Roberto Dallocchio

Barbara Sechi

Alessandro Dessì

Bezhan Chankvetadze

Sergio Cossu

Dr Victor Mamane
email: vmamane@unistra.fr

Robin Weiss

Patrick Pale

Dr Paola Peluso
email: paola.peluso@cnr.it

Enantioseparations of polyhalogenated 4,4'-bipyridines on polysaccharide-based chiral stationary phases and molecular dynamics simulations of selector-selectand interactions

Keywords: Bipyridines / electrostatic potential / enantiomer elution order / molecular dynamics / polysaccharide-based chiral stationary phases CSP, chiral stationary phase, DFT, density functional theory, EEO, enantiomer elution order, ESH, explicit σ-hole, MD, molecular dynamics, MP, mobile phase, NP, normal phase, TCIBP, 3,3',5,5

Introduction

In chiral chromatography, the basic components of the recognition process are chiral analyte, chiral stationary phase (CSP), and mobile phase (MP) [1]. In this molecular environment, the chromatographic separation process originates from consecutive single adsorption and desorption steps occurring on the CSP surface as the analyte moves along the column [2,3]. Intermolecular noncovalent interactions play a pivotal role in this process, and hydrogen bonds (HBs), halogen bonds (XBs), dipole-dipole, π-π stacking, steric repulsive, and van der Waals interactions underlie the adsorption process and the formation of transient diastereomeric assemblies between the chiral selector and the enantiomer pair [4]. The overall stereoselective contact between chiral selector and enantiomer originates from the sum of single noncovalent interactions, which is defined as steric, electrostatic or hydrophobic depending on the structural and electronic features of the interacting partners. MP polarity impacts the overall process, affecting electron density distribution and associated electrostatic potential (V) of the recognition sites [4,5] and, consequently, noncovalent interaction strength. A CSP represents a diffuse chirotopic environment. Indeed, as stated by Hirschmann and Hanson, chirality "is an all-pervasive property, as it affects all parts of a chiral structure" [6]. In the same perspective, Mislow and Siegel defined chirotopic "any atom, and, by extension, any point or segment of the molecular model [...] that resides within a chiral environment" [7]. On this basis, all sites of a CSP are in principle potentially able to participate in enantioselective contacts, contributing to enantiomer discrimination. This concept is particularly true for CSP with high density of chiral elements such as polysaccharide-based CSPs. Indeed, in addition to the presence of a large number of chiral centers, these polymeric CSPs are characterized by conformational chirality dependent on the helical twist generated by the specific glycosidic β-and α-1,4-linkages in cellulose and amylose chain, respectively [8]. Thus, a number of noncovalent interactions can potentially occur into the polymeric groove but, actually, only some of them act to recognize the enantiomers of a given chiral analyte, depending on its particular structure, size and shape, the sum of geometry and electronic distribution. Given this context, subtle variations of analyte and CSP structures, and MP polarity may deeply impact retention and enantioseparation on polysaccharide carbamate-based CSPs. For this reason, with the aim to detect noncovalent interactions and recognition patterns, molecular design can be fruitfully used to obtain specific structures suitable for recognition studies in liquid-phase environment. In this frame, we recently demonstrated that the structure of the 2'substituent has a pivotal impact on the enantioseparation of 2'-substituted TCIBPs (TCIBP = 3,3',5,5'-tetrachloro-2-iodo-4,4'-bipyridyl) (Fig. 1) on amylose-based CSPs [3]. Following this study, we report herein the comparative enantioseparation of both 2'-(4-pyridyl)-( 1) and 2'-(4-hydroxyphenyl)-TCIBPs (2) on four cellulose phenylcarbamate-based CSPs aiming to explore the effect of the polymer backbone, as well as the nature and position of substituents on the side groups/moieties on enantioseparability of these compounds. This issue is of interest because recently compounds 1 and 2 showed relevant inhibition activity against transthyretin fibrillation in vitro, the M-enantiomer being more effective than the P-enantiomer [9]. In the frame of this study, the impact of subtle variations of analyte and cellulose-based CSP structures, and MP polarity on retention and selectivity was evaluated. The effect of temperature on retention and selectivity was also considered, and overall thermodynamic parameters associated with the analyte adsorption onto the CSP surface were derived from van't Hoff plots. Finally, a theoretical investigation based on molecular dynamics (MD) simulations [10] was performed by using amylose and cellulose tris(3,5-dimethylphenylcarbamate) (A-3,5diMe and C-3,5diMe) nonamers, as virtual models of the polysaccharide-based selectors, with the aim of exploring the origin of the enantiodiscrimination processes at the molecular level.

Materials and methods

Chemicals

Compounds 1 and 2 were synthesized, purified and characterized as reported [9].

Chromatography

An Agilent Technologies (Waldbronn, Germany) 1100 Series HPLC system (highpressure binary gradient system equipped with a diode-array detector operating at multiple wavelengths (220, 254, 280, 360 nm), and a 20 μl loop) was employed. Data acquisition and analyses were carried out with Agilent Technologies ChemStation Version B.04.03 chromatographic data software. The UV absorbance is reported as milliabsorbance units (mAU). Lux Cellulose-1 (coated) (cellulose tris (3,5-

dimethylphenylcarbamate) (C-3,5diMe)), Lux Cellulose-2 (coated) (cellulose tris(3- chloro-4-methylphenylcarbamate) (C-3Cl,4Me)), Lux Cellulose-4 (coated) (cellulose tris(4-chloro-3-methylphenylcarbamate) (C-4Cl,3Me
)), and Lux i-Cellulose-5 (immobilized) (cellulose tris(3,5-dichlorophenylcarbamate) (C-3,5diCl)) were used as chiral columns (5 μm, 250 ×4.6 mm) (Phenomenex Inc., Torrance, CA, USA). HPLC grade n-hexane (Hex), isopropanol (IPA), and methanol (MeOH) were purchased from Sigma-Aldrich (Taufkirchen, Germany). Analyses were performed in isocratic mode at 25 °C. The flow rate (FR) was set at 0.8 ml/min. Dead time (t0) was measured by injection of tri-tertbutylbenzene (Sigma-Aldrich) as a non-retained compound [11]. The enantiomer elution order (EEO) was determined by injecting enantiomers of known absolute configuration [9]. The van't Hoff experiments were conducted at 5,10,15,20,25,30,35,40, and 45 °C by using a thermostat jacket equipped with a RE104 LAUDA circulating water-bath (Lauda, Königshofen, Germany). When the temperature was changed, the column was allowed to equilibrate for 1 h before injecting the sample. Thermodynamic parameters were derived from the slopes and the intercepts of the van't Hoff plots (see Supporting Information for details) by linear regression analysis. Statgraphics Centurion XVI (Statpoint Technologies, Inc., Warrenton, VA, USA) was used for all linear regression analyses.

Computationals

The 3D structures of compounds 1 and 2 and methyl 3,5-dimethylphenylcarbamate, methyl 3-chloro-4-methylphenylcarbamate, methyl 4-chloro-3methylphenylcarbamate and methyl 3,5-dichlorophenylcarbamate, as frameworks representing the CSP side chains, were prepared using the build function, and model kits and tools provided by Spartan '10 Version 1.1.0 (Wavefunction Inc., Irvine, CA, USA) [12] for building and editing organic molecules. On this basis, molecular structures were generated and their refinement was performed by a MMFF procedure.

Then, each structure was submitted to a conformational systematic search using MMFF, spanning all shapes accessible to the molecule without regard to energy. After the elimination of duplicates and high-energy conformers, a set of energetically accessible conformers was selected. For each conformer, geometry optimization was performed employing the DFT method with the B3LYP functional and the 6-311G* basis set, and finally the respective Boltzmann distribution was constructed. Geometry optimization and computation of electrostatic potential isosurfaces (VS) and related parameters (VS extrema, VS,max and VS,min values, given in au) were performed by using Gaussian 09 (DFT, B3LYP, 6-311G*) (Wallingford, CT 06492, USA) [START_REF] Frisch | Revision B. 01[END_REF]. Search for the exact location of such VS,max and VS,min was made through the Multiwfn code [START_REF] Lu | [END_REF] and through its module enabling quantitative analyses of molecular surfaces [15].

The AMBER18 Antechamber toolkit (University of California, San Francisco, USA) [START_REF] Case | AMBER 2018[END_REF] was used to assign the generalized Amber Force Field (GAFF) atom type and the AM1-BCC type of charge to 4,4'-bipyridines 1 and 2. The Gaussian 09 program (DFT, B3LYP, 3-21G*) [START_REF] Frisch | Revision B. 01[END_REF] was used for the ab initio geometry optimization calculation of the monomeric units of β-Dand α-D-glucose-1,4-dimethoxy-tris (3,5-dimethylphenylcarbamate). The optimized structures were used to build nonamers (9mer) of C-3,5diMe and A-3,5diMe, respectively [START_REF] Peluso | [END_REF]. C-3,5diMe was characterized by a left-handed threefold (3/2) helix according to the structure reported by Vogt and Zugenmaier [18], setting the dihedral angles of the units, defined by H1-C1-O-C4'(Φ) and H4'-C4'-O-C1(φ) to 60° and 0° (Supporting Information, Fig. S1A). A-3,5diMe was characterized by a 4/3 left-handed helical structure according to the structure reported by Okamoto and co-authors [19,20], setting the dihedral angles of the units, defined by H1-C1-O-C4'(Φ) and H4'-C4'-O-C1(φ) to -68.5° and -42.0° (Fig. S1B). The terminal residues of the polymers were closed with methoxyl groups. The polymer structures were energy-minimized using the GAFF force-fields with AM1-BCC charges assigned with the Antechamber toolkit. The atoms of the backbone were fixed in their positions during the simulations by assigning a force constant of 20 kcal/mol so that, starting from the setting initial values, the applied restriction restrained the rotation of backbone dihedral angles of residues 2-8 (Fig. S2). The energies and the structure of the polymers were first prepared using 100 ns MD simulations (see Supporting Information for details about MD stages) with Hex/IPA 90:10 as medium. This structure was used in the final MD simulations. The AMBER18 software [START_REF] Case | AMBER 2018[END_REF] was used to carry out 100 ns MD simulations. The initial positions of each enantiomer were determined by molecular docking (see Supporting Information for details). Solvent effect was taken into account by means of the explicit periodic solvent box (Hex:IPA 90:10). In this regard, the polysaccharide-analytes complexes were prepared for MD runs by solvating the system with an octahedral box with a 10 Å radius polysaccharide cutoff by using Packmol-memgen [21,22] and an in-house script to manage solvent mixtures.

100 ns of the trajectories from each case were considered for statistical analysis. The Chimera software (UCSF, San Francisco, USA) was used for visualization and analysis of the MD trajectories [23]. Interaction energies between the polysaccharide nonamer and the enantiomer were calculated, which include van der Waals (vdW) and electrostatic (el) energies.

Results and discussion

Electrostatic potential analysis of analytes and chiral selectors

For compounds 1 and 2, the electrostatic potential maxima (VS,max, Fig. 2, pale blue points) and minima (VS,min, Fig. 2, red points) values were computed in order to inspect the electron charge density distribution on the main electron-poor (electrophile, Lewis acid) and electron-rich (nucleophile, Lewis base) recognition sites, respectively (Fig. 2A,B). Recently, V analysis has been fruitfully used to gain insights on selector/analyte contacts by evaluating the electron charge density on molecular regions involved in noncovalent interactions [5,24,25]. In compounds 1 and 2, the distinctive substituents located at 2'-position are a 4-pyridyl ring in 1 and a 4-hydroxyphenyl group in 2.

Moreover, both compounds contain a common 3,3',5,5'-tetrachlorinated motif which represents a symmetric hydrophobic region surrounding the chiral axis. Another hydrophobic region is present at the 2-position where an iodine atom is located as substituent. This halogen may act as halogen bond (XB) donor interacting through its electrophilic σ-hole with the nucleophilic regions of the CSP (Fig. 2C). Higher polarization was induced by the 4-pyridyl substituent (1: VS,max = 0.0535 au) at the position 2' of the 4,4'-bipyridinyl scaffold compared to the 4-hydroxyphenyl substituent

(2: VS,max = 0.0496 au). In our previous studies, we demonstrated by chromatographic and computational analyses that the carbonyl oxygens of C-3,5diMe and A-3,5diMe are able, as Lewis bases, to form XBs with the electrophilic σ-hole regions of halogen substituents bound to the 4,4'-bipyridine rings [START_REF] Peluso | [END_REF]. HB sites are located on the aromatic substituents in 2'-position, a nitrogen as HB acceptor (VS,min = -0.0658 au) and a hydroxyl group as HB acceptor/donor (VS,min = -0.0421 au; VS,max = 0.1128 au), respectively. As the OH group in 2 is free to rotate around the C-O bond, the directionality of the HB sites on the OH may change, in principle making compound 2 more adaptable to the CSP chiral cavity than 1. In the latter case, the rotation of the 4-pyridyl substituent does not change the directionality of the HB involving the pyridyl nitrogen.

Chiral columns based on C-3,5diMe, C-3Cl,4Me, C-4Cl,3Me, and C-3,5diCl were selected for this study in order to evaluate the impact of aryl chlorination on their enantioseparation performances. All columns contain selectors based on the same cellulose backbone which is derivatized with distinctive side chains determining the stereoelectronic properties of each selector [8]. The effect of introducing chlorine in the CSP structure is to modify the electron charge density distribution on the side chain moieties, thus the electron charge density on both C=O and phenyl ring decreases (πacidity increases), whereas the acidity of the N-H increases [26]. This trend has been confirmed by calculating VS,max and VS,min values on pivotal regions of the side chains of C-3,5diMe, C-3Cl,4Me, C-4Cl,3Me, and C-3,5diCl (Table 1). It is known that the introduction of chlorine increases the fraction of free N-H groups [27], whereas the fraction of N-H involved in intramolecular HBs, contributing to maintain the high-ordered structure of the CSP, decreases. This could produce for the chlorinated CSPs a wider cavity available for the enantiomers with respect to the dimethylated selector, the overall enantioseparation resulting from the balance of carbamate polarity and intramolecular HB ability [27].

Chromatographic screening

The enantioseparability of TCIBPs 1 and 2 was tested on coated C-3,5diMe, C-3Cl,4Me, C-4Cl,3Me, and immobilized C-3,5diCl columns by using Hex/IPA 90:10 as MP. A comparison between the behaviours of the four columns is reported in Figure 3 (see Supporting Information, Table S2 for numerical data).

Good selectivity was achieved for the enantioseparation of 1 on C-3,5diMe (α = 2.82) exclusively, whereas lower selectivity values ranging from 1.07 to 1.15 were obtained in other cases. No enantioseparation was observed for 1 and 2 on C-3Cl,4Me and C-4Cl,3Me, respectively. Retention of both enantiomers was higher for 1 (average k1 = 4.9; average k2 = 6.4) compared to 2 (average k1 = 2.1; average k2 = 2.3) in almost all cases. The first eluted enantiomer of 2 showed higher retention only on C-3,5diMe (k1 (2) = 2.94 vs k2 (1) = 2.67). Given the presence of a HB acceptor (pyridyl nitrogen) as a distinctive recognition site, for compound 1 retention of the first eluted enantiomer tended to increase as the HB donor ability of the selector amidic N-H also increased (towards more positive VS,max values moving from C-3,5diMe to C-3,5diCl). The opposite trend was observed for compound 2 due to the presence of a HB donor (OH hydrogen) as distinctive recognition site. In this case, retention of both enantiomers increased as the N-H VS,max values and the electron charge density on the carbamate C=O decreased and increased (towards more negative VS,min values moving from C-3,5diCl to C-3,5diMe), respectively. As a particular case, retention of the second eluted enantiomer of compound 1 increased moving from C-3,5diMe (k2 = 7.54) to C-3,5diCl (k2 = 8.26), whereas the two chloromethyl substituted C-3Cl,4Me and C-4Cl,3Me provided lower k2 values (4.78 and 5.15, respectively). On the other hand, EEO reversal was observed on C-3Cl,4Me and C-4Cl,3Me (M-P) for both compounds compared to the 3,5-disubstituted C-3,5diMe and C-3,5diCl (P-M), this evidence revealing the occurrence of a different adsorption mechanism [3,[28][29][30][31] It is worth noting that EEO is also a key factor for the method development [32]. Indeed, as chiral separation methods are optimized for optical purity control of a chiral analyte, the possibility to modify the EEO may be advantageous in order to have the polluting enantiomer eluted first [28,31,32].

The addition of 5% MeOH to the MP was detrimental for retention and selectivity in almost all cases (Supporting Information, Table S3 and Fig. S3). However, for compound 1 on the C-3,5diMe the use of the mixture Hex/IPA/MeOH 90:5:5 contributed to reduce elution time (k1, -12%; k2, -52%) keeping selectivity value acceptable (α = 1.54) (Fig. S3A,B). In this regard, it is worth noting that the addition of MeOH to the MP impacted retention of the second eluted enantiomer of compound 1 more on the C-3,5diMe (k2, -52%) compared to the other chlorinated C-3Cl,4Me, C-4Cl,3Me, and C-3,5diCl (k2, -17.6%, -32.6%, -34.4%, respectively). This suggested that a second key interaction involving the carbamate C=O possibly affected by 5% MeOH addition may participate in chiral recognition. In this regard, the involvement of a XB between the 2-iodine of compound 1 as XB donor and the carbonyl of the CSP as XB acceptor could be envisaged, the VS,max value on 2-iodine being higher for 1 (0.0535 au) compared to 2 (0.0496 au). This chromatographic results confirmed previous observations showing that the anisotropic properties of chiral substituted 4,4'-bipyridines strongly depend on the stereoelectronic features of the 2,2',3,3',5,5'-substituents bore by the orthogonal heteroaromatic rings, as a consequence of the atropisomeric motif [33,34]. For 1 and 2, it was expected that the enantiodiscrimination degree should be related to the strength of noncovalent interactions involving both 2-and 2'positions, due to the symmetry of the 3,3',5,5'-tetrachloro pattern. On the other hand, the direct contribution to retention and selectivity of the 4,4'-bipyridine core was shown to be low, in particular due to the weakness of the pyridine nitrogens as HB acceptors (-0.0490 au ≤ VS,min ≤ -0.0426 au). However, in compound 1 three electron-withdrawing heteroaromatic substructures polarized iodine, thus contributing to its capability to exert XB.

Effect of temperature on enantioseparation

With the aim to explore the impact of temperature on enantioseparation, and compare the thermodynamic profiles of the cellulose-based CSPs as derived from van't Hoff analysis (see Supporting Information for details on van't Hoff and thermodynamic equations), retention and selectivity of compounds 1 and 2 on the four cellulose-based CSPs were determined at different temperatures from 5 to 45°C in 5°C increments (Supporting Information, Tables S4-S7) using Hex/IPa 90:10 as MP. Several papers have dealt with theory of adsorption phenomena in chromatography, and with methods for profiling temperature dependence of retention and selectivity and thermodynamic quantities associated with the adsorption of analytes on the CSP surface [35,36]. Some studies stressed that thermodynamic quantities derived from the classical van't Hoff equation are macroscopic entities which do not account for surface heterogeneity of the CSPs that determines individually achiral and chiral features of enantioseparation [37]. On the other hand, thermodynamic parameters depend on analyte, MP and the diffuse chiral (chirotopic) environment profile of the CSP.

Therefore, the nature of the analyte/CSP contact can be explored on the basis of thermodynamic considerations, and useful information can emerge by comparison of thermodynamic data of analogue analyte/CSP pairs as subtle variations of the chromatographic system occur. In addition, temperature is a useful variable to optimize enantioseparation [3,38,39].

The thermodynamic quantities derived from van't Hoff plots (Fig. 4) are reported in Table S8 (Supplementary information). On this basis, the following remarks emerged: i) compounds 1 and 2 showed different thermodynamic profiles, and the temperature dependence pattern was observed to be a function of the 2'-substituent structure and of the CSP type;

ii) for compounds 1 and 2 the enantioseparations were enthalpy-driven on the 3,5disubstituted CSPs because the temperature range was below the calculated TISO, and the thermodynamic ratio Q = ΔΔH/(298 ×ΔΔS) > 1 [40] (157°C ≤ TISO ≤ 587°C; 1.44 ≤ Q ≤ 2.85) (Fig. 4A,B,G,H). On the contrary, the enantioseparations were shown to be entropy-driven on the 3,4-disubstituted CSPs in almost all cases (Fig. 4C,E,F) (-70°C ≤ TISO ≤ 15°C; 0.68 ≤ Q ≤ 0.97). These different thermodynamic profiles could explain the EEO reversal from P-M to M-P observed as the substitution pattern of the CSP phenyl rings changes from the 3,5-to 3,4-disubstitution;

iii) for compound 1 partial separation was observed on C-3Cl,4Me in the range 30-45°C (1.017 ≤ α ≤ 1.035) (Supporting Information, Fig. S4A), whereas for compound 2 on the C-4Cl,3Me very low enantioseparation was detectable at 45°C exclusively (α = 1.018) (Fig. S4B); iv) in the case of compound 2 enantioseparation on C-3Cl,4Me, the thermodynamic profiles revealed the presence of two concurrent mechanisms in the range 5-45°C, an entropy controlled (TISO = -55°C, Q = 0.73) at low temperature and an enthalpy controlled mechanism (TISO = 97°C, Q = 1.24) at higher temperature. The two mechanisms coalesced between 30 and 20°C, providing at 25°C the best value of selectivity (α = 1.07), and a concave profile for the plot ln α = f(1/T) (Supporting Information, Fig. S5); v) on this basis, enantioseparation of compounds 1 and 2 could in some cases be optimized by varying the temperature. For compound 1 on C-3,5diMe, elution time could be reduced at 45°C maintaining good selectivity (α25°C→45°C = 2.83 → 2.32). In the other cases, enantioselectivity was almost independent of the temperature variation (Fig. S5). However, for 1 on C-4Cl,3Me, the enantioseparation under entropic control could be optimized at 45°C (α25°C→45°C = 1.11 → 1.13). For both compounds 1 and 2 on C-3,5diCl, enantioseparation could be optimized under enthalpic conditions at 5°C (α25°C→5°C (1) = 1.10 → 1.12; α25°C→5°C (2) = 1.14 → 1.16).

Molecular dynamics simulations

As depicted in Figure 5, C-3,5diMe and A-3,5diMe [3] showed complementary enantioseparation ability towards compounds 1 and 2. Indeed, compound 1 (Fig. 5A) (α = 2.82) was enantioseparated on the C-3,5diMe better than compound 2 (Fig. 5C) (α = 1.14), whereas 2 (Fig. 5D) (α = 1.26) was enantioseparated on the A-3,5diMe with selectivity higher than compound 1 (Fig. 5B) (α = 1.04). A backbone-dependent reversal of EEO was also observed, the elution sequence being P-M and M-P on C-3,5diMe and A-3,5diMe, respectively. In addition, thermodynamic analysis evidenced an enthalpic contribution to free energy difference (ΔΔG°) associated to the enantioseparations higher for C-3,5diMe (Q = 1.44, 1.60) compared to A-3,5diMe (Q = 1.04, 1.08). The enthalpic contribution to enantioseparation was higher for compound 2 compared to 1, the difference being more pronounced on C-3,5-diMe (ΔQ1,2 = 0.16) compared to the amylose-based selector (ΔQ1,2 = 0.02).

On this basis, with the aim to explore the molecular basis of these chromatographic behaviors, a theoretical investigation based on MD simulations was performed by using C-3,5diMe and A-3,5diMe nonamers as virtual models of the polysaccharidebased selectors.

The 100 ns MD simulations in the AMBER force field [41] were performed by using the mixture Hex/IPA 90:10 as a virtual solvent in accord with the experimental conditions used in the chromatographic studies. With the aim to confirm the hypothesis that a XB involving the 2-iodine substituent of the enantiomer (M)-1 could contribute to the high adsorption on C-3,5diMe (tR = 30.25 min), the explicit σ-hole (ESH) concept [42,43] was used to model the electrophilic electron charge density depletion on the iodine atom [START_REF] Peluso | [END_REF] (see Supporting Information for details). For both analytes, the simulations were performed with and without ESH in order to also evaluate the MD results when the electrophilic character of iodine is suppressed. The total interaction energies calculated for (M)-and (P)-enantiomers of 1 and 2 in their complexes with each of the polysaccharide nonamer are summarized in Table 2.

Table 2. Binding energies (Eint) (kcal/mol) and component contributions (Eel, EvdW) for the association of (M)-1, (P)-1, (M)-2, and (P)-2 with C-3,5diMe (EEOexp = P-M) and A-3,5diMe (EEOexp = M-P)

C-3,5diMe

A-3,5diMe The reported energies are mean values which were calculated from 5000 complexes obtained by snapshots taken every 20 ps from the 100 ns MD trajectories. The interaction energy (Eint) between enantiomer and selector is calculated on the basis of the energies of the selector-enantiomer complex, the selector and the enantiomer (eq.

1)

Eint = Etotal -Eenantiomer -Epolysaccharide-based selector (1) where the Eint term derived from the contributions of the van der Waals (vdW) and the electrostatic (el) interaction terms (eq. 2).

Eint = Eel + EvdW (2)
In Figure 6, representative snapshots and noncovalent interactions from the simulated MD trajectories of 1 and 2 complexes with C-3,5diMe (A-D) and A-3,5diMe (E-H) are depicted. The following remarks emerged: i) in accord with a previous observation [START_REF] Peluso | [END_REF], MD simulation provided a more compact structure for A-3,5diMe nonamer (with smaller cavities) compared to the C-3,5diMe;

ii) coherently, in all simulations involving the A-3,5diMe nonamers, the bulky iodine substituent protruded out of the polymer groove (Fig. 6F,G,H) or was oriented towards the void inside the groove (Fig. 6E), thus no XB was detected even if the ESH was introduced on the iodine. This finding is in accord with our previous observations on the detrimental effect of the compact structure of A-3,5diMe on XBs involving iodine [START_REF] Peluso | [END_REF];

iii) analogously, in all cellulose-based complexes involving the (P)-1, (P)-2, and (M)-2 enantiomers, modelled either with and without ESH, the iodine was oriented outside the polymer (Fig. 6A,C,D); iv) otherwise, a XB between the 2-iodine and the carbamate C=O was detected in the complex (M)-1 / C-3,5diMe as the ESH was introduced on the iodine of the analyte. In this case, the calculated EEO (Table 2) is fully consistent with the experimental elution sequence. On the contrary, the simulation performed without ESH correction provided a theoretical EEO not consistent with experimental EEO showing that the electrophilic feature of iodine has a pivotal role in the enantiodiscrimination. On this basis, the high retention of the enantiomer (M)-1 was related to a four-component noncovalent interaction pattern consisting of one HB, two π-π interactions and a XB (Fig. 6B); v) for each MD simulations, the EvdW component was found to be the major contribution to the interaction energy. Indeed, in all cases, hydrophobic contacts between the haloaromatic scaffold of the analyte and the surface of the polymer appeared to govern analyte / selector association along with distinctive HBs and π-π stacking interactions; 1.

  Fig. 2C, maxima a) and VS,min (Fig. 2C, minima b)

  vi) the fit of both enantiomers (P) on the C-3,5-diMe was very similar in accord with the close chromatographic retention values observed for the two P-enantiomers (tR (1) = 13.00 min, tR (2) = 14.00 min); vii) in both 2 / A-3,5diMe complexes, each enantiomer is bound to the polysaccharide surface with the 4-hydroxyphenyl part protruding deeply inside the groove, and with the hydroxyl group engaged in HBs with carbamate sites, while buried into the hydrophobic environment generated by the nonpolar regions of the polymer. This profile is consistent with the high retention of both enantiomers of 2 on A-3,5diMe (tR (M) = 26.31 min, tR (P) = 32.24 min)[3]; viii) finally, it is interesting to note that on A-3,5diMe the 2'-(4-pyridyl) substituent was found in the external part of the surface (Fig.6E;F), whereas the 2'-(4-hydroxyphenyl) substituent penetrated into the groove of the CSP, being blocked inside by HB interactions. Indeed, in this case, the hydroxyl group (and its associated recognition sites) is free to rotate around the C-O bond, protruding inside the chiral cavity as a molecular drill and making the analyte more adaptable compared to compound 1.5 Concluding remarksThe enantioseparation of TCIBPs 1 and 2 on 3,5-disubstituted (C-3,5diMe and C-3,5diCl) and 3,4-disubstituted (C-3Cl,4Me and C-4Cl,3Me) cellulose-based CSPs and related recognition mechanisms were explored through a multidisciplinary approach based on chromatographic and thermodynamic analysis, electrostatic potential analysis and MD simulations. Under NP elution conditions, lower selectivities were obtained in almost all cases compared to amylose-based selectors, which we had used in a previous study. The enantioseparation of 2'-(4-pyridyl)-TCIBP on C-3,5diMe represented an exception, and good selectivity could be obtained by using Hex/IPA 90:10 as MP (α = 2.82). Under these elution conditions, the analysis time was rather long (> 30 minutes). However, good selectivities could be obtained with shorter elution time by adding 5% MeOH to the MP (Hex/IPA/MeOH 90:5:5) (t < 20 min; α = 1.54) or by increasing elution temperature to 45°C (t < 22 min; α = 2.32).EEO reversals dependent on the substitution pattern of the phenyl group of the CSPs were observed, the elution sequence being P-M and M-P on the 3,5-and 3,4disubstituted CSPs, respectively, for both analytes. In particular, temperaturedependent enantioseparations performed in the range 5-45 °C allowed for identifying enthalpy-(TISO ≥ 157°C, Q > 1) and entropy-controlled (TISO ≤ 15°C, Q < 1) profiles for 3,5-and 3,4-disubstituted CSPs, respectively.The molecular bases of the complementary enantioseparation profiles and the backbone-dependent EEO reversal obtained for 1 and 2 on C-3,5diMe (P-M) and A-3,5diMe (M-P) were explored by MD simulations. Interaction energies calculated from 100 ns MD trajectories provided EEOs which were fully consistent with the experimental elution sequences. Analysis of calculated energies, analyte / selector complexes and noncovalent interaction patterns evidenced a) the more compact structure of the amylose-based polymer compared to the cellulose-based polysaccharide, and its capability to envelop the analytes which are able to penetrate into the cavity, b) the dominant contribution of van der Waals interactions to the overall analyte / selector binding, c) the pivotal role of the distinctive HB sites at the 2'-position of the TCIBP scaffold, inducing diverse adsorption mechanisms due to distinctive electronic and steric properties, d) the noncovalent interaction pattern causing the high adsorption of the enantiomer (M)-1 on C-3,5diMe, and finally e) the contribution of a XB interaction to the adsorption of (M)-1 on C-3,5diMe.

FIGURE CAPTIONS Figure 1 .

 CAPTIONS1 FIGURE CAPTIONSFigure 1. Structures of chiral 4,4'-bipyridines 1 and 2 and cellulose-based chiral stationary phases.

Figure 2 .

 2 Figure 2. Maps of potential recognition sites of compounds 1 (A) and 2 (B), and of carbamate side chain of C-3,5diMe (C) described in terms of VS,max (pale blue) and VS,min (red) (values are reported in au) representation. For values associated to the V extrema a and b see Table1.

Figure 3 .

 3 Figure 3. Correlation between retention range (k values) on four cellulose-based CSPs for compounds 1 and 2 and VS,max and VS,min values calculated on each cellulose carbamate recognition site (N-H and C=O).

Figure 4 .

 4 Figure 4. ln kM and ln kP vs 1/T plots for the enantioseparation of 1 and 2 on C-3,5diMe, C-3Cl,4Me, C-4Cl,3Me, and C-3,5diCl (Hex/IPA 90:10, FR = 0.8 ml/min, temperature range 278.15-318.15 K).

Figure 5 .

 5 Figure 5. Chromatograms of enantioseparations of compounds 1 and 2 on C-3,5-diMe (A and C, respectively) and A-3,5-diMe (B and D, respectively) [3], MP = Hex/IPA 90:10, FR = 0.8 ml/min, T = 25°C.

Figure 6 .

 6 Figure 6. Representative snapshots and noncovalent interactions from the simulated MD trajectories of 1 and 2 complexes with C-3,5diMe (A-D) and A-3,5diMe (E-H).

Table 1 .

 1 Cellulose carbamate-based CSPs/columns used in the study, and VS,max and VS,min values associated with the main recognition sites (carbamate N-H and C=O) Lux series columns (Phenomenex Inc., Torrance, CA, USA). b) VS values calculated at DFT/B3LYP/6-311G* level, VS,max (

	Column a)	Ar (R',R''-C6H4) Abbreviation VS,min C=O (au) b)	VS,max N-H (au) b)
	Cellulose-1	3,5-dimethyl	C-3,5diMe	-0.0630	0.0827
	Cellulose-2	3-chloro-4-methyl	C-3Cl,4Me	-0.0576	0.0902
	Cellulose-4	4-chloro-3-methyl	C-4Cl,3Me	-0.0578	0.0910
	i-Cellulose-5	3,5-dichloro	C-3,5diCl	-0.0532	0.0987
	a)

TABLE CAPTIONS Table 1 .

 CAPTIONS1 Cellulose carbamate-based CSPs/columns used in the study, and VS,max and VS,min values associated with the main recognition sites (carbamate N-H and C=O)

Table 2 .

 2 Binding energies (Eint) (kcal/mol) and component contributions (Eel, EvdW) for the association of (M)-1, (P)-1, (M)-2, and (P)-2 with C-3,5diMe (EEOexp = P-M) and A-3,5diMe (EEOexp = M-P)

Acknowledgements

This work has been supported by Università Ca' Foscari Venezia, Italy (Dipartimento di Scienze Molecolari e Nanosistemi DSMN, ADIR funds).

Conflict of interest

The authors have declared no conflict of interest.

Supporting information

Supporting information file: Additional HPLC, thermodynamic and computational data.


Characterisation of the peptides produced by the donor strains 1

Positive interactions between lactic acid bacteria mediated by peptides containing branched-chain amino acids

CONTEXT AND AIM STRATEGY

 1).  Among the peptides produced by Efa2412, Lla450 preferably used peptides containing more non-polar, small side chain, branched-chain, and aliphatic amino acids (Fig 4 ).  Similar results were observed with Llp1524. In a chemically defined medium composed of casein and lupin proteins as sole nitrogen sources, in which proteolytic and non-proteolytic lactic acid bacteria (LAB) strains grow in association, different interactions occurred, only impacting the receiving strains (Fig 1). They were mediated by the peptides and amino acids provided by the proteolytic strains. The aim of this study was to identify the characteristics of the peptides involved in these interactions.  Cluster 5 is associated in particular with high amounts in aliphatic, non-polar, and sulphurous side chain amino acids in contrast to cluster 1.  Cluster 3 is associated with peptides containing the highest percentage in essential amino acids for LAB and cluster 4 the lowest.

Table 1 Description of the clusters according to peptide characteristics and amino acids composition

Criteria that are significantly more/less represented in a specific cluster compared to the others. Example: cluster 1 contains peptides that have a significantly lower molecular weight than in clusters 4,5 and 6.

CONCLUSION

 Peptides containing non-polar and aliphatic amino acids, and more specifically branched-chain amino acids were involved in positive interactions.  Peptides containing branched-chain amino acids were associated with cluster 5 specific to Efa2412, which showed the strongest positive interactions with receiving strains. In contrast, peptides from cluster 1, specific to Lla244, which did not interact with receiving strains, contained less branchedchain amino acids.

 The peptide length and charge did not appear as important in the observed interactions.  In contrast to Lla244, the low amount of peptides containing branched-chain amino acids produced by Lla2125 is most likely compensated by the production of branched-chain free amino acids, which would explain why weak interactions were observed with receiving strains.  The amino acid composition of the proteins and the cleavage sites of the proteases of the donor strain are important factors to consider when in co-culture with proteolytic and non-proteolytic LAB strains.

Identification of the peptides used by the receiving strains

Figure 1 FigureFigure 3

 13 Figure 1 Three types of interactions observed between three proteolytic (=donor) lactic acid bacteria (LAB) strains in co-culture with non-proteolytic (= receiving) strains (Canon et al. 2021)



  Peptides significantly less abundant in the co-culture Lla2125 x Lla450 compared to Lla2125 monoculture were found in clusters 4, 3, 7 and 6 (Fig 5 & Table 1)  Among the peptides produced by Lla2125, Lla450 preferably used peptides containing non-polar amino acids (Fig 5).

Figure 4

 4 Figure 4 Clusters associated with the peptides used by Lla450 in co-culture with Efa2412 and characteristics of the peptides used compared to the all the peptides available Figure 5 Clusters associated with the peptides used by Lla450 in co-culture with Lla2125 and characteristics of the peptides used compared to the all the peptides available


Prof Robin P Weiss

Dr Emmanuel Aubert

Patrick Pale
email: ppale@unistra.fr

Victor Mamane
email: vmamane@unistra.fr

Dr V Mamane Pale

Lasyroc

Chalcogen Bonding Catalysis with Telluronium Cations

Keywords: chalcogen bonding, non-covalent interactions, organocatalysis, tellurium, Lewis acid O O Br

come   L'archive ouverte pluridisciplinaire

Although known since 1783, tellurium only slowly gained interest. [1] It is employed in some alloys, solar panels and semiconductors. Its implication in organic chemistry is more diverse, owing to the special properties of this element. [START_REF] Petragnani | Tellurium in Organic Synthesis[END_REF] Tellurium is the last stable element of the chalcogen group, [START_REF]The latest element of this series, polonium, is radioactive, with a half-live of 138 days for the -emitting 210 Po isotope and 128 years[END_REF] and as such, the largest [START_REF][END_REF] and the less electronegative, [5] that is the more polarizable and ionizable in this series. For these reasons, tellurium species exhibit the strongest non-covalent interaction with electron-rich atom through the so-called chalcogen bond (ChB), compared to their sulfur and selenium analogs. [6,7] ChBs are defined in analogy with halogen bonds (XB) as the interaction of an electron donor (Lewis base) with the electropositive region (-hole) located at the outermost end of a -bond including a chalcogen atom. [8] ChB has been sparingly investigated in the last few decades and mostly in solid state. [9] Following the development of XB applications in biology [10] and organic chemistry, [11] ChBs have very recently raised increasing interest in organic chemistry, especially in anion recognition and transport as well as in organocatalysis. [12] In the latter growing area, mostly S and Se derivatives have so far been investigated. [13] Nevertheless, in 2018, Matile studied perfluorophenylselenide and telluride as ChB donors in chloride-binding catalysis (Sch. 1, up) [14] and more recently, Huber used a bidentate triazolium-based tellurium catalyst in the same reaction and in Michael reactions with indoles (Scheme 1, middle). [15] While working on this manuscript, Gabbaï very recently demonstrated that telluronium species can bind chloride and even act as chloride transporter. [16] Engaged in XB and ChB study for applications in organocatalysis, we have recently designed new chiral catalysts. [17] To further explore this area and take benefit of the tellurium enhanced ChB property, we are currently designing tellurium-based catalysts. Inspired by the works of Lenardão [18] and Yeung [19] who demonstrated that selenonium could act as organic Lewis acid and promote reaction, we designed new telluronium salts and studied them, and we report here their ChB ability and for the first time the high efficiency of these telluronium species as organocatalysts in typical reactions (Sch. 1, bottom). To tune the expected ChB intensity, a series of telluronium derivatives carrying electron-rich or electron-poor substituent was prepared by alkylation of diaryltellurides. Addition of methyl triflate to diaryltellurides provided telluronium triflates but in modest yields (10-20%), while methyl iodide addition delivered unstable telluronium iodides. Adding silver triflate, tetrafluoroborate, nitrate, or hexafluoroantimonate together with methyl iodide readily gave the corresponding telluroniums 1a-b, 2a-b, 3a-b, 3de and 4a-b with excellent yields (Scheme 2). The tetrakis [3,5bis(trifluoromethyl)phenyl]borate (BArF) telluroniums 1c, 2c, 3c, 4c and tetrakis(pentafluorophenyl)borate telluronium 3f were quantitatively obtained by anion metathesis from the corresponding triflates. They proved stable at room temperature for months as solids, except for 3d, as well as in solution, except for 3e, which slowly evolved to the starting diaryltelluride. [20] Among them, 3a and 3e provided suitable crystals for X-ray analysis, confirming the structures of these new telluronium salts.

Scheme 2. Synthesis of the telluronium ChB donors.

In the solid state, 3a and 3e both crystallize with two independent cations in the asymmetric unit, all four molecules exhibiting a similar conformation (Fig. S9-S11 in S. I). In each structure, the telluronium cation exhibits close interaction with three neighbouring anions, one in each direction opposite to the CAr-Te and CMe-Te bonds, almost aligned with the Te -holes (Fig. 1a). With anion-Te distances ranging from 2.831 to 3.048 Å, well below the sum of the van der Waals radii of the two elements (O 1.52 Å, Te 2.06 Å) and with a nearly linear arrangement of the TfO/NO3••Te-C bonds (166 to 175°; Fig. S12-S15, S16-S17 and Table S10), these structures revealed the presence of ChB between each Te-C -hole and the non-bonding electrons of the triflate or nitrate oxygen atom (Fig. 1b). DFT calculations performed on the isolated telluronium cation 3 + confirmed the presence of three -holes along the three Te-C bonds, as shown by the positive electrostatic surface potential (ESP) around the tellurium centre (Fig. 1b). The corresponding Vs,max values perfectly reflect the electron-withdrawing or donating properties of the substituent (Table S23), and as expected, the CF3 group induces a significant increase (Vs,max (kcal/mol): 130.3 (3 + ) vs 113.0 (1 + ) vs 103.2 (2 + )) deepening the -holes. The Vs,max values of the two Te-Ar -holes are almost identical to one another whatever the telluronium, while the Te-Me -holes are always slightly lower. Superimpositions of the molecular packing around telluronium cations in 3a and of the positions of the ESP extrema (Fig. 1a and S16-S17) show that the position of the anions is governed by the electrostatic interaction with the cation, guided through the three -holes that are responsible for the VS,max observed on the molecular surface.

Encouraged by these positive structural results, we further studied the telluronium ChB in solution. For that, 17 O-labelled Ph3PO [21] was used as a probe in NMR, expecting that direct 17 O and 125 Te NMR monitoring of the interacting atoms would give key information on the interaction between the basic Ph3PO oxygen atom and the Te -holes. Using a phosphine oxide as ChB acceptor could further allow 31 P NMR monitoring [22] Once added to a telluronium d 2 -dichloromethane solution, Ph3PO induced significant shifts of the 17 O, 31 P and 125 Te NMR signals (Table 1).

Table 1. 125 Te, 17 O and 31 P NMR chemical shift differences from telluronium salts 1-3 in the presence of Ph3PO. [a] Entry Telluronium (R, X) 125

Te  [b] 17

O  [c] 31 P  [d] 1 1a (H, OTf) Expected to be driven by Te  holes deepness, the equilibrium occurring upon Ph3PO addition should be affected by the electronic effect of the telluronium aryl substituent, but also by the ability of the anion to act as a Lewis base and thus to compete with the added OPPh3. Such competition may be responsible for the variable peak broadening observed in some spectra for certain telluronium-OPPh3 complexes.

Therefore, electronic effect should best be detectable if no competition for Te  holes occurs. The telluronium series 1c, 2c, 3c and 4c, carrying the less coordinating anion BArF, was designed and prepared for these reasons in order to maximize Ph3PO••Te interaction. Rewardingly, these BArF derivatives exhibited the largest NMR shifts (entries 3, 6, 9 and 15), whatever the measured nucleus. Furthermore, good correlations between  and  Hammett constants were obtained in 17 O and 31 P NMR (R 2 = 0.9375 and 0.9846 respectively; see Fig. S4). The latter clearly confirms the formation of Ph3PO••Te complexes, and the role electronic effects played in this formation: the most effectively formed was obtained with the more electrodeficient telluronium 3 + , the one having the largest  holes (see Vs,max values above), inducing the largest 17 O and 31 P NMR shifts.

Alternatively, the anion effect could be best evaluated within the telluronium series which exhibits the largest  holes (see Table S21), i.e. the 3 + derivatives. Interestingly, the a index proposed by Alvarez to define the coordination ability of anions toward transition metal ions [23a] allowed to rationalize the observed variations. Remarkable correlations were indeed achieved with 17 O and 31 P NMR shifts for the 3a-3f telluronium salt series (entries 7-12; see Fig. S5, R 2 = 0.9607 and R 2 = 0.9420 respectively). These results confirm that coordination of the telluronium counterions compete with Ph3PO for the formation of Ph3PO••Te complexes.

It is worth noticing that, although a values were reported for transition metals, lanthanides, and s-block metals, [23] the present correlations suggest that they are also well suited for telluronium cations.

In contrast to O and P, only moderate to poor correlations could be achieved with Te, both for electronic and anion effects (see Fig. S6). This was expected because 125 Te NMR spectroscopy is highly sensitive to electronic and geometry changes in the tellurium environment. [24] Nevertheless, the correlation profile between Te and a for the telluronium salt series 3 + X -showed that Te is differently influenced if the anion is well or poorly coordinating. Thus, by considering only low (BArF and BC6F5) and medium (SbF6 and BF4) coordinating anions, a very good correlation (R 2 = 0.9931) was obtained between Te and a. With the more coordinating anions (NO3 and TfO), the effect of Ph3PO addition is balanced by other parameters, probably related to their competition for interaction with the telluronium and may be some modifications of the telluronium geometry if both the anion and Ph3PO interact with.

A complementary analysis of 125 Te chemical shifts was performed by calculating the chemical shielding of the tellurium in telluronium cations 1 + -4 + (see S. I., section VII). Interestingly, the good linear correlation between the chemical shifts of telluroniums 1c-4c measured in solution and the calculated chemical shielding of 1 + -4 + showed that the Te trend in this series can be well reproduced by DFT calculations (R 2 = 0.9076, Fig. S18). This result confirmed that Te in telluroniums 1c-4c is mostly influenced by electronic parameters. However, no correlation was observed between the calculated chemical shielding and the measured Te for the telluronium-OPPh3 adducts (Fig. S20). This was expected because the measured Te in solution corresponded to an equilibrium process whereas the chemical shielding were calculated for the adducts only.

It is interesting to note that the measured and calculated chemical shift values of the telluroniums reported here nicely fit with those recently reported (Fig. S19). [24d] To further probe this Ph3PO••Te interaction and to assess the formation of a monoadduct Ph3PO--Te+, we performed NOESY experiment (Fig. S7) on the solution containing Ph3PO and 3c, the most effective salt for such complex formation (see above). This experiment clearly established the spatial proximity of ortho protons of Ph3PO with the ortho protons of the 3,5di(trifluoromethyl)phenyl group of the telluronium entity (Fig. 2a). Furthermore, nOe was also detected between the ortho-protons of Ph3PO and the methyl group of the telluronium. Interestingly, calculations performed on a complex Ph3PO-3 + also highlighted the proximity of these protons (Fig. 2b andS25), with a very short distance between Te and O (2.570 Å), in line with the strongest 17 O NMR shift (21.1 ppm) observed with this complex. Its formation was further confirmed by in situ mass spectrometry, which allowed to detect the species present in the corresponding solution with a mass peak at 849.0448 within a set of peaks typical for the isotopic distribution of a 1:1 adduct between 3c and Ph3PO (Fig. 2c andS8). Overall, these effects established the presence of a ChB between Te and O of Ph3PO through interaction of the latter with the aryl-Te -hole located between the telluronium methyl and the other aryl group, as schematically shown in Fig. 2a.

As these observations clearly showed the Lewis acidity of the cationic chalcogen and its ability to activate a carbonyl-type derivative, it was worth evaluating their propensity to act as catalyst in some reactions. The bromination of anisole with Nbromosuccimide (NBS) was first used as benchmark reaction with catalysts 1-3 (Table 2). Interaction of the NBS carbonyl with the Te -hole should lower the N-Br bond LUMO and favour its attack by the electron-rich aromatic moiety of anisole.

At room temperature, no reaction occurred without telluronium (entry 1), but in the presence of catalytic amount of it (5 mol%), anisole bromination was achieved within 5 min. All the telluronium salts gave the expected 4-bromoanisole in high yields and no significant difference was noted between TfO and BF4 anions. As expected from the preceding studies, the BArF telluroniums 1c, 2c and 3c provided the best results, giving within 5 min the brominated product in high yields and almost quantitatively with 3c as catalyst (entries 4, 7 and 10). Based on these results and on the higher Vs,max values (Table S23), catalyst 3c was used for further catalytic tests (entries 11-15) and for other reactions (Tables 3 and4). Increasing or decreasing the catalyst amount did not change the catalyst efficiency (entries 11, 12 vs 10), and interestingly, as low as 0.5 mol% of catalyst still very efficiently promoted this reaction within 5 min. (entry 13). The catalyst 3c was then evaluated in competition with chloride, known to strongly interact with Te species. [7a,15a,16,25] Accordingly, the catalytic activity of 3c was suppressed in the presence of an equivalent amount of tetrabutylammonium chloride (entry 14). The possibility of hidden Brønsted catalysis was ruled out by adding potassium carbonate which did not modify the reaction course and its yield (entry 15). It is worth to mention that this reaction could be performed at mmol scale without significant change (entry 10 f ). The catalyst stability was also attested by NMR monitoring of the reaction; 1 H and 19 F NMR confirmed the structural integrity of the catalyst after reaction (see S.I. p 31-33). Entry [a] Catalyst loading Yield (%) [b] 1 None 0 2 1a 5 mol% 79 [c] 14 [d] 3c 1 mol% 0

15 [e] 3c 1 mol% 98

[a] Reactions were carried out with anisole (0.054 mmol), catalyst and NBS (0.056 mmol) in CD2Cl2 at rt for 5 min.; [b] Determined by 1 H NMR (see SI for details, NMR yield incertitude: ± 5%); [c] The same yield was obtained with 1a synthesized directly from MeOTf, thus ruling out catalysis by silver salts; [d] In the presence of 1 mol% of nBu4NCl after 24h; [e] In the presence of 10 mol% of K2CO3 after 5 min; [f] Isolated yield in brackets for a reaction performed at 1 mmol scale.

As NBS was efficiently activated by the telluronium catalyst in the aryl bromination described above, it seemed worth to examine another classical reaction induced by NBS, the bromolactonization reaction. A series of -unsaturated carboxylic acids was submitted to NBS in the presence of telluronium catalyst 3c (Table 3). Rewardingly, the expected bromolactones were obtained within 30 min in good to quantitative yields for 4 to 6 membered rings (entries 1-8), but in lower yields for larger rings (entries 9-10), while no reaction occurred without catalyst (entry 1). The -lactone was quantitatively formed (entries 5-6), even with only 1 mol% of catalyst (entry 5) but higher catalyst loading proved necessary for efficiently producing the -or -lactones, (entries 3-4 and 7-8 vs entry 5). For each case, the observed regioselectivity corresponds to what is expected from Baldwin 'rules'. [26] Here again, the reaction could be performed at mmol scale without significant change (entry 6 d ) and the catalyst remained stable during the reaction (see S.I. p 31-33), despite the role Lewis bases could play in bromo-and iodolactonisation. [START_REF] Denmark | Proc. Nat. Acad. Sci[END_REF] Table 3. Bromolactonisation catalyzed by telluronium.

Entry [a] Alkene x Lactone Yield [b,c] OMe OMe 98) 99 (92) [d] In order to further illustrate the -hole catalytic property of telluronium species, we evaluated the ability of the telluronium 3c to catalyze aza-Diels-Alder-type reaction. As an archetypal reaction, the Danishefsky's diene condensation with various imines was investigated (Table 4). Such reaction is known to follow a step-wise mechanism with soft Lewis acids or in protic solvents, driven by N-coordination or protonation. [28] Therefore, the soft telluronium 3c should here similarly activate the imine and induce Mannich-type addition of the Danishefsky's diene. All the examined imines readily afforded the expected N-phenyl 2,3dihydro-4-pyridinones in good to high yields with only 1 mol% of catalyst within 2 h at room temperature (entries 1, 3, 7). As expected, higher catalyst loading further improved the catalysis efficiency (entry 5 vs 4) and up to 91% of adducts could be obtained with 10 mol% of catalyst (entry 6 vs 4). Longer reaction times did not much improve yields (entries 2 vs 1, 4 vs 3 and 8 vs 7). A slight electronic effect was observed, with electron-poor imine being less reactive (entries 7,8) as expected from the envisaged mechanism. As in the preceding reactions, the reaction could be performed at mmol scale (entry 6 d ) and the catalyst stability could be assessed by 1 H and 19 F NMR (see S.I. p 31-33). Entry [a] Imine Catalyst loading Time Yield [b,c] 1 R = H 1 mol% 2 h 87 (84) (76) [d] 2 [a] Reactions were carried out with imine (0.14 mmol), Danishefsky's diene (0.18 mmol) and catalyst in CH2Cl2 at rt for 2 h; [b] Determined by 1 H NMR (SI for details, NMR yield incertitude: ± 5 %); [c] Isolated yield in bracket; [d] reaction performed at 1 mmol scale.

In conclusion, after having prepared new telluronium salts, we demonstrated the presence of three -holes on these telluronium and their importance in solution as indicated by Ph3PO binding studies through 17 O, 31 P and 125 Te NMR. The enhanced properties of these telluronium salts could be readily translated for the first time into high catalytic performance. These telluronium salts, especially the BArF telluronium carrying electron-withdrawing groups (3c), were able to efficiently catalyze various reactions, even at low loading. Quantitative reactions were thus achieved in the bromination of aromatic and in bromolactonization with only 0.5 mol% of catalyst. Aza-Diels-Alder-type reactions were also very effective (up to 90%) with 5 mol% of catalyst. The versatility of telluronium salts in their applications uncovered here opens up new routes/strategies in organic syntheses. Further works in those area are underway in our group.

Scheme 1 .

 1 Scheme 1. Te-based organocatalysis: The two sole examples known and those described here.

Figure 1 .

 1 Figure 1. a) Molecular packing about tellurium Te1 in telluronium triflate 3a (intermolecular Te•••O bond distances in Å) and its superposition with the location of the ESP extrema (VS,max, magenta dots; O•••Te•••VS,max angles are indicated in °); b) ESP map for the telluronium 3 + on DFT ρ=0.001 a.u. isodensity surface. Location of the three most positive extrema of ESP (VS,max points) are indicated with black arrows.

  [a] Conditions: 1:1 mixture (20 mM) of telluronium salts and Ph3PO in CD2Cl2 at 25 °C;[b] Te of the telluronium salts before and after addition of Ph3PO are = OMe, X = OTf (93%) 2b: R = OMe, X = BF 4 (97%) 3a: R = CF 3 , X = OTf (98%) 3b: R = CF 3 , X = BF 4 (99%) 3d: R = CF 3 , X = SbF 6 (96%) 3e: R = CF 3 , X = NO 3 (91%) 4a: R = t-Bu, X = OTf (96%) 4b: R = t-Bu, X = BF 4 (82%) 2c: R = OMe, X = BAr F 4 (96%) 3c: R = CF 3 , X = BAr F 4 (99%) 3f: R = CF 3 , X = B(C 6 F 5 ) 4 (84%) 4c: R = t-Bu, X = BAr F .I. (Tab. S1) as well as NMR incertitudes (Tab. S2);[c] O in the presence of 1-4 (given in S.I. (Tab. S1) as well as NMR incertitudes (Tab. S4)) compared to O of free Ph3PO (47.41 ppm);[d] P in the presence of 1-4 (given in S.I. (Tab. S1) as well as NMR incertitudes (Tab. S3)) compared to P of free Ph3PO(27.45 ppm).

Figure 2 .

 2 Figure 2. a) Representation of the interactions between OPPh3 and the telluronium protons of 3c as suggested from 17 O, 31 P and 125 Te NMR and NOESY experiment (only one single hydrogen was highlighted for clarity); b) B3LYP-D3/Def2TZVPP calculations of the 3 + -Ph3PO complex; c) mass spectrum of the 3 + -Ph3PO complex.

  Reactions were carried out with -unsaturated carboxylic acid (0.23 mmol), catalyst and NBS (0.23 mmol) in CH2Cl2 at rt for 30 min.;[b] Determined by 1 H NMR (see SI for details, NMR yield incertitude: ± 5 %);[c] Isolated yield in bracket;[d] reaction performed at 1 mmol scale.

  Te chalcogen bonding: New telluroniums salts exhibit enhanced properties, due to the presence of 3 -holes, and highlighted in solution by Ph3PO binding studies through 17 O, 31 P,125 Te NMR, MS and NOESY. These enhanced ChB properties translated into high catalytic performance, with low loading (0.5-5 mol%), short reaction time (5 min-2 h) and high yields.

Table 2 .

 2 Bromination of anisole catalyzed by telluroniums.

Table 4 .

 4 Telluronium-catalyzed condensation of imines with the Danishefsky's diene.

Acknowledgements

This research was funded by the International Center of Frontier Research in Chemistry (icFRC), the LabEx CSC (ANR-10-LABX-0026 CSC). RB thanks the LabEx CSC, Strasbourg, for a PhD fellowship. The EXPLOR mesocenter is thanked for providing access to their computing facility (project 2019CPMXX0984/wbg13).


Dr Robin Weiss

Prof. Dr Tamara Golisano

Dr Patrick Pale

Victor Mamane
email: vmamane@unistra.fr

Insight into the Modes of Activation of Pyridinium and Bipyridinium Salts in Non-Covalent Organocatalysis

Keywords: Charge transfer, Halogen bond, Organocatalysis, Hetero Diels-Alder, Pyridinium

A series of pyridinium and bipyridinium salts were prepared and their catalytic properties were evaluated in the aza-Diels-Alder between imines and Danishefsky diene. Depending on the substituents of the pyridinium/bipyridinium rings and on the nature of the counter-anion, two mechanisms of activation were demonstrated. In case of non-substituted rings, the substrate is activated through charge transfer involving the aryl ring on the C-side of the imine. When halogen atoms were introduced on the catalysts, the activation mode switched to halogen bond involving the imine nitrogen lone pair. Moreover, alternative activation modes based on hydrogen bonding and radical cation were ruled out.

This work allowed us to develop two families of catalysts whose potential was demonstrated in the cycloaddition of various imines with Danishefsky diene. The first family is composed of the simple methyl pyridinium triflate and dioctyl bipyridinium triflate. The former is active only with imines bearing a p-methoxyphenyl group on the C-side and the latter was found to be efficient with imines bearing different substituents on both the N-and C-sides of the imines. The second family is based on halogenated pyridinium salts which proved active with almost all considered imines.

Introduction

N-Substituted pyridinium salts are privileged structural units of significant importance in natural products and synthetic chemistry. [1] This scaffold is found in the nicotinamide adenine dinucleotide (NAD + ) cofactor essential to all organisms [2] and in some marine sponges [3] (Figure 1, top). Besides, pyridinium salts can serve as functional group transfer reagents, [4] phase-transfer catalysts [5] and ionic liquids. [6] Pyridinium salts have also been widely used as synthetic intermediates, for example in ring opening cascade reactions and dienal formation, [7] 1,3-dipolar additions [8] and photochemical transformations [9] (Figure 1, middle). In contrast, N,N'-disubstituted bipyridinium salts were far less studied. Although a few bipyridiniums have been proposed as antibacterial, [10] (Figure 1, bottom), the main application of bipyridinium salts is as functional materials, with viologen as archetypal compound. [11] Figure 1. Pyridinium and bipyridinium salts: selected natural products and applications.

However, the use of pyridinium and bipyridinium salts in organocatalysis is very limited. Indeed, only a handful of examples report the application of pyridiniums and even less of bipyridinium as catalyst. Surprisingly, different modes of action have been proposed for these catalysts, depending on the nature of the substituents on the heterocycle and the considered reaction (Figure 2). Deciphering the exact mechanism is not always straightforward, and represents a real challenge, not addressed so far. Berkessel proposed that catalyst 1a dissociates 1chloroisochroman via Coulombic interaction with chloride, thus allowing Mukaiyama aldol-type reaction with silyl ether nucleophiles. [12] Later, on the basis of X-ray data of 1a with halide counteranions (Cl -and Br -), Nagorny and Sun suggested that 1a could also act as hydrogen bond (HB) donor. [13] Activation through HB was also proposed by Bibal with catalyst 2 in the ring-opening polymerization of cyclic esters [14] and for aminolysis of epoxides. [15] However, in these cases, catalyst 2 was used in combination with two cocatalysts, rendering any interpretation less clear. With catalysts of type 1 bearing a phenyl (1b) or cyano group (1c), a mechanism based on alcohol addition to the catalyst and generation of a Brønsted acid was proposed by Connon for the acetalization of aldehydes and ketones [16] and by Berkessel for the addition of alcohols to glycals. [17] When iodine atom is introduced on the charged heterocycle, activation via halogen bond (XB)-based could operate. [18] Indeed, iodine electrophilicity, which is directly related to its -hole depth, [19] is increased due to the positive charge of the pyridinium heterocycle; iodine thus acts as a Lewis acid. [20] Although not used as a catalyst, compound 3 developed by Huber was the first iodo-bipyridinium able to promote the Ritter reaction through XB activation of benzhydryl bromide. [21] More recently, Yeung described that 4-iodopyridinium 4 was able to catalyse the intramolecular bromocarbocyclization of cinnamyl ethers and sulfonamides through XB activation of N,N-dibromodimethylhydantoin. [22] In 2020, Toy reported that bipyridinium 5 was a more efficient XB catalyst than its monomeric 3iodopyridinium analogue in Friedel-Crafts reaction with furans. [23] Pospíšil demonstrated that pyridiniumand isoquinolinium-based helquats 6 could catalyse Povarov reaction through a cation radical mechanism. [24] Mayer used bipyridiniums 7a,b as catalysts in imine aziridination and Povarov reaction, [25] but their mode of activation was not discussed.

Herein, in continuation to our efforts to develop efficient XB and related chalcogen bond donors for catalysis applications, [26] new polyhalogenated pyridinium and bipyridinium salts were designed to face/address the above mentioned challenge (Figure 2) and to gain real clues on their mode of action (Figure 3). To study the catalytic activity of these (bi)pyridinium salts, we selected the aza-Diels-Alder [27] reaction between Danishefsky's diene and imines. In the literature, the aza-Diels-Alder reaction between Danishefsky's diene and imines was shown to be catalysed through HB with tetraalkylammonium [28] and trialkylsulfonium [29] salts and through XB with iodotriazolium or iodoimidazolium salts. [START_REF]Yellow solid[END_REF] Recently, through theoretical calculations, the role of Coulombic (dipole-cation) interactions was also highlighted with onium catalysts. [31] Therefore, the polyhalogenated (bi)pyridinium salts reported here can in principle activate a substrate through the different modes of activation depicted in Figure 2.

We demonstrated here that Coulombic interactions are operative with naked heterocycles, whereas XB is the principal mode of activation when the heterocycles bear multiple halogens (Figure 3). Interestingly, HB is not operative with this class of compounds despite the acidity of the pyridinium hydrogens.

Results and Discussion

N-Alkyl pyridinium and bipyridinium salts were prepared by alkylation of their parent pyridines and 4,4'-bipyridines. Thus, addition of methyl iodide to the commercially available pyridines 8a-c and 8e as well as to 8d, obtained from the commercial 3,5dibromopyridine, furnished the iodide salts 9a-e. Analogously, pyridine 8a was reacted with methyl triflate to produce the N-methyl pyridinium triflate 10. Mono-alkylation of 4,4'-bipyridine 11 [32] with respectively methyl iodide, methyl triflate and octyl triflate delivered the iodide salt 12a and the triflate salts 12b,c. Finally, the double alkylation of 4,4'bipyridines 11, 13 and 14 [33] gave the bis-N-octylbipyridinium triflates 15a, 15b, 16 and 17, respectively (Scheme 1). Scheme 1. Synthesis of pyridinium and bipyridinium salts. a) CH3I or CH3OTf (2 eq.), CH3CN, MW 300W, 120°C, 2h; b) CH3I (1.35 eq.), Et2O, MW 300W, 50°C, 1h; c) CH3I (4 eq.), Et2O, 25°C, 24h; d) 12a: CH3I (1 eq.), CH3CN, 70°C, 48h; 12b: CH3OTf (1 eq.), Et2O, 25°C, 24h; 12c: n-OctOTf (1 eq.), Et2O, 25°C, 24h; e) 15a: n-OctI (4 eq.), CH3CN, 120°C, 24h; 15b and 16: n-OctOTf (4 eq.), CH3CN, 120°C, 24h; 17: n-OctOTf (4 eq.), CH3CN, 25°C, 24h.

The pyridinium and bipyridinium salt were tested in the reaction between Danishefsky's diene 18 and imines 19a-b in dichloromethane at room temperature (Table 1).

No reaction occurred without catalyst after 24h (entries 1 and 2) as well as with iodide 9a, even after addition of acetonitrile in order to increase its solubility (entries 3 and 4). Rewardingly, the reaction was complete within 5 min with 5 mol% of triflate 10 and the cyclized product 20a was quantitatively formed (entry 5). Complete conversion was also obtained by addition of K2CO3 in the reaction mixture containing catalyst 10; this result thus ruled out the possible role of hidden Brønstead acid (entry 6). A radical mechanism was also excluded by performing the reaction in the presence of butylated hydroxytoluene (BHT) with no change in the yield (entry 7). Moreover, tetrabutylammonium triflate (TBAOTf) was inactive in the reaction (entry 8); this interesting result showed that the triflate anion did not play a role in the catalytic activity of 10, but also that HB-catalysis driven by ammonium chains [28] was not operative under such conditions.

These first series of experiments suggests that dipole-cation interaction [31] or even charge transfer could be responsible for reaction initiation, but not HB. The inactivity of the iodide salt 9a could be due to the charge transfer occurring between the pyridinium and iodide, which do not allow the imine substrate to interact with the catalyst. In contrast, in the triflate salt 10, the interaction of the triflate anion with the charged heterocycle is weaker, the latter could thus be able to activate the substrate through charge transfer facilitated by the presence of a p-methoxy group in 19a.

To check this hypothesis, the imine substrate was switched from 19a (R = OMe) to 19b (R = Me). Rewardingly, the reaction was completely inhibited with this substrate, and no formation of the cyclized product 20b could be observed (entry 9). Charge transfer seems thus responsible for the activity of catalyst 10.

In a second series of experiments (entries 10-13), halogenated pyridinium iodides 9b-e were used as catalysts (5 mol%) in the aza-Diels-Alder reaction involving imine 19a in order to evaluate the possible implication of HB and/or XB modes of activation with these catalysts. In iodides salts 9b-e, two halogen atoms acting as electron-withdrawing substituents were added in 3-and 5-positions of 9a, thus increasing the acidity of the hydrogen atoms in 2-and 2'positions. Moreover, the -hole of these halogens is deepened because of the positive charge, except for fluorine whose -hole is rarely positive.

Pyridinium 9b bearing two fluorine atoms was completely inactive in the reaction despite the presence of acidic hydrogens, ruling out HB role (entry 10). A low yield of 40% was observed with 9c bearing chlorines as potential XB sites (entry 11). With pyridiniums 9d and 9e bearing bromine and iodine as XB donors, the yields reached about 60% (entries 12 and 13). In this series, the reactivity clearly increased with the -hole intensity of the halogen whereas it decreased with the hydrogen acidity. It is then likely that pyridiniums iodides 9c-e catalyze the reaction through XB activation of the imine substrate.

With the aim to further increase iodine -hole, pyridiniums 12 bearing an additional electronwithdrawing group in 4-position (3-bromo-5-iodo-4pyridinyl) compared to 9d, were prepared and evaluated as catalyst. Gratifyingly, the catalytic activity of these pyridiniums was greatly enhanced and in all cases, high to quantitative yields were obtained after only 5 min. Interestingly, shifting from iodide 12a to triflate 12b as catalyst increased reaction yields (77 vs 87%, respectively; entries 14 vs 15) as expected from stronger XB interaction of iodide compared to triflate. Quantitative yield was obtained with the more soluble N-octyl pyridinium triflate 12c after 5 min (entry 16). Interestingly, the same result was obtained when using imine 19b (entry 17). To confirm that halogen bonding is involved, we performed competitive experiments in the presence of tetrabutylammonium chloride (TBACl). As chloride anion is known to act as good -hole acceptor, [34] it can interact through XB with the -hole carried by the catalyst halogen(s) and thus compete with the substrate. This competition should lower the catalyst efficacy. [30b] With imine 19a, the catalytic activity of 12c was indeed lowered by addition of 10 mol% of TBACl (2 equiv. with regard to 12c) (entry 18) and completely suppressed with 20 mol% of TBACl (4 equiv. with regard to 12c) (entry 19). It is worth noting that after addition of the two first equivalents of chloride anion, the residual catalytic activity (38%) was probably due to the XB with the remaining halogens on the pyridine ring, as it is suppressed when one chloride per halogen was added (entry 19 vs 18 vs 17).

These set of results of this third series of experiments (entries 14-19) clearly suggest that the reaction is catalyzed through XB with pyridinium 12ac.

In order to have more insight on this XB mode of activation, crystals of pyridiniums 12a and 12b were grown and their solid-state structures were analyzed by X-ray diffraction (Figure 4). andc) using CYLView. [35] Color code: carbon in grey, hydrogen in white, bromine in orange, iodine in violet, nitrogen in blue, oxygen in red, sulphur in yellow and fluorine in green.

In the structure of 12a (Figure 4a), as expected, a XB was observed between the iodine on the pyridinium cycle and the iodide anion through a strong interaction revealed by a I---I distance (3.327 Ǻ) well below the sum of the van der Waals radii (I 1.98 Ǻ), and with a C-I---I angle of 175.8°. The iodide is probably a competitor in the reaction, thus explaining the lower activity of 12a compared to 12b.

In the structure of 12b, the triflate anion is also interacting through XB but with the pyridinyl iodine (Figure 4b). Interestingly, a strong XB was observed between the iodine on the pyridinium cycle and the pyridine nitrogen of another unit (N---I 2.864 Ǻ and N---C-I 176.9°) (Figure 4c). These data suggest that the pyridine nitrogen should be replaced in solution by the imine nitrogen in the XB observed in solid state. They also indicate that triflate 12b should better activate the imine substrate through XB than 12a, as observed.

Finally, in order to confirm the charge transfer or XB modes of activation of naked vs halogenated heterocycles, the bipyridinium salts 15-17 were also evaluated as catalysts in the same aza-Diels-Alder reaction.

As with the simple pyridiniums 9a and 10, the bipyridinium bis iodide 15a was inactive (entry 20) whereas the bis triflate salt 15b was (entry 21). The latter showed higher activity compared to pyridinium triflate 10 since complete conversion was obtained after only 5 min at room temperature (entry 21 vs 5).

Here again, hidden Brønsted acid (entry 22) and involvement of radical mechanism were ruled out (entry 23).

In contrast to its monomeric counterpart 10 (entry 9), bipyridinium 15b was able to activate imine 19b to generate the cyclized product quantitatively after 1h of reaction (entry 24 vs 9). This result could easily be explained by better charge transfer due to the structural analogy of 15b to viologen. As for charge transfer, the formation of a complex through -orbital interaction is required before charge transfer, we designed the 3,3',5,5'-tetramethylbipyridinium 16 in order to look at conformational effect on the catalytic activity.

Interestingly, reaction with imine 19a was completely suppressed by using 16 as catalyst (entry 25). The methyl groups in 16 thus prevent charge transfer between the pyridinium ring and the imine, probably by inducing the non-planarity of the two pyridinium moiety and/or by hindering each side of the pyridinium plane. When the methyl groups were replaced by halogens such as in 17, reaction occurred again and high yields were obtained for both imines 19a-b (entries 26 and 27), although these yields were slightly lower compared to the ones achieved with the pyridinium analogue 12c (entries 16 and 17). The role of XB in the activation of imines 19a-b by 17 was confirmed by TBACl addition which resulted in complete inactivation of the catalyst (entry 28).

To sum up, non-substituted pyridinium and bipyridinium triflates 10 and 15b activate imine substrates through Coulombic interaction and charge transfer [31] (Fig. 5a), whereas the halogenated ones, 9b-c, 12b-c and 17, are operating through XB (Fig. 5c). Activation through charge transfer could be inhibited by steric constrains (Fig. 5b).

After having demonstrated that pyridinium and bipyridinium act as catalysts in aza-Diels-Alder reaction and elucidated their respective modes of action, the reaction scope was studied by using different imines in the presence of 5 mol% of catalyst 10, 12c and/or 15b. For comparison purposes, all reactions were performed with Danishefsky's diene 18 over 1h (Table 2). The halogenated pyridinium 12c could efficiently catalyze the reaction of almost all considered imines with yields comprised between 72 and 91%, except for imine 19g bearing a p-dimethylamino group. The latter could not be converted to 20g, even after 18h of reaction and in the presence of 10 mol% of catalyst. In this case, the XB-driven catalyst is probably deactivated by the good XB acceptor ability of the dimethylamino group. As expected from this XB activation mode, a slight electronic effect could be observed with imines carrying electron-withdrawing substituent on the aryl group. Imines 19c and 19e were slightly less efficiently converted with catalyst 12c to adducts 20c and 20e than other imines (72-76% vs 82-93%). With pyridinium 10, as expected from the screening above, only imines 19a and 19i bearing pmethoxyphenyl substituent could be cyclized giving products 20a and 20i in good yields. A low yield of 20h was obtained with imine 19h containing the pmethoxyphenyl substituent on the nitrogen.

In contrast, the bipyridinium catalyst 15b readily catalyzed the reaction of all the imines tested 19a-g, the expected adducts 20a-g were always produced in good to high yields (61-95%). These results revealed the significant reactivity increase of the bipyridinium catalyst 15b compared to its simple pyridinium analog 10. Nevertheless, and as expected from the mode of action established above, electronic and steric effects affect the reaction outcome. Imines 19d-e bearing a methoxy substituent respectively in ortho and meta positions were cyclized with lower yields, and the cyclization of the dimethylamino-substituted imine 19g required higher catalyst loading (10 mol%) and prolonged reaction time (18h) to achieve a good yield of 20g.

Conclusion

A series of halogenated and non-halogenated pyridinium and bipyridinium catalysts were designed to explore the various activation modes in the organocatalyzed reaction of imines with Danishefsky's diene. The gained results allowed to discriminate the mode of activation of this family of catalysts with imine substrates. It was clearly demonstrated that non-halogenated derivatives activate the imine through charge transfer whereas the halogenated ones interact with the imine nitrogen through XB. Besides methyl pyridinium triflate catalyst (10) which showed good performance only with imines having a p-methoxyphenyl substituent on the imine carbon side, two powerful catalysts 12c and 15b based on the 4,4'-bipyridine scaffold were developed during this work. Further work is now underway in our laboratory in order to expand the scope of reactions that can be catalyzed by these molecules.

Experimental Section

General methods.

Et2O was distilled over Na/benzophenone and freshly used. CH2Cl2 and CH3CN were dried by passing through activated alumina under a positive pressure of argon using GlassTechnology GTS100 devices. Anhydrous reactions were carried out in flamedried glassware and under an argon atmosphere, using standard Schlenk techniques. Column chromatography was performed with silica gel from Merck (Kieselgel 60; 63-200 µm or 40-63 µm). 1 H, 13 C and 19 F NMR spectra were recorded on Bruker Advance 300, 400 or 500 spectrometers. Chemical shifts were determined by taking the solvent as a reference. Mass spectrometry (HRMS) experiments were performed on a Bruker Daltonics microTOF spectrometer (Bruker Daltonik GmgH, Bremen, Germany) by the Service de Spectrométrie de Masse de la Fédération de Chimie "Le Bel" (FR 2010). Pyridines 8a-c, 8e and bipyridine 13 were purchased and used as received.

3-bromo-5-iodopyridine (8d).

To a solution of 3-5 dibromo-pyridine (10.8 mmol, 2.60 g) in THF (50 mL), was added a solution of iPrMgCl . LiCl (11.8 mmol, 1.3 M in THF, 9.1 mL) at -10 °C. After 10 min of stirring a solution of iodine (11.8 mmol) in THF (16 mL) was added dropwise, keeping the temperature below -5 °C. After 10 min of stirring Et2O (200 mL) was added and was washed with NaHSO3 sat. (60 mL). Organic extract was washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated to give the desired product without further necessary purification. (3.05 g, 99% yield). White solid. 1 H NMR (600 MHz,J = 1.8,0.5 Hz,1H),8.62 (dd,J = 2.1,0.5 Hz,1H),1H).

General procedure for the synthesis of 9a and 10. Pyridine (0.5 mmol, 40 mg) was solubilized in CH3CN (2 mL) and placed in a specific microwave tube equipped with a magnetic stir bar, then was added the electrophile RX (1 mmol). The tube was placed in the microwave cavity (CEM Discover) and submitted to irradiation for 2 h at 120 °C using a maximum power of 300 W. After cooling to room temperature, the mixture was concentrated, Et2O (10 mL) was added and the suspension was filtrated, washed with Et2O (2 x 5 mL) and dried under vacuum overnight to give the desired product.

1-Methylpyridin-1-ium iodide (9a). [36] Yellow solid; 100 mg, yield: 94%. 1 H NMR (500 MHz, DMSO-d6) δ 9.01 (d, J = 6.1 Hz, 2H), 8.59 (t, J = 7.8 Hz, 1H), 8.14 (t, J = 7.1 Hz, 2H), 4.37 (s, 3H); 13 C NMR (126 MHz, DMSO-d6) δ 145. 4, 145.0, 127.6, 48.0.

1-Methylpyridin-1-ium trifluoromethanesulfonate (10). [37] White solid; 120 mg, yield: 97%. 1 H NMR (400 MHz, CD3CN) δ 8.66 (d, J = 5.2 Hz, 2H), 8.49 (t, J = 7.9 Hz, 1H), 8.04-7.96 (m, 2H), 4.31 (s, 3H); 19 F NMR (282 MHz, CD3CN) δ -78.16 (s, 3F).

3,5-Difluoro-1-methylpyridin-1-ium iodide (9b). [38] 3,5-Difluoropyridine (2.7 mmol, 310 mg) was solubilized in Et2O (4 mL) and placed in a specific microwave tube equipped with a magnetic stir bar, then was added the electrophile CH3I (3.6 mmol, 0.51 g). The tube was placed in the microwave cavity (CEM Discover) and submitted to irradiation for 1 h at 50 °C using a maximum power of 300 W. After cooling to room temperature, Et2O (30 mL) was added and the suspension was filtrated, washed with Et2O (2 x 15 mL) and dried under vacuum overnight to give the desired product (90 mg, 13% yield). Yellow solid. 1 H NMR (500 MHz, CDCl3) δ 9.26-9.11 (m, 2H), 8.71 (t, J = 8.3 Hz, 1H), 4.49 (s, 5H); 13 C NMR (126 MHz, CD3OD) δ 162.1 (dd, J = 257. 1, 12.3 Hz), 134.7, 123.3-122.2 (m), 50.2; 19 F NMR (471 MHz, CDCl3) δ -111.00 (t, J = 125. 5 Hz).

General procedure for the synthesis of 9c-9e. Halogenated pyridine (1 eq.) was solubilized in a Et2O (2 mL) and placed in a tube equipped with a magnetic stir bar. CH3I (4 eq.) was added and the tube was sealed with a screw cap. The mixture was stirred for 24 h at 25 °C. Then, the mixture was filtrated and washed with Et2O (2 x 5 mL) and dried under vacuum overnight to give the desired product.

3,5-Dichloro-1-methylpyridin-1-ium iodide (9c).

Starting from 3,5-dichloropyridine (0.20 g, 1.3 mmol) and CH3I (0.77 g, 5.4 mmol). 68 mg, yield: 18%. White solid, mp 204-206°C; 1 H NMR (500 MHz, CD3OD) δ 9.26 (d, J = 1.9 Hz, 2H), 8.93 (t, J = 1.9 Hz, 1H), 4.43 (s, 4H); 13

3-Bromo-5-iodo-1-methylpyridin-1-ium iodide (9d).

Starting from 3-bromo-5-iodopyridine (0.20 g, 0.70 mmol) and CH3I (0.17 g, 2.8 mmol). 78 mg, yield; 26%. Yellow solid, mp 188-190°C; 1 H NMR (500 MHz, D2O) δ 9.09 (s, 1H), 9.04 (s, 1H), 9.02 (s, 1H), 4.23 (s, 3H); 13 C NMR (126 MHz, D2O) δ 155. 5, 149.8, 145.5, 122.1, 92.9, 48.1; HRMS (ESI-TOF) m/z calcd for C6H6BrlN: 297.8723 [M] + ; found, 297.8730; m/z calcd for I: 126.9041 [M] -; found, 126.9050.

3-Bromo-5-fluoro-1-methylpyridin-1-ium iodide (9e).

Starting from 3-bromo-5-fluoropyridine (0.20 g, 1.14 mmol) and CH3I (0.64 g, 4.55 mmol). 96 mg, yield: 26%. Yellow solid, mp 215-218°C; 1 H NMR (500 MHz, Methanol-d4) δ 9. 39 -9.13 (m, 2H), 8.97 -8.89 (m, 1H), 4.45 (s, 3H); 13 C NMR (126 MHz, Methanol-d4) δ 161.3 (d, J = 258.4 Hz), 145.9, 137.2 (d, J = 21.3 Hz), 136.7 -135.9 (m), 124.2, 49.8; HRMS (ESI-TOF) m/z calcd for C6H6BrFN: 189.9662 [M] + ; found, 189.9654; m/z calcd for I: 126.9038 [M] -; found, 126.9050.

3,3'-Dibromo-5,5'-diiodo-1-methyl-[4,4'-bipyridin]-1ium iodide (12a). Bipyridine 11 (60 mg, 0.11 mmol) was solubilized in CH3CN (2 mL) and placed in a tube equipped with a magnetic stir bar. CH3I (16 mg, 0.11 mmol) was added and the tube was sealed with a screw cap. The mixture was stirred for 48 h at 70 °C. After cooling to room temperature, the mixture was filtrated and washed with Et2O (2 x 5 mL) and dried under vacuum overnight to give the desired product. 12 mg, yield: 16%. Orange solid, mp 230-234°C, 1 H NMR (500 MHz, DMSO-d6) δ 9.74 (s, 1H), 9.72 (s, 1H), 9.20 (s,1H), 9.06 (s, 1H), 4.37 (s, 3H); 13 C NMR (126 MHz,156.5,152.4,151.4,150.6,148.0,120.9,114.8,99.8,96.7,48.3 General procedure for the synthesis of 12b-12c. Bipyridine 11 (1 eq.) was solubilized in a Et2O (2 mL) and placed in a tube equipped with a magnetic stir bar. CH3OTf or n-Octyl-OTf (1 eq.) was added and the tube was sealed with a screw cap. The mixture was stirred for 24 h at 25 °C. Then, the mixture was filtrated and washed with Et2O (2 x 5 mL) and dried under vacuum overnight to give the desired product.

3,3'-Dibromo-5,5'-diiodo-1-methyl-[4,4'-bipyridin]-1ium trifluoromethanesulfonate (12b). Starting from bipyridine 11 (100 mg, 0.18 mmol) and methyltriflate (28 mg, 0.18 mmol). 72 mg, yield: 55%. White solid, mp 183-185°C; 1 H NMR (300 MHz, Acetone-d6) δ 9.88-9.82 (m, 1H), 9.80-9.77 (m, 1H), 9.22 (s, 1H), 9.04 (s, 1H), 4.75 (s, 3H); 13 C NMR (126 MHz,157.5,154.0,152.4,151.9,149.4,122.3,119.4,98.7,95.6,49.6;19 F NMR (282 MHz, 4.93 (m, 2H), 2.36-2.20 (m, 2H), 1.52-1.25 (m, 10H), 0.95-0.79 (m, 3H); 13 C NMR (126 MHz, Acetone-d6) δ 160. 9, 156.7, 152.2, 151.6, 151.0, 147.6, 122.1, 118.4, 98.5, 94.9, 62.8, 31.6, 31.0, 28.9, 28.8, 25.9, 22.4, 13.5; 19 General procedure for the synthesis of 15a,b, 16 and 17. Bipyridine 11, 13 or 14 (1 eq.) was solubilized in CH3CN (2 mL) and placed in a tube equipped with a magnetic stir bar. The electrophile (n-octyl iodide or n-octyl triflate) was added (3 eq.) and the tube was sealed with a screw cap. The mixture was stirred for 24 h at 120 °C. After cooling to room temperature, the mixture was filtrated and washed with Et2O (2 x 5 mL) and dried under vacuum overnight to give the desired product.

1,1'-Dioctyl-[4,4'-bipyridine]-1,1'-diium

bis(iodide) (15a). [39] Starting from 4,4'-bipyridine (0.2 g, 1.3 mmol) and n-octyl iodide (0.63 g, 2.6 mmol). 0.82 g, yield: 99%. White solid. 1 H NMR (500 MHz, CD3OD) δ 9.30 (d, J = 6.9 Hz, 3H), 8.70 (d, J = 6.3 Hz, 4H), 4.76 (t, J = 7.6 Hz, 4H), 2.10 (q, J = 7.6 Hz, 4H), 1.54 -1.21 (m, 20H), 0.91 (t, J = 6.7 Hz, 6H); 13 C NMR (126 MHz, CD3OD) δ 151.3, 147. 1, 128.4, 63.3, 32.9, 32.6, 30.2, 30.1, 27.3, 23.7, 14.4.

1,1'-Dioctyl-[4,4'-bipyridine]-1,1'-diium bis(trifluoromethanesulfonate) (15b).

Starting from 4,4'-bipyridine (0.2 g, 1.3 mmol) and n-octyl triflate (0.63 g, 2.6 mmol). 0.87 g, yield: 98%. 1 H NMR (500 MHz, DMSO-d6) δ 9.36 (d, J = 6.5 Hz, 4H), 8.76 (d, J = 6.4 Hz, 4H), 4.67 (t, J = 7.4 Hz, 4H), 1.97 (t, J = 7. 1 Hz, 4H), 1.40 -1.20 (m, 20H), 0.95-0.76 (m, 6H); 13 C NMR (126 MHz,145.8,126.7,120.7 (q,J = 322.3 Hz),61.0,31.2,30.8,28.5,28.4,25.5,22.1,14.0;19 3,3',5,5'-tetramethyl-4,4'-bipyridine (143 mg, 0.67 mmol) and n-octyl iodide (441 mg, 1.7 mmol). 0.49 g, yield: 99%. White solid, mp 168-171°C; 1 H NMR (500 MHz, Acetone-d6) δ 9. 21 (s, 4H), 4.86-4.74 (m, 4H), 2.28 (d, J = 0.7 Hz, 12H), 2.22-2.14 (m, 4H), 1.54-1.45 (m, 4H), 1.45-1.36 (m, 4H), 1.36-1.22 (m, 12H), 0.87 (t, J = 6.9 Hz, 6H); 13 C NMR (126 MHz,144.8,137.5,66.1,62.8,32.5,32.0,26.8,23.3,17.0,15.6,14.3;19 8,153.5,148.7,122.2 (q,J = 321.5 Hz),122.1,63.9,32.4,32.0,29.7,29.7,26.8,23.3,14.3;19 General procedure for the synthesis of imines 19. [40] A mixture of MgSO4 (1 g) in DCM (10 mL) was stirred for 10 minutes at room temperature. The relevant aldehyde (10 mmol, 1 eq.) and amine (10 mmol, 1 eq.) were sequentially added. The reaction was stirred under argon for 18 h. The mixture was diluted with DCM and then filtered. The filtrate was concentrated to give crude imine which was used without further purification. Purity > 95% was evidenced by 1 H NMR for all imines.

General procedure for the aza Diels-Alder reaction. In a round-bottom flask under argon at 25°C, the imine (0.5 mmol) was diluted in dry CH2Cl2 (2.5 mL) and trans-1methoxy-3-trimethylsiloxy-1,3-butadiene 18 was added. The catalyst (0.025 mmol) diluted in dry dichloromethane (0.5 mL) was added and the mixture was stirred for 1h. The reaction was quenched by addition of 1 mL of TBAF (1.0 M in THF) and the mixture was stirred for 15 min. Water (10 mL) was added and the phases were separated. The aqueous phase was extracted with CH2Cl2 (3 x 15 mL) then the organic phases were combined and dried over MgSO4. After filtration and concentration, the crude was purified by chromatography on silica gel (cyclohexane / ethyl acetate mixture) to give pure product.

1,2-Diphenyl-2,3-dihydropyridin-4(1H)-one (20a). [41] Yellow solid; 118 mg (95%) with 15b and 116 mg (93%) with 12c. 1 H NMR (500 MHz, CDCl3) δ 7.68 (dd, J = 8.0, 1. 4 Hz, 1H), 7.38 -7.20 (m, 5H), 7.11 (t, J = 7.3 Hz, 1H), 7.02 (d, J = 8.7, 2H), 5.33 -5.24 (m, 2H), 3.31 (dd, J = 16.4, 7.2 Hz, 1H), 2.79 (ddd, J = 16.4, 3.1, 1.2 Hz, 1H); 13 C{1H} NMR (126 MHz,CDCl3) δ 190.4,148.3,144.8,138.0,129.7,129.1,128.0,126.3,124.5,118.6,103.2,61.8,43.6.

1-Phenyl-2-(p-tolyl)-2,3-dihydropyridin-4(1H)-one (20b

). [41] Yellow solid; 122 mg (93%) with 15b and 118 mg (90%) with 12c. 1 H NMR (500 MHz, CDCl3) δ 7.67 (dd, J = 7.8, 1.2 Hz, 1H), 7.30 (dd, J = 8.7, 7. 5 Hz, 2H), 7.17 -7.08 (m, 5H), 7.03 (dd, J = 8.7, 1.2 Hz, 2H), 5.31 -5.23 (m, 2H), 3.28 (dd, J = 16.3, 7.0 Hz, 1H), 2.77 (ddd, J = 16.3, 3.4, 1.2 Hz, 1H), 2.31 (s, 3H); 13 C{1H} NMR (126 MHz,CDCl3) δ 190.5,148.3,144.9,133.7,134.9,129.8,129.7,126.2,124.5,118.6,61.6,43.7,27.0,21.2.

2-(4-Nitrophenyl

)-1-phenyl-2,3-dihydropyridin-4(1H)one (20c). [41] Yellow solid; 104 mg (71%) with 15b and 111 mg (76%) with 12c. 1 H NMR (500 MHz, CDCl3) δ 8.20 (d, J = 8.9 Hz, 2H), 7.71 (d, J = 8.0 Hz, 1H), 7.47 (d, J = 8.9 Hz, 2H), 7.32 (t, J = 7.7 Hz, 2H), 7.15 (t, J = 7.5 Hz, 1H), 6.98 (d, J = 7.6 Hz, 2H), 5.42 -5.36 (m, 1H), 5.33 (d, J = 8.0 Hz, 1H), 3.36 (dd, J = 16.6, 7.3 Hz, 1H), 2.78 (dd, J = 16.6, 2.0 Hz, 1H); 13 C{1H} NMR (126 MHz, CDCl3) δ

Figure 2 .

 2 Figure 2. Pyridinium and bipyridinium salts in organocatalysis and their proposed mode of activation (counterions are not shown for clarity).

Figure 3 .

 3 Figure 3. Pyridinium and bipyridinium salts considered in this study (Y = I or OTf).

  mmol), in a) CH2Cl2 or b) CH2Cl2/CH3CN 9/1 (2 mL), hexamethylbenzene as internal standard (78 L of a 0.1 M solution in CH2Cl2); c) Determined by 1 H NMR.

Figure 4 .

 4 Figure 4. X-Ray structures of pyridiniums 12a (a) and 12b (band c) using CYLView.[35] Color code: carbon in grey, hydrogen in white, bromine in orange, iodine in violet, nitrogen in blue, oxygen in red, sulphur in yellow and fluorine in green.

Figure 5 .

 5 Figure 5. Schematized modes of activation of pyridinium organocatalysts: Activation by Coulombic interaction and charge transfer (a) or by halogen bond (b); inhibition of charge transfer due to steric constrains (c).

  C NMR (126 MHz, CD3OD) δ 144.7, 144.0, 135.4, 48.3; HRMS (ESI-TOF) m/z calcd for C6H6Cl2N: 161.9871 [M] + ; found, 161.9879; m/z calcd for I: 126.9042 [M] -; found, 126.9050.

  3,3'-Dibromo-5,5'-diiodo-1-octyl-[4,4'-bipyridin]-1-ium trifluoromethanesulfonate (12c). Starting from bipyridine 11 (100 mg, 0.18 mmol) and n-octyltriflate (47 mg, 0.18 mmol). 137 mg, yield: 92%. White solid, mp 156-160°C; 1 H NMR (300 MHz, Acetone-d6) δ 9.93 (d, J = 1.3 Hz, 1H), 9.87 (d, J = 1.3 Hz, 1H), 9.20 (s, 1H), 9.02 (s, 1H), 5.02-

3

  ,3',5,5'-Tetramethyl-1,1'-dioctyl-[4,4'-bipyridin]-1diium bis(trifluoromethanesulfonate) (16). Starting from

Table 1 .

 1 Aza-Diels-Alder Reaction Catalysed by Pyridinium salts.

	Entry Imine Catalyst Additive a)	Time Yield (%) c)
	1	19a	None	-	24h	0
	2	19a	None	-b)	24h	0
	3	19a	9a	-	24h	0
	4	19a	9a	-b)	24h	0
	5	19a	10	-	5min 99
	6	19a	10	1 eq.	1h	99
				K2CO3
	7	19a	10	0.05 eq.	1h	99
				BHT
	8	19a	TBAOTf -	1h	0
	9	19b	10	-	1h	0
		19a	9b	-b)	1h	0
		19a	9c	-b)	1h	40
		19a	9d	-b)	1h	55
		19a	9e	-b)	1h	60
		19a	12a	-	5min 77
		19a	12b	-	5min 84
		19a	12c	-	5min 99
		19b	12c	-	5min 99
		19a	12c	0.1 eq.	5min 38
				TBACl
		19a	12c	0.2 eq.	5min 0
				TBACl
		19a	15a	-	1h	0
		19a	15b	-	5min 99
		19a	15b	1 eq.	5min 99
				K2CO3
		19a	15b	0.05 eq.	5min 99
				BHT
		19b	15b		1h	99
		19a	16	-	1h	0
		19a	17		5min 87
		19b	17		5min 90
		19a	17	0.2 eq.
				TBACl

0 Conditions: Imine 19a or 19b (0.14 mmol), diene 18 (0.17

Table 2 .

 2 Scope of the charge-transfer or XB-catalyzed aza-Diels-Alder reaction.

	-	95%	93%
	0	93%	90%
	0	71%	76%
	0	65%	91%
	0	61%	72%
	77%	85%	82%
	0	70%	0
	10%	-	84%
	92%	-	89%

  ; HRMS (ESI-TOF) m/z calcd for C11H7Br2I2N2: 578.7060 [M] + ; found, 578.7091; m/z calcd for I: 126.9050 [M] -; found, 126.9042.

  F NMR (471 MHz, DMSO-d6) δ -77.8. HRMS (ESI-TOF) m/z calcd for C26H42N2: 191.1669 [M] 2+ ; found, 191.1666; m/z calcd for CF3O3S: 148.9526 [M] -; found, 148.9526.

-Dibromo-5,5'-diiodo-1,1'-dioctyl-[4,4'-bipyridine]- 1,1'-diium bis(trifluoromethanesulfonate) (17).

  F NMR (471 MHz, Acetone-d6) δ -78.89; HRMS (ESI-TOF) m/z calcd for C30H50N2: 219.1981 [M] 2+ ; found, 219.1978; m/z calcd for CF3O3S: 148.9526 [M] -; found, 148.9510.

	3,3'Starting
	from bipyridine 11 (54 mg, 0.10 mmol) n-octyltriflate (100
	mg, 0.38 mmol). 60 mg, yield: 58%. White solid, mp 197-200°C; 1 H NMR (500 MHz, Acetone-d6) δ 9.99 (s, 2H),
	9.93 (s, 2H), 5.03-4.93 (m, 4H), 2.35-2.23 (m, 4H), 1.55-
	1.49 (m, 4H), 1.45-1.37 (m, 4H), 1.37-1.23 (m, 12H), 0.90-0.82 (m, 6H); 13 C NMR (126 MHz, Acetone-d6) δ 159.

  F NMR (282 MHz, Acetone-d6) δ -78.84; HRMS (ESI-TOF) [M] + m/z calcd for C26H38Br2I2N2: 578.7060 [M] + ; found, 578.7032.

Acknowledgements

This research was funded by the International Center Frontier Research in Chemistry (icFRC), the LabEx CSC (ANR-10-LABX-0026 CSC). RB thanks the LabEx CSC, Strasbourg, for a PhD fellowship.