Noura Metri

Xavier Sallenave

Cedric Plesse

Layla Beouch

Pierre-Henri Aubert

Fabrice Goubard

Claude Chevrot

Gjergji Sini
email: gjergji.sini@u-cergy.fr.

Cédric Plesse

Processable Star-Shaped Molecules with Triphenylamine Core as Hole-Transporting Materials: Experimental and Theoretical Approach

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

INTRODUCTION

Solar cells are one of the most promising alternatives for energy conversion. Among various strategies, dye-sensitized solar cells (DSSCs) 1 have attracted a lot of attention, and a large amount of research and development has been dedicated to this field over the past two decades. In a DSSC device, light is absorbed by the dye anchored on the TiO 2 surface, and then electrons from the excited dye are injected into the conduction band of the TiO 2 , which generates an electric current. The ground state of the dye is regenerated by the electrolyte which contains a redox couple I 3 -/I -mediator to give efficient charge separation. For a decade, a promising type of DSSC has been developed: contrary to the electrolyte cell, it is based on a heterojunction between the dye-sensitized TiO 2 and an organic holeconducting material. Recently an overall efficiency of 6% was achieved. 2 While dye properties are very important for these devices, we focus here on the role of the hole-transporting materials. A variety of organic hole-transporting materials have been described, and efficiencies higher than 5% have been obtained by using compounds such as amorphous 2,2′,7,7′-tetrakis-(N,Ndi-p-methoxyphenylamine)9,9′-spirobifluorene (spiro-OMe-TAD) as a hole-transporting material. 3,4 Among the various classes of organic hole-transporting materials, 5 special attention has been paid to compounds derived from triphenylamine (TPA). [6][7][8] These compounds (possessing three-dimensional geometry) can lead to amorphous materials with interesting optical and charge-transport properties as shown by many research groups who have designed such materials for a variety of photovoltaic devices. [9][10][11][12][13] Different π-conjugated ring molecules can be used to extend the π-conjugated system of the TPA core. Star-shaped molecules containing TPA core and thiophene (Th) derivatives have yielded interesting results in DSSC applications. [14][15][16] Another promising candidate, which can be combined with these star-shaped molecules, is the thieno [3,2-b]thiophene (TTh) group which contains two fused thiophene rings. A c t u a l l y ,c o m p a r e dt oi t sa n a l o g u e ,T h -Th, the TTh compound offers better π-conjugation and smaller geometric relaxation energy upon oxidation.

Based on these observations, we recently reported the synthesis of five star-shaped molecules containing various combinations of Th and thieno [3,2-b]thiophene-C 9 H 19 (TTh-C 9 ) located in the arms linked to the TPA core (Chart 1). 17 Preliminary results concerning the optical properties of these molecules have shown promising properties for use in photovoltaic applications.

From a theoretical point of view, the hole-transport properties in these materials are mainly discussed in terms of disorder models. 18 While mobility calculations based on firstprinciples are still a challenge, 19,20 determination of chargetransfer rate constants (k HT ) based on the Marcus theory can provide very useful information. [21][22][23][24] Limiting the discussion to the case of amorphous materials, the elementary step of the charge transport can be seen as a charge transfer (CT) between two adjacent molecules. In such a case, the rate constant of a hole-transfer reaction can be described by the "hopping" mechanism and calculated using the following equation: [21][22][23][24] = π πλ

-Δ °+λ λ ⎡ ⎣ ⎢ ⎢ ⎤ ⎦ ⎥ ⎥ k h kT t G kT 41 4 exp () 4 HT 2 B 2 2 B (1)
In this equation, t is the electronic coupling between two adjacent molecules, ΔG°is the free energy of the hole-transfer reaction (which is zero in the case of hole transfer between identical molecules), and λ is the reorganization energy. This parameter is the sum of two terms: (i) λ s , containing the contribution from the medium polarization energy, and (ii) λ i , representing the energetic effort due to the intramolecular geometric relaxations related to the hole transfer between two adjacent molecules. Knowledge of the three parameters is necessary in order to determine the k HT constant between two adjacent molecules, and still additional parameters need to be known in order to evaluate hole mobility. However, in a series of similar compounds, each of these parameters considered individually can provide trends which could be helpful in the design of new materials. In the case of a family of similar amine compounds for instance, a good correlation between the trends of the λ values and the experimental hole mobilities has been observed. 25 Other molecular electronic parameters such as the HOMO and LUMO level energies and their space distributions within the molecular backbone are extensively used in order to evaluate the optical properties of these materials and the efficiency of the film-electrode (film-dye) contact properties but also to predict their charge-transport properties. [26][27][28][29] In the present work, we report a detailed investigation of some of these properties by comparing experimental and theoretical results. UV-vis measurements and electrochemical cyclic voltammetry were performed in order to assess various parameters (HOMO and LUMO level energies and their band gap, spectral absorption domains, and extinction coefficients). Calculations based on various density functional theory (DFT) 30 methods (especially B3LYP 31,32 ) were also carried out in order to understand the impact of the structural and electronic properties of these molecules on their optical properties, geometrical relaxation energies (λ i ), and their potential use as hole-transporting materials in solar cell applications. 17 The structures of these molecules are shown in Chart 1.

EXPERIMENTAL SECTION

2.2. Instruments and Measurements. UV-vis absorption spectra were taken with a Jasco (V-570) UV-vis-NIR spectrophotometer. The cyclic voltammetry was carried out in a glovebox using a Princeton Applied Research-Potentiostat/ Galvanostat model 273A (EG&G, The Netherlands). The electrochemical setup was a three-electrode single cell, with a glassy carbon (GC) disk (2 mm diameter) coated with the sample film as the working electrode, a Pt wire as counter electrode, and a silver wire pseudoreference electrode. Ferrocenium/ferrocene (Fc/Fc + ) redox potential was measured at the end of each experiment in order to calibrate the pseudoreference electrode as recommended by IUPAC. 33 Compounds 1-5 (C = 1.0 × 10 -3 M in tetrahydrofuran) were drop-casted onto the GC-electrode and then dried. The GC-electrode was immersed into the electrochemical cell containing acetonitrile with0 . 1Mt e t r a b u t y l a m m o n i u m hexafluorophosphate (TBAPF 6 ). The compounds were electrochemically reduced prior to being oxidized between -3.0 V and +1.5 V at a scan rate of 20 mV/s.

2.3. Computational Methodology. Density functional theory (DFT) methods and especially the B3LYP one are extensively used in this kind of study. However, B3LYP and other hybrid functionals containing low percentage of Hartree-Fock exchange (%HF) are known to miss the dispersion interactions 34,35 which are important when considering the πstacking interactions. This can affect for instance the dihedral angles between the conjugated rings of TPA-Th (TTh) molecules. In this case, the use of DFT methods containing higher %HF exchange may be necessary. In order to take into Chart 1. Triphenylamine (TPA) Derivatives (1-5) Bearing Different π-Conjugated Arms account this effect, the M05-2X functional containing 56% of exact exchange was also considered. 36 The comparison between the B3LYP and M05-2X frontier orbital energies is shown in Figure S1 (Supporting Information). Due to the better agreement with the experimental results, only the B3LYP results will be discussed in the following.

The DFT calculations employing the B3LYP and M05-2X functionals were carried out by using the Gaussian 03 program. [START_REF] Frisch | Gaussian 03[END_REF] The geometries of all the molecules were optimized by imposing a C 3 symmetry and by using the 6-31G* basis set. Frequency calculations were carried out to ensure that true minima were obtained. Tests for symmetry-relaxed geometry optimizations were also carried out, and negligible energy differences were observed. The geometry optimizations of the cationic radical species were carried out at the restricted open shell level.

The spectroscopic properties of the molecules were calculated by mean of time-dependent density functional theory method (TDDFT) [START_REF] Gross | [END_REF][39][40][41][42] at the TDDFT/6-31+G*// DFT/6-31G* level. The HOMO and LUMO energies reported in this study were also obtained with the 6-31+G* basis set. The influence of greater basis sets (6-311++G**) on the HOMO and LUMO energies was also tested for the compounds 1 and 2, and the results show only a small effect as compared to 6-31+G*.

The calculation of the internal reorganization energies (λ i )is shown schematically in Figure 1. Accordingly, the λ i values are calculated as shown in the following equation 43 λ =λ +λ =-

+- + ++ + EE EE {} {} ii 1 i 2 M GeometryM M GeometryM M GeometryM M GeometryM (2)
where the quantity E M GeometryM + for instance corresponds to the energy of the neutral molecule M in the geometry of the cationic species M + .

In view of the complexity of our molecules, the 6-31G* basis set was used for the calculation of the λ i values which was shown to give negligible changes as compared to the 6-31G** basis set. 44

RESULTS AND DISCUSSION

3.1. Geometries. The optimized geometries for all the molecules, along with some geometrical parameters, are presented in Figure 2. As generally observed for TPA derivatives, the three N-C bonds are coplanar, which is characteristic of enamines and indicates some degree of conjugation between the arms. Steric hindrance between the phenyl rings, grafted onto the central nitrogen atom, produces the typical propeller shape of the molecule. The dihedral angles between the adjacent conjugated rings in the compounds 1-5 (Figure 2) are in good agreement with previous calculations for these types of systems. 8,44 The influence of the CH 3 group (mimicking the steric effect of the experimental nonyl group in our compounds) is reflected in the greater Ph-TTh dihedral angle obtained in the case of compound 1 as compared to compound 2 (42.8°and 27.6°, respectively). The same effect is observed when comparing compounds 4 and 5. It can be expected that this difference should influence their packing properties during the formation of the solid films.

Finally, we note that all the dihedral angles in the cationic state are reduced by roughly 10°-15°, with the exception of the Ph-Ph dihedral angles which undergo a slight reduction of only ∼1-2°(Figure 2).

3.2. Electronic Structure and Electrochemical Properties. Cyclic voltammetry was performed to investigate the electrochemical properties and HOMO and LUMO energy levels of compounds 1-5. These compounds were drop-casted onto the electrode from a THF solution resulting in a thin layer of 1-5 on the working electrode. As shown in Figure 3, all compounds exhibited n-and p-doping/dedoping (reduction/ oxidation) processes. The n-doping was realized prior to the pdoping (as described in the Experimental section). In fact, pdoping was subsequently followed by a polymerization process (CV shown in Figure S2), since the α-carbon (in the vicinity of the sulfur atom) of the external thiophene ring is free. This was somewhat surprising because our calculations performed on the charged molecules (radical cations) reveal spin density mainly localized on the TPA core (58-34% for 1-5, respectively, B3LYP/6-31G* level), whereas only 8% and 3% of the total spin density (compounds 1 and 5, respectively) were found on each terminal heterocycle. In fact, during the p-doping, the reverse potential was chosen systematically far beyond the onset potential, hence bringing out the production of high concentration of radical cations in the layer and favoring the coupling of vicinal radical cation into a polymeric system. Hence the reverse scan of the p-doping process exhibits the reduction of the polymeric system. In the case of the n-doping, 4 and 5 exhibited a quasi-reversible reduction process while the three other compounds were irreversibly reduced. The origin of the reversibility of compounds 4 and 5 stands in their LUMO levels which are lower than those of compounds 1-3, thus stabilizing the radical anion. Hence, the n-doping appears quasireversible.

The onset oxidation (or reduction) potential measures the potential at which the electron is withdrawn from the HOMO (or given to the LUMO). In the case of p-doping, the onset potentials, for 1-5, range between 0.58 and 0.41 V vs Fc/Fc + (-2.68 and -2.35 V for the n-doping, Table 1). The HOMO and LUMO energy levels were calculated from the onset oxidation and reduction potentials. 45,46 The electrochemical data are summarized in Table 1.

As expected, adding TTh and Th conjugated groups to the TPA core in 1-5 makes the HOMO (LUMO) energies increase (decrease) resulting in decreasing HOMO-LUMO gaps. The same trends are obtained from the B3LYP calculations on the isolated molecules (Figure 4 and Table 1), indicating that the molecular packing forces do not significantly modify the intramolecular electronic structure. The differences between the theoretical and electrochemical values are a well-known consequence of performing calculations that lack medium-related factors. In spite of this, both approaches show similar trends in the evolution of the HOMO and LUMO levels.

In order to understand the difference in the way the HOMO and LUMO levels change, we focus on the nature of the frontier orbitals. The HOMO and LUMO pictograms for compounds 3-5 are presented in Figure 5 (Figure S3 for compounds 1 and 2). The HOMO orbitals are mostly developed in the TPA core with vanishing influence from the peripheral Th or TTh groups. This can be explained by considering, for instance, the HOMO of compound 2 as an antibonding combination of the local HOMO (TPA) core and the local HOMO (TTh): the HOMO (TPA) core is higher in energy than the TTh counterpart (-5.24 eV versus -6.09 eV, B3LYP/6-31+G* level) and will, therefore, provide a greater contribution to the total HOMO orbital. The energy difference between the local HOMO orbitals (the HOMO of a new Th or TTh group and the HOMO of TPA-TTh-(Th) n ) increases with the number of heterocycles in the arms, further reducing the contribution of the new heterocycles in the global HOMO orbital of the molecule. This effect should be reinforced by the presence of dihedral angles between the rings. The overall effect seems to be that, after three additional rings, the space extension of the total HOMO is almost negligible (Figure 5) indicating that the major influence of the peripheral heterocycles on the HOMO energy is predominantly inductive in nature.

Given that a good hole transfer implies a good overlap between the HOMO orbitals of two adjacent molecules, it is reasonable to suppose that increasing the number of conjugated heterocycles beyond a total of four in each arm should have little effect on the hole transfer.

As for the LUMO orbitals, there are, in each case, two quasidegenerate virtual orbitals. In Figure 5, only the lower ones are presented for each compound. The extension of these orbitals mainly in the arms can be discussed in a similar way as for the HOMO orbitals. As a result, it is reasonable to expect that the influence of the last rings in the arms should be more significant for the LUMO energies, meaning that they are more influential on the spectroscopic properties and eventually on the electron mobility.

3.3. Optical Properties. The absorption spectra for the compounds 1-5 in films spin-coated from CHCl 3 solution (C = 1.0 × 10 -5 M) are shown in Figure 6, while the optical characteristics are provided in Table 2.

Broad absorption bands covering the wavelength range from 300 to 510 nm can be observed for all compounds in CHCl 3 solution, exhibiting λ max which are red-shifted by roughly 74 nm when going from 1 to 5. The absorption spectra measured in tetrahydrofuran (THF) exhibit the same trend as in chloroform and are presented in Table S1 (Supporting Information).

Compound 1 has the shortest λ max value, probably due to the fact that it has the shortest conjugated chain length but also to the severe deplanarization caused by the steric hindrance induced by the nonyl chain. Displacing the nonyl chain to an external position in compound 2 induces a bathochromic effect of 32 nm due to a lower deviation in the molecular geometry from planarity. The λ max values of 2, 3, and 4 show a progressive bathochromic shift when the length of the conjugated system is increased, exhibiting absorption maxima ranging from 386 nm for 2 to 422 nm for 4 in CHCl 3 . This trend is in agreement with the trend previously reported in the case of homologous series of conjugated oligothiophene. 47,48 We finally note that the comparison of absorption signals between compounds 4 and 5 in CHCl 3 , in which TTh-C 9 and Th units are combined in different orders, reveals only minor differences with a slightly longer λ max for compound 5.

Compared to their dilute solutions, the absorption bands in solid films are slightly red-shifted (10-28 nm). This red-shift can be related to the geometrical changes that occur in the solid films: the dihedral angles between adjacent rings in these molecules are reduced as is always observed when comparing gas-phase geometries to crystal structures 49 which, consequently, improves the π-conjugation and decreases the HOMO-LUMO gaps.

The theoretical UV-vis spectra for compounds 1-5 (Figure S4, Supporting Information) indicate that the λ max values (410, 420, 478, 500, and 490 nm, respectively) are dominated by S 0 → S 1 electronic transitions. The influence of higher excited states of the λ max values is visible only for compounds 4 and 5 but does not modify the trend deduced from the S 0 → S 1 transitions alone. The S 0 → S 1 transition energies for compounds 1-5 are presented in Table 2 and show the same global trend as compared to the experimental λ max values. Comparison between the relative experimental extinction molar coefficients and relative oscillator strengths (Table 2) also shows good overall agreement.

There is, however, a small ambiguity in the experimental results for compounds 4 and 5. The experimental UV-vis spectra in solution suggest that the efficiency of the πconjugation increases in the order of 1-2-3-4-5, while the UV-vis spectra for solid films suggest a permutation in the order between 4 and 5. The TD-DFT calculations and also the HOMO-LUMO gaps deduced from electrochemical measurements yield results which are in agreement with the UV-vis spectra in solid films.

These somewhat surprising results might be related to the conformational twists along the conjugated arms in solution where rotational barriers between the heterorings can be as low as ∼10 kJ/mol. 50 The HOMO pictograms of compounds 4 and 5 (Figure 5) indicate that, in the case of 4, there are more possible rotations between rings contributing significantly in the HOMO orbital. These conformational twists are not taken into account in the calculations and should also be less pronounced in solid state thus yielding the agreement between the respective results. Nevertheless, we note that the differences observed between the HOMO-LUMO gaps of compounds 4 and 5 are small (0.04 eV roughly) and may also be due to other factors.

Evaluation of Hole-Transfer Properties.

In this paragraph we present a preliminary comparison of compounds 1-5 with respect to their intramolecular properties as holetransporting materials. We remember that, in order to obtain high hole-transfer rate constants (k HT ), the electronic coupling parameter (t) should be maximized whereas the reorganization energy (λ = λ s + λ i ) should be minimized (eq 1).

3.4.1. Internal Reorganization Energy (λ i ). The λ i values for compounds 1-5 (calculated using eq 2) decrease from 0.219 eV (compound 1) to 0.150 eV for the compound 4 (B3LYP/6-31G* level, Table S2). In terms of a monoelectronic description, this global trend suggests that the intramolecular geometry-relaxation energy should be smaller for systems with larger space delocalization in the HOMO orbital. While this observation might hold true for compounds with planar πsystems, 43,51 other factors could play in the opposite sense. Indeed, the λ i values of compounds 1-5 are all higher than the value of 0.11-0.12 eV found for the TPA core (this work and ref 8, respectively). The reason may be related to the fact that adding Th or TTh groups increases the number of dihedral angles between adjacent rings which are very sensitive to changes in the number of electrons in the π-system. The decrease of the dihedral angles upon oxidation is coupled to changes in the bond lengths due to the increase in the πconjugation efficiency, thus resulting in increased λ i values (see also refs 8 and 44).

The importance of this effect can also be seen when comparing for instance the Ph-TTh dihedral angles of compounds 1 and 2 (42.8°and 27.6°respectively, neutral state) which could explain the corresponding λ i values (0.219 and 0.188 eV, respectively). The same trend is observed when comparing the compounds 5 (λ i = 0.173 eV) and 4 (λ i = 0.150 eV) for which the TTh-Th dihedral angles are 40.1°and 19.2°, respectively. The smaller impact of this effect in the latter case could be explained by the smaller contribution of the peripheral rings on the HOMO orbital of 4 and 5 as compared to 1 and 2 (Figure 5).

Based on these λ i values it can be deduced that the higher k HT values should be observed for the compounds 3, 4, and 5. Actually, the k HT values estimated by considering a fixed value of the electronic coupling (t = 0.05 eV) 52 for all the compounds are multiplied by a factor of 3 when going from 1 to 5 (Table S2, Supporting Information calculations based on the Marcus-Levich-Jortner equation 53 ). Admittedly, this change is small but shows the impact of λ i on the relative hole-transfer rate constants of compounds 1-5.

3.4.2. Electronic Coupling. Due to the size and shape of compounds 1-5, the calculation of the electronic coupling Table 2. Energy of the S 0 → S 1 Transitions from the TD-B3LYP/6-31+G* Calculations (E S 0 →S 1 ), Absolute ( f) and Relative ( f/ f(1)) (with Respect to 1) Oscillator Strengths from the TD-B3LYP Calculations, Experimental λ max Values, and Absolute (ε) and Relative (ε/ε(1)) (with Respect to 1) Extinction Molar Coefficients from the UV-Vis Spectra in Chloroform (1.0 × 10 -5 M) TDB3LYP/6-31+G* (gas phase) UV-vis parameter (t) is not straightforward. However, the comparison of the space extension of their HOMO orbitals gives a good insight in this respect. Indeed, the HOMO orbitals of 1-5 are mostly developed in the TPA core while the influence of the Th or TTh groups vanishes with the distance from the center (Figure 5 and Figure S3). This implies that, in order for compounds 1-5 to exhibit good hole-transfer properties, a good overlap should be established between the central parts of the HOMO orbitals of two adjacent molecules. This observation suggest that the presence of the nonyl group in the proximity of the TPA core is expected to prevent significant overlap between the HOMO orbitals of adjacent molecules which, in turn, should have a negative influence on the transport properties. It can be then expected that compounds 3, 4, and 5, in which the nonyl group is situated relatively far from the TPA core, exhibit efficient overlaps and non-negligible t values, which, according to the Marcus-Levich formula (eq 1), corresponds to faster hole transfer. We finally note that both parameters discussed here (λ i and t) work in "concert" and suggest that, from the molecular standpoint, the hole-transport properties of compounds 3, 4, and 5 should be better as compared to those of 1-2.

E S0→S1 (eV) ff /f(1) λ max [CHCl 3 ] a (nm) λ max [film] a (nm) ε (M -1 .cm -1 ) ε/ε(1) 1

CONCLUSIONS

Five star-shaped molecules with TPA core and various conjugated linkers such as thiophene and thieno[3,2-b]thiophene-C 9 H 19 have been investigated using a combination of experimental and theoretical methods. Electrochemical measurements show the HOMO energy levels gradually increasing from -5.38 to -5.13 eV with the addition of Th or TTh groups while LUMO values decrease from -2.12 to -2.45 eV. Both interval values suggest for these compounds promising contact efficiency with common dyes or gold electrodes. In addition, the absorption bands for these materials are found to extend only slightly into the visible region.

The HOMO orbitals of compounds 4 and 5 are mostly localized in the central part of these molecules and exhibit negligible contribution from the peripheral Th or TTh groups, suggesting that adding another heterocyclic ring in compounds 4 or 5 would have minimal impact on their hole-mobility properties.

The above conclusions suggest that, in order to obtain good electron coupling between two adjacent molecules, a good overlap is necessary between the central parts of the frontier orbitals in these molecules. The presence of the nonyl group in the proximity of the TPA core is expected to negatively affect the transport properties.

Finally, the calculated reorganization energies (λ i ) are found to be relatively small and suggest better charge-transfer properties for compounds 3, 4, and 5, which is in line with the conclusions drawn on the basis of the larger space extension of the HOMO orbitals and the positioning of the nonyl group in these molecules.

Based on this analysis it is concluded that the compounds 3, 4, and 5 possess interesting hole-transporting properties and can be good candidates for use in hybrid solar cells.

■ ASSOCIATED CONTENT

2. 1 .

 1 Materials. All chemicals were purchased from Aldrich or Fischer Scientific without further purification. All solvents, unless otherwise stated, were of analytical grade, purist quality and used as received from VWR international. Tris(4-(3nonylthieno[3,2-b]thiophen-2-yl)phenyl)amine 1,t r i s ( 4 -( 3nonylthieno[3,2-b]thiophen-5-yl)phenyl)amine 2, tris(4-(5-(3nonylthieno[3,2-b]thiophen-5-yl)thiophen-2-yl)phenyl)amine 3, tris(4-(5-(5-(3-nonylthieno[3,2-b]thiophen-5-yl)thiophen-2yl)thiophen-2-yl)phenyl)amine 4, and tris(4-(5-(3-nonyl-2-(thiophen-2-yl)thieno[3,2-b]thiophen-5-yl)thiophen-2-yl)phenyl)amine 5 were synthesized according to the procedure described in the literature.

Figure 1 .

 1 Figure 1. Schematic representation for the calculation of the internal reorganization energy, λ i . The curves represent the potential energies of the neutral and the cationic species.

Figure 2 .

 2 Figure 2. Optimized structures for the compounds 1-5. Some typical dihedral angles (absolute values in degree) for the neutral and cationic (in parentheses) species are shown. Nitrogen, sulfur, carbon, and hydrogen atoms are shown respectively in blue, yellow, gray, and white colors.

Figure 3 .

 3 Figure 3. Cyclic voltammograms of thin films prepared by dropcasting from the compounds 1-5 on glassy carbon disk in 0.1 M TBAPF 6 in ACN.

Figure 4 .

 4 Figure 4. Comparison of the electrochemical and theoretical (B3LYP/ 6-31+G* level) HOMO and LUMO energies of compounds 1-5.

Figure 5 .

 5 Figure 5. HOMO and LUMO pictograms for compounds 3-5 (B3LYP/6-31+G*). The encircled parts correspond to the TPA core.

Figure 6 .

 6 Figure 6. UV-vis absorption spectra for the compounds 1-5 (solid state) spin-coated from CHCl 3 solutions (1.0 × 10 -5 M). The compound number is indicated on the top of each curve.

Table 1 .

 1 Onset Oxidation Potentials Obtained from the Electrochemical Measurements for Compounds 1-5 and the Corresponding HOMO and LUMO Energies and HOMO-LUMO Gaps (E g ec ) a

		E ox	E red
		onset (V)	onset (V)	E HOMO (eV)	E LUMO (eV)	E g ec (eV)
	1	0.58	-2.68	-5.38 (-5.15)	-2.12 (-1.54)	3.26 (3.60)
	2	0.56	-2.46	-5.36 (-5.05)	-2.34 (-1.68)	3.02 (3.37)
	3	0.54	-2.45	-5.34 (-4.98)	-2.35 (-2.02)	2.99 (2.96)
	4	0.33	-2.27

-5.13 (-4.94) -2.53 (-2.22) 2.60 (2.72) 5 0.41 -2.35 -5.21 (-4.96) -2.45 (-2.16) 2.76 (2.80) a In parentheses are presented the theoretical values calculated at the B3LYP/6-31+G* level.


Suzanne Daignan Fornier

Alexandre De Saint

Pascal Retailleau

Jean-Paul Pillot

Quentin Taulera

Lucile Andna

Laurence Miesch

Soizic Rochange

Jean-Bernard Pouvreau

François-Didier Boyer

Suzanne Daignan Fornier

= Me

Noncanonical Strigolactone Analogues Highlight Selectivity for Stimulating Germination in Two Phelipanche ramosa Populations

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

2

ABSTRACT. Strigolactones (SLs) are plant hormones exuded in the rhizosphere with a signaling role for the development of arbuscular mycorrhizal (AM) fungi and as stimulants of seed germination of the parasitic weeds Orobanche, Phelipanche and Striga, the most threatening weeds of major crops worldwide. Phelipanche ramosa is present mainly on rape, hemp and tobacco in France. P. ramosa 2a preferentially attacks hemp while P. ramosa 1 attacks rapeseed. The recently isolated cannalactone (14) from hemp root exudates has been characterized as a non-canonical SL that selectively stimulates the germination of P. ramosa 2a seeds in comparison with P. ramosa 1. In the present work, (-)-solanacol (5), a canonical orobanchol-type SL exuded by tobacco and tomato, was established to possess a remarkable selective germination stimulant activity for P. ramosa 2a seeds. Two cannalactone analogues, named (±)-SdL19 and (±)-SdL118, have been synthesized. They have an unsaturated acyclic carbon chain with a tertiary hydroxy group and a methyl or a cyclopropyl group instead of a cyclohexane A-ring, respectively. (±)-SdL analogues are able to selectively stimulate P. ramosa 2a revealing that these minimal structural elements are key for this selective bioactivity. In addition, (±)-SdL19 is able to inhibit shoot branching in Pisum sativum and Arabidopsis thaliana, and induces hyphal branching in AM fungus Rhizophagus irregularis, like SLs.

Strigolactones (SLs) are a class of compounds first identified in 1966 1 as stimulants of seed germination of the root parasitic weeds Orobanche, Phelipanche and Striga. More recently, SLs have been discovered as the 9 th class of plant hormone that control plant architecture including shoot branching stem secondary growth, plant height, root architecture, and adventitious roots. [2][3][4][5] They are biosynthesized in plant roots in trace concentrations, and are partly excreted into the rhizosphere. They belong to the apocarotenoid family (Figure 1). The structural core of SLs is a tricyclic lactone (ABC part, canonical SLs) or an unclosed BC-ring (non-canonical SLs) connected via an enol ether bridge to an invariant α,β-unsaturated furanone moiety (D ring). All natural SLs have the same R-configuration at the C-2ʹ position. Canonical SLs are divided into strigol (1) and orobanchol (2) types, corresponding to a β-[e.g., 5-deoxystrigol (4), sorgolactone (7)] or an α-oriented C-ring [e.g., orobanchyl acetate (3), solanacol (5), solanacyl acetate (6), fabacyl acetate (8)], respectively, and with A and B rings bearing various substituents (Figure 1A). 6 Non-canonical SLs [e.g., avenaol (11), zealactone (12), bryosymbiol (13), and cannalactone (14)] can be structurally more diverse than canonical SLs (Figure 1B). Figure 1. Natural strigolactones (SLs), of the canonical (A), non-canonical (B) types and artificial SL analogues and mimic (C). For bryosymbiol (13), the absolute configuration at C-4 and C-5 could be interchangeable. 7 7 SLs have been shown to play an additional rhizospheric signaling role by stimulating spore germination and hyphal proliferation of arbuscular mycorrhizal (AM) fungi. 34,35 The hypothesis is that plant SLs boost fungal metabolism prior to root infection. 36 The AM symbiosis arose very early in land plant evolution and is thought to have been instrumental in allowing the successful plant conquest of terrestrial environments. 37 It has been proposed that an ancestral role of SLs was to attract these highly beneficial fungal symbionts, 7 with root parasitic plants having highjacked this system much later to detect their host. 25 Biological activities of SLs could be detected at concentrations as low as 10 - 13 M on AM fungi, 10 -12 M on seeds of parasitic weeds, and 10 -8 M on lateral buds as plant hormones.

Phelipanche ramosa seeds recognize allelochemical signals and interact with their hosts depending on their genetic structure, as reported recently. 38,39 Three main genotypic groups were described in European P. ramosa populations from the Mediterranean basin. P. ramosa of genetic group 1 (P. ramosa 1) is present exclusively in western France. It parasites essentially rapeseed but also melon, tobacco, and sunflower. P. ramosa 2a attacks preferentially hemp but also tobacco and tomato in France and Italy. P. ramosa 2b is widespread in Europe and Turkey and attacks mainly tobacco but also various crops like rapeseed, hemp and tomato. Seed sensitivity to germination stimulants as SLs emitted by host plants is proposed as a marker of genetic groups and their adaptation to hosts. In rapeseed, no SL has been yet identified. 40 Tobacco plants exude canonical strigol-type and orobanchol-type SLs 41,42 and tomato plants exude orobanchol-type SLs. 43 The non-canonical SL cannalactone (14) has been recently characterized from hemp (Cannabis sativa). [START_REF] Hamzaoui | Proceedings of the 15th World Congress on Parasitic Plants[END_REF] Differential sensitivities to synthetic GR24 enantiomers and cannalactone (14) for germination of P. ramosa seeds of different genetic groups have been reported. 39,[START_REF] Hamzaoui | Proceedings of the 15th World Congress on Parasitic Plants[END_REF][START_REF] De Saint Germain | [END_REF] The SL receptor in seed plants DWARF14 (D14) 46 has been identified as a member of the superfamily of the α/β-hydrolases. 4,47 In Arabidopsis, a paralogue of AtD14, KARRIKIN INSENSITIVE2/HYPOSENSITIVE TO LIGHT (AtKAI2 /AtHTL), has also been described. It encodes a protein that shows a global structure very similar to AtD14 and has conserved the catalytic triad (Ser, His, Asp). AtKAI2 has been shown to regulate Arabidopsis seed germination. 48 Interestingly, the KAI2 gene family has expanded during the evolution of parasitic plant genomes. Some members of these expanded families in witchweeds and broomrapes have evolved the capacity to bind SLs, making them good candidates for SL receptors. Six of the 11 S. hermonthica KAI2 paralogues, belonging to the divergent clade, were identified as being highly sensitive to SLs. [49][50][51] These results indicated the involvement of one or several α/β-hydrolases in SL perception in parasitic plants. In P. ramosa 1, among the 5 KAI2 paralogues, our group established recently that PrKAI2d3 encodes a SL receptor that is also able to interact with isothiocyanates (ITCs), other germination stimulants of P. ramosa seeds. 52 The objective of the present work was to analyze the biological activity of various cannalactone analogues in comparison with natural cannalactone (14), natural SLs and especially SLs produced by tobacco 41,42 and tomato 43 [(-)-orobanchol (2), (-)-orobanchyl acetate (3), 5deoxystrigol (4), (-)-solanacol ( 5) and (-)-solanacyl acetate (6)], the SL mimic contalactone [START_REF] De Saint Germain | [END_REF] and well-known GR SL analogues. Through a structure-activity relationship (SAR) study, key minimal structural elements were deciphered for the selective germination activity of P. ramosa seeds focused on P. ramosa 1 vs. 2a, 38 which attack selectively major crops as rapeseed and hemp, respectively. Moreover, the results have shown that these novel non-canonical SL analogues possess hormonal activity in the pea and induce hyphal branching in arbuscular mycorrhizal (AM) fungi.

RESULTS AND DISCUSSION

Synthesis of Cannalactone Analogues. Two types of cannalactone analogues were designed (Figure 2). Type I possesses an unsaturated acyclic chain instead of the cyclohexane A-ring of cannalactone (14), and type II bears an unsaturated acyclic carbon chain with a tertiary hydroxy group and a methyl or a cyclopropyl group (R 2 group) replacing the cyclohexane A-ring. The starting material for type I and type II analogues is (±)-4-vinyldihydrofuran-2(3H)-one (19) or Taniguchi lactone, 53 prepared in a multigram-scale from the commercially available cis-2-butene-1,4-diol via formation of a ketene acetal and its [3,3]-sigmatropic rearrangement.

Formylation of (±)-Taniguchi lactone and one-pot coupling with D-Br 20 at -78 °C led to the formation of the diastereomeric type-I cannalactone analogues (±)-SdL4F1 and (±)-SdL4F2 that are easily separable by silica gel chromatography in 41% overall yield (Scheme 2). SdL7, another Type-I cannalactone analogue was obtained by cross metathesis (CM) of (±)-SdL4F1 with a large excess of methyl acrylate at 50 °C with Hoveyda-Grubbs II (HG II) initiator in 57%

O O O O O R 1 R 1 = H, CO 2 Me
Type I cannalactone analogue Cannalactone ( (±)-SdL19 and (±)-SdL21 in non-optimized 53 and 89% yields, respectively. However, these conditions for the silylated cyclopropyl derivative (±)-SdL118F1 were inefficient but the targeted alcohol (±)-SdL127 was produced with scandium(III) triflate and Ac 2 O 57 in poor 19% yield and without detection of the acetylated product. The tertiary alcohols (±)-SdL19 and (±)-SdL21 were acylated using standard conditions to furnish acetylated derivative (±)-SdL51 and the 4-nitrobenzoates (±)-SdL33 and (±)-SdL34. In addition, the formation of the 3,5nitrobenzoate (±)-SdL50 from (±)-SdL21 furnished a crystalline compound for which the X-ray diffraction properties were suitable for establishing unambiguously the relative configurations of stereogenic centers C-2ʹ and C-4 of (±)-SdL50 as 2ʹR and 4S, respectively (Figure 3) and, by consequence, the relative configurations of stereogenic centers for the other SdL analogues, where R 1 = Me (Scheme 3). For (±)-SdL127, (±)-SdL118F1, and (±)-SdL118F2 (R 1 = Cy) the relative configurations at C-4 and C-2ʹ were proposed according to the order of elution on SiO 2 in comparison with the series where R 1 = Me. With these putative SL analogues in our hands, their biological activity was studied. Bioactivity and Selectivity of SdL Derivatives toward Phelipanche ramosa 1 vs. 2a. The germination stimulant activities of SdL analogues on P. ramosa 1 and 2a seeds were determined and compared to (±)-GR24, (±)-GR5 and natural cannalactone (14) by measuring the maximum of germination stimulant activity as well as half maximal effective concentrations (EC 50 ) using a concentration range from 10 -13 to 10 -6 mol.L -1 (Figure 4A, Figure S1, Supplementary Information). 58 Germination stimulant activities of all SdL analogues and the putative cannalactone biosynthetic precursor, (±)-MeCLA, reached the maximum induced by (±)-GR24, which makes them germination stimulants, as expected for SL analogues (Figure S2A, Supplementary Information). The bioactivities of these chemicals based were compared based on their EC 50 values.

Our group reported previously that cannalactone (14), a non-canonical SL, possesses a stronger germination stimulation activity on P. ramosa 2a than on P. ramosa 1. In contrast, (±)-GR24, an artificial canonical SL, possesses a more potent germination stimulation activity for P. ramosa 1 rather than for P. ramosa 2a. [START_REF] Hamzaoui | Proceedings of the 15th World Congress on Parasitic Plants[END_REF] These results have led to the proposal that P. ramosa 1 could be more sensitive to canonical SLs and P. ramosa 2a to non-canonical SLs. To illustrate the selectivity of seed germination between P. ramosa 1 and P. ramosa 2a, the ratio of EC 50 (P.

ramosa 1/ P. ramosa 2a) (r EC50 ) of each molecule was calculated (Figure 4B). For example, r EC50

[(±)-GR24] = 0.014 and r EC50 (14) = 410.

A set of various canonical SLs were studied belonging to the strigol-[(±)-1, (±)-4, (±)-7)] and orobanchol-types [(±)-2, (±)-3, (-)-5, (-)-6, (±)-8] as well as (±)-contalactone (Figure 1), [START_REF] De Saint Germain | [END_REF] a recent SL mimic discovered during the preparation of (±)-GR24. All canonical SLs (1-4, 7-8)

and (±)-contalactone [START_REF] De Saint Germain | [END_REF] were found less active than (±)-GR24, (+)-GR24 and (-)-2ʹ-epi-GR24, [START_REF] De Saint Germain | [END_REF] possessing configurations found in natural canonical SLs (strigol-type and orobanchol-type, respectively). Their r EC50 (< 1) reflect selectivity for P. ramosa 1, except for the cases of (-)solanacol ( 5) and (-)-solanacyl acetate (6) where high germination activity and selectivity for P. ramosa 2a were recorded (r EC50 [(-)-5] = 164) (Figure 5, Figure S2B, Supplementary Information). Although high germination activities were observed with (-)-solanacyl acetate (6) and the 7,8-desmethyl solanacol derivatives 15 and 16, selectivity was reduced, highlighting the importance of the hydroxy group but also the methyl group on the A aromatic ring for selectivity. High levels of (-)-solanacol ( 5) and (-)-solanacyl acetate (6) were detected in tobacco root exudates 41 and in tomato root extracts. 43 Notably, tobacco and tomato plants are also hosts for P. ramosa 2a, 38,39 in accordance with the sensitivity of P. ramosa 2a seeds to (-)solanacol ( 5) and (-)-solanacyl acetate (6) shown herein.

The germination stimulation activities of the SL biosynthetic intermediates were also studied (Scheme 1). For all-trans-β-carotene (17), a SL biosynthetic precursor without any D-ring, no germination was recorded, demonstrating that this compound is not bioactive and/or that no enzyme of the SL biosynthesis pathway was sufficiently present in plant parasitic seeds (Figure S2, Supplementary Information). For racemic MeCLA (10) and CL (9), which are SL biosynthetic precursors possessing a D-ring but no C ring and having an A ring substituted with a tertiary hydroxy group, their germination stimulation activities were drastically reduced in comparison with 14 and (±)-GR24. A small difference was observed between P. ramosa 1 and P. ramosa 2a for MeCLA (10) (r EC50 = 0.47), the putative biosynthetic precursor of cannalactone ( 14) [START_REF] Hamzaoui | Proceedings of the 15th World Congress on Parasitic Plants[END_REF] (Figure 5B). A specificity of biological response appeared from the compounds downstream of MeCLA in the biosynthetic pathway. Isothiocyanates (ITCs) as glucosinolatebreakdown products are germination stimulants of P. ramosa seeds secreted in the soil by host plants as Brassicaceae. 31,40,59 As for (±)-MeCLA (10), a small difference of germination was observed between P. ramosa 1 and P. ramosa 2a (0.1 < r EC50 < 1.1 for 2-phenylethyl isothiocyanate). 39,45 ramosa 1)/EC 50 (P. ramosa 2a) for each SL and (±)-contalactone. [START_REF] De Saint Germain | [END_REF] ND = not determined.

On comparing the germination stimulation activities of (±)-GR5 to that of (±)-GR24, the loss of rings A and B led to bioactivity reductions but also lessened the difference between P. ramosa 1 and P. ramosa 2a (r EC50 [(±)-GR5] = 0.10) (Figure 4B). All (±)-SdL analogues were bioactive but less than (±)-GR24 for P. ramosa 1. (±)-SdL118F1 and (±)-SdL127F2 proved to be more bioactive toward P. ramosa 2a while all the other SdLs were less bioactive than cannalactone even if they have high activity for this population. The SdL analogues exhibited EC 50 values varying between 5.6×10 -10 -4.8×10 -8 M for P. ramosa 1, and 6.4×10 -12 -2.1×10 -7 M for P. ramosa 2a. Removal of the A and B rings and replacement by an unsaturated acyclic carbon chain at the C-3a position induced a loss of bioactivity toward P. ramosa 1 for all SdL analogues compared to (±)-GR24. Moreover, no significant difference was observed between "cis" (2ʹR*,4R*) and "trans" (2ʹR*,4S*) SdL analogues toward P. ramosa 1. On the other hand, for P. ramosa 2a seeds, SdL analogues with an acyclic unsaturated carbon chain gave bioactivities similar to that of (±)-GR24. For the genetic group 2a, it was also interesting to find that the (±)-trans-SdL analogues were more active than the (±)-cis-SdL analogues. It was therefore possible to classify SdL analogue bioactivity using the r EC50 diagram (Figure 4B). The most active and selective molecules toward P. ramosa 2a are thus (±)-SdL118F1 (EC 50 = 5.6×10 -10 M, r EC50 = 87.5), (±)-SdL19 (EC 50 = 1.5×10 -10 M, r EC50 = 18.7) and (±)-SdL7 (EC 50 = 2.4×10 -10 M, r EC50 = 10.8). The analogue (±)-SdL19 showed a similar selectivity for the germination of P. ramosa 2a vs. P. ramosa 1 as cannalactone ( 14), and a similar bioactivity toward P. ramosa 2a (EC 50 1.5×10 -10 M 20 vs. 1.0×10 -10 M) (Figure 4A). Consequently, due to the ease of preparation of compound (±)-SdL19 and its specificity, the rest of this study was focused on this compound.

SLs and mimics are sensitive to hydrolysis, giving for (±)-GR24 poorly active or inactive 5hydroxy-3-methylbutenolide (D-OH) 52 and inactive ABC=CHOH tricyclic analogues. 23 Their sensitivity to hydrolysis has been tested routinely in aqueous solutions and compared to that of (±)-GR24, 23,60 even though the assay was not performed at biologically active concentrations.

Therefore, the chemical hydrolysis of (±)-SdL19 in comparison with (±)-GR24 was evaluated in a mixture of ethanol/water at pH 6.8, as previously reported for other SL analogues and mimics. 23,60 Under these conditions, a slightly higher stability of (±)-SdL19 was recorded in S3, Supplementary Information).

comparison with (±)-GR24 [(±)-SdL19 t 1/2 ≈ 120 h, (±)-GR24 t 1/2 ≈ 100 h] (Figure

The Cannalactone Analogue (±)-SdL19 Interacts weakly with the SL Receptor

PrKAI2d3. The PrKAI2d3 protein has been characterized recently as a SL receptor from P. ramosa 1 among five PrKAI2 candidates, and its potent interaction with (±)-GR24 has been demonstrated. 52 The interaction between (±)-SdL19 and PrKAI2d3 was tested using nano differential scanning fluorimetry (nanoDSF). NanoDSF allows an evaluation of the interaction between a receptor protein and a ligand by monitoring the melting temperature of the receptor protein. In nanoDSF, changes in the protein fluorescence (ratio 350 nm/330 nm) are recorded and do not require a dye. This technique can highlight interactions that induce minor conformational changes. Compared to (±)-GR24, incubation with (±)-SdL19 at low concentrations did not affect the melting temperature of PrKAI2d3. However, the use of high concentrations of (±)-SdL19 lowered the inflection point of the melting curve to indicate the binding of the ligand to the protein (Figure S4, Supporting Information). The binding affinity of (±)-SdL19 towards PrKAI2d3 was also studied by intrinsic fluorescence but no Kd value was measurable in comparison with (±)-GR24 (Figure 6). The lower affinity of (±)-SdL19 for PrKAI2d3 in comparison with (±)-GR24 confirmed the nanoDSF data. These results are in accordance with the lower bioactivity of (±)-SdL19 on seeds of P. ramosa 1 in comparison with (±)-GR24. The characterization of SL receptor(s) from P. ramosa 2a is still required and would be applicable to discuss the SAR results presented herein in light of the ligand-protein interactions. the apparent Kd values. The plots represent the mean of two replicates and the experiments were repeated at least three times.

Cannalactone Analogues Efficiently Inhibit Bud Outgrowth in Pea and Arabidopsis, and

Interact with SL Receptor D14 Proteins. The biological activities of (±)-SdL analogues as plant hormones were evaluated using a pea branching assay with the highly branched SLdeficient rms1-10 mutant. 61 All (±)-SdL analogues showed activity at a concentration of 1 µM but were found to be notably less active than (±)-GR24, since they were not significantly bioactive at 10 nM or below (Figure 7, Table S1). This confirms the low specificity of SL reception in the pea in which a wide range of SLs, and their analogues and mimics are bioactive. 23,60 Moreover, (±)-SdL analogues were inactive on the branching of the pea rms3-4 perception mutant (Figure 7A, Table S2). These results suggest that (±)-SdL analogues, such as (±)-GR24, are bioactive SL analogues and inhibit bud outgrowth via the RMS3 receptor, an orthologue of D14 in pea, and not because of toxicity. To investigate potential species differences, the effect of (±)-SdL19 on Arabidopsis shoot branching was examined. Thus, (±)-SdL19 and (±)-GR24 were applied to hydroponically grown max3-11 plants. max3-11 is a SLdeficient mutant. 62 Treatment with all concentrations of (±)-SdL19 inhibited outgrowth of axillary buds in max3-11. However, a significant difference between (±)-SdL19 and (±)-GR24 was observed at the low concentrations tested (< 3 µM) (Figure 7B). These results indicated that (±)-SdL19 is less active than (±)-GR24 in Arabidopsis for shoot branching inhibition when applied to the roots in a hydroponic culture.

( ± ) -G R 2 4 ( ± ) -S d L 1 9 ( ± ) -S d L 7 ( ± ) -S d L 1 1 8 F 1 C T L 0 W T N T ( ± ) -G R 2 4 ( ± ) -S d L 1 9 ( ± ) -S d L 7 ( ± ) -S d L 1 1 8 F 1 C T L 0
Length of bud/branch (mm) In order to validate that (±)-SdL analogues are perceived by the pea SL receptor RMS3, differential scanning fluorimetry (DSF) was performed and this revealed a shift in RMS3 melting temperature in the presence of (±)-SdL19, corresponding to a protein destabilization as for the other SL bioactive analogues 4 (Figure 8A-B). It was shown that the destabilization of RMS3 by (±)-SdL19 was recorded at higher ligand concentrations in comparison with (±)-GR24, suggesting a lower affinity. For AtD14, an orthologue of D14 in Arabidopsis, DSF recordings revealed an increased sensitivity for (±)-SdL19, inversely for (±)-GR24, as previously reported for cannalactone ( 14) [START_REF] Hamzaoui | Proceedings of the 15th World Congress on Parasitic Plants[END_REF] and in contrast to RMS3 (Figure 8C-D). These results are in accordance with the fact that in pea, the main SLs detected are canonical SLs, 63 while in Arabidopsis, no canonical SL has been detected but only non-canonical SLs or their biosynthetic precursors such as methyl carlactonoate [(+)-MeCLA (10)] and HO-MeCLAs. 13,14 Then, the binding affinity of (±)-SdL19 towards RMS3 and AtD14 was estimated by intrinsic fluorescence and a measurable Kd value (98.65 ± 10.86 μM) was determined only for AtD14 (Figure 6B). The lower affinity of (±)-SdL19 for RMS3 in comparison with AtD14 highlighted differences between biochemical and in planta results where in both Arabidopsis and pea, (±)-SdL19 is less bioactive than (±)-GR24 for bud outgrowth inhibition. Each line represents the average protein melt curve for three technical replicates and the experiment was carried out three times.

The Cannalactone Analogue (±)-SdL19 Promotes Hyphal Branching in the Arbuscular

Mycorrhizal (AM) Fungus Rhizophagus irregularis. The SL analogues produced in the present study can also help to address SAR requirements in AM symbiosis. SLs are known to induce a number of developmental, cellular and metabolic responses in AM fungi. [34][35][36] In particular, the stimulation of hyphal branching is commonly used in bioassays to detect the bioactivity of SLs on these fungi. Using this approach, a number of SLs and related compounds have been tested

for their activity on the AM fungus Gigaspora margarita. Taken together, these studies highlighted the complexity of structural requirements in the rings ABC moiety. GR5 was shown to be inactive, and early studies proposed that a minimal structure would contain the ring C-D moiety fused to at least one ring system. 35,64 This proposal is consistent with the recent discovery of bioactive bryosymbiol (13) carrying A, C and D rings. 7 However, the results obtained with CL derivatives later indicated that some compounds without a C ring could retain activity. 26 Combined with reports on natural non-canonical SLs bearing variations on the A or C rings, 6 these studies indicate that requirements for bioactivity on AM fungi can be fulfilled in several divergent ways.

In the present study, an in vitro assay was first used, which allowed a quantification of hyphal branching 65 in the AM fungus Rhizophagus irregularis. As shown in Figure 9A, (±)-SdL19 stimulated hyphal branching (i.e., the formation of first-order and higher-order branches) in a similar fashion to (±)-GR24. This indicates that a ring C-D moiety combined with an acyclic carbon chain is sufficient for bioactivity against this AM fungus.

The hyphal branching response triggered by SL analogues is used commonly as a proxy measure for the stimulation of the fungal symbiont, but this response does not always reflect the ability of these compounds to facilitate the initiation of symbiosis. 65 To further document the ability of (±)-SdL19 to stimulate R. irregularis in a true symbiotic context, an additional test was used. This assay involves Medicago truncatula plants mutated in the SL biosynthesis gene CCD8. These mutants are strongly affected in their ability to initiate symbiosis with R. irregularis, and this phenotype can be rescued by exogenous application of SL analogues. 65 Like other bioassays involving SL biosynthesis plant mutants and designed to investigate the hormonal activities of SL analogues, this test records the ability of these analogues to "replace" natural SLs in a physiological context. Using this assay, the bioactivity of cannalactone ( 14) was previously shown in AM symbiosis, [START_REF] Hamzaoui | Proceedings of the 15th World Congress on Parasitic Plants[END_REF] indicating that the B ring is dispensable. In the present study, (±)-SdL19 exhibited an activity similar to (±)-GR24 (Figure 9B). This extends previous observations by showing that symbiotic activity can be conserved when both the A and B rings are absent and replaced by an unsaturated acyclic carbon chain. In conclusion, with this study comprehensive SAR data were provided for two genetic groups of P. ramosa, comprising routes for synthesis of 14 analogues (SdL) of the non-canonical SL cannalactone (14). The SdL analogues with the best specificity towards the population 2a were found to be the compounds with a polar moiety on the side carbon chain. In addition to noncanonical SL cannalactone ( 14) recently identified from hemp by our group, [START_REF] Hamzaoui | Proceedings of the 15th World Congress on Parasitic Plants[END_REF] the natural canonical SLs (-)-solanacol ( 5) and (-)-solanacyl acetate (6) exuded by tobacco 41,42 and tomato, 66 host plants for P. ramosa 2a, 38,39 are reported here as potent selective germination stimulants for P. ramosa 2a seeds, contrary to other SLs such as orobanchol (2), orobanchyl acetate (3), and 5deoxystrigol (4), also detected in tobacco root exudates. 42 The present data further extend basic knowledge on SL analogues that are involved in plant and AM fungi development. Further studies on the interaction of those substances with specific receptors in AM fungi, key players for a sustainable agriculture, and in P. ramosa 2a, will help to fully resolve the structure-activity relationships in these species. 65 Moreover, these highly active non-canonical SLs [e.g., (±)-SdL19] could be useful for the study of other organisms which putatively produce only noncanonical SLs such as the moss Physcomitrium patens 6,67,68 and Arabidopsis thaliana. 14

EXPERIMENTAL SECTION

General Experimental Procedures. IR spectra were recorded on a PerkinElmer Spectrum 100 FT-IR spectrometer, in reciprocal centimeters (cm -1 ). Nuclear magnetic resonance spectra

( 1 H; 13 All separations were carried out under flash-chromatographic conditions on silica gel (prepacked column, 230 -400 mesh) at medium pressure (20 psi) with an Armen fraction collector and pump. Buffers and aqueous mobile phases for UPLC were prepared using water purified with a Milli-Q system.

All-trans-β-carotene ( 17) is commercially available from Sigma-Aldrich. (±)-GR24 was prepared according to known procedures 69 and careful purification (HPLC). [START_REF] De Saint Germain | [END_REF] (±)-GR5 was synthesized by known procedures. 22 (±)-MeCLA ( 10) is available via the synthesis described in Abe et al. 70 (±)-Carlactone (9) was synthesized by Adrian Scaffidi. 71 Natural cannalactone (14) was isolated as described in Hamzaoui et al. [START_REF] Hamzaoui | Proceedings of the 15th World Congress on Parasitic Plants[END_REF] (±)-Strigol ( 1), (±)-5-deoxystrigol ( 4), (±)sorgolactone ( 5), (±)-orobanchol (2) are commercially available from OlChemIm. Orobanchyl acetate (3) was synthesized from (±)-orobanchol by acetylation with acetic anhydride in pyridine as described in Boutet et al. 72 (-)-Solanacol ( 5) and (-)-solanacyl acetate (6) are obtained as described in Boyer et al. 23 and (±)-7,8-desmethyl solanacol ( 15) and (±)-7,8-desmethyl solanacyl acetate (16) as described in Chen et al. 73 13 The combined organic layers were dried with Na 2 SO 4 , filtered, and evaporated under reduced pressure to afford crude product 22a as a brown oil (381.8 mg, 96%). This was used in the next step without further purification but can also be purified by chromatography on silica gel Preparation of Cyclopropylmagnesium Bromide. 75 To a stirred suspension of magnesium turnings (300 mg, 12.30 mmol) in dry THF (2.0 mL) under argon was added commercially available cyclopropyl bromide (0.800 mL, 9.99 mmol) in dry THF (12.2 mL) dropwise at room temperature for 30 min. During this operation, the reaction continued refluxing without heating.

After the addition of the halide was completed, the mixture was stirred at room temperature for 5 h. The titration was performed as described in Sugano et al. 75 (E)-4- previously. 81 The rms1-10 (M3T-884) and rms3-4 (M2T-30) mutants were obtained from the dwarf cv Térèse. Plants were grown in a greenhouse under long days as described in Braun et al. 82 All Arabidopsis thaliana plants used in this study originated from the Columbia (Col-0) ecotype background and have been described previously: max3-11. 62 Plants were grown as described in Cornet et al. 83 for a hydroponic assay (see also below).

(E)-4-(3-Methyl-3-[(trimethylsilyl)oxy]but-1-en-1-yl)dihydrofuran-2(3H)-one (23a

(E)-4-(3-Cyclopropyl-3-[(trimethylsilyl)oxy]but-1-en-1-yl)dihydrofuran-2(3H)-one (23b

Two batches of parasitic plant seeds were used in this study. A population of seeds of Phelipanche ramosa associated to genetic group 1 (P. ramosa 1) was collected from Saint Martin-de-Fraigneau, France, on broomrape parasitizing winter rapeseed (Brassica napus) in June 2015. Seeds of P. ramosa from genetic subclade 2a (P. ramosa 2a) were harvested at Saint Martin-de-Bossenay, France, on broomrape developed on hemp (Cannabis sativa) in August 2012. 38 Seeds were surface sterilized and conditioned according to Pouvreau et al. 58 (dark condition; 21 °C).

Pea Shoot Branching Assay. The compounds to be tested were applied directly to the axillary bud with a micropipette as 10 µL of a solution containing 0.1% DMSO with 2% polyethylene glycol 1450, 50% ethanol and 0.4% DMSO. 23,[START_REF] Muñoz | In Strigolactones: Methods and Protocols[END_REF] The control-0 is the treatment with 0.1% DMSO without compound. Altogether, 24 plants were sown per treatment in trays (2 repetitions of 12 plants). The treatment was generally performed 10 days after sowing, on the axillary bud at node 3. The branches at nodes 1 to 2 were removed to encourage the outgrowth of axillary buds at the nodes above. Nodes were numbered acropetally from the first scale leaf as node 1 and cotyledonary node as node 0. Bud growth at node 3 was measured with digital callipers 8 to 10 days after treatment. Plants with a damaged main shoot apex or showing a dead white treatedbud were excluded from the analysis. The SL-deficient rms1-10 and SL-reception rms3-4 pea mutants were used for all experiments and WT Térèse was used as control.

Hydroponic Assay on Arabidopsis. The hydroponic assay was adapted from Cornet et al. 83 Seeds were surface-sterilized for 8 min in a solution of ethanol (95%)-Bayrochlore (Bayrol, Mundolsheim, France) (10%), and were rinsed twice with ethanol (100%). Each seed was sown on top of a cut 0.5 mL Eppendorf tube filled with agar medium containing 0.65% agar and 10% nutritive solution 5 mM NO 3 . Tubes were soaked in water and stored in the dark at 4 °C for 2 days. Twelve plants per pipette tip box (13 × 9 × 7 cm) were grown and supplied with nutrient solution as in Boyer et al. 60 at a concentration of 5 mL.L -1 (750 mL of solution per box). Each week the nutrient solution was renewed and every ten days when the molecules were added into the solution. The first treatment occurred at day 27 after sowing when plants started to bolt. The number of rosette branches was performed at day 42.

Germination Stimulation Activity Assay on Root Parasitic Plant Seeds. Germination stimulant activity (GS) of chemicals on seeds of parasitic plants was determined using a method described previously. 58,[START_REF] Pouvreau | In Strigolactones: Methods and Protocols[END_REF] Chemicals were suspended in DMSO, except all-trans-β-carotene (17) in THF, at 10 mmol.L -1 , then diluted with water at 1 mmol.L -1 (water/DMSO; v/v; 9/1). Dilutions of 1×10 -5 mol.L -1 to 1×10 -12 mol.L -1 were then performed in water/DMSO (v/v; 9/1). For each compound, a range of concentrations from 10 -13 to 10 -6 mol.L -1 (water/DMSO; 99/1) was applied to the conditioned parasitic seeds. DMSO (1%) was used as a negative control (seed germination < 1%) and (±)-GR24 at a concentration of 1 µmol.L -1 was used as a positive control and induced 72-87% of seed germination for P. ramosa 1 and 80-90% for P. ramosa 2a. To avoid variations related to sterilization events percentages of germination are reported as a ratio relative to the positive control [(±)-GR24, 1 µmol L -1 ] included in each germination assay. Each dilution and germination assay was repeated at least three times. For each compound tested, dose-response curves [Germination Stimulation = f(c), Germination Stimulant activity relative to (±)-GR24 1 µmol.L -1 ; c : concentration (mol.L -1 )], half maximal effective concentration (EC 50 ), and maximum of germination stimulant activity were determined using a Four Parameter Logistic Curve computed with SigmaPlot ® 10.0.

Fungal Material. Rhizophagus irregularis spores (strain DAOM197198) were purchased from Agronutrition (France). They were rinsed twice with sterile water before use.

Analysis of Hyphal Branching in Vitro.

Experiments were carried out as described in Taulera et al. 65 Spores were placed on plates containing M medium [START_REF] Bécard | [END_REF] and supplemented with SL analogues (100 nM.L -1 ), or 0.1% DMSO for mock treatments. Plates were incubated at 30 °C under 2% CO 2 for 12 days. Germ tubes were then identified as the longest hypha coming out of each spore. The number of hyphal branches of the 1 st order (growing from the germ tube) and higher order (growing from 1 st -order branches) was scored using a dissecting microscope for each germinated spore. The average number of branches of each order was calculated for all the germinated spores on a plate (typically 25 to 35 spores). For statistical analysis, each plate was

Figure 2 .

 2 Figure 2. Cannalactone analogues Types I and II designed in this work. Numbering is provided

Scheme 2 .

 2 Scheme 2. Preparation of Type-I Cannalactone Analogues: (±)-SdL4F1, (±)-SdL4F2 and

  of alcohols 22a,b in neat trimethylsilyl imidazole furnished the protected derivatives 23a,b in 70% yield, which led by formylation and coupling with D-Br 20 to a separable mixture of diastereomers in moderate yields (34-54%) [(±)-SdL18F1/(±)-SdL18F2, (±)-SdL118F1/(±)-SdL118F2]. A mild deprotection of the separated silylated derivatives [(±)-SdL18F1 and (±)-SdL18F2] with catalytic Lewis acid [Sc(OTf) 3 ] and H 2 O 56 led to both alcohols

Scheme 3 .q

 3 Scheme 3. Preparation of Type-II Cannalactone Analogues (±)-SdL18F1, (±)-SdL18F2, (±)-

HCO 2 13 Figure 3 .

 2133 Figure 3. ORTEP view of the asymmetric unit of (±)-SdL50 (solvent molecule omitted) with

Figure 4 .BFigure 5 .

 45 Figure 4. Bioactivity on Phelipanche ramosa 1 vs. Phelipanche ramosa 2a seed germination of

Figure 6 . 3 K

 63 Figure 6. Biochemical analysis of the interaction between the PrKAI2d3, RMS3 and AtD14

Figure 7 .

 7 Figure 7. Bioactivity in pea and Arabidopsis. Bioactivity in pea of (±)-SdL7, (±)-SdL19 and (±)-

Figure 8 .

 8 Figure 8. Biochemical analysis of the interaction RMS3/PsD14, AtD14 proteins with (±)-GR24

Figure 9 .

 9 Figure 9. Activity of (±)-SdL19 on the development of R. irregularis and its symbiotic ability.

  C NMR (75 MHz, CDCl 3 ) δ 197.5 (C-2), 175.4 (C-8), 142.9 (C-6) 132.5 (C-7), 71.4 (C-5), 38.6 (C-4), 34.0 (C-3), 27.8 (C-9); UPLC-TQD (MS) m/z 155.2 ([M + H] + , 100%).(E)-4-(3-Hydroxy-3-methylbut-1-en-1-yl)dihydrofuran-2(3H)-one (22a). Ground CeCl 3 wasdried by heating at 140 °C/30 rpm for 2 h. THF (20 mL) was added to anhydrous CeCl 3 (800.0 mg, 3.24 mmol, 1.4 equiv.) at 0 °C. The mixture was stirred overnight under argon at room temperature. At this temperature, a solution of ketone 21 (357.3 mg, 2.32 mmol) in THF (11.5 mL) was added and stirred for 1 h. Then it was cooled to -70 °C and a solution of methylmagnesium chloride in THF (1.1 mL (3 M), 3.24 mmol, 1.4 equiv.) was added by syringe.The reaction mixture was stirred until TLC indicated disappearance of starting material. The reaction was quenched with an aqueous 10% acetic acid solution (20 mL) and extracted with EtOAc (3 × 10 mL). The organic layer was washed with water (2 × 10 mL) and brine (10 mL).

( 1 (

 1 heptane-EtOAc, 40:60) to afford pure alcohol 22a as an oil: IR (film) ν max 3446, 2973, 2921, 1774, 1677, 1464, 1420, 1374, 1170, 1043, 1015, 975, 908, 839, 796, 690 cm -1 ; 1 H NMR (300 MHz, CDCl 3 ) δ 5.75 (1H, dd, J = 1.0, 15.5 Hz, H-6), 5.59 (1H, dd, J = 8.0, 15.5 Hz, H-7), 4.41 (1H, dd, J = 8.0, 10.0 Hz, H-5a), 3.97 (1H, t, J = 8.5 Hz, H-5b), 3.19 (1H, sext, J = 8.5, H-4), 2.65 (1H, dd, J = 8.5, 17.5 Hz, H-3a), 2.34 (1H, dd, J = 9.5, 17.5 Hz, H-3b), 1.30 (6H, s, H-9 and H-10); 13 C NMR (75 MHz, CDCl 3 ) δ 176.7 (C-2), 141.3 (C-7), 124.5 (C-6), 72.8 (C-8), 70.7 (C-5), 38.8 (C-4), 34.8 (C-3), 30.0 (C-9 or C-10), 20.4 (C-9 or C-10); UPLC-TQD (MS) m/z 93.100%), 153.2 ([M -H 2 O] + , 90%).

( 3 -

 3 Cyclopropyl-3-hydroxybut-1-en-1-yl)dihydrofuran-2(3H)-one (22b). Ground CeCl 3 (455.5 mg, 1.80 mmol, 1.4 equiv.) was dried by heating at 140 °C/30 rpm for 2 h. Dry THF(11 mL) was added at 0°C. The mixture was stirred overnight under argon at room temperature. At this temperature, a solution of ketone 21 (204.8 mg, 1.32 mmol) in 4 mL of dry THF was added and left to stir for 1 h. Then it was cooled to -70 °C and cyclopropylmagnesium bromide (5.6 mL, 0.32 M, 1.80 mmol, 1.4 equiv.) was added by syringe. The reaction mixture was left to stir until TLC indicated that no starting material remained. The reaction was quenched with an aqueous 10% acetic acid solution (10 mL) and extracted with EtOAc (3 × 10 mL). The organic layer was washed with water (2 × 10 mL) and brine (10 mL). The combined organic layers were dried (Na 2 SO 4 ). Solvent was removed to afford crude product 22b (266.4 mg, quantitative), which was used in the next step without further purification, but also could be purified by chromatography on silica gel (heptane-EtOAc, 40:60) to afford pure alcohol 22b as an oil: IR (film) ν max 3446, 2921, 2851, 1777, 1462, 1423, 1373, 1171, 1045, 1016, 977, 737 cm -1 ; 1 H NMR (300 MHz, CDCl 3 ) δ 5.64 (1H, s, H-7), 5.62 (1H, s, H-6), 4.40 (1H, t, J =8.5 Hz, H-5a), 3.97 (1H, t, J = 8.0 Hz, H-5b), 3.19 (1H, sext, J = 8.0 Hz, H-4), 2.64 (1H, dd, J = 8.5, 17.5 Hz, H-3a), 2.33 (1H, dd, J = 9.0, 17.5 Hz, H-3b), 1.23 (3H, s, H-9), 1.01-0.92 (1H, m, H-10), 0.40 (2H, d, J = 8.0 Hz, H-11 or H-12), 0.28 (2H, d, J = 5.5 Hz, H-11 or H-12); 13 C NMR (75 MHz, CDCl 3 ) δ 176.6 (C-2), 139.0 (C-7), 125.8 (C-6), 72.8 (C-5), 71.6 (C-8), 38.9 (C-4), 34.9 (C-3), 27.5 (C-10), 21.6 (C-9), 1.1 (C-11 or C-12), 0.97 (C-11 or C-12); HRESIMS m/z 197.1180 [M + H] + (calcd for C 11 H 17 O 3 , 197.1178).

; 1 H

 1 NMR (300 MHz, CDCl 3 ) δ 5.66 (1H, dd, J = 1.0, 15.5 Hz, H-6), 5.46 (1H, dd, J = 8.0, 15.5 Hz, H-7), 4.38 (1H, dd, J = 8.0, 9.0 Hz, H-5a), 3.95 (1H, t, J = 9.0 Hz, H-5b), 3.16 (1H, sext, J = 8.5 Hz, H-4), 2.62 (1H, dd, J = 8.0, 17.5 Hz, H-3a), 2.31 (1H, dd, J = 9.5, 17.5 Hz, H-3b), 1.26 (6H, s, H-9 and H-10), 0.07 (9H, s, TMS); 13 C NMR (75 MHz, CDCl 3 ) δ 176.7 (C-2), 142.2 (C-7), 123.8 (C-6), 73.2 (C-8), 72.8 (C-5), 38.9 (C-4), 34.7 (C-3), 30.5 (C-9 and C-10), 2.7 (TMS); UPLC-TQD (MS) m/z 153.3 ([M -OTMS] + , 100%), 93.2 (90%).

; 1 H

 1 NMR (300 MHz, CDCl 3 ) δ 5.62 (1H, d, J = 15.5 Hz, H-6), 5.52 (1H, dd, J = 7.5, 15.5, H-7), 4.40 (1H, dd, J = 8.0, 9.0, H-5a), 3.97 (1H, t, J = 8.5, H-5b), 3.18 (1H, sext, J = 8.0, H-4), 2.64 (1H, dd, J = 8.5, 17.5 Hz, H-3a), 2.33 (1H, dd, J = 9.5, 17.5 Hz, H-3b), 1.25 (3H, s, H-9), 0.88-0.78 (1H, m, H-10), 0.35-0.25 (4H, m, H-11 and H-12), 0.06 (9H, s, TMS); 13 C NMR (75 MHz, CDCl 3 ) δ 176.7 (C-2), 140.4 (C-7), 125.0 (C-6), 74.0 (C-8), 72.8 (C-5), 38.9 (C-4), 34.8 (C-3), 27.3 (C-10), 22.2 (C-9), 2.7 (C-11 or C-12), 1.3 (C-11 or C-12); HRESIMS m/z 179.1070 [M -OTMS] + (calcd for C 14 H 24 O 3 Si, 179.1072). (2ʹR*,4R*)-SdL18F1 and (2ʹR*,4S*)-SdL18F2. A mixture of compound 23a (363.4 mg, 1.5 mmol), ethyl formate (0.12 mL, 1.50 mmol, 1.0 equiv.) in THF (3 mL) was stored at -78 °C. Potassium tert-butoxide was added (190.7 mg, 1.70 mmol, 1.13 equiv.). The mixture was stirred for 2 h. A solution of bromide derivative 20 (286.7 mg, 1.60 mmol, 1.1 equiv.) in THF (3 mL) was added. The mixture was stirred at room temperature for 12 h. The mixture was diluted with EtOAc and saturated NH 4 Cl aqueous solution. The organic phase was washed with water and brine. The combined organic layers were dried with Na 2 SO 4 , filtered, and then evaporated under reduced pressure. The mixture was purified by chromatography on silica gel (heptane-EtOAc, 80:20 to 70:30 for 20 min) to afford the products (±)-SdL18F1 and (±)-SdL18F1 as brown oils [(±)-SdL18F1: 100.7 mg (18%) and (±)-SdL18F2: 86.0 mg (16%), total: 34%]. (±)-SdL18F1: IR (film) ν max 2967, 2925, 1785, 1754, 1683, 1380, 1344, 1250, 1183, 1082, 1031, 956, 840, 752 cm -1 ; 1 H NMR (300 MHz, CDCl 3 ) δ 7.50 (1H, d, J = 2.5 Hz, H-6ʹ), 6.82 (1H, t, J = 1.5 Hz, H-3ʹ), 6.08 (1H, t, J = 1.5 Hz, H-2ʹ), 5.64 (1H, d, J = 15.5 Hz, H-7), 5.48 (1H, dd, J = 7.5, 15.5 Hz, H-6), 4.43 (1H, t, J = 9.0 Hz, H-5a), 4.06 (1H, q, J = 4.0 Hz, H-5b), 3.82-3.74 (1H, m, H-4), (1H, d, J = 15.5 Hz, H-7), 5.58 (1H, dd, J = 7.5, 15.5 Hz, H-6), 4.43 (1H, t, J = 9.0 Hz, H-5a), 4.07 (1H, dd, J = 4.5, 9.0 Hz, H-5b), 3.83-3.76 (1H, m, H-4), 1.99 (3H, t, J = 1.5 Hz, H-7ʹ), 1.29 (6H, s, H-9 and H-10); 13 C NMR (75 MHz, CDCl 3 ) δ 171.4 (C-2), 170.2 (C-5ʹ), 151.6 (C-6ʹ), 140.9 (C-3ʹ), 140.3 (C-7), 135.8 (C-4ʹ), 123.7 (C-6), 110.2 (C-3), 100.6 (C-2ʹ), 71.2 (C-5), 70.5 (C-8), 39.3 (C-4), 29.8 (C-9 and C-10), 10.7 (C-7ʹ); HRESIMS m/z 317.

  (±)-Fabacyl acetate(7) was kindly furnished by Koichi Yoneyama. (±)-4-Vinyldihydrofuran-2(3H)-one(19) was prepared by a known procedure.53 butoxide was added (240 mg, 2.14 mmol). The mixture was stirred for 2 h at -78 °C. A solution of bromide derivative 2074 (350 mg, 1.96 mmol) in THF (3.2 mL) was added. The mixture was stirred at room temperature for 12 h. The mixture was diluted with EtOAc and saturated NH 4 Cl aqueous solution and then separated. The organic phase was washed with water and brine. The combined organic layers were dried with Na 2 SO , 979, 890, 838 cm -1 ; 1 H NMR (300 MHz, CDCl 3 ) δ 6.66 (1H, dd, J = 7.5, 15.5 Hz, H-6), 6.17 (1H, dd, J = 1.0, 16.0 Hz, H-7), 4.48 (1H, dd, J = 7.5, 11.5 Hz, H-5a), 4.08 (1H, dd, J = 7.5, 11.5 Hz, H-5b), 3.38 (1H, sext, J =8.0 Hz, H-4), 2.74 (1H, dd, J = 7.5, 16.0 Hz, H-3a),

	General Procedures for Compound Synthesis.
	(2ʹR*,4R*)-SdL4F1 and (2ʹR*,4S*)-SdL4F2. A mixture of alkene 19 (200 mg, 1.78 mmol) and ethyl formate (144 µL, 1.96 mmol) in THF (4 mL) was placed at -78 °C. Potassium tert-5a), 4.07 (1H, q, J = 4.5 Hz, H-5b), 3.83-3.74 (1H, m, H-4), 1.97 (3H, t, J = 1.5 Hz, H-7ʹ); 13 C NMR (75 MHz, CDCl 3 ) δ 171.5 (C-5ʹ), 170.4 (C-2), 151.7 (C-6ʹ), 141.1 (C-3ʹ), 135.9 (C-4ʹ), 135.4 (C-6), 116.7 (C-7), 109.9 (C-3), 100.7 (C-2ʹ), 71.1 (C-5), 40.6 (C-4), 10.9 (C-7ʹ); HRESIMS m/z 237.0762 [M + H] + (calcd for C 12 H 13 O 5 , 237.0763). (±)-SdL4F2: IR (film) ν max 3089, 2917, 2850, 1775, 1747, 1679, 1342, 1181, 1082, 1003, 950 cm -1 ; 1 H NMR (300 MHz, CDCl 2.44 (1H, dd, J = 7.5, 16.0 Hz, H-3b), 2.25 (3H, s, H-9);

4 , filtered, and evaporated under reduced pressure. The mixture was purified by chromatography on silica gel (heptane-EtOAc, 80:20 to 60:40 for 30 min) to afford pure (±)-SdL4F1 (87.5 mg, 21%) and (±)-SdL4F2 (85.7 mg, 20%) as brown oils. (±)-SdL4F1: IR (film) ν max 3092, 2983, 2919, 2850, 1781, 1752, 1683, 1344, 1207, 1184, 1083, 1026, 955, 866, 801, 745 cm -1 ; 1 H NMR (300 MHz, CDCl 3 ) δ 7.51 (1H, d, J = 2.0 Hz, H-10), 6.88 (1H, t, J = 1.5 Hz, H-6ʹ), 6.12 (1H, t, J = 1.5 Hz, H-2ʹ), 5.81-5.70 (1H, m, H-6), 5.12 (1H, dt, J = 1.0, 6.0 Hz, H-7a), 5.08 (1H, d, J = 1.0 Hz, H-7b), 4.41 (1H, t, J = 9.0 Hz, H-3 ) δ 7.48 (1H, d, J = 2.0 Hz, H-10), 6.88 (1H, t, J = 1.5 Hz, H-6ʹ), 6.12 (1H, t, J = 1.5 Hz, H-2ʹ), 5.79-5.68 (1H, m, H-6), 5.10 (1H, dt, J = 1.0, 6.0 Hz, H-7a), 5.06 (1H, d, J = 1.0 Hz, H-1162, 1015

  ). A mixture of alcohol 22a (137 mg, 0.80 mmol) and TMS-imidazole (7.4 mL, 50.39 mmol, 62.6 equiv.) were stirred at 50 °C under argon for 1 h. The mixture was stirred at room temperature for 1 h and diluted with heptane (20 mL). The organic layer was washed with brine (2 × 10 mL), dried with Na 2 SO 4 , filtered, and evaporated under reduced pressure to afford pure product 23a as an oil

(136.2 mg, 70%): IR (film) ν max 2966, 2918, 1783, 1377, 1362, 1250, 1165, 1038, 1019, 972, 839, 755 cm -1

  ). A mixture of alcohol 22b (240.7 mg, 1.23 mmol) and TMS-imidazole (7.22 mL, 49.20 mmol, 40 equiv.) were stirred at 50 °C under argon for 1 h. The mixture was stirred at room temperature for 1 h and diluted with 20 mL of heptane. The organic layer was washed with brine (2 × 10 mL), dried over Na 2 SO 4 and evaporated. The mixture was purified by chromatography on silica gel (4 g, heptane-EtOAc, 80:20) to afford pure product 23b as an oil (222.4 mg, 67%): IR (film) ν max 3007, 2958, 2894, 1785, 1702, 1420, 1372, 1250, 1209, 1165, 1095, 1065, 1018, 975, 887, 839, 754, 685 cm -1

  Potassium tert-butoxide was added (142.5 mg, 1.27 mmol, 2.26 equiv.). The mixture was stirred for 2 h. A solution of bromide derivative 20 (106.2 mg, 0.60 mmol, 1.08 equiv.) in THF to 2θmax = 144°. However, the tiny crystals obtained by dissolving in ethyl acetate (48 electrons)/ heptane (58 electrons), and slow cooling of the solution turned out to be poor diffractors, so a compromise was chosen between reasonable exposure time and exploitable data for model solution. 10732 measured reflections up to the θ value matching the iUCR criteria, and 4562 independent reflections (Rint = 0.138), of which only 687 had |F o | > 4σ (F o ), using the CrystalClear 2.0 suite. Data were corrected for Lorentz and polarization effects, and also for absorption (T min , max : 0.448, 1.000). Despite the small signal detected beyond the 1.4 Å resolution limit, the structure could be readily solved either by dual methods (SHELXD),76 or intrinsic phasing methods (SHELXT program); 77 all other calculations regarding the model refinement based on F using full-matrix least-squares methods with the weight scheme of 1/[σ 2 (F Crystal data for SdL50: C 22 H 20 N 2 O 11 [+solvent], M = 488.40, a = 15.593(5) Å, b = 5.9014(18) Å, c = 29.216(9) Å, α = 90°, β = 94.304(9)°, γ = 90°, V = 2680.8(14) Å 3 , T = 203 (2) K, space

	(2ʹR*,4R*)-SdL33. To a solution of (±)-SdL19 (15.6 mg, 0.05 mmol) in 0.3 mL of CH 2 Cl 2 was amorphous solid): IR (film) ν max 2919, 2850, 1784, 1754, 1722, 1683, 1607, 1527, 1464, 1349, NMR (125 MHz, CDCl 3 ) δ 206.3 (C-11), 171.2 (C-5ʹ), 170.2 (C-2), 152.0 (C-6ʹ), 148.8 (C-12),
	added pyridine (0.09 mL, 1.06 mmol, 20 equiv.), 4-nitrobenzoylchloride (49.2 mg, 0.26 mmol, 5 1289, 1184, 1101, 1032, 1013, 955, 875, 842, 721 cm -1 ; 1 H NMR (300 MHz, CDCl 3 ) δ 8.25 (2H, 141.1 (C-3ʹ), 135.8 (C-7), 132.3 (C-14 and C-14ʹ), 129.5 (C-13 and C-13ʹ), 127.4 (C-6), 122.4
	equiv.), and DMAP (tip of spatula). The mixture was stirred overnight at 40 °C. The mixture was dt, J = 2.0, 8.5 Hz, H-14 and H-14ʹ), 8.10 (2H, dt, J = 2.0, 8.5 Hz, H-13 and H-13ʹ), 7.50 (1H, d, (C-15), 109.9 (C-4ʹ), 100.9 (C-2ʹ), 71.1 (C-8), 67.2 (C-5), 39.5 (C-4), 29.9 (C-3), 27.2 (C-9 or C-group P2 1 /n, Z = 4, μ(Cu Kα) = 0.848 mm -1 , 4848 reflections measured, 1013 independent
	co-evaporated with toluene, extracted with EtOAc, washed with NaHCO 3 and dried with J = 2.5 Hz, H-6ʹ), 6.92 (1H, t, J = 1.5 Hz, H-3ʹ), 6.11 (1H, t, J = 1.5 Hz, H-2ʹ), 5.96 (1H, dd, J = 10), 26.8 (C-9 or C-10), 10.9 (C-7ʹ); HRESIMS m/z 552.1226 [M + CH 3 CN + Na] + (calcd for reflections (R int = 0.1141). The final R 1 values were 0.1010 (I > 2σ(I)). The final wR(F 2 ) values
	0994 [M + Na] + (calcd Na 2 SO 4 , filtered, and evaporated under reduced pressure. The product was purified by 1.0, 15.5 Hz, H-7), 5.60 (1H, q, J = 8.0 Hz, H-6), 4.46 (1H, t, J = 8.5 Hz, H-5a), 4.09 (1H, t, J = C 24 H 23 N 3 O 11 Na, 552.1230). were 0.2686 (I > 2σ(I)). The final R 1 values were 0.1317 (all data). The final wR(F 2 ) values were
	for C 15 H 18 O 6 Na, 317.1001). preparative TLC (heptane-EtOAc, 60:40) to afford pure (±)-SdL33 (18.2 mg, 77%, yellow 7.5 Hz, H-5b), 3.87-3.79 (1H, m, H-4), 2.02 (3H, s, H-7ʹ), 1.24 (6H, t, J = 7.0 Hz, H-9 and H-(2ʹR*,4R*)-SdL51. To a mixture of (±)-SdL19 (21.8 mg, 0.07 mmol) and pyridine (250 µL, 0.3086 (all data). The goodness of fit on F 2 was 1.238.
	(2ʹR*,4R*)-SdL21. To a solution of scandium trifluoromethanesulfonate (0.56 mg, 1.14 µmol) amorphous solid): IR (film) ν max 2919, 2850, 1785, 1755, 1722, 1683, 1607, 1527, 1464, 1349, 10); 13 C NMR (175 MHz, CDCl 3 ) δ 171.3 (C-2), 170.3 (C-5ʹ), 163.5 (C-11), 151.8 (C-6ʹ), 150.6 3.09 mmol, 41.7 equiv.) was added acetic anhydride (250 µL, 2.64 mmol, 35.7 equiv.), and Plant Material. Pea (Pisum sativum) branching mutants used in this study were derived from
	in CH 3 CN (1.3 mL) was added silyl derivative (±)-SdL18F2 (83.5 mg, 0.33 mmol) in CH 3 CN (C-15), 141.2 (C-3ʹ), 136.6 (C-7), 135.4 (C-4ʹ), 130.8 (C-13 and C-13ʹ), 126.6 (C-6), 125.2 (C-1290, 1185, 1102, 1033, 1014, 956, 876, 842, 720 cm -1 ; 1 H NMR (300 MHz, CDCl 3 ) δ 8.26 (2H, various cultivars of pea after ethyl methanesulfonate (EMS) mutagenesis, as described
	(0.29 mL) and water (2.0 µL, 0.11 µmol) at room temperature. The resultant mixture was stirred dt, J = 2.0, 8.5 Hz, H-14 and H-14ʹ), 8.11 (2H, dt, J = 2.0, 8.5 Hz, H-13 and H-13ʹ), 7.51 (1H, d, 12), 123.7 (C-14 and C-14ʹ), 110.1 (C-3), 100.7 (C-2ʹ), 82.2 (C-8), 71.1 (C-5), 39.7 (C-4), 27.1
	for 1 h at room temperature and quenched with a phosphate buffer (pH 7). The organic materials J = 2.5 Hz, H-6ʹ), 6.89 (1H, t, J = 1.5 Hz, H-3ʹ), 6.10 (1H, t, J = 1.5 Hz, H-2ʹ), 5.98 (1H, dd, J = (C-9 or C-10), 27.0 (C-9 or C-10), 10.9 (C-7ʹ); HRESIMS m/z 507.1389 [M + CH 3 CN + Na] +
	were extracted with dichloromethane (3 × 5 mL), and the combined extracts were washed with 1.0, 15.5 Hz, H-7), 5.63 (1H, q, J = 8.0 Hz, H-6), 4.45 (1H, t, J = 8.5 Hz, H-5a), 4.10 (1H, t, J = (calcd for C 24 H 24 N 2 O 9 Na, 507.1380).
	brine, dried with Na 2 SO 4 , filtered, and evaporated under reduced pressure to afford pure (±)-7.5 Hz, H-5b), 3.87-3.79 (1H, m, H-4), 2.02 (3H, s, H-7ʹ), 1.23 (6H, t, J = 7.0 Hz, H-9 and H-(2ʹR*,4S*)-SdL50. To a solution of (±)-SdL21 (34.3 mg, 0.117 mmol) in CH 2 Cl 2 (0.6 mL) was
	SdL21 as an oil (59.7 mg, 89%): IR (film) ν max 3488, 2969, 2925, 1784, 1751, 1682, 1343, 1263, 10); 13 C NMR (175 MHz, CDCl 3 ) δ 171.4 (C-2), 170.3 (C-5ʹ), 163.5 (C-11), 151.8 (C-6ʹ), 150.6 added pyridine (0.189 mL, 2.34 mmol, 20 equiv.), 3,5-dinitrobenzoyl chloride (134.9 mg, 0.58 15.5 Hz, H-7), 5.48 (1H, dd, J = 8.0, 15.5 Hz, H-6), 4.43 (1H, t, J = 8.5 Hz, H-5a), 4.03 (1H, t, J
	1185, 1084, 1026, 1013, 955, 863, 802, 730, 701 cm -1 ; 1 H NMR (300 MHz, CDCl 3 ) δ 7.50 (1H, (C-15), 141.1 (C-3ʹ), 136.4 (C-7), 131.5 (C-4ʹ), 130.7 (C-13 and C-13ʹ), 126.8 (C-6), 123.8 (C-mmol, 5 equiv.), and DMAP (tip of spatula). The mixture was stirred at 40 °C overnight. The = 5.0 Hz, H-5b), 3.83-3.74 (1H, m, H-4), 2.02 (3H, s, H-7ʹ), 1.95 (3H, s, H-12), 1.47 (3H, s, H-9
	d, J = 2.5 Hz, H-6ʹ), 6.89 (1H, t, J = 1.5 Hz, H-3ʹ), 6.11 (1H, t, J = 1.5 Hz, H-2ʹ), 5.68 (1H, d, J = 12), 123.7 (C-14 and C-14ʹ), 110.0 (C-3), 100.6 (C-2ʹ), 82.2 (C-8), 71.2 (C-5), 39.6 (C-4), 27.3 mixture was co-evaporated with toluene, extracted with EtOAc, washed with NaHCO 3 and dried or H-10), 1.45 (3H, s, H-9 or H-10); 13 C NMR (175 MHz, CDCl 3 ) δ 171.4 (C-11), 170.5 (C-2),
	15.5 Hz, H-7), 5.54 (1H, dd, J = 7.5, 15.5 Hz, H-6), 4.43 (1H, t, J = 9.0 Hz, H-5a), 4.04 (1H, dd, J = 4.5, 9.0 Hz, H-5b), 3.82-3.74 (1H, m, H-4), 1.97 (3H, t, J = 1.5 Hz, H-7ʹ), 1.22 (6H, s, H-9 and H-10); 13 C NMR (75 MHz, CDCl 3 ) δ 171.6 (C-2), 170.4 (C-5ʹ), 151.7 (C-6ʹ), 141.1 (C-3ʹ), 140.7 (C-7), 135.9 (C-4ʹ), 123.7 (C-6), 110.6 (C-3), 100.7 (C-2ʹ), 71.4 (C-5), 70.7 (C-8), 39.4 (C-4), 29.9 (C-9 and C-10), 10.8 (C-7ʹ); HRESIMS m/z 317.1000 [M + Na] + (calcd for C 15 H 18 O 6 Na, 317.1001). (C-9 or C-10), 27.1 (C-9 or C-10), 11.0 (C-7ʹ); HRESIMS m/z 466.1131 [M + Na] + (calcd for C 22 H 21 NO 9 Na, 466.1114). (2ʹR*,4S*)-SdL34. To a solution of (±)-SdL21 (15.6 mg, 0.05 mmol) in 0.3 mL of CH 2 Cl 2 was added pyridine (0.09 mL, 1.06 mmol, 20 equiv.), 4-nitrobenzoylchloride (49.17 mg, 0.265 mmol, 5 equiv.), and DMAP (tip of spatula). The mixture was stirred overnight at 40 °C. The mixture was co-evaporated with toluene, extracted with EtOAc, washed with NaHCO 3 and dried with with Na 2 SO 4 . The product was purified by preparative TLC (heptane-EtOAc, 45:55) to afford pure (±)-SdL50 as a white solid (17.7 mg, 31%): IR (film) ν max 2985, 1737, 1447, 1373, 1301, 1233, 1098, 1043, 938, 918, 847, 786 cm -1 ; 1 H NMR (300 MHz, CDCl 3 ) δ 9.19 (1H, t, J = 2.0 Hz, H-15), 9.04 (2H, d, J = 2.0 Hz, H-13 and H-13ʹ), 7.52 (1H, d, J = 2.5 Hz, H-6ʹ), 6.94 (1H, t, J = 1.5 Hz, H-3ʹ), 6.12 (1H, t, J = 1.5 Hz, H-2ʹ), 5.96 (1H, dd, J = 1.0, 16.0 Hz, H-7), 5.65 (1H, dd, J = 7.5, 15.5 Hz, H-6), 4.46 (1H, t, J = 9.0 Hz, H-5a), 4.08 (1H, t, J = 5.0 Hz, H-5b), 3.89-170.0 (C-5ʹ) 151.9 (C-6ʹ), 141.4 (C-3ʹ), 137.3 (C-7), 135.8 (C-4ʹ), 125.8 (C-6), 109.7 (C-3), 100.7 (C-2ʹ), 80.0 (C-8), 71.3 (C-5), 39.6 (C-4), 27.3 (C-9 or C-10), 27.1 (C-9 or C-10), 14.4 (C-12), 10.9 (C-7ʹ); HRESIMS m/z 359.1109 [M + Na] + (calcd for C 17 H 20 O 7 Na, 359.1107). (2ʹR*,4R*)-SdL118F1 and (2ʹR*,4S*)-SdL118F2. A mixture of compound 23b (150.8 mg, 0.56 mmol), ethyl formate (0.09 mL, 1.12 mmol, 2 equiv.) in THF (1.2 mL) was placed at (C-H = 0.94-1.00 Å) and refined as riding on their parent atoms, with Uiso(H) values constrained to 1.2 Ueq(C) or 1.5 Ueq(Cmethyl). Molecular graphics were computed with MERCURY. 80 Crystallographic data for the reported structure have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 2082797. -78°C. restraints throughout the structure, while hydrogen atoms were placed in calculated positions Copies of the data can be obtained free of charge by request to CCDC, 12 Union Road,
	Na 2 SO 4 , filtered, and evaporated under reduced pressure. The product was purified by
	preparative TLC (heptane-EtOAc, 60:40) to afford pure (±)-SdL34 (6.8 mg, 25%, yellow
	40

3.81 (1H, m, H-4), 2.02 (3H, s, H-7ʹ), 1.65 (3H, s, H-9 or H-10), 1.62 (3H, s, H-9 or H-10);

13

C DMAP (tip of spatula). The mixture was stirred at room temperature under argon overnight. The product was co-evaporated with toluene and purified by preparative TLC (heptane-EtOAc, 45:55) to afford pure (±)-SdL51 as an oil (10.2 mg, 41%): IR (film) ν max 2985, 1737, 1447, 1373, 1301, 1234, 1097, 1043, 938, 918, 847, 786 cm -1 ; 1 H NMR (300 MHz, CDCl 3 ) δ 7.51 (1H, d, J = 2.5 Hz, H-6ʹ), 6.95 (1H, t, J = 1.5 Hz, H-3ʹ), 6.09 (1H, t, J = 1.4 Hz, H-2ʹ), 5.83 (1H, dd, J = 1.0, o ) + 0.2(F o 2 )] were performed with SHELXL system 77 and PLATON 78 programs, keeping meaningful X-ray diffraction. A potential solvent accessible region with disordered electron density was detected within the crystal structure. SQUEEZE 79 was used to model the unresolved electron density likely resulting from disordered crystallizing solvent molecules, representing a total of 44 electrons per unit cell. This contribution was not included in the crystal data. All the nonhydrogen atom displacements were refined in anisotropic manner applying RIGU(sd 0.02) Cambridge CB 1EZ, U.K. [fax: int. +44(0) (1223) 336 033); e-mail: deposit@ccdc.cam.ac.uk].

Type II cannalactone analogue

R 2 OR 3

st order branches Higher-order branches

We are grateful to Hemp it adn for financial support. The IJPB benefits from the support of Saclay Plant Sciences-SPS (ANR-17-EUR-0007). This work was supported by IJPB's Plant Observatory technological platforms. A.d.S.G. received support of the EU through the Marie-Curie FP7 COFUND People Programme, through an AgreenSkills/AgreenSkills+ fellowship and from Saclay Plant Sciences-SPS (ANR-17-EUR-0007) through a fellowship. The work of S.R. and Q.T. was supported by the TULIP LabEx (ANR-10-LABX-41). The CHARM3AT LabEx program (ANR-11-LABX-39) is also acknowledged for support. The authors thank Catherine Rameau for statistical analyses and Adrian Scaffidi for the generous gift of a sample of (±)carlactone. The authors thank Jean-Marie Beau, Catherine Rameau and Sandrine Bonhomme for commenting on the manuscript.

fluorescence signal was measured as a function of increasing temperature with a Prometheus NT.48 fluorimeter (NanoTemper Technologies), with 55% excitation light intensity and 1 °C/min temperature ramp. Analyses were done on capillaries filled with 10 µL of the respective samples. Intrinsic fluorescence signals expressed by the 350 nm/330 nm emission ratio that increased as the proteins unfolded, were plotted as a function of temperature (Figure S4A and Figure S4B, Supplementary Information). The plots showed one of three independent data collections performed for each protein.

Intrinsic Tryptophan Fluorescence Assays and Determination of the Dissociation

Constant K D .. These experiments were performed as described previously in de Saint Germain et al. 61 using a Spark Multimode Microplate Reader (Tecan).

Hydrolysis of (±)-SdL19 and (±)-GR24 in Aqueous Solution. (±)-GR24 and (±)-SdL19 were tested for their chemical stability in an aqueous solution. Aqueous solutions of the compound to be tested (50 μg/mL) were incubated at 22 °C in the HPLC vials. The compounds were first dissolved in DMSO (2 mg/mL). Then, 25 μL of these solutions [(±)-GR24 and (±)-SdL19] were diluted to the final concentration with H 2 O (750 μL) and EtOH (175 μL) and the solution adjusted to pH 6.8. Aqueous solutions of the compounds to be tested (50 μg/mL) were incubated at 22 °C in the HPLC vials. (±)-1-Indanol (25 μL of a 1 mg/mL solution in DMSO) was added as internal standard to each solution. The samples were subjected to reverse-phase-ultraperformance liquid chromatography (RP-UPLC)-MS analyses by means of UPLC system equipped with a PDA and a TQD mass spectrometer (Acquity UPLC-TQD, Waters). RP-UPLC (HSS C 18 column, 1.8 µm, 2.1 mm × 50 mm) involved 0.1% (v/v) formic acid in CH 3 CN and 0.1% (v/v) formic acid in water (aq. FA, 0.1%, v/v, pH 2.8) as eluents [10% CH 3 CN, followed by linear gradient from 10% to 100% of CH 3 CN (4 min)] at a flow rate of 0.6 mL.min -1 . The detection was done using a PDA and with the TQD mass spectrometer operated in an electrospray ionization-positive mode at 3.2 kV capillary voltage. To maximize the signal, the cone voltage and collision energy were optimized to 20 V and 12 eV, respectively. The collision gas was argon at a pressure maintained near 4.5 10 -3 mBar. The relative quantity of remaining (non degraded) product was determined by integration in comparison with the internal standard.

Statistical Analyses. Since deviations from normality were observed for axillary bud length after SL treatment, the Kruskal-Wallis test was used to compare treatments using R Commander version 1.7-3. 89 For bioassays with Arabidopsis thaliana, data were analyzed with the Shapiro-Wilkinson normality test. For bioassays with AM fungi, data were analyzed using Statgraphics Centurion software (SigmaPlus). Non-parametric tests were used because normality or homoscedasticity criteria were not met. Datasets were analyzed using the Kruskal-Wallis test, followed by pairwise comparisons with Mann-Whitney U test.

ASSOCIATED CONTENT

The Supporting Information is available free of charge at https://pubs.acs.org/doi/xxx. Bioactivity data (supplementary data for Figure 7, Tables S1-S2, Figure S1-S2), stability of SdL19 (Figure S3), nanoDSF data (Figure S4) and 1 H and 13

AUTHOR INFORMATION

Notes

The authors declare no competing financial interest. ACKNOWLEDGMENT