Ivan Scodeller

Karine De Oliveira Vigier

Eric Muller

Chengming Ma

Frédéric Guégan

Raphael Wischert

François Jerome

A Combined Experimental-Theoretical Study on Diels-Alder Reaction with Bio-Based Furfural: Towards Renewable Aromatics

Keywords: Aromatics, Bio-based furanics, Diels-Alder, DFT, Renewables

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

Introduction

The production of aromatics, such as Benzene, Toluene and Xylene (BTX) represent an enormous market, with 45 million tons of benzene produced per year, for instance. [1] A myriad of valuable downstream aromatic chemicals can be obtained from BTX, such as terephthalic acid, cyclohexane, cumene, phenol, and styrene, to mention a few only. Industrially, BTX are mainly produced by catalytic reforming of naphtha or steam cracking of hydrocarbons. With the exponential increase of the world population, chemistry will face difficulty to supply aromatics in the future and alternative routes are nowadays explored. In this context, there is a growing interest in the synthesis of aromatics from biomass. [2] A wide range of aromatics can be theoretically obtained from lignocellulosic biomass. For instance, alkyl phenols can be produced by the catalytic lignin-first depolymerization process [3] or from tannins present in lignocellulosic biomass. [4] Aromatics can be also produced from hemicellulose or cellulose through the furfural [5][6][7][8][9][10][11][12][13] or 5-hydroxymethylfurfural platform, [14,[START_REF] Aj | Proc. Nat. Acad. Sci[END_REF] using a Diels-Alder (DA) / aromatization sequence. A similar strategy has been reported, using pinacol [16] or limonene as other bio-based platforms. [17] While mainly para-substituted aromatics are obtained from lignin, ortho-and meta-substituted aromatics can be obtained from sugars and pinacol, further demonstrating the potential of biomass for the synthesis of aromatics. Considering the current low price of fossil feedstocks, the production of bio-based aromatics with a higher value than BTX is a more reasonable approach. Functionalized aromatics such as benzaldehyde, benzylamine, benzonitrile, benzyl alcohol are typical examples with important applications in the field of fragrances, flavors, specialty polymers, agriculture, etc. As mentioned above, renewable aromatics can be synthesized by reaction of bio-based furanic derivatives with olefins through a Diels-Alder/aromatization reaction.

In this context, 5hydroxymethylfufural (HMF), "the sleeping giant" among sugarderived chemicals, has received a growing interest, in particular for the synthesis of terephthalic acid. HMF is poorly reactive in the Diels-Alder (DA) reaction and is often reduced to 2,5dimethylfuran [14] or oxidized to furan dicarboxylic esters [START_REF] Aj | Proc. Nat. Acad. Sci[END_REF] before undergoing a DA reaction. Although valuable aromatics were produced in high yields from HMF, the lack of cost-competitive processing technologies to convert low cost sugars to HMF currently hampers the industrial deployment of these routes. Note that BASF, Avantium and Avabiochem have recently communicated on the production of HMF at a pilot scale, indicating that mature technologies may be available in a close future for such application. In contrast to HMF, furfural is a low cost furanic derivative (1-2€/kg) nowadays industrially produced in large scale from biomass waste (> 300 kT/year). [18] Like HMF however, furfural is poorly reactive in DA reactions. The electron withdrawing -CHO group reduces the nucleophilicity of the furanic ring (as shown by a severe lowering of the highest occupied molecular orbital (HOMO) energy vs. the parent furan), thus making the DA reaction unfavourable. [5] To circumvent this problem, furfural can be decarbonylated to furan before the DA reaction. [6,7] For instance, Hubert reported the synthesis of toluene and benzene by co-feeding ethylene or propylene with furfural at 450-600°C in the presence of ZSM-5, with in situ decarbonylation of furfural. [6a] Alternatively, to preserve the chemical functionality of furfural while decreasing the HOMO-LUMO gap, furfural can be derivatized to amines, [8] acetals, [9] alcohols, [10] phenolic-type resins [11] or amides, [12] making the DA reaction thermodynamically favorable This strategy was employed to synthesize furfuralderived polymers which can be depolymerized via a retro-DA reaction at the end of life. The aromatization of these furfural-derived cycloadducts has been investigated only sporadically. In 2016, Hailes reported the DA reaction of furfural dimethylhydrazone with highly activated dienophiles such as Nalkyl-maleimides at 80°C in water, followed by aromatization to the corresponding phthalimide derivatives. [13] However, starting from cheaper and more readily available olefins such as acrylonitrile or dimethyl maleate, the reaction yields were drastically lower (< 25% yield). Guided by DFT calculations, we recently reported the synthesis of meta-xylylenediamine (MXD), an important target in polymer chemistry, from furfural and acrylonitrile, two cheap and abundant chemicals. [5] Furfural was first derivatized with ethylene glycol, restoring the nucleophilicity of the furanic ring (upshift of the HOMO by 0.4 eV compared to furfural), and allowing the DA cycloadducts to be obtained with a yield of 75%. In addition, our strategy improved upon previous work in that (i) the subsequent aromatization step was catalyzed at low temperature (30°C) by bases, thus avoiding the competition with the retro-DA, and (ii) the reaction could be selectively driven to meta-substituted aromatics only, by playing with the rate of the aromatization reaction. Although this route was an expedient protocol to synthesize renewable MXD, predicting and controlling the ortho : meta selectivity during the Diels-Alder reaction remains highly desirable to widen the scope of this protocol for the synthesis of renewable aromatics. The present work addresses this fundamental scientific challenge. Through a combined experimental-theoretical approach, we rationalize the impact of the substituents on furfural-derived diene and various dienophiles on the ortho : meta selectivity of the Diels-Alder reaction. Further, we present an improved protocol to aromatize the so-obtained Diels-Alder adducts to aromatics.

Results and Discussion

Reaction between 2-(furan-2-yl)-1,3-dioxolane and acrylonitrile.

As described in our previous report, [5] furfural was first converted to 2-(furan-2-yl)-1,3-dioxolane by reaction with ethylene glycol in the presence of a catalytic amount of Amberlyst-15. Next, the asobtained 2-(furan-2-yl)-1,3-dioxolane was reacted with a 5-fold excess of acrylonitrile at 80°C, affording the corresponding DA cycloadducts (mixture of ortho and meta regioisomers, and corresponding endo and exo stereoisomers) in a combined yield of 73%, after 72 h of reaction (Fig. 1). The selectivity to the DA cycloadducts was 98%, the missing 2% corresponding to the deprotection of 2-(furan-2-yl)-1,3-dioxolane to furfural and ethylene glycol, presumably due to traces of water (stabilizer) in acrylonitrile (0.2-0.5% w/w). Extending the reaction time to 120 h resulted in a minor change of the yield (76%), indicating that the reaction had reached the DA vs. retro-DA equilibrium (Fig. 1).

Selectivity of the Diels-Alder reaction.

At equilibrium (measured after 120 h, see Fig. S1), the ortho: meta ratio reached a value of 52 : 48. After 120 h, the endo to exo ratios were 70 : 30 and 60 : 40 for the ortho and meta isomers, respectively. These features are captured reasonably well by DFT calculations (Tables S10-S11). First, activation barriers are in all cases (ortho or meta, exo or endo) found in a narrow energy range, ca 31.4-32.5 kcal/mol in free enthalpy. Thus, little to no kinetic selectivity is expected. Similarly, reaction free enthalpies range from +3.7 to +4.6 kcal/mol, suggesting low thermodynamic selectivity. Using a Maxwell-Boltzmann statistical model on relative reaction free enthalpies affords an ortho : meta ratio of 50 : 50 at 60°C, in line with experiments. Calculated endo : exo ratios are 67 : 33 and 46 : 54 for the ortho and meta isomers, respectively, again in good agreement with experiment. These findings suggests that DFT calculations can be used to predict not only the feasibility but also the selectivity of DA reactions, which will provide very helpful throughout this work. Before going further, it is important to mention that from a practical perspective we are interested in ortho : meta ratios. Indeed, the endo : exo selectivity is lost in the aromatization step, while the ortho : meta selectivity is preserved. Therefore, in what follows, we will only discuss ortho : meta ratios; additional experimental and theoretical data on the endo : exo ratios is provided in the SI.

Effect of the reaction temperature.

Increasing the reaction temperature from 80 to 100 and 120°C resulted in a much faster reaction, expressed as the time to reach the highest achievable (equilibrium) DA yield, but yields were lower with 52% after 24 h, and 42% after 8 h, at 100 and 120°C, respectively (Table 1, entries 1-3; Fig. 1). This means that at temperatures higher than 80°C, the retro-DA clearly became the dominating reaction. Conversely, a decrease of the temperature to 60°C resulted in a slower reaction but the maximum yield of the DA cycloadduct was identical to that obtained at 80°C (76%, Table 1, entry 4; Fig. 1). A further decrease of the reaction temperature to 40°C was possible but decreased the reaction rate to an unacceptable level (Table 1, entry 5; Fig. 1). After ca. 24 h the ortho : meta ratio stabilized at ~50 : 50 for reaction temperatures of 40-60°C, decreasing to ~40 : 60 at 80-120°C, suggesting that the meta product is thermally slightly more stable than the ortho (Fig. S1). This trend is reproduced by DFT calculations, as temperature-dependent endergonic corrections from electronic energies to free enthalpies are slightly smaller for meta products than for ortho products (Table S11). 8 60 1 48 24 [d] [a] time to reach maximum (equilibrium) yield; [b] maximum (equilibrium) yield; [c] other detected product (2%) are furfural and ethylene glycol; [d] not at reaction equilibrium, reactions were stopped after 48 h.

Effect of the dienophile to diene molar ratio.

Reactions were performed at 60°C and stopped after 48 h to assess the effect of the dienophile : diene molar ratio on the reaction rate. Increasing the acrylonitrile / 2-(furan-2-yl)-1,3dioxolane molar ratio from 5 to 20 : 1 had no effect. However, a decrease from 5 to 1 slowed down the reaction, with only half the yield obtained after 48 h (Table 1, entries 6-8, Fig. S2). For practical reasons (the boiling point of acrylonitrile is 77°C), a temperature of 60°C was selected in the following experiments, together with an acrylonitrile : 2-(furan-2-yl)-1,3-dioxolane molar ratio of 5 : 1.

Effect of the catalyst.

Without catalyst, the DA reaction is quite slow at 60°C (Fig. 1). For instance, after 25 h of reaction, the DA cycloadducts were obtained in only 26 % yield (Table 2, entry 1). To increase the formation rate of the DA cycloadducts, different Lewis acid catalysts (10 mol%), mostly metal chlorides, were screened (at 60 °C). The choice for Lewis acid catalysts was motivated by the fact that Brønsted acid catalysts led to tar-like materials as major products. In all cases, anhydrous Lewis acid catalysts were tested to limit the deprotection of 2-(furan-2-yl)-1,3-dioxolane to furfural and ethylene glycol.

Metal chlorides (MCln with M = Ga, In, Sc, Cu, Hf and Nb) led to the formation of unidentified black soluble and insoluble materials as main product (Table 2, entries 2-7). With M = Mn, Cd, Sr, Bi, Sn, Li and Al, the reaction took place, but no improvement of the yield was observed after 25 h of reaction, compared to catalystfree conditions (Table 2, entries 8-14). Even worse, these metal Entry Catalyst DA cycloadduct yield (%) [a] acrylonitrile/2-(furan-2-yl)-1,3-dioxolane molar ratio = 5,; [b] tar-like materials were formed as major products.

Deprotection (furfural yield) (%) 1 - 29 2 2 [b] GaCl3 - - 3 [b] ScCl3 - - 4 [b] InCl3 - - 5 [b] CuCl2 - - 6 [b] HfCl4 - - 7 [b] NbCl5 - - 8
chlorides catalyzed the concomitant deprotection of 2-(furan-2-yl)-1,3-dioxolane (4-39% yield) with moisture from the air atmosphere and/or water contained in acrylonitrile (0.2-0.5% w/w). In contrast, in the presence of Co or Zn chloride, significantly higher yields of 51 and 68 %, respectively, were reached after 25 h (and 75% after 48 h of reaction, Fig. S3, Table 2, entries 15-16). Unlike with the other metal chlorides, the deprotection of 2-(furan-2-yl)-1,3dioxolane was limited (~10% yield), although it was still higher than under catalyst-free conditions (2%). To explore the effect of the counter anion, Zn(OTf)2 and ZnI2 were also screened, affording the DA cycloadducts in 57 and 75% yield, respectively, after 25 h (Table 2, entries 17-18). The low yield obtained with Zn(OTf)2 is probably due to its high hygroscopicity, promoting in turn deprotection to furfural (23%, Table 2, entry 17). ZnI2 was found to be the most efficient catalyst screened, as further confirmed by a kinetic profile which showed that the equilibrium yield was reached slightly earlier than with ZnCl2 (Fig. S3). Addition of strong complexing agents such as 1,2bis(diphenylphosphino)ethane (DPPE) or 1,2bis(dimethylphosphino)ethane (DMPE) to ZnI2 or ZnCl2 inhibited their catalytic activities, further confirming the catalytic role of these Lewis acids (Table 2, entries 19-20). Finally, it should be noted that the ortho : meta ratio was not significantly affected by the presence of the catalyst (Fig. S4). For the remainder of the study, ZnCl2 was preferred over ZnI2, because it is cheaper and it does not lead to yellow coloration of the reaction products, while its catalytic activity is very similar to that of ZnI2.

Effect of the protective group on furfural. Different protective agents were used to derivatize furfural, with the aim of assessing the effects on yield and isomer ratio. To this end, furfural was reacted with different diols, amino-alcohols and also 1,2-ethanedithiol (see SI for details of synthesis and characterization). The as-obtained furfural derivatives were reacted with acrylonitrile under the same conditions as 2-(furan-2yl)-1,3-dioxolane, i.e. 60°C, 10 mol% ZnCl2, and 5 equiv. of acrylonitrile (Table 3, entries 1-5). The reaction with 2-(furan-2-yl)-1,3-dioxolane is shown for comparison in entry 1. Additional experiments were also carried out in the absence of catalysts (Table S3). DFT calculations suggested the feasibility of all these reactions with HOMO-LUMO gaps similar to those found for the successful combination of 2-(furan-2-yl)-1,3-dioxolane with acrylonitrile (6.89-7.39 eV, Table S3). Calculated ortho:meta ratio (%) [c] Thermodynamic selectivity [d] Kinetic selectivity [e] 1 [a] 75 67 73 [ f] 39 : 61 91 : 9 37 : 63 90 : 10 As expected from the DFT predictions, these furfural derivatives underwent reaction in all cases (Table 3, entries 1-5). The yields at equilibrium were similar to what was found for 2-(furan-2-yl)-1,3-dioxolane (67-85%, Table 3, entries 2,5). The selectivity to the DA cycloadducts always exceeded 89% (95% without ZnCl2), with furfural and the protecting agent being formed as the only side products (by deprotection of the furfural derivate). Note, that using amino alcohols (ethanol amine, Nmethylethanolamine) as derivatizing agents of furfural led to an unexpected reaction outcome (Table S3). Instead of forming DA adducts, the starting oxazolidine-derived furfural was converted, but a complex mixture of unidentified products was formed, tentatively attributed by NMR spectroscopy to uncontrolled Michael-type reaction with acrylonitrile.

At equilibrium, the ortho : meta ratio of DA adducts was around 50 : 50 for all alcohol-protected furfural derivatives (Table 3, entries 1-4), albeit with a slightly higher amount of the meta isomer in the case of 2-(furan-2-yl)-4,4,5,5-tetramethyl-1,3dioxolane (pinacol derivative) (Table 3, entry 4). For the thiolderivative 2-(1,3-dithiolan-2-yl) furan, an even higher amount of meta isomer was observed (Table 3, entry 5). Note, that in the absence of a catalyst, similar trends were observed (Table S3).

The experimental product ratios are in excellent agreement with equilibrium product ratios obtained from DFT-calculated relative free enthalpies of reaction (Table 3, entries 1 d -5 d , and Tables S10-S19), confirming that the thermodynamic equilibrium had been reached in all cases. In contrast, poorer agreement is found with ratios calculated from DFT-calculated relative free enthalpies of activation, representing kinetic conditions. We now turn our attention to isomer ratios at the beginning of the reaction, where such kinetic selectivity should manifest. Noteworthy, both with and without catalyst, the ortho DA cycloadduct is initially formed in a higher amount than the meta adduct (Table S3, Fig. S6). This effect is most evident for 2-(furan-2-yl)-4,4,5,5-tetramethyl-1,3-dioxolane and 2-(1,3dithiolan-2-yl) furan, with initial amounts of ortho adduct of 65% and 74%, respectively, at 30-40% conversion (Table S3). When the reaction time is extended, the ortho : meta ratios converge to the values obtained at the reaction equilibrium (Table S3, Fig. S6), strongly suggesting that the ortho adduct is the kinetic product. DFT modelling, based on activation free enthalpies at 60°C, nicely reproduces these observations with a kinetic selectivity for the ortho adduct, markedly more pronounced for 2-(1,3-dithiolan-2-yl) furan. To support this hypothesis, we carried out the reaction of 2-(1,3-dithiolan-2-yl) furan at 30°C in the presence of ZnCl2. As expected, under these conditions, the ortho DA cycloadduct was the major product (91%, see Table 3, entry 5). Note that, due to the lower temperature, leading to inhibition of the retro-DA reaction, the yield slightly increased from 67 to 73%, (Table 3, entry 5, Figs. S5 andS6). The experimental and calculated endo : exo ratios were, within experimental or computational error, identical for the meta isomer of all DA adducts, showing a slight preference for the endo isomer. This preference was more pronounced in the case of the ortho isomer, Table S4). Again, a similar trend was observed in the absence of catalyst, although the reaction equilibrium had not been reached yet (Table S3).

Reaction of 2-(furan-2-yl)-1,3-dioxolane with other dienophiles.

Since the DA reaction of 2-(furan-2-yl)-1,3-dioxolane and acrylonitrile had been successful, other relevant dienophiles were also tested, i.e. acrolein, methyl vinyl ketone, and methyl acrylate. The calculated HOMO-LUMO gaps for the combination of these dienophiles with 2-(furan-2-yl)-1,3dioxolane (6.89-7.47 eV, Table S5) were comparable to that found for the combination with acrylonitrile (ΔE = 7.39 eV), suggesting that these reactions Table 4. Reaction of 2-(furan-2-yl)-1,3-dioxolane with different dienophiles. [a] Entry -R Yield (%) [b] Experimental ortho : meta ratio (%) [b] Calculated ortho : meta ratio (%) [c] Experimental ortho : meta ratio (%) < 15 % conv.

Calculated kinetic ortho : meta ratio (%) [d] 1 should be feasible as well. Indeed, the reactions were successful (carried out without catalyst, see Fig. S7 for kinetic profiles). At equilibrium, the DA reaction plateaued at 28-40% yield with methyl acrylate, methyl vinyl ketone, and acrolein compared to 75% with acrylonitrile (Table 4 and Fig. S7). The selectivity of the reaction remained close to 100%, suggesting that the retro-DA reaction was less favorable for acrylonitrile than for the other dienophiles. We also tested allyl amine as an example of an electron-rich dienophile. The HOMO-LUMO gap calculated by DFT was high (9.24 eV), indicating that the DA should not proceed, as indeed observed experimentally. This result shows that only electron poor dienophiles are eligible for DA reaction with 2-(furan-2-yl)-1,3-dioxolane.

The nature of the dienophile also impacts the ortho : meta ratio.

Although in all cases the ortho cycloadduct was the kinetic product, at equilibrium the meta product was predominant (Table 4 and Fig. S8), in particular in the case of methyl vinyl ketone, with 87% meta isomer (Table 4, entry 2). Again, the experimental findings are well reproduced by DFT calculations, with a clear thermodynamic meta selectivity for methyl vinyl ketone, methylacrylate and acrolein with an ortho : meta ratios of 14:86, 18:82 and 28:72, respectively, using free enthalpies of reaction (Table 4, entries 2-4, and Tables S20-S32). As observed earlier, for all ortho and meta isomers, a slight preference for the endo isomer was observed, both experimentally and theoretically (Table S5).

Reaction of other furanic dienes with acrylonitrile.

To assess the molecular diversity of DA cycloadducts that could be obtained through this pathway, other-furfural-derived dienes such as 2-furonitrile, furfuryl amine, furfuryl alcohol, ethylfurfuryl ether, and 2-methylfuran, nowadays obtained on a large scale from furfural, were screened. Like furfural, 2furonitrile contains an electron-withdrawing substituent (the -CN group), and consequently the HOMO-LUMO gap calculated by DFT (ΔE = 7.77 eV) suggested an unreactive combination (for comparison, ΔE = 7.72 eV for the unsuccessful combination of furfural and acrylonitrile). In contrast, the other furfural derivatives contain an electron-donating group which should permit the reaction with acrylonitrile, as corroborated by HOMO-LUMO gaps (6.94-7.09 eV, Table S6), comparable to the reactive combination of 2-(furan-2-yl)-1,3-dioxolane and acrylonitrile (ΔE = 7.39 eV).

Consistent with the prediction from DFT, no DA reaction was observed for 2-furonitrile, while it was successful for furfuryl alcohol, ethyl furfuryl ether and 2-methyl furan, with equilibrium yields of 81%, 54% and 69%, respectively (Table 5). In the case of furfuryl amine (Table 5, entry 2), a mixture of different products was obtained, due to concomitant Michael addition of the -NH2 group on acrylonitrile. The DA cycloadduct was observed by NMR (again consistent with the prediction from DFT) but, due to significant formation of side products, it was not possible to accurately determine the corresponding yield by 1 H NMR. Noteworthy, in the case of 2-furonitrile, attempts to perform an inverse electron demanding DA reaction by reaction with allyl amine also failed (HOMO-LUMO gap = 7.94 eV). [a] based on a Maxwell-Boltzmann statistical model (see SI for details), using DFT-calculated relative free enthalpies of reaction at 60°C; [b] based on relative free enthalpies of activation; [c] n.d. means not determined, furfuryl amine successfully reacted with acrylonitrile but a complex mixture of compounds was obtained due to parasite Michael addition of furfuryl amine to acrylonitrile, making impossible the determination of the reaction yield; [d] the reaction was stopped after 48 h because extending the reaction produced tar-like material; [e] the calculations were performed on a simplified model (ethyl group replaced by methyl). At equilibrium, the ortho : meta ratio varied from 60 : 40 to 66 : 34, with 2-methyl furan showing the highest ortho selectivity (Table 5, entries 3-5), higher than in all other cases explored so far. Kinetic profiles (Fig. 2, Fig. S9) show that the ortho DA cycloadduct is the kinetic product, like in all other cases. At < 15% conversion, the ortho DA was formed in different amounts: 2-methylfuran (85%), furfuryl alcohol (72%), ethylfurfuryl ether (66%) and 2-(furan-2-yl)-1,3-dioxolane (52%), indicating that substituents on the furanic ring also impact the initial formation rate of the ortho and meta DA cycloadducts (Fig. 2, Fig. S9). DFT calculations once again nicely retrieve the experimental findings, both in terms of thermodynamic and kinetic selectivity (Table 5, entries 3-5, Tables S33-S40). Again, for all DA cycloadducts, the endo isomers were formed preferentially (Table S6).

Optimal combination of diene and dienophile.

Again guided by HOMO-LUMO gaps determined by DFT calculations (Table S7), furfuryl alcohol, ethylfurfuryl ether and 2methylfuran were next reacted with different dienophiles (acrolein, methyl vinyl ketone and methyl acrylate) to further improve molecular diversity and to further assess how substituents impact the yield and ortho or meta selectivity. As suggested by DFT calculations, all tested combinations were successful, affording a library of DA cycloadducts with different chemical functionality (Fig. 3). As found with 2-(furan-2-yl)-1,3-dioxolane, the highest equilibrium yields of DA cycloadducts were obtained using acrylonitrile as a dienophile, confirming again that the -CN group has the strongest promoting effect on the yield of the DA cycloadduct.

In line with what was discussed earlier for the combination of methyl vinyl ketone with 2-(furan-2-yl)-1,3-dioxolane (Table 4), at equilibrium, the amount of meta DA cycloadducts was the highest with methyl vinyl ketone (87%), followed by methyl acrylate (67%), indicating that the ortho : meta ratio was mostly governed by the substituent present on the dienophile. As observed for acrylonitrile, the ortho cycloadduct was always the kinetic product (Fig. 4).

Elements of rationalization.

As we have seen, DFT calculations proved successful in reproducing the experimental ortho : meta selectivity, both under kinetic and thermodynamic conditions. But beyond the reproduction and prediction of data, modelling offers insight into the fundamentals of this selectivity. Chemical intuition suggests that meta addition should minimize the steric repulsion between the pending group of the diene (R1) and the pending group of the dienophile (R2), as well as the deformation energy required to reach the transition state and product geometries. On the other hand, in the spirit of the Klopman-Salem model of reactivity, [19] charge control would result in the interaction of the most charged sites of both reagents (Scheme 1). Natural population analysis (NPA) [20] shows that the two carbon atoms of the diene carry positive partial charges with different relative magnitude (+ and ++), depending on the substituent R1, while the two carbon atoms of the dienophile carry negative partial charges (-and --), again differing in magnitude, depending on the substituent R2. The highest partial charges on both diene and dienophile are always found on the carbon atoms linked to the respective substituents (Table S8). In the DA reaction, the coulombic interaction between the reaction partners is maximized when the most charged atoms interact, i.e. the most positive partial charge on the diene will attract the most negative partial charge on the dienophile. Hence, the functional groups of the resulting DA adduct end up in an ortho arrangement (Scheme 1).

Scheme 1. Competition between electronic and steric interactions: ++ / + and --/ -indicate the relative magnitude of the positive and negative partial charges of the carbon atoms involved in bond formation during the DA reaction.

For the reaction of a given dienophile with different dienes, the higher the charge difference () between the pro-ortho and prometa C atoms of the diene, the higher is the expected ortho selectivity (and by definition the lower is the meta selectivity). This is indeed the case, with a reasonable correlation between  and the experimental ortho : meta ratio at equilibrium found in the reaction of acrylonitrile with different furfural derivatives (Figure 5). In the same spirit, for the reaction of a given diene with different dienophiles, the charge difference between the two olefinic carbons on the dienophile should impact the selectivity: for methyl vinyl ketone (trans isomer: = 0.009 e) methyl acrylate (trans isomer: = 0.036 e) and acrolein (trans isomer: = 0.049 e) is significantly lower than that of acrylonitrile (= 0.091 e) (Fig. 6, Figure 6. Experimental equilibrium meta selectivity as a function of the charge difference (δ) between the two C atoms of the double bond of the dienophile for various combinations of dienophiles and dienes. For the sake of clarity, only the trans isomer of the respective dienophile is represented. See SI for more information on the cis isomer (Table S8).

Table S8). Hence, the difference in electronic interaction between furfural-derived chemicals and these dienophiles is expected to be lower than with acrylonitrile, and formation of the ortho cycloadduct should be less favored. This claim is supported by Figure 6 showing that, irrespective of the starting diene, the lower the  on the dienophile, the higher the amount of meta product. Note, that while steric effects cannot be properly quantified here, it is reasonable to assume that such effects now take over, thus favoring the formation of the meta product.

To summarize, we may propose a "rule of thumb" to account for the experimental observations: here, the observed selectivity stems from a competition between steric interaction, favoring meta addition, and electronic interactions, favoring ortho addition (Scheme 1). The model also has the additional benefit that it can be used by non-experts. Instead of time-consuming explicit calculations of reaction pathways, only partial charges of the reactants are necessary, acting as convenient descriptors for selectivity.

Aromatization reaction

As previously reported by us, the DA cycloadducts derived from furfural can be quantitatively aromatized, with the loss of water, in the presence of a catalytic amount of sodium tert-butoxide and at only 30°C, thus preventing the unwanted competitive retro-DA reaction. [5] This protocol relies on DMSO as a solvent in order to produce in situ a superbase, by solvation of sodium by DMSO. [21] We found here that, except in the case of 2-(1,3-

Electronic interaction Steric interaction

dithiolan-2-yl) furan for which no aromatization was observed, for all other tested protective agents, the aromatization reaction proceeded well (70-80% yield), always with the meta aromatic being formed more rapidly than the ortho isomers (Fig. S10, Table S9), a mean to produce consecutively, but separately, the ortho and meta aromatics, as previously shown by us. [5] Scheme 2. Base-catalyzed aromatization of the DA cycloadducts.

Although this protocol is an expedient way to produce various functionalized renewable aromatics in nearly quantitative yields, the use of DMSO as a solvent may raise ecological issues. However, since the role of DMSO is to solvate tBuONa, we surmised that it should be possible to use it in a small amount (as a co-solvent). In what follows, the aromatization of the DA cycloadducts obtained from 2-(furan-2-yl)-1,3-dioxolane and acrylonitrile was selected as a case study (Scheme 2). DMSO was first employed as a co-solvent with toluene. In a mixture of DMSO and toluene, the aromatization was observed, but the yields of aromatics were lower than in pure DMSO; 42% and 10% in a mixture DMSO/toluene 1/1 and 1/3, respectively (vs 90% in neat DMSO) (Table 6, entries 2,3). These results can be explained by the inhibition of the DMSO/ tBuONa superbase by released water. Indeed, in neat DMSO, the initial production rate of aromatics (raromatic) was 1.83 mmol/h (Fig. 7). However, when 5 eq. of water (relative to the DA cycloadduct) were initially added into DMSO, raromatic decreased to 0.42 mmol/h and the aromatization reaction stopped at 57% conversion. A further incremental increase of the initial amount of water in DMSO from 5 to 10 and 20 eq. stopped the reaction at yield of only 48% and 29%, respectively, and further decreased the aromatization rate to 0.18 and 0.05 mmol/h, respectively, reflecting the deactivation of the DMSO/ tBuONa superbase by water.

Figure 7. Effect of water on the total yield of aromatics (30°C, DMSO, 20 mol % t BuONa).

To maintain the catalytic activity of the DMSO / t BuONa superbase while limiting its deactivation by released water, the aromatization reaction was therefore conducted under reflux in toluene, in a Dean-Stark apparatus, and in the presence of only 2.5% of DMSO. Pleasingly, with continuous removal of water, aromatics were formed in 81% yield after only 10 min of reaction.

Here again, the meta aromatic was always produced faster than the ortho isomer (Table 6, entry 4).

Conclusion

We show that the Diels-Alder reaction of cheap alkenes with industrially available bio-based furfural and its derivatives is a promising pathway for the synthesis of functionalized renewable aromatics. Various furfural-derived dienes were successfully coupled at 60°C to various dienophiles featuring electronwithdrawing groups (-CHO, -CN, -COCH3, -COOCH3), affording the corresponding DA cycloadducts with yields ranging from 28 to 85% at the thermodynamic equilibrium. In all cases, the DA reaction proceeds with a selectivity higher than 98%. The reaction can be accelerated by ZnCl2 or ZnI2 catalysts, while maintaining the reaction selectivity higher than 90%. Pleasingly, it is possible to control the ortho : meta ratio by a suitable choice of the substituents on diene and dienophile and the reaction conditions (kinetic or thermodynamic). The ortho product is the kinetic product, in the sense that it forms faster than the meta adduct. Conversely, under thermodynamic conditions, the meta product is more stable in most cases. DFT calculations were carried out to not only predict the feasibility of the DA reaction for the different diene and dienophile combinations, but also to understand how the ortho : meta ratio depends on the substituents on the dienes and dienophiles. We find excellent agreement between experimental and calculated product ratios under both thermodynamic and kinetic conditions, underlining the value of DFT calculations as predictive tool for organic chemistry and biomass valorization. We find that the substituents on the dienes and dienophiles impact the reaction outcome in two principal ways. First, the (thermodynamic) feasibility of the reaction is conditioned by the HOMO-LUMO gap between the reactants, which in turn depends on the electron-withdrawing or -donating nature of the substituents. The calculated HOMO-LUMO gap can thus be used as a convenient descriptor for the feasibility of the reaction. Second, the ortho : meta selectivity of the reaction depends on the balance between steric repulsion, favoring the meta isomers, and electronic interactions (characterized by charges), orienting towards the ortho products. As a result, the highest selectivity to the ortho isomer is obtained for the reaction of acrylonitrile and methyl furan, while the highest meta selectivity is observed with Reaction time (h) methyl vinyl ketone, independent of the furan derivative. The calculated charges of the reactants thus allow a quick estimation of the ortho : meta selectivity, as alternative to the calculation of full reaction pathways. This work also points to an optimization of the aromatization step. Notably, by continuous removal of released water, it was possible to perform the base-catalyzed aromatization reaction is a toluene/DMSO (97.5:2.5 mass ratio) mixture, instead of neat DMSO as previously described by us. By providing efficient synthetic and predictive tools to overcome the low reactivity of bio-based furfural in Diels-Alder reaction, we do believe this work complements the scope of biomass for the supply of renewable aromatics. While para-substituted aromatics are mainly produced from lignin, the present work opens the door to ortho-and meta-substituted aromatics from sugarderived furfural.

Figure 1 .

 1 Figure 1. Total yield of DA cycloadducts as a function of time for the reaction of 2-(furan-2-yl)-1,3-dioxolane with a 5-fold excess of acrylonitrile at different temperatures.

[ a ]

 a Reaction time = 48 h;[b] reaction time = 25 h;[c] based on a Maxwell-Boltzmann statistical model using DFT-calculated energies (see SI for details);[d] based on relative free enthalpies of reaction at 60°C;[e] based on relative free enthalpies of activation at 60°C;[f] reaction temperature = 30 °C.

Figure 2 .

 2 Figure 2. Effect of the substitution of the furanic ring on the ortho : meta ratio. Acrylonitrile : derivatized furfural molar ratio = 5, reaction temperature = 60°C, without catalyst.

Figure 3 .

 3 Figure 3. Combination of various furfural-derived chemicals and dienophiles. Dienophile : derivatized furfural molar ratio = 5 : 1, reaction time = 120 h (except for the combination of acrylonitrile with ethylfurfuryl ether for which the reaction time was 48 h).

Figure 4 .

 4 Figure 4. ortho : meta molar fraction as a function of the combined yield of DA cycloadducts, using methyl acrylate and methyl vinyl ketone as dienophiles. Dienophile : diene molar ratio = 5, reaction temperature = 60°C.

  -COOCH 3 (dienophile = methyl acrylate)

Figure 5 .

 5 Figure 5. Experimental equilibrium meta selectivity as a function of the calculated charge difference (δ) between the two C atoms of the double bond of the diene, for the reaction of acrylonitrile with different dienes.



  (charge difference on the dienophile) ortho meta

Table 1 .

 1 Effect of the reaction temperature (entries 1-5) and dienophile to diene molar ratio (entries 6-8) on the DA reaction of 2-(furan-2-yl)-1,3-dioxolane with acrylonitrile.

	Entry	Temp.	Acrylonitrile / 2-(furan-	Time	Yield
		(°C)	2-yl)-1,3-dioxolane	(h) [a]	(%) [b,c]
			ratio
	1	80	5	72[ a]	76
	2	100	5	24 [a]	52
	3	120	5	8 [a]	42
	4	60	5	120 [a]	76
	5	40	5	120 [a]	30
	6	60	5	48	48 [d]
	7	60	20	48	44 [d]

Table 2 .

 2 Effect

of Lewis acid catalysts on the DA reaction of 2-(furan-2-yl)-1,3dioxolane with acrylonitrile.

[a]

Table 3 .

 3 Reaction of acetal and thioacetal derivatives of furfural with acrylonitrile.

	Entry	Diene	Yield (%)	Experimental ortho : meta ratio (%)

Table 5 .

 5 Reaction of furan derivatives with acrylonitrile.

	Entry	-R	ΔE(HOMO-LUMO) (eV)	Yield (%)	Experimental ortho : meta ratio (%) at equilibrium	Calculated thermodynamic ortho : meta ratio (%) [a]	Experimental ortho : meta ratio conv. (%) at 15 %	Calculated kinetic ortho : meta ratio (%) [b]
	1	-CN	7.77	-	-	-	-	-
	2	-CH2NH2	7.09	n.d. [c]	-	-	-	-
	3	-CH2OH	7.03	81	60 : 40	57 : 43	72 : 28	82 : 18
	4 [d]	-CH2OEt	7.04	54	64 : 36	63 : 37 [e]	66 : 34	81 : 19 [e]
	5	-CH3	6.94	69	66 : 34	61 : 39	85 : 15	91 : 9

Table 6 .

 6 Influence of solvents on the base-catalyzed aromatization of the DA cycloadduct obtained from 2-(furan-2-yl)-1,3-dioxolane and acrylonitrile.

	Entry	Solvent	Total yield of aromatics (%)	ortho : meta ratio (%)
	1	DMSO	93	30: 70
	2	DMSO/Toluene (1/1)	42	9 : 91
	3	DMSO/Toluene (1/3)	10	5: 95
	4	Toluene + 2.5% DMSO	81	50 : 50

Acknowledgements

The authors are grateful to the CNRS, the University of Poitiers, the Région Nouvelle Aquitaine, and SOLVAY for financial support. The International Consortium on Eco-conception and Renewable Resources (FR CNRS INCREASE 3707) and the chair "TECHNOGREEN" are also acknowledged for their funding?

Experimental Section

General procedure for Diels-Alder reaction: 50 mmol of furan derivative was mixed with 250 mmol of a dienophile and heated under N2 atmosphere at 60°C for 25-48 h in a carousel flask equipped with a magnetic stirring bar and a condenser. At the DA equilibrium, the excess of dienophile was removed by distillation under vacuum. The asrecovered DA cycloadduct can be directly used as collected for the subsequent aromatization reaction. For characterization purposes the DA cycloadduct was purified by flash chromatography (silica gel, EtOAc/cyclohexane).

Catalytic Diels-Alder reactions were performed in a similar way, with the addition of 0.1 eq. of catalyst, preferentially ZnCl2 or ZnI2. In such case, the catalyst needs to be removed (by purification over silica gel) before engaging the DA cycloadduct in the aromatization step.

Aromatization reaction:

The reaction was performed as described in our previous work. [5] Computational details: All calculations were performed using Gaussian 16 rev B, at the M06-2X/6-311++G (d,p) level of theory. The M06-2X functional indeed provides reliable reaction paths for Diels Alder additions, both in terms of selectivity and reactivity, [22] while inclusion of polarization and diffuse functions was shown to be necessary in the calculation of sulphur-containing compounds. [START_REF] Wah | Quantum Chemical Calculations of Sulfur-Rich Compounds in ElementalSulfur and Sulfur-Rich Compounds II; Elemental sulfur and sulfur-rich compounds[END_REF] All geometries were fully optimized and frequency calculations were performed in order to confirm the nature of the stationary points. Unless stated otherwise, all calculations used an implicit solvation model (PCM), with a dielectric constant of ε = 33.0 (25°C, 1 bar value for acrylonitrile). HOMO-LUMO energy values were extracted from the last single point evaluation on the optimization calculations. Endo/exo selectivity were evaluated for both ortho and meta attacks on the dienophile, and two conformers of the diene were considered (conformation of the furan substituting group on position 2). This led to 8 transition state and 8 product structures in the case of acrylonitrile. For the other dienophiles (methyl acrylate, acrolein, methyl vinyl ketone), additional geometries were considered because of the cis/trans isomerism around the conjugated bond, resulting in 16 transition state and 16 product structures. Atomic charges were calculated for the optimized structures, using the Natural Population Analysis method as implemented in NBO version 3.1 for Gaussian. The computed natural charges for the reagents are provided in Table S8.

Entry for the Table of Contents

We report here the synthesis of renewable aromatics from bio-based furfural and various alkenes, using a Diels-Alder/aromatization sequence. This investigation aims at rationalizing, and even predicting, not only the feasibility of the Diels-Alder reaction but also the effect of substituents on the ortho : meta selectivity.

Electronic interaction

Steric interaction ortho meta -H 2 O


Shachar Gat

Camille Simon

Clément Campillo

Anne Bernheim-Groswasser

Cécile Sykes
email: cecile.sykes@curie.fr

Finger-like membrane protrusions are favored by heterogeneities in the actin network †

Finger-like protrusions in cells are mostly generated by an active actin cytoskeleton pushing against the cell membrane. Conventional filopodia, localized at the leading edge of the cells, are long and thin protrusions composed of parallel actin filaments that emanate from a branched actin network. In contrast, dendritic filopodia, precursors of dendritic spines in neurons, are entirely filled in with a branched actin network. Here, we investigate in vitro how the dynamics of branched actin structures, polymerized at a membrane surface, trigger the formation of both protrusion types. Using supported bilayers and liposomes, we show that a decrease in the amount of activation sites at the membrane surface leads to the appearance of heterogeneities in the actin network coverage. Such heterogeneities promote the formation of membrane protrusions, and the size of heterogeneity patches matches the one of the protrusion base. Protrusion shape, cylindrical or conical, directly correlates with the absence or the presence of actin branches, respectively.

Cell membranes deform during cell processes such as division, motility, endocytosis, under the combined action of membrane proteins and the cytoskeleton 1 . In particular, the growth and assembly of the actin cytoskeleton are activated at membranes where it polymerizes and branches, thus generating a force sufficient to deform the membrane. One single actin filament, in principle, is able to generate a sufficient force (1-9 pN) to produce a finger-like membrane deformation if the actin filament is fixed at its non-growing end 2,3 . Moreover, a homogeneous branched network growing at a membrane was shown to generate inward and outward membrane deformations 4 .

Inward and outward deformations of the cell membrane refer, respectively, to endocytosis and filopodia formation. For both types of deformation, the membrane curves inward or outward with a radius of curvature on the order of about 100 nm. There-

fore, the details of the composition of the membrane that may explain its curvature has attracted most attention, whereas the role of actin dynamics in pushing or pulling on the membrane has been less quantified, although in cells, actin is always present at sites where the cell membrane deforms.

Endocytosis is the process of external material uptake by a cell. It involves, at its onset, an inward membrane curvature at the site of contact. External material can be substances the cell needs for its metabolism, or bacteria, or even viruses. Thus, the initial membrane curvature for endocytosis spans from micrometers to below tens of nanometers, evoking a limited number of proteins or lipids involved in the process. Research has therefore focused a lot on lipidomic and proteomic approaches to reach an understanding of the endocytic machinery. As usual in such complex biological phenomena, many proteins and lipids have been found to play a crucial role, and endocytosis is nowadays understood as a whole orchestra piece with proteins and lipids working in concert [START_REF] Idrissi | Proceedings of the National Academy of Sciences[END_REF] . Actin-based forces have occupied physicists for a while, to understand how a single filament [START_REF] Mogilner | [END_REF] or a network of filaments 7 could deform a membrane. Mimicking experiments, using purified proteins and non-charged lipids have allowed to address the details of the physical mechanism of membrane deformation: the actin network has elastic and viscous properties, and its growth close to the membrane is able to deform it very efficiently 4,8,9 . Experiments in yeast revealed that the growth of actin filaments is, indeed, what deforms the membrane for endocytosis 10 , whereas lipids and proteins that precede the de-formation assemble in "plateforms", plane membrane structures that support the recruitment of actin polymerization activators at the membrane 11 . A lipodomic/proteomic approach of liposomes bathed in cell extracts 12 led to two distinct hypothesis that could explain membrane deformations, driven either by spontaneous membrane curvature, or by actin filament growth. Using experimentally controlled systems mimicking the cell membrane and its underlying cytoskeleton, in the absence of any curvature protein, tubular membrane deformations are obtained 4 that are strikingly reminiscent of the ones obtained in 12 . Moreover, experiments based on the synthetic cell approach reveal that actin polymerization into a branched network can also push the membrane into structures that are similar to the ones that deform and even rupture the nuclear membrane in starfish oocytes 4,13 .

Filopodia refer to long, actin-rich, cellular protrusions that are believed to sense cell environment. As for endocytosis, a large effort has been dedicated to decipher the molecular players of filopodia formation, as biochemistry and molecular biology tools became available. Two "models" have been proposed to explain filopodia formation, the "tip-elongation" one, in which proteins, localized at a membrane spot, nucleate actin filament elongation that pushes the membrane, whereas the "convergent-elongation" model relies on the convergence of branched actin filaments into a bundle through elastic interactions with the membrane 2,14,15 . However, different filopodia are observed in vivo in the synapse of neurons and in vitro in reconstituted actin cortices 16 that do not contain specifically parallel actin filaments, but a branched, dendritic actin network 17 .

Asters of actin filaments form under the action of the branching nucleator, the Arp2/3 complex, both in vitro 18 and in vivo 19 . They can reorganize into stars in the presence of bundling agents, such as fascin, and parallel filament bundles appear that can push the membrane and are reminiscent of conventional filopodia 18,[20][21][22][23] . Their mechanism of formation relies on parallel filament bundles that emanate from dendritic branched networks. Bundled actin filaments then continue to grow leading to protrusion elongation. We reproduce here branched actin structures in vitro growing from membrane with two geometries, planar and spherical, to decipher how different structures (dendritic and parallel networks) emanate from a branched growing network and form, develop, and deform the membrane. We look into how the pure effect of actin polymerization through branching, triggered at the membrane surface, generates a large variety of shapes depending on the initial heterogeneity in the actin network.

Methods

Chemicals and proteins

Chemicals are purchased from Sigma Aldrich (St. Louis, MO, USA) unless specified otherwise. L-alpha-phosphatidylcholine (EPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl polyethylene glycol 2000] (biotinylated-PEG lipids) are purchased from Avanti polar lipids (Alabaster, USA). TexasRed c 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, triethylammonium salt (TexasRed-DHPE) is from Thermofisher (Waltham, USA). Actin and porcine Arp2/3 complex are pur-chased from Cytoskeleton (Denver, USA) and used with no further purification. Alexa-Fluor 488 actin is obtained from Molecular Probes (Eugene, Oregon, USA). Mouse α1β 2 capping protein (CP) is purified as in 24 . Untagged human profilin and the nucleation promoting factor S-pVCA (Streptavidin-C-terminal active domain of Wiskott-Aldrich syndrome protein (WASP)) are purified as in 25 . Porcine Arp2/3 complex is fluorescently labeled with Alexa Fluor 488 C5-maleimide (ratio of 1:10 protein:label) at pH 7.2 for 3h on ice and then purified on a PD Minitrap G-25 column. A solution of 30 µM monomeric actin containing 15 mol % of labeled Alexa-488 actin is obtained by incubating the actin solution in G-Buffer (2 mM Tris, 0.2 mM CaCl 2 , 0.2 mM DTT, 0.2 mM ATP, pH 8.0) overnight at 4 • C . All proteins except actin, i.e. S-pVCA, profilin, and CP are diluted in a TPE buffer (25 mM imidazole, 70 mM sucrose, 1 mM Tris, 50 mM KCl, 2 mM MgCl 2 , 0.1 mM DTT, 2 mM ATP, 0.02 mg/mL β -casein, pH 7.4, 200 mOsm), before using them in the actin polymerization solution for triggering the formation of the branched actin network.

Preparation of liposomes and Supported lipid bilayers (SLBs)

Liposomes are prepared using the electroformation technique 26 . Briefly, 10 µL of a lipids mixture of (mol %) 99.8 % EPC, 0.1 % biotinylated-PEG lipids, and 0.1 % fluorescently labeled lipids (TexasRed-DHPE) with a concentration of 2.5 mg/mL in chloroform/methanol 5:3 (v/v) are spread onto indium tin oxide (ITO)coated plates under vacuum for 2 h. A chamber is formed using the ITO plates (their conductive sides facing each other) filled with TPI buffer (0.2 M sucrose, 2 mM Tris, pH 7.4, 200 mOsm) and sealed with hematocrit paste (Vitrex Medical, Denmark). Liposomes are formed by applying an alternate current voltage (10 Hz, 1 V rms) for 2 h. Liposomes are then coated with S-pVCA by mixing the liposomes with a solution of S-pVCA for 15 min at room temperature. The S-pVCA molecules bind to the liposome surface via streptavidin-biotin links. We varied the concentration of S-pVCA in the incubating solution between 0.035 µM -3.5

µM. Liposomes are used immediately at the end of the incubation step. Supported lipid bilayers (SLBs) are formed by flowing 1 mg/mL solution of small unilamellar vesicles (SUVs) in membrane buffer (10 mM Tris-HCl, pH 7.4, 2 mM MgCl 2 and 50 mM KCl) in a flow-cell chamber prepared using piranha-treated glass slide and glass coverslip 27 . The chamber volume is ∼ 40 µL. The SUVs are 50 nm in diameter and have the same lipid composition as that of the liposomes described above. After 15 min of incubation at 37 • C, the excess of vesicles are washed with 2 volumes of membrane buffer. A solution of S-pVCA is then loaded into the chamber and incubated for 15 min at room temperature. Excess S-pVCA is rinsed with 3 volumes of TPE buffer. We use the same S-pVCA concentrations as those used in the liposome system.

Formation of a branched actin network

To promote polymerization on the liposome surface, a volume of 5 µL S-pVCA-coated liposomes is mixed with 25 µL of an actin polymerization solution, leading to final concentrations of 1.5 mM Tris-HCl, 40 mM KCl, 1.5 mM MgCl 2 , 1.5 mM ATP, 75

µM DTT, 0.02 mg/mL β -casein, 57 mM sucrose, 20 mM imidazole, 3 µM G-actin (15 % actin labeled with Alexa-Fluor 488), 3

µM profilin, 0.037 µM Arp2/3 complex, and 0.025 µM CP. Actin polymerization on SLBs is promoted by flowing one volume of the actin polymerization solution in the SLB-coated flow chambers. In both systems, the actin polymerization solution is used immediately after mixing (defined as t = 0).

Image acquisition and analyses of liposomes and SLBs

Liposomes are imaged by phase contrast and epifluorescence microscopy. Imaging is performed using an IX70 Olympus inverted microscope with a 100x or a 60x oil-immersion objective. Images are collected by a charge coupled device CCD camera (CoolSnap, Photometrics, Roper Scientific). For three-dimensional acquisition, confocal microscopy is performed using an inverted confocal spinning disc Roper/Nikon with a 100x oil-immersion objective and lasers with wavelengths of 491 nm for Arp2/3 complex and 561 nm for lipids. A FITC filter cube (excitation filter: 478-495 nm/emission filter: 510-555 nm) and a TxRed filter cube (excitation filter: 560-580 nm/emission filter:600-650 nm) are used to acquire respectively Arp2/3 complex and lipids fluorescence. Images are collected by a CCD camera (CoolSnap HQ2, Photometrics, Roper Scientific). Z-stacks are acquired using the software Metamorph on each wavelength with a z-interval of 0.3 µm. SLBs are imaged by total internal reflection fluorescence microscopy (TIRFm) on a Leica DMI6000B microscope. Samples are excited by total internal reflection illumination 488 and 568 nm (lasers excitation wavelength), and images are captured with an Andor back-illuminated DU-897 EMCCD camera controlled by Leica software (LAS-AF-6000, Leica Microsystems CMS GmbH, Germany). The illumination settings, i.e. the lasers intensity, camera acquisition time, and the decay penetration length, are kept the same in all experiments. Image analysis is performed with ImageJ (NIH), Origin (Origin-Lab Corp.) and Matlab (MathWorks). For images at low S-pVCA concentrations (35 and 100 nM), we first remove background fluorescence by applying a Gaussian filter (σ = 15) and subtracting the Gaussian filtered image pixel by pixel from the original image. We then apply a median filter (2,2) to the subtracted image to remove high frequency noise 28 . We finally generate a binary image by histogram equalization, where we set the background fluorescence (intensity of pixels located in between individual actin asters) to zero and the rest of the pixels is set to 1. For images at S-pVCA concentrations ≥ 350 nM, we replace the Gaussian filter step with a rolling ball algorithm with radius of curvature of 50 pixels (as described in 29 ) to correct possible uneven illumination. Then the original image is subtracted pixel by pixel from the smoothened image. The binary image is then obtained by setting to zero the 5% of all pixels with the lowest intensity and the remaining 95 % of the pixels is set to 1.

To determine the actin aster area and number of individual actin asters in a given image during actin network growth (at t = 30, 80, and 110 s) and at steady state (t = 30 min), we use the Particle Analyzer algorithm as plug-in from ImageJ. From the analysis, we determine the distribution of aster area and mean number of asters per image area (each image contains 512x512 pixels which corresponds to an area of 117x117 µm 2 ). We set the minimal size of an aster as 0.05 µm 2 (i.e. 1 pixel). Errors in number of asters per image correspond to the standard deviation of experimental values. The fraction of surface coverage of actin asters was calculated from the total number of pixels with nonzero intensity (n) divided by the total number of pixels per image (N 0 = 512x512 pixels). The percentage of surface coverage per image is thus given by: %coverage = n N 0 * 100. Errors correspond to the standard deviation of experimental values. Estimating the number of lipid clusters per SLB surface at various stages of the experiment. To remove background fluorescence, we use the same method as for actin signal images with a Gaussian filter (σ = 5) and a median filter (5,5). Using the same Particle Analyzer algorithm as described above, we determine the number of lipid clusters per image area (each image has 470x470 pixels which corresponds to an area of 108x108 µm 2 ). Errors in number of lipid clusters per image at steady state correspond to the standard deviation of experimental values. Presence of the Arp2/3 complex inside spikes. For each spike, we measure the mean intensity of the Arp2/3 complex fluorescence inside the spike, I spike , on a two-pixel width line that spans from the base to the tip. We then subtract the mean background intensity of the Arp2/3 complex, I s,background , evaluated using the same line positioned in the bulk solution. We then evaluate the mean intensity of the Arp2/3 complex fluorescence within the clump of actin network observable at the base of the spike, I clump , on a two-pixel width line along the base width, and subtract the mean background fluorescence I c,background , evaluated outside the liposome the same way as above. We then calculate the relative difference in intensity as ∆I = I spike -I s,background

I clump -I c,background .

Results

Experimental system

Cell mimicry is achieved here with a lipid membrane supporting a dynamically growing actin network. We follow how the actin network grows and how it correlates with membrane deformation. For that, we study two geometries in parallel. Spherical giant unilamellar vesicles (here called liposomes) allow us to visualize membrane indentations whereas planar supported lipid bilayers (SLBs) permit the detailed observation of actin network growth at the membrane. Liposomes and SLBs are coated with a nucleation promoting that activates actin polymerization through the branching agent, the Arp2/3 complex. The nucleation promoting factor we use is the fragment pVCA of WASP with a streptavidin tag to link it to the biotinylated lipids present in the membrane (see Methods). We call this fragment S-pVCA. A branched actin network grows from the activated membrane with the use of an actin polymerization solution in which the S-pVCA-coated liposomes are placed, or which is introduced in a flow chamber containing the S-pVCA-coated SLB (Methods and 25 ). Incubating liposomes with 350 nM of S-pVCA (referred to as the 'reference condition', or 'ref') triggers the growth of a branched actin network once placed in the actin polymerization solution (Fig. 1A, ref condition) 4,25 . In reference condition, symmetry breaking events happen and prove that our system is efficient to generate an actively polymerizing actin network 25 . Likewise, an actin network forms at the surface of SLBs pre-incubated with 350 nM of S-pVCA (Fig. 1B). Rarely, but observably around liposomes, elongated structures of membranes filled in with actin, hereafter called "spikes", point inward and are reminiscent of filopodia 4 . In the absence of actin, inward membrane deformations are not observed (Fig. 1A, 0 nM S-pVCA). This excludes that spontaneous membrane deformations may be induced by our liposome production method, as we use electroformation with non-charged lipids 30 . Additionally, spikes collapse when the actin network is photodamaged 4 . Altogether, these results confirm that the spikes observed here rely exclusively on actin network growth.

In order to assess if such spike formation may depend on the quantity of nucleation promoting factor and the actin network structure, we vary the nucleation promoting factor concentration, below and above 350 nM S-pVCA, in both liposome and SLB sys-tems.

Variation of nucleation promoting factor concentration

We incubate the membrane with lower (35 nM, 100 nM) and higher (1 µM, 3.5 µM) concentrations of S-pVCA as compared to the reference condition. Strikingly, at lower S-pVCA concentrations, when the liposome surface is not saturated with bound S-pVCA molecules (Supplementary Information), we observe more liposomes displaying spikes, and the actin layer formed around the liposomes appears less homogeneous than in reference condition (Fig. 1A). Even more visible on SLBs, the actin network appears noticeably patchy at low S-pVCA concentrations (Fig. 1B). In contrast, at higher S-pVCA concentrations, no spikes are observed (Fig. 1A andC) and the actin network grown on SLBs is homogenous (Fig. 1B andD). Since the SLB is confined to a rigid support, inward membrane deformations (such as spikes) cannot occur. We do observe though lateral reorganization of the membrane in the form of lipid clusters, but it is already present before actin is added (Fig. S1). Note that actin polymerization does not promote additional lipid clusterization (Fig. S1B).

Quantitative analysis of actin growth dynamics

We characterize the growth dynamics of actin on SLBs for all concentrations of S-pVCA by following actin fluorescence intensity and actin network structural organization as a function of time (Fig. 2A). The branched nucleation of actin at the SLB surface promotes the formation of actin "asters" 18 (white arrow-head in Fig. 2A). More asters are formed at higher concentrations of S-pVCA, as the fraction of covered pixels at early time increases with concentration (Fig. 2B). Indeed, 30 seconds after addition of the actin polymerization solution, only a few actin nucleation sites are observed at low S-pVCA concentrations (35 nM, 100 nM) whereas at higher S-pVCA concentrations (≥ 350 nM) numerous asters are already formed (Fig. 2A). Individual asters grow and densify with time as revealed by the increase in the fluorescence intensity (Fig. 2A, fluorescence intensity profiles) and aster size (Fig. 2B). Moreover, we observe the nucleation of new actin asters with time which increases the fraction of actin network surface coverage (Fig. 2B). This increase is far more prominent as the concentration of S-pVCA is elevated as depicted in Fig. 2B. Quantitative analysis of the intensity of individual asters versus time reveals that the temporal evolution of branched actin network density follows a sigmoidal function characterized by a characteristic time constant, τ (see Methods). The higher the S-pVCA concentration, the faster the steady state is reached (i.e. the smaller is τ, Fig. 2C), the denser is the actin network (i.e. higher average actin fluorescence intensity, Fig. 1D and Fig. 2C). Consistently, compared to reference conditions, lower concentrations of S-pVCA produce less fluorescence of the actin network around liposomes (Fig. 1A). At higher concentrations of S-pVCA for liposomes, the membrane is saturated with S-pVCA molecules (Supplementary Information) which leads to bulk polymerization and explains the fluorescent background of liposome images (Fig. 1A, last column).

Comparison of aster area (SLB) and spike base area (Liposome) at steady state for low S-pVCA concentrations

We determine the area of the asters formed on supported lipid bilayers at steady state at S-pVCA concentrations of 35 and 100 nM (see Methods), which corresponds to the conditions for which spikes form on liposomes. For comparison, we determine the area of the spike base. We observe that the distribution of aster area on SLBs is narrower at 35 nM S-pVCA as compared to 100 nM S-pVCA, and so is the distribution of spike base area (Fig. 3A andB). Although the distribution of aster areas for a given S-pVCA concentration is wider than that of spike base areas, on average, aster and spike base areas are of the same order of a few micrometer size. On supported bilayers, the total number of asters increases with S-pVCA concentration, as can be already observed in Fig. 2. We find indeed that the average number of asters per image at 35 nM is N = 590 ± 50 and at 100 nM is N = 820 ± 70 (mean ± SD, Fig. 3C). Strikingly, we observe the opposite tendency in number of spikes per liposome: at the lower concentration of S-pVCA (35 nM), liposomes display more spikes than in reference condition (Fig. 3D). This observation reveals that even if more asters are present at a higher concentration of S-pVCA, only a fraction of them turn into spikes around liposomes. Conversely, a low concentration of S-pVCA is very efficient in producing spikes. This suggests that lower density of actin branches and smaller asters are more efficient in producing spikes. Note that spike length at steady state is not affected at S-pVCA concentrations of 35 and 100 nM (Fig. S4A).

Estimate of the Arp2/3 complex content of spikes at low S-pVCA concentrations

A closer look at the structure of spikes at low S-pVCA concentrations reveals that they display different structures, either thin and long, or conical with a wider base (Fig. 4 and Fig. S4B). In order to characterize the branching degree of the network inside those different spikes, we use a fluorescent Arp2/3 complex to visualize the actin network, as actin and the Arp2/3 complex are intimately linked, and estimate the amount of branches through the network (Methods). The fluorescence intensity of Arp2/3 complex within the spikes is compared to that in the bulk solution and is normalized to the basal branched network intensity (i.e. the clump of actin at the base of the spike, as previously reported, 4 Methods). Negative values correspond to an absence of the Arp2/3 complex, as the spike contains less Arp2/3 fluorescence than the bulk solution, whereas positive values reveal the presence of the Arp2/3 complex within the spike. We find values that span from negative to positive values (Fig. 4), indicating that for around 10 % of spikes, the internal network is devoid of the Arp2/3 complex. Such spikes correlate with a thinner base, as is observed in the intensity/width diagram (Fig. 4B and Fig. S4B). Therefore, spikes can grow as membrane deformations, both in the absence or in the presence of the Arp2/3 complex available in the network. Moreover, we find that higher amounts of the Arp2/3 complex in the spike correlates with a wider spike base (Fig. 4).

Discussion

A homogeneous actin network that grows towards the membrane is able to deform it locally in a spike, as previously shown 4 . The continuous model used in 4 considers the actin network as a homogeneous viscous medium and therefore does not apply when this medium is heterogeneous. We investigate here experimentally the role of network heterogeneities in the mechanism of membrane deformations induced by actin polymerization 16 . At 350 nM S-pVCA and higher concentrations the system forms a percolated actin network at steady state, confirmed by the 90 % of the SLB surface covered with a dense actin layer, above the percolation threshold of 50 % for 2D systems 31 . Individual actin asters, i.e. actin network heterogeneities, persist only at the lowest S-pVCA concentrations of 35 and 100 nM. In these conditions, more spikes are observed around liposomes. The increase in the capacity of liposomes to form spikes is correlated with the appearance of individual actin asters i.e., more spikes are observed in the conditions of isolated asters. Furthermore, aster area correlates with spike base area (Fig. 3), thereby suggesting that spikes have their origin in asters. This positive correlation between heterogeneities in the actin coverage and occurrence of membrane de-formations is consistent with the formation of actin patches that generate membrane protrusions in reconstituted actin cortices 16 or that precede the formation of dendritic filopodia in the spinal cord of chicken embryos 32 .

Strikingly, spikes with different morphologies and structures are observed (Fig. 4). Elongated, cylindrical shape protrusions are devoid of actin branches whereas conical shape protrusions contain branches, as evidenced by fluorescent markage of the Arp2/3 complex (Fig. 4). Cylindrical protrusions, reminiscent of conventional filopodia, are explained by actin filaments that emanate from the branched network and grow as a parallel bundle through bundling effect of the membrane, as previously reported, 2 without the need for any actin crosslinker. In the absence of the membrane, bundlers such as fascin are necessary to orient parallel filaments emanating from a branched network 22,33 . Conical shape protrusions, reminiscent of dendritic filopodia in neurons, rely on constant actin filament branching at their membrane surface 4 . Their mechanisms of formation could rely either on a dynamic effect of actin filament growth 16 or an enhanced local branching on the surface of the initial heterogeneity 34,35 . Therefore, our experimental results recapitulate the variety of protrusion structures, with a different branching degree within the actin network, reported in in vitro systems 2,4,16 .

Conclusions

We show here that spikes can form either from a continuous actin network or from a patchy, localized branched actin struc- ture. More spikes are formed when the concentration of actin nucleation promoting factors is lowered, pointing to a facility of spikes to form when the actin network is not homogeneous. In these conditions of a patchy, branched network, both dendritic and conventional filopodia-like membrane deformations are observed. Therefore, cell membranes underlined by a branched cytoskeleton increase their ability to deform into filopodia-like structures in the presence of inhomogeneities of the actin network. Then they have multiple ways to deform under the effect of actin filament growth, either into branches or into parallel filaments that exclude the Arp2/3 complex.

1. 4 . 1

 41 SLB system. Local changes in actin network and lipid membrane structural organization. To obtain fluorescence intensity (density) profiles of the lipids and the actin, lines of 3 pixel thick and 30 µm long are drawn across the SLBs at various time points of the experiment. Growth dynamics of branched actin networks. For each S-pVCA concentration, we measure the changes in actin fluorescence (which provides a measure of actin filament density) with time. To avoid edge effects, we use the central two third of the image where sample illumination is uniform. For each time point, we determine the mean actin fluorescence intensity in 10 regions of 10 µm 2 . The data points are fitted to a sigmoidal I = A 1+Be -t τ , where A and B are constants and τ is a characteristic time constant for actin network growth. Errors in mean actin fluorescence intensity correspond to standard deviation of experimental values. Intensity values were divided by a factor of 6000. Data is presented in arbitrary units. Estimating actin aster (or actin patch) area, number of actin asters per image, and the fraction of actin network surface coverage.

1. 4 . 2

 42 Liposome system. Occurrence of spikes. The occurrence of spikes equals the number of liposomes displaying spikes (visible in both membrane and actin fluorescence signal) divided by the total number of liposomes. Error bars correspond to the standard deviation of two independent experiments. Estimating spike base and tip width, spike length and spike base area. The base (resp. tip) width and length of a spike are measured from the fluorescent signal of the membrane. A line of 5 pixel thick is drawn at the base (resp. tip) of the spike (or from spike base to tip) to obtain a fluorescence profile which is fitted with a polynomial function. The first maximum and the second minimum of the fit derivative, corresponding to inflexion points of the profile, determine the membrane edges. The base (resp. tip) width (or spike length) is then the distance between the two edges. The base area is equal to the area of a disc of diameter the width of the spike base i.e. π( spike base width 2 ) 2 .

Fig. 1

 1 Fig. 1 Impact of S-pVCA concentration on branched actin network structural organization around liposomes (A, C) and on flat supported lipid bilayers (B, D) at steady state. (A) Phase contrast (upper row), fluorescence imaging of the lipid membrane (middle row) and the actin network (bottom row). White arrow heads point to "spikes". (B) TIRFm imaging of the supported lipid membrane (upper row) and the branched actin network (lower row). (C) Number of liposomes displaying spikes. (35 nM, N = 77; 100 nM, N = 93; 350 nM, N = 53; 1000 nM, N = 40; 3500 nM, N = 36). (D) Average intensity of actin fluorescence as a function of S-pVCA concentration (see Methods and Fig. S2C). Errors correspond to standard deviation of experimental values. Images are acquired 30 min after addition of the actin polymerization solution. Bars are 10 µm.

Fig. 2

 2 Fig. 2 Branched actin network nucleation and growth on SLBs. (A) Dual TIRFm imaging of the lipid membrane and the branched actin network at increasing amounts of S-pVCA. The lipid membrane is labeled with TexasRed and actin is labeled with Alexa-Fluor 488. (Left to right, refers to columns 1-5). (Column 1): lipid membrane after TPE buffer wash to remove unbound (excess) S-pVCA and prior the addition of the actin polymerization solution. (Columns 2-4). Time-lapse TIRFm imaging of actin network assembly from mixing, i.e., t = 0, to t = 110 s. White arrow-heads mark the nucleation of actin asters (shown for image clarity at t = 30 s only). Bars are 10 µm. (Column 5) Intensity (density) profiles of the actin network along the lines drawn in columns 2-4 at three time points. (B) Average area of actin asters as a function of the fraction of surface coverage estimated at different time points. Shown are the results for the various S-pVCA concentrations. The grey dotted line corresponds to surface coverage of 0.5. (C) Average intensity of actin fluorescence as a function of time. The lines represent sigmoidal fits (R 2 ∼ 0.99) to the experimental values (symbols). The decay time constant characterizing actin network growth is given by τ (see Methods). Conditions: the actin polymerization solution and membrane lipid composition are the same as in Fig. 1.

Fig. 3

 3 Fig. 3 Aster area in supported bilayers (SLB) and spike base area in liposomes (Lip) at steady state for low S-pVCA concentrations. Distribution of aster area (A) and spike base area (B) for S-pVCA concentrations of 35 and 100 nM. (C) Mean number of asters per SLB surface area. Errors correspond to standard deviation of experimental values. (D) Number of spikes per liposome. The black column is for 350 nM S-pVCA above which no spikes form.

Fig. 4

 4 Fig. 4 Presence of the Arp2/3 complex inside spikes at lower concentrations of S-pVCA (35 nM, 100 nM). (A) Representative images of the presence (two first rows, green and blue) or absence (third row, red) of the Arp2/3 complex inside spikes. The fourth row (yellow) shows an ambiguous case. First three columns: sum of z-stack confocal images of the membrane, the Arp2/3 complex and false colour image of the Arp2/3 complex. Last two colums: 3D reconstruction for z-stack confocal images (membrane in red, Arp2/3 complex in green). White double arrows indicate the clump of actin at the base of the spike. White arrow heads point toward spikes. Scale bars: 5 µm. (B) Arp2/3 complex fluorescence intensity values as a function of spike width. The colored points (green, blue, red, yellow) refer to rows in (A). Squares (resp. circles) refer to 35 nM (resp. 100 nM) S-pVCA. Note that a different batch of S-pVCA is used for these experiments compared to main figures. Due to a difference in protein activity, spikes are obtained in the 100 nM condition for actin and profilin set to 1 µM. N=25 liposomes, n = 27 spikes.

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1-9 J o u r n a l N a me , [ y e a r ] , [ v o l . ] ,

Acknowledgements

This work was supported by the MAIMONIDE Hubert Curien Franco-Israeli program, the French Agence Nationale pour la Recherche (ANR), grant ANR-14-CE090006 and ANR-12-BSV5001401, and by the Fondation pour la Recherche Médicale (FRM), grant DEQ20120323737. A.B.-G. thanks the Israel Science Foundation (grant 1618/15) for financial support. Ca.S. was supported by a Labex Celtisphybio transition fellowship.

Conflicts of interest

There are no conflicts to declare.