended with PBzMA via RAFT dispersion polymerisation, in an ethanol/water binary mixture, at 70˚C, to produce a series of PDMA 88 -PBzMA x diblock copolymer nanoparticles. Reactions were conducted at a total solids content of 20 % w/w with the target DP, x, for PBzMA varied between 120 and 1000 (Figure S1). Each polymerisation was carried out for 24 h and then analysed by 1 H NMR, THF SEC, DLS and TEM; the results are summarised in Table S1. As described in a previous publication the dispersion polymerization of BzMA was conducted in a mixture of ethanol/water to reach high monomer conversion in a short time. 15 This rate enhancement is due to stronger partitioning of the BzMA monomer, which is soluble in ethanol but insoluble in water. This means that, following the onset of micellisation, unreacted BzMA preferentially enters the micelles, increasing the local monomer concentration and thus increasing the rate of BzMA polymerization. Addition of water is also beneficial in this work since it results in the protonation of some of the PDMA chains (acquiring positive charge). 19,20 As reported previously these positive charges force the growing PDMA-PBzMA chains to self-assemble into spherical particles rather than higher order morphologies. 20 As expected and shown in Figure S1, the five different PBzMA block length led to the formation of spherical particles, the size of which increases linearly (average hydrodynamic diameter from 65 nm to 260 nm) with the DP of the core forming PBzMA block. As seen in Figure S1(f), the obtained hydrodynamic diameter is slightly bigger than the average diameters calculated from TEM images. This slight difference arises from the fact that the stabilizing chains are collapsed on the surface of the particles at dry state and does not contribute to the calculated particle size. Scheme 1. Synthesis of a poly(2-dimethylamino)ethyl methacrylate (PDMA) macro-CTA by RAFT solution polymerisation, using PETTC as CTA, and subsequent chain extension with benzyl methacrylate (BzMA) by RAFT dispersion polymerisation to produce sterically stabilised PDMA-PBzMA diblock copolymer nanoparticles; and silica deposition through sol-gel reaction.

The rest of the study was performed using the two batches of largest particles (PBzMA DPs of 500 and 1000) because it is easier to obtain clear images with bigger particles. Thin film membranes deposited onto a commercially available Nylon support were prepared from these spherical particles using spin-coating. Figure 1 show the surface and the cross section of the prepared membranes obtained using AFM and SEM. As it can be seen monodisperse spherical particles form a homogeneous thin layer in a compact hexagonally packed arrangement. The two batches of PDMA 88 -PBzMA x spherical particles (x= 500 and 1000) were then silicified using an acid catalysed protocol. Particles were diluted down to 10 % w/w solids in a 2:1 ethanol/water binary mixture. The pH of the solution was adjusted to 4 using acetic acid.

TMOS was then added and the reaction mixture was stirred at 25˚C overnight (17 h). TEM studies (Figure 2) confirmed that small silica particles were deposited on the surface of the PDMA-PBzMA particles. It is important to note that the TEM images of these particles were obtained without using any stain. This is an additional proof that the silica is present on the surface of the particles as it is the high electron contrast conferred by the silica that makes the imaging possible.

Our attempts to measure and calculate the silica shell thickness was unsuccessful.

Calculations based on the diameter of the template (spherical particles) and the amount of TMOS added did not lead to a meaningful trend as not all the produced silica was deposited on the spherical particles. The shell thickness measured from the TEM images (measurements done on 100 particles using ImageJ software) suggest that the shell thickness increased from 2 to 9 nm for smaller particles (DP PBzMA = 500) and from 2 to 11 nm for the bigger particles (DP PBzMA = 1000) when the TMOS/copolymer ratio was increased from 1.1 to 5 (Table S2).

TGA analysis was also used to get the silica content of the coated particles. The obtained silica mass % from TGA analysis indicates that the amount silica deposited on the surface of the particles increased as the TMOS/copolymer mass ratio was increased (for both particle sizes, see Table S2). The accuracy of these values is very low because: (1) all of the added TMOS may not have reacted (2) all the silica sols generated may not have deposited on the particles (secondary nucleation). Hence these values should be considered only as an approximation. Four thin film membranes prepared from colloidal solution containing PDMA 88 -PBzMA 500 and PDMA 88 -PBzMA 1000 particles (Figure 1) and their silicified counterparts were prepared on a nylon support (average pore size of 0.2 µm) using spin-coating under inert atmosphere.

Membranes prepared from naked (non-silicified) samples had to be dried slowly under controlled atmosphere and kept moist to avoid fissures. In contrast, membranes prepared from silicified particles could be dried at ambient temperature and atmosphere and could be stored in dry state. SEM images (Figure 3) of the top surface of the membranes prepared from silicified particles show a homogeneous layer. The cross sections of these membranes (Figure 3b and 3d) display a compact arrangement of the silica coated spherical particles throughout the thickness of the membranes. A closer look at the SEM images of the coated particles (inset Figure 3a and 3c) at a higher magnification, reveals a coarse surface (almost raspberry like). This roughness could hinder the formation of regular pores in the membrane. The AFM images of the top surface of the membranes also confirmed the compact assembly of the particles into a thin film (Figure S5). The porosity of the membranes was tested via filtration of pure water using a dead-end filtration cell (Millipore -10 mL). The dead-end filtration cell was filled with water at pH 7.1 and connected to a pressurized water reservoir. To ensure the stability of the membranes, they were kept at each pressure for 30 minutes prior to data collection. The setup pressure was increased gradually from 0 to 3.5 bars. The flux values were calculated based on Darcy's law (Eq. S1) using data obtained for 3 full cycles (increasing from 0 to 3.5 bar and then decreasing to 0 bar). Figure 4a shows the evolution of flux (Jv) for membranes prepared from naked and silicified PDMA 88 -PBzMA 500 spherical particles. The membranes prepared from naked spheres had a flux value of 40 L.h -1 .m -2 at 3.5 bars. At the same pressure the flux value was almost double (80 L.h -1 .m -2 ) when the equivalent silicified particles were used. The membranes prepared from bigger particles (PDMA 88 -PBzMA 1000 ) followed a similar trend. The uncoated particles had a flux value of about 100 L.h -1 .m -2 at 3.5 bar while membranes prepared from silica-coated particles had a flux of 160 L.h -1 .m -2 (Figure 4b). Membranes prepared from bigger spherical particles (PDMA 88 -PBzMA 1000 ) were unsurprisingly more permeable than the membranes with smaller particles (PDMA 88 -PBzMA 500 ) since their pore size is smaller (compact packing of bigger spheres will lead to larger pores). Silica-coated particles possessed a rougher surface (raspberry like morphology) due to the deposition of very small silica sols. This roughness could lead to bigger pore sizes when forming the thin layer membrane since the particles with rough surface may not be able to form a perfect triangle (the space between 3 spherical particles with touching edges). Another reason for this increase in the flux values is due to the higher resistance to compression of the silica-coated particles under the high pressure of the filtration test. The soft uncoated polymer particles are easily compressed during the filtration test (this phenomena was observed for (PSAN-b-PEO-b-PSAN) particles with PSAN core with higher T g than PBzMA (T g ~54°C) See Ref. 22). This compression would lead to reduced pore size and to the decrease in the flux value. In contrast, membranes prepared with silica coated particles are less prone to deformation under pressure.

Conclusions

In summary, a relatively long PDMA macro-CTA (DP=88) was synthesized and used to prepare a series of PDMA 88 -PBzMA x spherical nanoparticles in an ethanol/ water binary mixture of 80/20 using RAFT mediated PISA. The resulting spherical particles were fully characterized using NMR, SEC, DLS and TEM. Particles formed using PBzMA 500 and PBzMA 1000 were used as templates to deposit silica sols, and form silica-coated particles.

Thin film membranes were prepared from these silica-coated particles and from naked particles via spin-coating on a mechanical support (Nylon). Filtration tests at different pressures were performed and water flux values were calculated. The results suggest, as expected, that smaller spherical particles (DP PBzMA = 500, 126 nm) form smaller pores compared to the particles with bigger hydrodynamic diameter (DP PBzMA = 1000, 258 nm).

When comparing the flux values of membranes prepared from coated and uncoated particles, it is evident that the membranes prepared from silica-coated particles display higher flux values. This may be due to the silica shell around the spherical particles that confers higher Table S2. TMOS/ diblock copolymer ratios, DLS intensity-average particle diameters, particle diameters calculated from TEM images, calculated SiO 2 shell thickness and SiO 2 mass % obtained from TGA for PDMA 88 -PBzMA 500 coated with silica.

Eq (1):

Flux (J v )=V P / (t*S) (L. h -1 .m -2 )

Permeability (L P )= J v / ∆P (L. h -1 .m -2 .bar -1 ) b) a)

  CDCl 3 and CD 2 Cl 2 were purchased from Eurisotop, Saint Aubin, France. Synthesis of poly(2-(dimethylamino)ethyl methacrylate) (PDMA) macro-CTA. A roundbottomed flask was charged with 2-(dimethylamino) ethyl methacrylate (DMA; 10.0 g, 63.6 mmol), PETTC (191 mg, 0.6 mmol), ACVA (32 mg, 0.1 mmol) and THF (10.0 g) (target DP = 90

  H NMR spectroscopy (in CDCl 3 or CD 2 Cl 2 ) by comparing the integrated signals corresponding to the aromatic protons at 7.2 -7.4 ppm with those due to the methacrylic polymer backbone at 0.4 -2.5 ppm.Synthesis of poly(2-(dimethylamino)ethyl methacrylate)-poly(benzyl methacrylate)(PDMA-PBzMA) diblock copolymer particles via dispersion polymerisation in ethanol/H 2 O mixture.

Figure 1 .

 1 Figure 1. AFM and SEM images of films prepared from PDMA 88 -PBzMA x diblock copolymer spheres (a, b) film top surface x= 500 (c) film cross section x= 500 (d, e) film top surface x= 1000 (f) film cross section x= 1000.

Figure 2 .

 2 Figure 2. TEM images of silica coated PDMA 88 -PBzMA x spherical particles prepared using different ratios of TMOS to Diblock copolymer (a) x= 500, 1.1: 1 (b) x= 500, 2.5: 1 (c) x= 500, 5: 1 (d) x= 1000, 1.1: 1 (b) x= 100, 2.5: 1 (c) x= 100, 5: 1.

Figure 3 .Figure 4 .

 34 Figure 3. SEM images of thin films prepared from silica coated PDMA 88 -PBzMA x diblock copolymer spheres using [TMOS]/ [diblock copolymer] of 2.5: 1 (a) film top surface x= 500 (b) film cross section x= 500 (c) film top surface x= 1000 (d) film cross section x= 1000.

  x= 120 (b) x= 200 (c) 220 (d) 500 (e) 1000 and (f) the particle diameter obtained from DLS and calculated from TEM images (based on 100 particles analysed using ImageJ).

Figure S2 .

 S2 Figure S2. SEM images of virgin Nylon support with average pore size of µm (a) top surface (b) cross section.

Figure S5 .

 S5 Figure S5. AFM images of membrane prepared from silica-coated ([TMOS]/ [diblock copolymer] of 2.5: 1) spherical nanoparticles (a) PDMA 88 -PBzMA 500 and (b) PDMA 88 -PBzMA 1000 before filtration.

Figure S6 .Figure S7 .

 S6S7 Figure S6. SEM images of membrane prepared from silica-coated ([TMOS]/ [diblock copolymer] of 2.5: 1) spherical nanoparticles (a) PDMA 88 -PBzMA 500 top surface (b) PDMA 88 -PBzMA 500 cross section (c) PDMA 88 -PBzMA 1000 top surface (d) PDMA 88 -PBzMA 1000 cross section after 3 cycles of filtration (1 cycle = increasing pressure up to 3.5 bar and then decreasing to 0).

Acknowledgements:

The authors would like to thank the European Institute of Membrane (IEM) and CNRS for the financial support and Dr. S. L. Boy for fruitful discussions.

resistance to compression and deformation under the pressure applied during filtration tests.

The presence of silica hard shell around the soft polymer particles leads to the formation of more durable and robust thin film membranes.

Supporting Information

All the diblock copolymer nanoparticle details, TEM, SEM, AFM images, SEC and DLS data are reported in the Electronic Supporting Information that is available from the Wiley Online Library. PISA prepared spherical nanoparticles of different size were used as templates for silica deposition through sol-gel using TMOS as precursor. These naked (soft) and silica-coated (hard) nanoparticles were used to prepare thin film porous membranes via spin-coating. The porous nature and performance of the resulting membranes were evaluated using imaging techniques and water flux experiments.

Supporting Information