domain: chim sdv

text:
Eleftherios Zarkadas

Hong Zhang

Wensheng Cai

Gregory Effantin

Jonathan Perot

Jacques Neyton

Christophe Chipot

Guy Schoehn

Francois Dehez
email: francois.dehez@univ-lorraine.fr

Hugues Nury
email: hugues.nury@ibs.fr

The binding of palonosetron and other antiemetic drugs to the serotonin 5-HT3 receptor

Keywords: Cryo-EM, receptor, serotonin, nausea, vomiting, antiemetics, 5-HT Results & discussion

ouverte pluridisciplinaire HAL, est destinée au dépôt

Introduction

In the realm of serotonin receptors, the 5-HT3 receptor stands apart as the only ion channel [START_REF] Lummis | 5-HT3 Receptors[END_REF] . It belongs to the family of pentameric ligand-gated ion channels (pLGICs) [START_REF] Nemecz | Emerging Molecular Mechanisms of Signal Transduction in Pentameric Ligand-Gated Ion Channels[END_REF] . The pLGIC family comprises, in mammals, members activated by serotonin, acetylcholine, glycine and ɣ-aminobutyric acid. They share a common architecture: neurotransmitters bind to extracellular interfaces between subunits; the transmembrane domain (TMD) possesses 5x4 helices and features a central gated pore; the dispensable intracellular domain (ICD) is for a good part intrinsically disordered. The interest in 5-HT3 receptor pharmacology surged when chemotherapies became first available (e.g. cisplatin approved in 1978 for ovarian and testicular cancers). At the time of their discovery in the 1980s, the first competitive antagonists were characterized as the "most potent drugs of any pharmacological class so far described" [START_REF] Richardson | Identification of Serotonin M-Receptor Subtypes and Their Specific Blockade by a New Class of Drugs[END_REF] , and less than a decade later the antiemetic research on 5-HT3 receptors was considered done by the pharma industry with numerous clinically and commercially successful drugs (coined -setrons) available [START_REF] Sanger | A History of Drug Discovery for Treatment of Nausea and Vomiting and the Implications for Future Research[END_REF] . Beyond emesis, fundamental and pre-clinical research indicate that the 5-HT3 receptor plays a role in depression [START_REF] Fakhfouri | 5-HT3 Receptor Antagonists in Neurologic and Neuropsychiatric Disorders: The Iceberg Still Lies beneath the Surface[END_REF] and in body weight control [START_REF] Weber | Treatment with the 5-HT3 Antagonist Tropisetron Modulates Glucose-Induced Obesity in Mice[END_REF][START_REF] Oh | Regulation of Systemic Energy Homeostasis by Serotonin in Adipose Tissues[END_REF] ; alosetron is used clinically to treat irritable bowel syndrome [START_REF] Chey | Irritable Bowel Syndrome: A Clinical Review[END_REF] , vortioxetine is an antidepressant non-selectively targeting the serotonin transporter and receptors [START_REF] Bang-Andersen | Discovery of 1-[2-(2,4-Dimethylphenylsulfanyl)phenyl]piperazine (Lu AA21004): A Novel Multimodal Compound for the Treatment of Major Depressive Disorder[END_REF] . The molecular mode of action of antiemetics has been explored extensively through binding, mutagenesis and electrophysiology studies, while structural studies on soluble model proteins gave approximate poses for the bound drugs [START_REF] Kesters | Structural Basis of Ligand Recognition in 5-HT(3) Receptors[END_REF][START_REF] Price | Palonosetron-5-HT3 Receptor Interactions As Shown by a Binding Protein Cocrystal Structure[END_REF] . Recently cryo-EM structures of the 5-HT3 receptor have revealed the conformations of the apo state, of the tropisetron-bound inhibited and of two serotonin bound states [START_REF] Basak | Cryo-EM Structure of 5-HT3A Receptor in Its Resting Conformation[END_REF][START_REF] Polovinkin | Conformational Transitions of the Serotonin 5-HT3 Receptor[END_REF][START_REF] Basak | Cryo-EM Reveals Two Distinct Serotonin-Bound Conformations of Full-Length 5-HT3A Receptor[END_REF] . However, the limited resolution of the tropisetron-bound structure precluded a thorough analysis of its binding. Here, we report a new cryo-EM structure of the 5-HT3 receptor in its inhibited state, binding palonosetron, and solved at a 2.8-Å resolution, germane for structural pharmacology. Palonosetron possesses the highest affinity in the -setron family, a slow dissociation rate and nanomolar IC50s (~0.2 nM when pre-incubated, ~80 nM when co-applied) [START_REF] Lummis | Agonists and Antagonists Induce Different Palonosetron Dissociation Rates in 5-HT₃A and 5-HT₃AB Receptors[END_REF] . Leaning on this structure, and that recently reported for the granisetron-bound 5-HT3 receptor [START_REF] Basak | Molecular Mechanism of Setron-Mediated Inhibition of Full-Length 5-HT3A Receptor[END_REF] , we set out to provide a comprehensive structural framework that established the mode of action of antiemetics. Our palonosetron-bound structure served as a template for molecular dynamics (MD) simulations not only with palonosetron, but also with cilansetron, dolasetron, granisetron and ondansetron to elucidate the general rules that underlie binding of the setron family to the 5-HT3 receptor. We further establish a relationship between the body of biochemical and pharmacological data available in light of our findings.

2.8 Å reconstruction with well-defined densities throughout the three domains of the protein (Figure 1 and SI1) -except at the level of the intrinsically disordered region of the intracellular domain (residues 329 to 399, see Figure SI2 for topology and numbering), for which the density is averaged out. The global conformation corresponds to that of previously determined inhibited states (4PIR by a nanobody form [START_REF] Hassaïne | X-Ray Structure of the Mouse Serotonin 5-HT3 Receptor[END_REF] , 6HIS by tropisetron [START_REF] Polovinkin | Conformational Transitions of the Serotonin 5-HT3 Receptor[END_REF] , 6NP0 by granisetron [START_REF] Basak | Molecular Mechanism of Setron-Mediated Inhibition of Full-Length 5-HT3A Receptor[END_REF] ). Compared with the previous structures, this one and the granisetron-bound one stand out in quality and may be used as templates in future modeling work, where the inhibited state is relevant. Non-proteinic densities in the orthosteric sites could be attributed non-ambiguously to palonosetron molecules (Figure 2b).

General features of the -setron binding site

Palonosetron binds tightly in the classical neurotransmitter site at the subunit/subunit interface (Figure 1). The occluded surface area between two subunits, for the extracellular domain (ECD) only, amounts to ~1400 Å 2 . Palonosetron acts as a molecular glue, each molecule locking the conformation of the interface and shielding an extra ~170 Å 2 of the principal subunit and ~210 Å 2 of the complementary subunit from the solvent. MD simulations essentially provide the same picture (Figure SI3). The conformational dynamics of the drugs within their binding sites induce variability in the shielding of the principal subunit, concomitant with the dynamics of the H-bond network (see below). Like every -setron drug, palonosetron possesses two moieties (Table SI1). The azabicyclo ring penetrates deep into the binding pocket, while the isoquinoline ring sits in a cavity at the surface of the complementary subunit, capped by loop C of the principal subunit (Figure 2b). The drug remains partly solvent-accessible between loop C and loop F (and one could imagine derivatives with probes grafted there). Four aromatic residues (W156, Y126, Y207, W63) forming the so-called 'aromatic cage' tightly wrap around the azabicyclo ring (Figure 2b, 3a). Mutants harboring non-aromatic side chains at any of these four positions show either much decreased inhibition by palonosetron, or simply no response to serotonin [START_REF] Del Cadia | Exploring a Potential Palonosetron Allosteric Binding Site in the 5-HT(3) Receptor[END_REF] . Even conservative mutations such as W156Y or Y126F 1 result in a 180-and 20-fold loss of inhibition, respectively [START_REF] Del Cadia | Exploring a Potential Palonosetron Allosteric Binding Site in the 5-HT(3) Receptor[END_REF] . The palonosetron isoquinoline moiety is constrained by hydrophobic interactions with Y126, W63, I44 and I201 (Figure 2b, 3a). It also interacts with R65, the conformation of which is particularly interesting: the arginine extends over palonosetron, possibly engaging in a cation-π interaction, as reported for granisetron page 2 (the conformation of loop D residues is further discussed in Figure SI4). A dense network of interactions stabilizes the side chains of Y126, R65, Y67, D42, R169, D177 on the complementary side (Figure 3 and Figure SI5). During simulations of all drugs, the D42-R65 and D42-R169 salt bridges are always established, whereas the D177-R169 one is occasionally disrupted. While the side-chain conformations in the binding loops of the complementary subunit strikingly resemble those of the serotonin-bound receptor, it is worth noting that both D42-R65 and D177-R169 (to a lesser extent) can break in simulations of the apo or serotonin-bound receptor (Figure SI5e,f).

Conformational dynamics of the drugs, H-bonding patterns

The protonated nitrogen of the azabicyclo ring forms a H-bond with the main chain carbonyl of W156 (Figure 7). This H-bond is consistently observed throughout the MD trajectory for four palonosetron molecules, whereas the fifth one engages in a long-lived H-bond with N101 (Figure 4b,c). The equivalent nitrogen of granisetron steadily interacts with W156 in four subunits and is left unpaired in the last subunit (Figure SI7). In trajectories generated for ondansetron and cilansetron, the nitrogen also forms H-Bonds with W156 and N101, with a preference for the latter residue (Figure SI9). This trend is more pronounced for ondansetron, whereas cilansetron also occasionally forms an H-Bond with S155 (Figure SI8). Dolasetron engages in a long-lived H-Bond with N101 through its ring nitrogen. It is also transiently H-Bonded to W156 but through its carboxyl oxygen, at variance with the other -setrons. Interestingly, dolasetron also forms an H-Bond between the nitrogen of its aromatic moiety and W63 (Figure SI6). The H-bond dynamics observed at the binding sites along the MD trajectories seem correlated with the conformational dynamics of the inhibitors (Figure 4a and SI6-SI9). It essentially involves a rotation around one (two for cilansetron) dihedral angle connecting the two rigid moieties of the -setron drugs, resulting in distinct conformations, with populations possibly modulated by the environment, which are observed for all drugs in solution or bound to the 5-HT3 receptor. The free-energy profile along the dihedral connecting the palonosetron rigid moieties, computed both in water and in the protein, revealed four minima (labeled A-D in Figure 4a) separated by barriers amounting up to 4 kcal/mol (Figure SI10). It echoes the 1993 report on the discovery of palonosetron, where the authors noted: "An affinity of this magnitude could only be associated with a compound that binds at or near its global minimum" [START_REF] Clark | 2-(Quinuclidin-3-yl)pyrido[4,3-B]indol-1-Ones and Isoquinolin-1-Ones. Potent Conformationally Restricted 5-HT3 Receptor Antagonists[END_REF] . The inset shows the distributions of the dihedral angle between the two moieties of palonosetron (drawn in red), either bound to the receptor or free in water. The position of the aromatic part is relatively conserved in the protein indicating that the conformations sampled result from the conformational dynamics of the azabicyclo ring in the binding site. b. The two distance distributions depict the H-Bonds between the azabicyclo ring -NH group and the backbone carbonyl of W156 (the major interactant) and the sidechain carbonyl of N101 (note the sharp peak at 2.5 Å distance) monitored over the MD trajectory for the 5 subunits. c. Three snapshots illustrate the main conformations sampled by palonosetron. page 3

What interacts with the ligand H-bond acceptor?

The presence of an H-bond acceptor in between the aromatic moiety and the cycle-embedded nitrogen moiety seems a defining feature of 5-HT3 receptor antagonists [START_REF] Thompson | Recent Developments in 5-HT3 Receptor Pharmacology[END_REF] . Yet, no obvious interaction for this chemical group is immediately visible when observing the palonosetron-bound cryo-EM structure (or the granisetron-bound one). In all simulations, we observed the formation of an H-bond between a water molecule and the oxygen of the ligand central carbonyl. This water molecule also engages in two H-bonds with the protein, one with the backbone carbonyl of Y64 and one with the -NH group of the W156 indole. In experimental densities, a faint peak is recorded and may correspond to a water molecule, this signal being stronger in the granisetron structure (visual inspection of the 6NP0 density map). We placed a water molecule and conducted new MD simulations. Indeed, this molecule remained trapped most of the time in the case of palonosetron (Figure 5), and at various levels for the other drugs (Figure SI11).

A structural framework for functional data

Over nearly three decades, numerous studies have probed how mutations in, or around, the orthosteric binding site influenced anti-emetics inhibition of the 5-HT3 receptor. We briefly discuss the literature, in light of our structure and simulations, for each stretch of the receptor participating in the binding site.

Loop A Among loop A residues, only N101 lies close enough to palonosetron (and to other ligands in other structures) for a direct interaction (Figure 6). In our simulations, the distance distributions of N101 with -setron drugs feature a main peak at 5 Å with two additional peaks at 2.5 Å and at ~8 Å, indicating transient H-bonding of the ligands with this residue (Figures SI6-SI9). Palonosetron is 10-fold more potent with the N101A mutant, while N101Q remains similar to the wild type [START_REF] Price | Palonosetron-5-HT3 Receptor Interactions As Shown by a Binding Protein Cocrystal Structure[END_REF] , in line with the idea that the absence of H-bonding capacity would stabilize the azabicyclo moiety in the conformation where the N interacts with W156 only. Interestingly, mutations of N101 into a variety of amino acids (A, D, E, L, Q, R, V) does not affect [3H]-granisetron binding [START_REF] Sullivan | Defining the Roles of Asn-128, Glu-129 and Phe-130 in Loop A of the 5-HT3 receptor[END_REF] . Accordingly, simulations show an absence of H-bond with the drug at this residue. [3H]-granisetron-binding experiments illuminate a drastic effect of mutations of E102, with a complete loss of binding at the subnanomolar range for A, D, G, K, N (data obtained on oocytes) [START_REF] Price | A Hydrogen Bond in Loop A Is Critical for the Binding and Function of the 5-HT3 Receptor[END_REF] ; or 60-to 80-fold decrease in K d for E102D/N (data obtained on HEK cells) [START_REF] Boess | Analysis of the Ligand Binding Site of the 5-HT3 Receptor Using Site Directed Mutagenesis: Importance of Glutamate 106[END_REF] . E102 is not oriented towards the binding site and interacts with S150 and T152 (β7), while its main chain carboxyl forms an H-bond with A134 (β6) (Figure 6). Thus, one possible role for E102 is to pack the β-sandwich and to position properly loops A and B. In addition, E102 creates an electronegative patch next to the binding site entrance, which might participate in ligand attraction, in line with the observation that the deleterious effect of introducing a net positive charge in loop A (e.g. N101K, E102Q) can be assuaged by removal of the positive charge at K211(regardless of H-bonding ability at position 211) [START_REF] Mosesso | A Triad of Residues Is Functionally Transferrable between 5-HT3 Serotonin Receptors and Nicotinic Acetylcholine Receptors[END_REF] . At position 103, granisetron binding appears only marginally affected by F103Y/N substitutions [START_REF] Steward | Importance of Phenylalanine 107 in Agonist Recognition by the 5-hydroxytryptamine(3A) Receptor[END_REF] , while it was decreased ~5-fold by F103A/W mutations [START_REF] Sullivan | Defining the Roles of Asn-128, Glu-129 and Phe-130 in Loop A of the 5-HT3 receptor[END_REF] .

Loop B

Loop B harbors in most pLGIC a pivotal tryptophan residue (here W156), which forms the back wall of the binding site (Figure 7) and establishes cation-π interactions with ligands [START_REF] Beene | Cation-Pi Interactions in Ligand Recognition by Serotonergic (5-HT3A) and Nicotinic Acetylcholine Receptors: The Anomalous Binding Properties of Nicotine[END_REF] . Unsurprisingly, alanine substitution abolishes granisetron and palonosetron binding [START_REF] Del Cadia | Exploring a Potential Palonosetron Allosteric Binding Site in the 5-HT(3) Receptor[END_REF][START_REF] Thompson | Loop B Is a Major Structural Component of the 5-HT3 Receptor[END_REF] , while a more conservative mutation to tyrosine increases 10-and 180-fold the respective K d of these drugs. Nearby alanine substitutions are relatively innocuous. L157A has a 10-fold effect on granisetron binding, maybe because the smaller side chain does not hinders the possible motions of Y126. L157I is wild-type-like. page 4

Loop C

Loop C appears to be the most variable loop (with loop F) in multiple sequence alignments of pentameric ligand-gated channels. It undergoes deformation upon ligand binding, which remains modest in the 5-HT3 receptor structures. As a heritage from studies of stiff acetylcholine binding proteins (AChBP), where loop C was the most evident moving element [START_REF] Celie | Nicotine and Carbamylcholine Binding to Nicotinic Acetylcholine Receptors as Studied in AChBP Crystal Structures[END_REF][START_REF] Gao | Agonist-Mediated Conformational Changes in Acetylcholine-Binding Protein Revealed by Simulation and Intrinsic Tryptophan Fluorescence[END_REF] , its position is often referred to as a hallmark of the receptor state. Legitimate emphasis on loop C should not blunt other relevant markers of states (see below). Yet, it remains worth underscoring the simultaneous motion of the flexible loop C and ECD/TMD interface loops. Loop C connects by the rigid β9 and β10 strands, stabilized further by the N-linked carbohydrates (Figure SI12). The coupling between ligand binding and pore opening is a complex process [START_REF] Lee | Principal Pathway Coupling Agonist Binding to Channel Gating in Nicotinic Receptors[END_REF][START_REF] Purohit | Loop C and the Mechanism of Acetylcholine Receptor-Channel Gating[END_REF] , but a structurally intuitive hypothesis is that motion of loop C in response to ligand binding might be transmitted to the ECD/TMD interface by means of a central β-sheet rigid hinge. I201 and Y207 are the loop C residues that point towards palonosetron (Figure 8). Only aromatic mutations are tolerated at Y207, with Y207F yielding a 3-fold increase of K d 18 (11-fold increase of the granisetron K d [START_REF] Suryanarayanan | The Loop C Region of the Murine 5-HT3A Receptor Contributes to the Differential Actions of 5-Hydroxytryptamine and M-Chlorophenylbiguanide[END_REF] ). Interestingly an extra density is present at the tip of Y207 (at low contour level), which might correspond to a water molecule linking this residue to the complementary side via Y126 and Q124. I201 is not conserved in the human receptor. It corresponds to M228, the mutation to alanine of which produces a 6-fold decrease of K d [START_REF] Del Cadia | Exploring a Potential Palonosetron Allosteric Binding Site in the 5-HT(3) Receptor[END_REF] , in line with the van de Waals interaction observed in the structure (I201A also deteriorates 5-fold granisetron binding in the mouse receptor [START_REF] Suryanarayanan | The Loop C Region of the Murine 5-HT3A Receptor Contributes to the Differential Actions of 5-Hydroxytryptamine and M-Chlorophenylbiguanide[END_REF] ). From a structural standpoint, loop C is stabilized by interactions of E198 and S206, and the presence of a negative charge at its tip (D202) might participate in attracting the azabicyclo moiety during binding. In line with a much broader spatial rearrangement of loop C following serotonin binding, mutations of loop C residues tend to have stronger effects on serotonin binding and activation than on binding of granisetron or palonosetron (for instance F199Y does not change the binding of both antagonists, but decreases the serotonin K i by a 180-fold, while D202A entails a 140 K i decrease and 12-fold EC 50 increase).

Loop D (and G)

Two residues of loop D, W63 and R65, are in direct contact with palonosetron, sandwiching its isoquinoline ring (Figure 3). As mentioned previously, W63A is nonfunctional, while W63Y exhibits wild-type properties both for serotonin activation and palonosetron inhibition [START_REF] Price | Palonosetron-5-HT3 Receptor Interactions As Shown by a Binding Protein Cocrystal Structure[END_REF] . The conformation of R65 seems in line with a cation-π interaction both in our palonosetron-bound structure and in the granisetron-bound one [START_REF] Basak | Molecular Mechanism of Setron-Mediated Inhibition of Full-Length 5-HT3A Receptor[END_REF] , and this interaction has been demonstrated experimentally with fluorinated derivatives of granisetron [START_REF] Kesters | Structural Basis of Ligand Recognition in 5-HT(3) Receptors[END_REF] . It is noteworthy that both R65A and R65C mutations yield different effects for different antiemetics. R65A reduces the binding affinity for granisetron, but produces no significant change for palonosetron [START_REF] Price | Palonosetron-5-HT3 Receptor Interactions As Shown by a Binding Protein Cocrystal Structure[END_REF][START_REF] Yan | Structural Features of the Ligand-Binding Domain of the Serotonin 5HT3 Receptor[END_REF] . R65C abolishes granisetron binding, yet marginally affects binding of tropisetron [START_REF] Ruepp | The Binding Orientations of Structurally-Related Ligands Can Differ; A Cautionary Note[END_REF] . The differential effect was linked to putatively different orientations of the aromatic moieties of the drug, which is not the case for palonosetron and granisetron. In our previous, lower-resolution structure in complex with tropisetron [START_REF] Polovinkin | Conformational Transitions of the Serotonin 5-HT3 Receptor[END_REF] , we modeled its aromatic ring perpendicular to the ones of palonosetron or granisetron. However, ligand building in 4.5-Å maps is tricky, and better data would be required to ascertain this difference in orientation. In loop G, D42C abolishes tropisetron binding, albeit barely modifies that of granisetron [START_REF] Ruepp | The Binding Orientations of Structurally-Related Ligands Can Differ; A Cautionary Note[END_REF] .

Loop E

Mutations at Y126 affect both granisetron (Y126A, 8-fold) [START_REF] Venkataraman | Identification of Critical Residues page 9 in Loop E in the 5-HT3ASR Binding Site[END_REF] and palonosetron binding (Y126A/F, 20-30-fold) [START_REF] Del Cadia | Exploring a Potential Palonosetron Allosteric Binding Site in the 5-HT(3) Receptor[END_REF] , in line with the proximity of the side chain to palonosetron and the implication of the terminal hydroxyl in intra and inter-subunit interactions (Figure page 5 3). The necessity of H-bonding capacity at Y126 has also been established by nonnatural amino-acid mutagenesis [START_REF] Beene | Tyrosine Residues That Control Binding and Gating in the 5-hydroxytryptamine3 Receptor Revealed by Unnatural Amino Acid Mutagenesis[END_REF] . In addition, mutagenesis suggests roles of neighboring residues in shaping the binding site properly, as alanine mutation of any residues in the 124-127 range produces a 6 to 8-fold decrease in the granisetron K d [START_REF] Venkataraman | Identification of Critical Residues page 9 in Loop E in the 5-HT3ASR Binding Site[END_REF] .

Figure 9. Loop F. On a cartoon representations, loop F residues (orange) which mutations are discussed in the text are represented as sticks.

Loop F

The extensive network of interactions on the complementary side, together with mutagenesis, points to an essential role of the precise positioning of loop F. Mutants at positions W168(Y), P171(H), L167(A), W168(A), R169(A) and K178(A) yield >5-fold increase of granisetron K d , while D177(A) shows no affinity [START_REF] Thompson | Mutagenesis and Molecular Modeling Reveal the Importance of the 5-HT3 Receptor F-Loop[END_REF] (Figure 9). The interaction of D42, R169 and D177 readily explains the mutant phenotypes at these positions: if disrupted, free charged side chains would impede the drug binding. Interestingly enough, D177N/E mutations, preserving the H-bonding capacity, yield unchanged granisetron binding, while D177A abolishes it. Serotonin activation is also lost for D177A. On top of its interaction with R169, D177 also positions the bottom of loop F through an interaction with the main chain of S179. This idea that the precise position of loop F is crucial for function is further reinforced by the effect at K178, which anchors the loop to the β9 strand at the main chain of L190. Conversely, mutation of R175 does not produce any change, in line with its orientation towards the solvent.

Would palonosetron bind at heteromeric interfaces?

Five homologous subunits of the 5-HT3 receptor are encoded by the human genome, while rodents possess only two of them. In heteromeric receptors, the activation interface is usually thought to be only the A(+)/A(-) one, i.e., the one where palonosetron fits tightly in the present structure. Early studies have concluded that some ligands bind differently to different subpopulations of receptors [START_REF] Tang | Neurochemistry in Clinical Application[END_REF] , and one wonders whether palonosetron may also bind to other interfaces. Mutations at position W156, R65 and Y126 yield no or only poor binding of palonosetron. Thus, one can infer that interfaces involving a B(+), C(+), D(+) or E(+) subunit would not bind the drug (they all lack the tryptophan in loop B, at the position equivalent to W156). Similarly B(-), C(-) or E(-) interfaces lack an arginine at positions equivalent to R65, and D(-) interfaces lack a tyrosine at position equivalent to Y126. Put together, we hypothesize that only A(+)/A(-) interfaces are responsible for palonosetron binding and inhibition.

The singularity of palonosetron?

Discovered in 1993 [START_REF] Clark | 2-(Quinuclidin-3-yl)pyrido[4,3-B]indol-1-Ones and Isoquinolin-1-Ones. Potent Conformationally Restricted 5-HT3 Receptor Antagonists[END_REF] and marketed in 2003, palonosetron was coined a "second-generation antagonist", obviously because it came after granisetron, tropisetron and ondansetron, but also because, unlike these molecules, it was reported to prevent not only acute-, but also delayed-CINV in clinical studies [START_REF] Grunberg | Prevention of Delayed Nausea and Vomiting (D-CINV): Carryover Effect Analysis of Pooled Data from 2 Phase III Studies of Palonosetron (PALO)[END_REF] . Palonosetron distinctive features (higher potency [START_REF] Wong | The Interaction of RS 25259-197, a Potent and Selective Antagonist, with 5-HT3 Receptors, in Vitro[END_REF] and longer plasma half-life [START_REF] Stoltz | Pharmacokinetic and Safety Evaluation Palonosetron, a 5-hydroxytryptamine-3 Receptor Antagonist, in US and Japanese Healthy Subjects[END_REF] ) cannot in principle explain this unique effect on delayed CINV: other drugs administered more often and at higher doses would also prevent it. In attempts to identify other singularities that might rationalize the advertised clinical outcome, it was also proposed that palonosetron 1) shows cooperativity and binds to allosteric sites [START_REF] Rojas | Palonosetron Exhibits Unique Molecular Interactions with the 5-HT3 Receptor[END_REF] , 2) triggers 5-HT3 receptor internalization [START_REF] Rojas | Palonosetron Triggers 5-HT(3) Receptor Internalization and Causes Prolonged Inhibition of Receptor Function[END_REF] and 3) influences the crosstalk between 5-HT3 receptors and the neurokinin 1 receptor in a way that elicits a synergistic effect of antagonists at each receptor [START_REF] Rojas | The Antiemetic 5-HT3 Receptor Antagonist Palonosetron Inhibits Substance P-Mediated Responses in Vitro and in Vivo[END_REF] (neurokinin 1 inhibitors such as aprepitant are widely used in combination with -setron drugs for CINV). Our data unequivocally shows that palonosetron binds to the orthosteric site; no secondary site was apparent from the inspection of the residual densities, in line with mutagenesis experiments disproving the existence of an allosteric site underneath the neurotransmitter one [START_REF] Del Cadia | Exploring a Potential Palonosetron Allosteric Binding Site in the 5-HT(3) Receptor[END_REF] . Granisetron, tropisetron, and palonosetron bind to the same site and apparently stabilize the same conformation of the homomeric 5-HT3A receptor: In this respect, the clinical singularity of palonosetron is not apparent in current structural studies. We have however underscored some of the palonosetron unique features in terms of binding and dynamics: the preeminence of its conformation of minimal energy (Figure 4a, SI10); the continuous presence of a water molecule next to its H-bond acceptor (Figure 5). In addition, the notion of "restricted" conformational freedom of the aromatic moiety, which guided the synthesis of palonosetron [START_REF] Clark | 2-(Quinuclidin-3-yl)pyrido[4,3-B]indol-1-Ones and Isoquinolin-1-Ones. Potent Conformationally Restricted 5-HT3 Receptor Antagonists[END_REF] , is clearly echoed here. The distribution of the angle between the aromatic moiety and the main axis of the protein is narrower for palonosetron compared to other drugs (Figure SI13). page 6

Serotonin-bound ECD conformations clearly differ from antagonist-bound ones

The orthosteric site of pentameric ligand-gated channels lies at the interface between subunits, and quaternary reorganization of extracellular domains is key to their activation [START_REF] Polovinkin | Conformational Transitions of the Serotonin 5-HT3 Receptor[END_REF] . To get a sense of the spread of quaternary arrangements of available 5-HT3 receptor structures, we superimposed their complementary subunits and looked at their principal subunits (Figure 10). Two groups are clearly distinguished. The average pairwise RMSD within the group of inhibited structures is 0.49±0.06 Å, while the average pairwise RMSD between inhibited and activated structures is 0.91±0.22 Å. At the binding site level, the activated structures possess tighter cavities where -setron binding would not be possible (Figure 10c). The apo structure rather lies within the cluster of inhibited ones, following the allosteric theory hypothesis stating that ligands stabilize preexisting conformations [START_REF] Monod | ALLOSTERIC TRANSITIONS: A PLAUSIBLE MODEL[END_REF] . However , as already noted by Basak et al. [START_REF] Basak | Molecular Mechanism of Setron-Mediated Inhibition of Full-Length 5-HT3A Receptor[END_REF] , the apo structure presents moderate deviations compared to the inhibited ones, which are most apparent at the level of strands β8 and β9, and of loop F. In line with the closed phenotype in the absence of agonist, no deviation is observed at the ECD/TMD interface, a key region for the transduction of the signal from the orthosteric site to the pore gate.

5-HT3 receptor inhibitors beyond emesis?

5-HT3 receptor antagonists are widely used for the control of emesis. Their potential in other therapeutic areas, by themselves or as adjunct drugs, has been explored in preclinical and clinical studies with equivocal results. A significant fraction of these studies were carried out more than two decades ago and it is worth combining older [START_REF] Costall | 5-HT3 Receptors[END_REF] and more recent reviews [START_REF] Sanger | A History of Drug Discovery for Treatment of Nausea and Vomiting and the Implications for Future Research[END_REF][START_REF] Fakhfouri | 5-HT3 Receptor Antagonists in Neurologic and Neuropsychiatric Disorders: The Iceberg Still Lies beneath the Surface[END_REF][START_REF] Gupta | 5HT3 Receptors: Target for New Antidepressant Drugs[END_REF] to get a sense of the yet unmet promises of 5-HT3 receptor inhibition in areas such as depression, anxiety, schizophrenia and drug withdrawal.

In parallel, roles of 5-HT3 receptors in a variety of tissues continue to be uncovered, for instance in the lacrimal gland [START_REF] Imada | Serotonin Hormonally Regulates Lacrimal Gland Secretory Function via the Serotonin Type 3a Receptor[END_REF] , in the development of the urinary system [START_REF] Ritter | Serotonin Receptor 5-HT3A Affects Development of Bladder Innervation and Urinary Bladder Function[END_REF] , as well as in adipocytes [START_REF] Oh | Regulation of Systemic Energy Homeostasis by Serotonin in Adipose Tissues[END_REF] . These and future similar studies may one day help reinterpret older observations from (pre)clinical studies. In our era of drug repurposing, -setron molecules, which enjoy a good safety profile and a relative absence of adverse effects, might spur renewed interest.

Conclusion

Detailed knowledge of antiemetics mode of action with the 5-HT3 receptor has remained hitherto fragmentary. We have solved using Cryo-EM the palonosetron-bound structure of a mouse 5-HT3 receptor, which amid the very few related structures available, provides valuable new information towards understanding inhibitory mechanisms at the atomic level. Combining our structural and computational data, we put forth a convenient framework for rationalizing the host of biochemical and pharmacological information accrued since the first-setron drug synthesis. This framework is well-suited for correlating specific mutations to observed phenotypes, and for establishing a general structure-activity relationship for antiemetic drugs. As more ligand-bound structures of serotonin 5-HT3 receptors will likely be obtained at increasing resolutions, our grasp of pharmacology and conformational equilibrium will deepen. In the coming years we also anticipate that experimental data on asymmetry (either in homomeric or heteromeric receptors) and on conformational variability will continue to reshape the current view on the modus operandi of these fascinating allosteric neuroreceptors.

Figure 10. The inhibited conformations form a group that differ from the active ones . Because the quaternary organization is key to the gating of the 5-HT3 receptor, we here represent the principal subunit ECD after alignment of the complementary subunit ECD. Active conformations are depicted in red, inhibited in blue, while the apo one is in green. All the inhibited conformations are clearly grouped, all the active ones as well. The inhibited group is closer to the apo conformation, and similar at the ECD/TMD interface while differences are seen in β8 and β9. a. and b. panels are two different orientations where the subunit is seen from the point of view of its two neighbors. c. Palonosetron (yellow spheres) fits tightly in its binding site (surface of loop C in blue) but would clash with the binding site of the active conformation (red surface of loop C). page 7

Methods

No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.

Protein production and purification

The wild-type mouse 5-HT3A receptor was expressed in a stable and inducible cell line, and purified in the C12E9 detergent as previously described [START_REF] Polovinkin | Conformational Transitions of the Serotonin 5-HT3 Receptor[END_REF][START_REF] Hassaïne | Expression, Biochemistry, and Stabilization with Camel Antibodies of Membrane Proteins: Case Study of the Mouse 5-HT3 Receptor[END_REF] .

Electron microscopy and image analysis

The most homogeneous fractions after size-exclusion chromatography were concentrated to ~1.5 mg/ml, mixed with lipids (0.01% phosphatidic acid, 0.01% cholesterol hemisuccinate, 0.01% brain phosphatidylcholine; Avanti Polar Lipids) and 1 mM palonosetron (gift from C. Ulens) and incubated for 10 minutes on ice. 3.5 μl were deposited on a glow-discharged (25 mA, 50 s) Quantifoil copper-rhodium R 1.2/1.3 grid, blotted at 8 o C for 10 s at force 0 using a Mark IV Vitrobot and plunge-frozen in liquid ethane. The dataset was recorded on a Titan Krios electron microscope at the ESRF 51 (Table SI2). Movies (40 frames) were acquired with EPU on a Gatan K2 Summit camera mounted after an energy filter (20eV slit width). The 3'037 raw movies were aligned and summed with MotionCor2 52 , within Relion 3.08 [START_REF] Scheres | Implementation of a Bayesian Approach to Cryo-EM Structure Determination[END_REF] . CTF estimation for non dose-weighted sums was calculated using GCTF [START_REF] Zhang | Gctf: Real-Time CTF Determination and Correction[END_REF] . Particles were picked with the FPM algorithm [START_REF] Estrozi | Fast Projection Matching for Cryo-Electron Microscopy Image Reconstruction[END_REF] using as templates 2D projections of the serotonin-bound 5-HT3 receptor (PDB code: 6HIQ), low-pass filtered at 30 Å. All subsequent steps were performed in Relion. A total of 514'057 images were extracted, 4x4 binned and sorted in 20 classes by 2D classification. Class averages that resembled a pentameric ion channel were selected, amounting 348'500 particles. Selected particles were sorted by 3D classification without imposing symmetry and using as a reference model the low-pass filtered 6HIQ map. The classes that presented typical 5-HT3 receptor features and some density for the ICD were selected. The selected subset, amounting 198'076 images, was re-extracted at full size and used for 3D auto-refinement with imposed C5 symmetry, resulting in an initial map at 3.6 Å resolution. The particles from this refinement job were further sorted in four classes by focused 3D classification, with a mask around the ICD, without image alignment. The best class, amounting 149'317 particles, was subjected to cycles of 3D auto-refinement, Bayesian polishing and CTF refinement resulting in a 3.2 Å unmasked map. The 'shiny' particle stack from the last polishing job was imported to, and final steps were performed in, cryoSPARC [START_REF] Punjani | cryoSPARC: Algorithms for Rapid Unsupervised Cryo-EM Structure Determination[END_REF] . After 2D classification cleaning (Figure SI14), 127'971 particles were used for an ab initio reconstruction, followed by three rounds of Non-Uniform refinement, with C5 symmetry, yielding a final masked map at 2.82 Å resolution. Local resolutions were estimated with the ResMap software [START_REF] Kucukelbir | Quantifying the Local Resolution of Cryo-EM Density Maps[END_REF] (Figure SI14).

Simulation details

The cryo-EM structure was the initial point for the simulation of palonosetron. A pure palmitoyl-oleyl-phosphatidylcholine (POPC) bilayer, consisting of 120 units in each leaflet, was generated using the CHARMM-GUI interface [START_REF] Jo | CHARMM-GUI: A Web-Based Graphical User Interface for CHARMM[END_REF][START_REF] Wu | CHARMM-GUI Membrane Builder toward Realistic Biological Membrane Simulations[END_REF] . The complex was solvated with 38386 TIP3P water molecules [START_REF] Jorgensen | Comparison of Simple Potential Functions for Simulating Liquid Water[END_REF] and 150 mM KCl. The initial structures of granisetron-, ondansetron-, dolasetron-, and cilansetron-bound 5-HT3 pentamers were obtained by replacing the palonosetron by these ligands. The aromatic rings of the -setron ligands were aligned with the aromatic plane of palonosetron. The parameters for all ligands were generated by CHARMM general force field (CGenFF) [START_REF] Vanommeslaeghe | CHARMM General Force Field: A Force Field for Drug-like Molecules Compatible with the CHARMM All-Atom Additive Biological Force Fields[END_REF] . The protein and lipid were described by CHARMM36 protein [START_REF] Mackerell | All-Atom Empirical Potential for Molecular Modeling and Dynamics Studies of Proteins[END_REF][START_REF] Best | Optimization of the Additive CHARMM All-Atom Protein Force Field Targeting Improved Sampling of the Backbone ϕ, ψ and Side-Chain χ1 and χ2 Dihedral Angles[END_REF] and lipid [START_REF] Klauda | Update of the CHARMM All-Atom Additive Force Field for Lipids: Validation on Six Lipid Types[END_REF] force fields, respectively. Hydrogen mass repartition [START_REF] Hopkins | Long-Time-Step Molecular Dynamics through Hydrogen Mass Repartitioning[END_REF] was used for all drug-receptor complex simulations, allowing for using a time step of 4 fs. Program NAMD 2.13 [START_REF] Phillips | Scalable Molecular Dynamics with NAMD[END_REF] was employed for all the simulations. The temperature and pressure were maintained at 1 atm and 300K by the Langevin piston method [START_REF] Feller | Constant Pressure Molecular Dynamics Simulation: The Langevin Piston Method[END_REF] and the Langevin thermostat, respectively. Long-range electrostatic interactions were evaluated by the particle-mesh Ewald (PME) algorithm [START_REF] Darden | Particle Mesh Ewald: An N⋅log(N) Method for Ewald Sums in Large Systems[END_REF] . Integration was performed with a time step of 8 and 4 fs for long-and short-range interactions, respectively, employing the r-RESPA multiple time-stepping algorithm [START_REF] Tuckerman | Reversible Multiple Time Scale Molecular Dynamics[END_REF] . The SHAKE/RATTLE [START_REF] Ryckaert | Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of N-Alkanes[END_REF][START_REF] Andersen | Rattle: A "velocity" Version of the Shake Algorithm for Molecular Dynamics Calculations[END_REF] was used to constrain covalent bonds involving hydrogen atoms to their experimental lengths, and the SETTLE algorithm [START_REF] Miyamoto | Settle: An Analytical Version of the SHAKE and RATTLE Algorithm for Rigid Water Models[END_REF] was utilized for water. For each molecular assembly, a 100 ns pre-equilibration was performed using soft harmonic positional restraints on heavy atoms, followed by a 400 ns production run with no restraints. The VMD program was employed to analyze and render the MD data [START_REF] Humphrey | Visual Molecular Dynamics[END_REF] .

Model refinement and structure analysis

Cycles of real-space refinement in Phenix [START_REF] Liebschner | Macromolecular Structure Determination Using X-Rays, Neutrons and Electrons: Recent Developments in Phenix[END_REF] were performed, alternating with manual rebuilding in Coot [START_REF] Emsley | Features and Development of Coot[END_REF] . The model comprises residues 8-329 and 397-459. Palonosetron (numbered 903) was placed in the orthosteric site. Even though uncommon at this resolution, residues 67 and 168 were modeled in two alternate conformations (see text and Figure SI4) with occupancies set at 0.5. The model is 5-fold symmetric, as the reconstruction used to build it possesses C5 symmetry. Validation was performed with Molprobity 76 (Table SI2). Visual information about the model and its fit in the map are found in figures 1a,b, 2b, SI1, SI4, SI12 and SI15. Figures were prepared with An alternate conformation at the level of Y67 and W168? Above R65, the density map appears more noisy than in the surroundings (middle panel, zone encircled in purple), which may indicate that W168 and Y67 and W168 can adopt dual conformations. The first one (represented in black from the 6np0 granisetron-bound model, or in limon) features a four-layer stack (drug-R65-W168-Y67) where Y67 lies far from the drug, while in the second one (represented in yellow), W168 moves out to permit a direct interaction of Y67 with R65. Mutagenesis seems to indicate that the ability to form hydrogen bond at position 67 impacts palonosetron binding (Y67F shows a 6-fold decrease in Ki) [START_REF] Price | Palonosetron-5-HT3 Receptor Interactions As Shown by a Binding Protein Cocrystal Structure[END_REF] . We ran molecular dynamics simulations of the two possible palonosetron-bound conformations and observed a better stability of the drug in conformation #1. In the granisetron-bound map of Basak et al. (depicted in the right panel), only the conformation #1 seems present. Cysteine substitution at position 67 affects slightly granisetron binding but drastically tropisetron binding [START_REF] Ruepp | The Binding Orientations of Structurally-Related Ligands Can Differ; A Cautionary Note[END_REF] , implying that the relative position of loop D side chains and of the drug aromatic moiety varies from drug to drug. The inset shows the distributions of the dihedral angle between the two moieties of ondansetron (drawn in red), either bound to the receptor or free in water. The position of the aromatic part is relatively conserved in the protein indicating that the conformations sampled result from the conformational dynamics of the azabicyclo ring in the binding site. b. The four distance distribution depict the H-Bonds between the ring -NH group and the backbone carbonyl of W156, the backbone carbonyl of S155, the backbone carbonyl of T154 and the side chain carbonyl of N101 monitored over the MD trajectory for the 5 subunits. c. Four snapshots illustrate the main conformations sampled by ondansetron. page 16

Figure 1 .

 1 Figure 1. Cryo-EM structure of the mouse 5-HT3 receptor in complex with palonosetron. a. View parallel to the membrane plane. Three subunits are represented as cartoons while the cryo-EM map is depicted for the two last subunits. Palonosetron molecules are depicted as yellow spheres. b. View of a slab (fuschia lines) perpendicular to the membrane (as indicated by the arrow). c. Chemical formula of palonosetron, with its aromatic moiety in blue, its ring-embedded protonated nitrogen in green and its H-bond acceptor oxygen group in red. The 's' letters next to chiral carbon atoms indicate stereochemistry.

Figure 2 .

 2 Figure 2. Close-up of the palonosetron binding site. a. Electrostatic potential mapped on the molecular surface of the 5-HT3 receptor, with stick representation of palonosetron in the neurotransmitter binding site. The view is from outside, approximately perpendicular to the membrane plane. b . Cartoon and stick representation, approximately parallel to the membrane. Side chains within 4 Å of the drug are depicted as sticks. The protein is colored using the same code throughout the report (on the principal side, loop A: maroon, loop B: green, loop C: yellow; on the complementary side, loop D: purple, loop E: orchid, loop F: orange, loop G: forest green). The cryo-EM density for palonosetron is depicted as a grey mesh.

Figure 3 .

 3 Figure 3. Network of side chain interactions on the complementary subunit. a. Cartoon and stick representation, approximately parallel to the membrane. Side chains within 4 Å of the drug are depicted as sticks. Yellow dashes indicate H-bonds or salt bridges. The dual conformation of residues Y67 and W168 (see Methods) is depicted as transparent sticks. b . Distance distributions between atoms forming the three salt bridges D42-R65, D42-R169 and D177-R169 (black, red and transparent cyan respectively) sampled during the MD simulation in the presence of palonosetron.

Figure 4 .

 4 Figure 4. H-bonding pattern and dynamics of palonosetron during MD simulations. a.The inset shows the distributions of the dihedral angle between the two moieties of palonosetron (drawn in red), either bound to the receptor or free in water. The position of the aromatic part is relatively conserved in the protein indicating that the conformations sampled result from the conformational dynamics of the azabicyclo ring in the binding site. b. The two distance distributions depict the H-Bonds between the azabicyclo ring -NH group and the backbone carbonyl of W156 (the major interactant) and the sidechain carbonyl of N101 (note the sharp peak at 2.5 Å distance) monitored over the MD trajectory for the 5 subunits. c. Three snapshots illustrate the main conformations sampled by palonosetron.

Figure 5 .

 5 Figure 5. A trapped water molecule links palonosetron H-bond acceptor and the receptor. a. Representative snapshot illustrating the water mediated H-Bond network linking the palonosetron central carbonyl to protein residues W156 and Y64. b . Pair radial distribution functions between the oxygen of the palonosetron central carbonyl and the water oxygen, characterizing the probability density to find the two species at a given distance. The peak located at ~3 Å indicates that for every subunit, there is always one water molecule H-bonded to palonosetron.

Figure 6 .

 6 Figure 6. Loop A. On a cartoon representation, the three most important residues of loop A are shown as sticks, together with neighbors of E102. The interactions of E102 described in the text are represented as dashes.

Figure 7 .

 7 Figure 7. Loop B. On a cartoon representation, residues of loop B facing palonosetron (blue sticks) are shown as green sticks. The interaction of W156 main chain with the protonated nitrogen of the ligand is depicted as a dashed line.

Figure 8 .

 8 Figure 8. Loop C. The serotonin-bound activated structure is superimposed to the palonosetron inhibited one (on the complementary subunit) to highlight the yellow→cyan capping motion of loop C. a. View parallel to the membrane plane of loop C. Palonosetron and residues which mutations are discussed in the text are represented as sticks b. View perpendicular to the membrane plane. Here, serotonin present in the activated structure is also represented as sticks.

Figure SI2 .

 SI2 Figure SI2. Nomenclature of the orthosteric site and sequence alignment. a. Ribbon representation of a full subunit, and two adjacent extracellular domains, with colored stretches for the binding elements making up the neurotransmitter site: 3 loops named A-C on the principal subunit, 3 strands and a loop named D-G on the complementary subunit. b. Multiple sequence alignment of the mouse and human 5-HT3 receptors, with binding elements highlighted.

Figure SI3 .

 SI3 Figure SI3. Normalized distribution of interaction areas sampled along the MD trajectories. Left column. Interface area between ECDs. Middle column. Interface area between the drug and the complementary subunit. Right column. Interface area between the drug and the principal subunit.

Figure SI4.

  Figure SI4. An alternate conformation at the level of Y67 and W168?Above R65, the density map appears more noisy than in the surroundings (middle panel, zone encircled in purple), which may indicate that W168 and Y67 and W168 can adopt dual conformations. The first one (represented in black from the 6np0 granisetron-bound model, or in limon) features a four-layer stack (drug-R65-W168-Y67) where Y67 lies far from the drug, while in the second one (represented in yellow), W168 moves out to permit a direct interaction of Y67 with R65. Mutagenesis seems to indicate that the ability to form hydrogen bond at position 67 impacts palonosetron binding (Y67F shows a 6-fold decrease in Ki)[START_REF] Price | Palonosetron-5-HT3 Receptor Interactions As Shown by a Binding Protein Cocrystal Structure[END_REF] . We ran molecular dynamics simulations of the two possible palonosetron-bound conformations and observed a better stability of the drug in conformation #1. In the granisetron-bound map of Basak et al. (depicted in the right panel), only the conformation #1 seems present. Cysteine substitution at position 67 affects slightly granisetron binding but drastically tropisetron binding[START_REF] Ruepp | The Binding Orientations of Structurally-Related Ligands Can Differ; A Cautionary Note[END_REF] , implying that the relative position of loop D side chains and of the drug aromatic moiety varies from drug to drug.

Figure SI5 :

 SI5 Figure SI5: Evolution of the salt bridges during the MD trajectories. Representative snapshots and distance distributions between atoms forming the three salt bridges D42-R65, D42-R169 and D177-R169 (black, red and transparent cyan respectively) sampled during the MD simulation in the presence of (a) ondansetron, (b) granisetron, (c) dolasetron, (d) cilensentron, (e) serotonin and (f) without drug.

Figure

  Figure SI6. H-bonding pattern and dynamics of dolasetron during MD simulations. a. The inset shows the distributions of the dihedral angle between the two moieties of dolasetron (drawn in red), either bound to the receptor or free in water. The position of the aromatic part is relatively conserved in the protein indicating that the conformations sampled result from the conformational dynamics of the azabicyclo ring in the binding site. b. The six distance distributions depict the H-Bonds between the dolasetron central carbonyl and the -NH group of W156 (W156-O), the ring -NH group and the backbone carbonyl of W156 (W156-N) or the sidechain carbonyl of N101 (N101-N), the carbonyl of the nitrogen cage and the-NH group of N101 (N101-O) and the -NH group of the dolasetron aromatic ring and the carbonyl of W63 or Y64 monitored over the MD trajectory for the 5 subunits. c. Four snapshots illustrate the main conformations sampled by dolasetron.

Figure

  Figure SI7. H-bonding pattern and dynamics of granisetron during MD simulations. a.The inset shows the distributions of the dihedral angle between the two moieties of granisetron (drawn in red), either bound to the receptor or free in water. The position of the aromatic part is relatively conserved in the protein indicating that the conformations sampled result from the conformational dynamics of the azabicyclo ring in the binding site. b. The distance distribution depicts the H-Bonds between the ring -NH group and the backbone carbonyl of W156 monitored over the MD trajectory for the 5 subunits. c. Two snapshots illustrate the main conformations sampled by granisetron.

Figure

  Figure SI8. H-bonding pattern and dynamics of cilansetron during MD simulations. a.The inset shows the distributions of the two dihedral angles between the two moieties of cilansetron (drawn in red), either bound to the receptor or free in water. The position of the aromatic part is relatively conserved in the protein indicating that the conformations sampled result from the conformational dynamics of the azabicyclo ring in the binding site. b. The three distance distributions depict the H-Bonds between the ring -NH group and the backbone carbonyl of W156, the backbone carbonyl of S155 or the side chain carbonyl of N101 monitored over the MD trajectory for the 5 subunits. c. Four snapshots illustrate the main conformations sampled by cilansetron.

Figure

  Figure SI9. H-bonding pattern and dynamics of ondansetron during MD simulations. a.The inset shows the distributions of the dihedral angle between the two moieties of ondansetron (drawn in red), either bound to the receptor or free in water. The position of the aromatic part is relatively conserved in the protein indicating that the conformations sampled result from the conformational dynamics of the azabicyclo ring in the binding site. b. The four distance distribution depict the H-Bonds between the ring -NH group and the backbone carbonyl of W156, the backbone carbonyl of S155, the backbone carbonyl of T154 and the side chain carbonyl of N101 monitored over the MD trajectory for the 5 subunits. c. Four snapshots illustrate the main conformations sampled by ondansetron.

Figure SI10 .

 SI10 Figure SI10. Potential of mean-force (PMF) associated with the palonosetron dihedral angle defined in Fig. 4.a. Free-energy calculations were performed using the e-ABF algorithm implemented in NAMD together with the colvar module. (black) PMF determined for the palonosetron in complex with 5-HT3 (200ns) (red) PMF determined for the palonosetron in water (400ns).

Figure SI11 .

 SI11 Figure SI11. A trapped water molecule links (a) ondansetron, (b) granisetron, (c) dolasetron and (d) cilansetron H-bond acceptor and the receptor. Upper panel. Representative snapshot illustrating the water mediated H-Bond network linking the drug central carbonyl to protein residues W156 and Y64. Lower panel .Pair radial distribution functions between the oxygen of the drug central carbonyl and the water oxygen, characterizing the probability density to find the two species at a given distance. The peak located at ~3 Å indicates that for every subunit, there is always one water molecule H-bonded to all drugs

Figure SI13 .

 SI13 Figure SI13. Distribution of the angle between the aromatic moiety and the pseudo-symmetry axis of the protein. The red line on the left panel corresponds to the value for the palonosetron cryo-EM structure .

Figure SI14 . 19 Figure SI15 .

 SI1419SI15 Figure SI14. Image analysis workflow. a. A representative micrograph (out of 3037 micrographs) of the pre-incubated with palonosetron 5HT3A receptor in vitreous ice. b. Gold-standard Fourier shell correlation (FSC), calculated by comparing the two independently determined half maps in the final Non-Uniform refinement in cryoSPARC. The dotted line represents the 0.143 FSC threshold, which indicates a nominal resolution of 3.6 Å for the unmasked (red) and 2.8 Å for the masked (blue) reconstruction. c. Model-to-map Fourier shell correlation (FSC). The dotted line represents the 0.5 FSC threshold, which indicates a nominal resolution of 3.23 Å for the unmasked (red) and 3.13 Å for the masked (blue) reconstruction. d. The selected 2D class averages of the particles that were used for the final reconstruction. e. Side view of the unfiltered and unsharpened 3D density map colored according to the local resolution estimated in ResMap. f. Heat map of the angular distribution of particle projections for the final reconstruction calculated in cryoSPARC and colored-coded depending the approximate number of particles for each orientation.

  [START_REF] Kesters | Structural Basis of Ligand Recognition in 5-HT(3) Receptors[END_REF]

  -residue to facilitate the reading, without specifying if they are mutants of the human or of the mouse 5-HT3 receptor (as long as both receptors have the same amino-acid at a given position of course).

	1	Throughout	the	report,	mutants	are	simply	noted
	residue-number

Table SI2 . Cryo-EM data collection, refinement and validation statistics for the Palonosetron-bound 5-HT3 receptor, EMD-10673, PDB 6Y1Z page 18

 SI2

	Electron exposure (e -/Å 2 )	53	Model composition
			Non-hydrogen atoms	16'610
			Protein	16'095
			Palonosetron	110
			Carbohydrates	405
	Defocus range (μm)	-0.8 to -2.5	B factors (Å 2 ) min/max/mean
			Protein	60.17/158.56/96.97
			Ligand	76.35/133.10/104.23
	Pixel size (Å)	1.067	R.m.s. deviations
			Bond lengths (Å)	0.003
			Bond angles (°)	0.537
	Symmetry imposed	C5	Validation
			MolProbity score	1.56
			Clashscore	3.46
			Poor rotamers (%)	2.21
	Initial particle images (no.)	514'057	Ramachandran plot
			Favored (%)	97.11
			Allowed (%)	2.89
			Disallowed (%)	0
	Final particle images (no.)	127'971
	Map resolution (Å)	2.8
	FSC threshold	0.143

Acknowledgements

We acknowledge access to ESRF CM01 Krios microscope. We thank L. Estrozi and A. Desfosses for advice in image analysis; members of the Nury group for discussions. The work was funded by ERC Starting grant 637733 Pentabrain. It used the platforms of the Grenoble Instruct-ERIC center (ISBG ; UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-05-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). The electron microscopy facility is supported by the Rhône-Alpes Region, the FRM, the FEDER and the GIS-IBISA. F.D. acknowledge the State-Region Plan "Technological Innovations, Modeling and Personalized Medical Support" (IT2MP) and the European Regional Development Funds (ERDF) for generous support. H.Z. gratefully thank the financial support from China Scholarship Council (No. 201806200074).

Supplementary information

page 11


Maxime Munch

Dr Mathieu Curtil

Prof Pauline M Vérité

Dr Denis Jacquemin
email: denis.jacquemin@univ-nantes.fr

Dr Julien Massue
email: massue@unistra.fr

Gilles Ulrich
email: gulrich@unistra.fr

Ethynyl-Tolyl Extended 2-(2'-Hydroxyphenyl)benzoxazole Dyes: Solution and Solid-state Excited-State Intramolecular Proton Transfer (ESIPT) Emitters

Keywords: Fluorescence, Hydroxybenzazole, ESIPT, TD-DFT calculations

Dual solution/solid-state emissive fluorophores based on a 2-(2'-Hydroxyphenyl)benzoxazole (HBO) core bearing one or two ethynyl-tolyl moieties at different positions were synthesized via an expedite two-step synthetic procedure. HBO derivatives are known to display intense Excited-State Intramolecular Proton Transfer (ESIPT) emission in the solid-state but are mildly emissive in solution due to the detrimental flexibility of the excited-state opening efficient non-radiative pathways. The sole introduction of a rigid ethynyl moiety led to a sizeable enhancement of the fluorescence quantum yield in solution, up to a 15-fold increase in toluene as compared to unsubstituted HBO dyes while keeping the high solidstate fluorescence efficiency. The position of the substitution on the π-conjugated core led to subtle fine-tuning of maximum emission wavelengths and quantum yields. Moreover, we show that the ethynyl tolyl substituent at the para position of the phenol ring is a suitable moiety for an efficient stabilization of the corresponding emissive anionic HBO derivatives in dissociative solvents like DMF THF or EtOH. These observations were confirmed in CH3CN by a basic titration. For all dyes, the nature of the excited-state involved in the fluorescence emission was rationalized using ab initio calculations.

Introduction

Photoinduced proton transfer is one of the most important chemical reactions in nature and plays a pivotal role in a wide range of biological processes. 1 Depending on environmental dynamics, proton transfer can occur in many different ways; therefore, understanding and controlling such processes is of upmost importance not only for academic purposes but also to target valuable applications. Excited-State Intramolecular Proton Transfer (ESIPT) processes consist of a photoinduced proton transfer occurring within a molecular entity. Consequently, ESIPT leads to the presence of different tautomers in the ground and excited-state which induces large Stokes shifts, i.e. a strong spectral separation between absorption and emission bands. 2 ESIPT was first evidenced in natural heterocyclic structures 3 and further extended to synthetic chromophores, including notably 2-(2'-hydroxyphenyl)benzazole (HBX) derivatives 4 as well as many others. 5 While the range of organic structures prone to display ESIPT is wide, a common feature to all ESIPT dyes lies in the presence of a strong intramolecular H-bond. which can involve various donors and acceptors and form five, six, seven or eightmembered rings. 6 Many structural and environmental parameters can impact the H-bond energy and thus the ESIPT process efficiency, leading to single or dual emitters, depending on the strength and nature of the H-bond. 7 Additionally, physical factors such as temperature, viscosity, pH or polarity/proticity in the close vicinity of the dye have a strong impact on both the position and intensity ratio of the tautomeric emission bands. 8 As a consequence, each ESIPT dye displays a unique, characteristic environment-sensitive emission pattern which has been beneficially utilized in various areas where the microenvironment is prone to undergo subtle changes, such as cellular imaging 9 or fluorescence sensing. 10 ESIPT luminescence is however rather faint in solution, owing to detrimental motions occurring in the excited-state, triggering efficient non-radiative pathways and consequently leading to a strong quenching of fluorescence radiations. A trademark of ESIPT probes is their typical solid-state brightening due to a beneficial restriction of molecular torsions which contributes to a significant decrease of non-radiative deactivations. 11 The engineering of ESIPT dyes displaying fluorescence emission both in solution and in the solid-state still appear as a serendipitous challenge. 12 In this article, we demonstrate that the sole introduction of ethynyl tolyl substituents at the periphery of 2-(2'-hydroxyphenyl) benzoxazole (HBO) dyes is sufficient to reach a significant enhancement of quantum yield (ΦF) in the solution-state (multiple solvents were considered) while maintaining a strong fluorescence intensity in the solid-state. Furthermore, we show that the extension of conjugation via a triple bond at the 5 position of the phenol ring leads to the stabilization of an emissive anionic species in dissociative solvents or upon addition of a base in CH3CN. The general structure of the dyes studied is presented on Figure 1. All these observations are rationalized using ab initio calculations.

Results and Discussion

The synthesis of HBO dyes 1-2 and 5-6 is described on Schemes 1 to 4 while the structure of reported HBO dyes 3 13 and 4 13 is presented on Figure 2. The synthesis of HBO dyes 1-6 is very straightforward, consisting of two synthetic steps starting from commercially available reagents. The HBO core was constructed via two alternative routes depending on substitution. HBO 7 and 10 bearing a bromine atom were formed by reacting the appropriate 2-aminophenol and salicylaldehyde derivatives in the presence of an excess of phenylboronic acid and potassium cyanide in methanol at room temperature. HBO 9 and 11 bearing iodine atoms were formed by refluxing the corresponding reagents in ethanol followed by oxidation at room temperature in dichloromethane with a slight excess of 2,3-dichloro-5,6dicyano-1,4-benzoquinone (DDQ). It is necessary to protect the phenol ring in HBO 10 and 11 with an acetate group in order to avoid Pd-catalyzed intramolecular cyclization leading to the formation of an undesired benzofuran derivative. 14 Sonogashira cross-coupling reactions on the halogenated intermediates 7 and 9-11 were performed in a mixture of toluene and triethylamine using a catalytic amount of Pd(II) catalyst, either PdCl2(PPh3)2 or Pd(dppf)Cl2 and copper iodide to afford the target HBO dyes 1-2 and 5-6 after purification by column chromatography on silica gel. All compounds were characterized by 1 H and 13 C NMR spectroscopy, HR-MS and FT-IR (see the experimental section and Supporting Information, ESI). The photophysical properties of HBO 1-6 measured in solution and in the solid-state are gathered in Table 1. The absorption and emission spectra of all dyes in solution are respectively presented in Figures 3a and3b while absorption and emission spectra of HBO dye 6 in a range of solvents are respectively depicted in Figures 4a and4b. Full list of spectral data can be found in the ESI. The absorption spectra of HBO derivatives 1-6 in toluene feature similar trends such as a broad, rather unstructured band assigned to the S0-S1 transition, with a maximum wavelength comprised between 332 and 368 nm. The additional absorption bands observed at higher energies (below 300 nm) are assigned to the π-π * transitions of the phenyl rings. Molar absorption coefficients are in the 15000 to 45000 M -1 .cm -1 range, which is typical for compact polyaromatic dyes (Figure 3a). The absorption spectra in ethanol display very similar features as those observed in toluene (see the SI), which indicates that the optical properties in the ground state are not strongly influenced by the substitution pattern nor the nature (dipolarity) of the solvent. Upon excitation in the lowest-energy absorption band in toluene (λexc = 330-350 nm), all HBO dyes 1-6 display a single intense emission band whose maximum wavelength spans from 489 to 550 nm depending on the position of the ethynyl tolyl substituent(s) on the HBO core. In all cases, the excitation spectrum matches well the absorption one, excluding the presence of emissive aggregates in the excited-state (see the SI). The Stokes' shifts (SS) are in the range 8000-11000 cm -1 which is typical of ESIPT emitters. These SS values, larger than in standard organic fluorophores, are consistent with the formation of the keto tautomer in the excited-state (K*), as a consequence of the phototautomerization process.

Table 1. Optical data of HBO dyes 1-6 and 12 measured in aerated solution at room temperature or dispersed in potassium bromide pellets.

[a] A poor match was observed between absorption and excitation spectra evidencing the formation of aggregates in the excited-state, hence the impossibility to accurately calculate the fluorescence quantum yield of the molecular species. [b] Stokes' shift [c] Quantum yield determined in solution by using Rhodamine 6G as a reference (λexc = 488 nm, Φ = 0.88 in ethanol) or as an absolute value using a spectrofluorimeter fitted with an integration sphere. [d] Fluorescence lifetime [e] kr (10 8 s -1

) and knr (10 8 s -1

) were calculated using the following equations: kr = ΦF/τ, knr = (1-ΦF)/τ. [f] Embedded in potassium bromide pellet. Concentration around 10 -4 M.

In the case of HBO 3, a very small shoulder can be observed in the higher energies (λem = 397 nm) which can be assigned to the residual presence of the enol tautomer (E * ) in the excited-state, evidencing a partially frustrated ESIPT process. The E * band is indeed characterized by a smaller SS, consistent with a significantly least reorganization of the molecular structure in the excited-state. As can be observed in Figure 3b, the position of the ethynyl tolyl fragment on the HBO core has a subtle but non negligible influence on the maximum emission wavelength. HBO 4, bearing a substituent at the 4 position of the phenol ring presents the most blueshifted emission of the series (λem = 489 nm); an observation consistent with a weakly polarized excited-state. HBO 1,2 and 3 with a respective substitution at the 3', 6 and 5 position of the HBO scaffold display similar maximum emission wavelengths at 504, 519 and 514 nm respectively. HBO 5 and 6 which share a common substitution at position 3 of the HBO core are the most bathochromically shifted in the series (λem = 550 nm), highlighting the beneficial impact of the ethynyl tolyl fragment at the ortho position of the phenol ring for a red-shifted emission. This can be also indicative of the presence of a weak but sizeable internal charge transfer (ICT) state in these dyes.

The most striking influence of the position of the functionalization on the optical properties of HBO dyes 1-6 lies in the fluorescence quantum yield values in solution. As listed in Table 1 and schematically represented on Figure 3c, drastic differences can be observed in the isomers series. As a general observation, the introduction of a single ethynyl tolyl group on the phenol ring leads to a strong enhancement of ΦF as compared to unsubstituted HBO derivatives, with values ranging from 10 to 32% in toluene. As a reference, 4methyl-2-(2'-hydroxyphenyl)benzoxazole 12, bearing a methyl group at the 4 position was synthesized 15 and its optical properties investigated in toluene and ethanol (Table 1 and Figure 4). ) λem (nm) SS [b] (cm -1

) Φ F [c] τ(ns) [d] Kr (10 HBO 12 displays blue-shifted absorption and emission spectra compared to HBO dyes 1-6 (λabs/λem = 321/406-490 nm and 317/397-481 nm for 12 in toluene and ethanol respectively). HBO 12 is barely emissive in solution, as shown of Figure 4c (ΦF = 2% in toluene and ΦF = 1% in ethanol, leading to Kr/Knr values of 0.06/2.72 and 0.04/3.54 10 8 s -1 in these two solvents). HBO 5 and 6 which include a single or a double ethynyl tolyl substitution at position 3 and/or 5 are the most emissive dyes in the series with quantum yields reaching 32 and 30% in toluene, respectively; a 16/15-fold enhancement with respect to unsubstituted HBO 12. HBO 1, the only dye in the series substituted on the benzoxazole side stand out as weakly emissive (ΦF = 4% in toluene), further highlighting the beneficial role of the substitution on the phenol ring to gain fluorescence intensity. This luminescence enhancement is correlated with a strong increase of the radiative constant values (Kr = 0.06, 0.85 and 1.39 10 8 s -1 for HBO 12, 6 and 5, respectively). The non-radiative constant rates along with the fluorescent lifetimes remain however in the same range regardless of substitution (Knr = 2.72, 2.09 and 2.96 10 8 s -1 , τ = 3.6, 3.4 and 2.3 ns for HBO 12, 6 and 5, respectively). We wish to underline that these purely organic solution-state emissive ESIPT emitters can be obtained in only two or three synthetic steps from commercially available starting materials, allowing a facile synthesis on a multigram scale in the laboratory. Moreover, it is worth mentioning that these results highlight the interest of using oxygen-based rather than sulfur-based heterocycles, the latter more efficiently leading to intersystem crossing and, consequently smaller ΦF. 16 Triple bond extended conjugation on the phenol ring, along with the presence of a benzoxazole scaffold seem to be of paramount importance in order to reach improved luminescence intensity in solution. Moreover, the substitution at the specific 3 position of the HBO core, i.e. adjacent to the hydroxy group leads to the largest quantum yield in the series. In ethanol, all dyes with the notable exception of 5 and 6 display a dual E*/K* emission, as a result of the stabilization of the enol tautomer (E*) in the excited-state owing to the formation of intermolecular H-bonds. Interestingly, the present dyes remain emissive in ethanol (ΦF = 17 and 24% respectively), whereas protic environments are known to often quench ESIPT emission. 17 Dye 3 stands out by the presence of an additional emission band at 450 nm in ethanol (Figure S3.6) which is reminiscent of what was observed for reported HBO derivatives featuring a highly acidic phenolic proton where the deprotonated conjugated base D* was found to be both stable and emissive. 18 With an extended conjugation at the para position of the hydroxy group, HBO dye 3 displays a triple E*/D*/K* emission behavior with, however a quenching of the fluorescence (ΦF = 4% in ethanol). It is interesting to note that this triple emission is not observed in dye 6 in ethanol which also presents a para substitution. In order to shed more light on the photophysical properties of these para-substituted dyes, the optical behavior of HBO dyes 3 and 6 were studied in a range of solvents of different dipolarity and proticity (Figures S3.4-S3.7 and Figure 5, respectively). For HBO dye 3, the maximum absorption wavelength is not very sensitive to the nature of the solvent (λabs = 338-351 nm) but one can clearly notice in DMF the appearance of an additional small band at 417 nm, attributed to the deprotonation of the phenol group in the ground state (D band). Excitation in this absorption band leads to the appearance of an intense D * emission band at 467 nm but also to random aggregation processes as evidenced by a poor match between absorption and excitation spectra. As can been observed on Figure 5a, the absorption profile of HBO dye 6 in all solvents is slightly red-shifted as compared to 5 (λabs = 365-371 nm) but is not significantly influenced by the nature of the solvent except in DMF which leads to the appearance of an additional red-shifted absorption band (λabs = 460 nm) and an extra band at 361 nm as well. The lowestenergy band can be again assigned to the stabilization of the anionic species formed by deprotonation of the HBO dye 6 by DMF, the most basic of all solvents studied. Stabilization of related deprotonated HBO derivatives in DMF has already been observed and beneficially used for the engineering of white light emission. 19 In all solvents except DMF, HBO dye 6 exhibits a bright green/yellow emission upon photoexcitation at 350 nm with quantum yields in the range 21-35% (Figure 5c). Notably, the fluorescence efficiency remains high in the whole range of solvents studied, regardless of dipolarity or proticity. The K * emission band maximum wavelength is not dependent on the nature of the solvent (λem = 540-550 nm). In DMF, however, upon photoexcitation in the lowest energy absorption band (λem = 450 nm), a distinctive intense blue emission is observed at 495 nm, attributed to the emission of the excited deprotonated species D * (Figure 5c). The emission of the D * state is stronger than that from the K * state, as evidenced by a fluorescence quantum yield of 49%, calculated in DMF, the highest in the series for HBO dye 6. It is important to note that unlike 5, HBO 6 does not feature aggregates as evidenced by a good match between absorption and excitation spectra in DMF, evidencing the beneficial presence of the tertiobutyl group on the benzoxazole side but most importantly that of the ethynyl substitution at the 3-position of the phenol in order to enhance fluorescence intensity but also to decrease aggregation processes. The emission bands observed at 470 and 474 nm in THF and acetonitrile respectively are also assigned to the same D * species. These photophysical processes are schematically summarized in Figure 6.

To ascertain the formation of the stabilized phenolate anion of HBO dye 6 in DMF, a large excess of potassium tert-butoxide ( t BuOK) was added and the optical properties recorded (Figure S3.12). No spectral difference can be observed in the absorption and emission profile of HBO dye 6, upon addition of a large excess of base, further evidencing the intrinsic presence of the phenolate in both the ground and excitedstates in DMF solution. It is noteworthy that the addition of an excess of hydrochloric acid in a DMF solution of HBO dye 6 leads to the appearance of a UV centered emission likely originating from the protonation of the benzoxazole ring. In order to investigate the formation and stability of the D * band, absorption and emission titrations were performed on HBO dye 6 upon addition of increasing equivalent of a base, tetramethylammonium hydroxide (Me4NOH) in acetonitrile (Figure 7). Acetonitrile was chosen as a solvent to ensure a good solubility of both HBO dyes 6 and Me4NOH. In the absence of base, an absorption band with a maximum wavelength at 365 nm was recorded. Upon excitation at 360 nm, a dual emission profile was observed, consisting of a major band at 545 nm, assigned to the K * tautomer and a minor (shoulder) band at 474 nm, attributed to the D * form. Upon addition of increasing amounts of Me4NOH, the absorption band at 365 nm was found to gradually decrease and lead to the appearance of new bands at 352 nm and 446 nm, which is reminiscent of the absorption profile of fully deprotonated HBO dye 6 in DMF (λabs = 361/460 nm). In the successive recorded emission spectra, the fluorescence stemming from the D * state at 474 nm rose, as a result of the deprotonation of HBO 6 while the K * band at 545 nm showed a significant intensity decrease.

Plotting the ratio of intensity of D * (474 nm)/K * (545 nm) vs. the number of base equivalents gives a good correlation (Figure 7c). The intensity ratio between the two emission bands D * /K * gradually increases upon addition of increasing amounts of base, until around 7 equivalents; after which a plateau is reached, corresponding to the full deprotonation of HBO 6. This gradual deprotonation of 6 in basic medium is also accompanied by the appearance of a small additional emission band at 388 nm which was also found in DMF in the absence of base. We assign the presence of this band to the minor formation of a benzofuran side-product obtained by an intramolecular cyclization in basic conditions. 14 To confirm the nature of this side-product whose in situ formation was observed during the titration, the synthesis of dye 13 was undertaken in one step from bis-iodo HBO derivative 11.

Without protection of the hydroxy group, Pd-catalyzed crosscoupling reaction between HBO 11 and p-tolyl acetylene in a mixture of toluene and triethylamine lead to the sole formation of dye 13 in 25% yield (Scheme 5). The photophysical data of compound 13 in acetonitrile are presented on Figure S3.13. A major absorption band at 300 nm is observed, along with an emission band at 390 nm upon photoexcitation, which corresponds to the emission wavelength of the side-product observed during the titration. This experiment further confirms the formation of compound 13 in situ during the basic titration. Unfortunately, the presence of this undesired compound impeded the accurate calculation of a pKa in the ground and excited state.

Outside their promising optical properties in solution, HBO dyes 1-6 appeared to be very bright as powders under irradiation at 365 nm by a bench UV-lamp by the naked eye (Figure 8b) which prompted us to investigate their photophysical behavior in the solid-state as dispersed in potassium bromide pellets. Their emission profiles appeared to be very similar to those observed in toluene, i.e. a single K * emission band ranging from 489-547 nm depending on the position of substitution (Figure 8a). HBO dyes 3 and 6 bearing a mono-or disubstitution at the 5 or 3,5 position of the phenol ring respectively, are the most red-shifted in the series (λem = 530-547 nm for 3 and 6 vs. λem = 489-504 nm for all other dyes). Photoluminescent quantum yields, calculated as absolute using a spectrofluorimeter fitted with an integration sphere are significantly higher (Φ =48-88%) than those in solution; a feature encountered in the large majority of ESIPT emitters. HBO 1, which was poorly emissive in the solution-state (Φ =4% in toluene) appears to be the brightest solid-state emitter in the series (Φ =88%). To obtain complementary insights into the involved excitedstate processes, ab initio calculations were performed following a protocol detailed in section S4. The results are collected in Table 2. The computed fluorescence wavelengths for the K * emission in toluene very nicely match their experimental counterparts of Table 1, both quantitatively and qualitatively, with the most redshifted (blueshifted) emission observed for HBO dyes 5 and 6 (1 and 4), clearly confirming that this emission band originates from the keto form in the apolar solvent. This is further substantiated by computed driving forces for the ESIPT process (G K*-E* ) process that are all significantly negative indicating a favorable situation for proton transfer. The proton transfer is also computed to be barrierless by theory, with a transition state lying below the E * form once entropic corrections are accounted for. Before analyzing the ethanol results, it should be recalled that the theoretical approach used for model solvent effects is a continuum model and therefore does not account for specific solute-solvent interactions but only for polarity effects. It turns out that the negative solvatochromism of the K * band, evident from the measurements, is well reproduced by theory, so that this effect is probably mainly driven by the relative polarities of the excited states. In contrast, the computed ESIPT driving forces all remain strongly negative in the calculation, which is not in line with the emergence of dual emission experimentally observed in ethanol. One can therefore conclude that the frustration of the ESIPT process in ethanol is due to the formation of hydrogen bonds between the dye and the surrounding ethanol molecules.

Table 2. Theoretical data computed for dyes 1-6 in toluene and ethanol (DMF for 6): emission wavelength and oscillator strengths determined for both the enol and keto tautomers, difference of free energy between the K * and E * forms, the ESIPT transition-state (TS-PT * ) and the E * tautomer, and the twisting transitionstate (TS-TW * ) and the K * forms. See figure S3 in the SI for a representation of these key points. All values are obtained with TD-DFT and corrected with ADC(2), but the oscillator strength and twisting barrier that are the non-corrected TD-DFT results.

As stated above, the fluorescence quantum yields in solution are generally limited by the presence of efficient non-radiative routes. Indeed, as can be seen in Table 2, all dyes present significant oscillator strengths, i.e. only very bright states are at play. For ESIPT dyes, one of the most well-known deactivating pathway is the twisting around the central interring bond after proton transfer (Figure S4.2.1), that can ultimately lead to a conical intersection with the ground state, and hence the emission quenching. To obtain first insights into that pathway, the transition state characterizing this twisting from the K * tautomer was determined. As can be seen, positive barriers are systematically obtained, consistent with the presence of fluorescence. If one excludes HBO 2 in which the ethynyltolyl fragment is non-planar due to the interaction with the furan, it is noticeable in Table 2 that the largest barriers are determined for HBO dyes 5 (0.27 eV) and 6 (0.31 eV) in toluene, which are also the two compounds with the smallest Knr (and largest ΦF) of the series experimentally (Table 1). Conversely, the barriers for the twisting are very small for both dyes 2 and 5 in ethanol and these dyes indeed present very large Knr and trifling ΦF experimentally. This qualitative correlation therefore confirms that the twisting of the K * tautomer is a relevant deactivation pathway, which is also consistent with the large increase of emission quantum yield in the solid state, where this large amplitude motion is hampered. Finally, we have also modeled the anionic form of HBO 6 in DMF. Interestingly, our computational protocol predicts a bright emission (f=1.00) at 500 nm, that is in between the E * and K * band, which nicely fits the experimental value (495 nm), and therefore confirms the experimental assignment.

Conclusions

In summary, a series of ESIPT emitters based on ethynyltolyl-extended HBO scaffolds has been synthesized in a twosteps expedite synthesis. The photophysical properties were explored in multiple solvents where these dyes exhibit a single K * or a dual E * /K * emission band with fluorescence quantum yield values reaching 35%. These values represent up to a 16-fold increase as compared to the unsubstituted reference dye, highlighting the beneficial introduction of these spacers on the molecular core. Depending on the position of the substitution, the stabilization of an anionic species was evidenced in a selection of dissociative solvents and by a basic titration in CH3CN. These dyes keep their intense fluorescence emission in the solid-state, as embedded in KBr matrix, where a fine-tuning of the emission wavelength is observed. The nature of the excited-states was rationalized by ab initio. The origin of these enhanced optical properties is currently unclear but could be attributed to the restriction of the twisting around the central interring bond in the excited state. Transient absorption experiments along with timeresolved fluorescence spectroscopy are currently under investigation to shed more light on the excited-states dynamics in these systems.

Experimental Section

All reactions were performed under a dry atmosphere of argon. Chemicals were purchased from commercial sources and used without further purification. Reaction solvents were distilled according to common procedures. Thin layer chromatography (TLC) was performed on silica gel or aluminum oxide plates coated with fluorescent indicator. Chromatographic purifications were conducted using 40-63 μm silica gel. All mixtures of solvents are given in v/v ratio. spectra were obtained by using a Horiba S2 Jobin Yvon Fluoromax 4. All fluorescence spectra were corrected. Solvents for spectroscopy were spectroscopic grade and were used as received. All fluorescence spectra were corrected. The fluorescence quantum yield (Φexp) was calculated from eq (1).

I denotes the integral of the corrected emission spectrum, OD is the optical density at the excitation wavelength, and η is the refractive index of the medium. Quantum yield is determined in solution by using quinine sulfate as a reference (λ exc = 366 nm, Φ = 0.55 in 1N H2SO4), for dyes emitting below 480 nm, Rhodamine 6G as a reference (λexc = 488 nm, Φ = 0.88 in ethanol), for dyes emitting between 480 and 570 nm or cresyl violet (λexc = 546 nm, Φ = 0.55 in ethanol) as a reference for dyes emitting above 570 nm. Solid-State emission was recorded using an integration sphere fitted to a spectrophotometer. Luminescence lifetimes were measured on an Edinburgh Instruments spectrofluorimeter equipped with a R928 photomultiplier and a PicoQuant PDL 800-D pulsed diode connected to a GwInstect GFG-8015G delay generator. No filter was used for the excitation. Emission wavelengths were selected by a monochromator. Lifetimes were deconvoluted with FS-900 software using a light-scattering solution (LUDOX) for instrument response. The excitation source was a laser diode (λexc = 320 nm). HBO 3 13 and 4 13 and salicylaldehyde 8 20 were synthesized according to reported procedures.

General procedure for the synthesis of HBO dyes 7 and 10

The relevant salicylaldehyde (0.6 mmol) and 2-aminophenol derivatives (0.66 mmol, 1.1 equivalent), boronic acid (0.72 mmol., 1.2 equivalent) and potassium cyanide (1.8 mmol, 3 equivalents) were stirred overnight at room temperature in 40 mL of MeOH. The solvents were removed in vacuo and the crude mixture purified by column chromatography on SiO2 (CH2Cl2/Pet. Ether. 1:9 to 2:8) to afford HBO dyes 7 and 10 as white powders. HBO dye 7.65%. 1 H NMR (400MHz, CDCl3) δ (ppm): 11.16 (s, 1H, OH), 7.99 (dd, 1H, CH, 3 J = 8Hz, 4 J = 1.6Hz), 7.86 (s, 1H, CH), 7.42-7.48 (m, 3H, CH), 7.10 (dd, 1H, CH, 3 J = 8.4Hz, 4 J = 0.7Hz), 7.00 (t, 1H, CH, 3 J = 8.3Hz). 13 C NMR (100.5Hz, CDCl3) δ (ppm):164.0, 158. 9, 148.2, 141.6, 134.1, 128.4, 127.2, 122.2, 119.7, 117.8, 117.6, 111.9, 110. 3, 155.6, 149.5, 139.8, 137.0, 126.5, 126.1, 125.5, 120.6, 119.7, 111.9, 111.5, 111.0. ESI-HRMS: calcd for C13H9BrNO2: 291.9792 (M+H), found 291.9775 (M+H). IR (ν, cm -1 ): 2869, 1628, 1579, 1543, 1452, 1402, 1343, 1289, 1241, 1146, 1071, 813, 758, 747, 736.

General procedure for the synthesis of HBO dyes 9 and 11

To a solution of 2-aminophenol or 4-tertiobutyl-2-aminophenol (0.5 mmol) in absolute ethanol was added salicylaldehyde 8 or 3,5-diiodo-2-hydroxybenzaldehyde (0.5 mmol) and the resulting suspension was refluxed for one hour before an orange precipitate formed. After cooling down, the precipitate was filtered and washed with ethanol. It was then redissolved in dry dichloromethane and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (0.6 mmol) was added as a concentrated dichloromethane solution. The resulting dark mixture was stirred at room temperature overnight. After solvent evaporation, the crude residue was purified by silica gel chromatography eluting with CH2Cl2/Pet. 8, 148.7, 138.8, 133.8, 133.5, 125.9, 125.4, 119.2, 117.9, 114.2, 111.1, 91.2. ESI-HRMS: calcd for C13H9INO2: 337.9673 (M+H), found 337.9663 (M+H). IR (ν, cm -1 ): 1617, 1572, 1522, 1445, 1310, 1281, 1176, 1085, 1041, 1002, 948, 770, 748, 735. HBO dye 11. 75%. 1 H NMR (400MHz, CDCl3) δ (ppm): 12.45 (s, 1H, OH), 8.27 (s, 1H, CH), 8.14 (s, 1H, CH), 7.72 (s, 1H, CH), 7.46-7.52 (m, 2H, CH), 1.39 (s, 9H, CH3). 13 C NMR (100.5Hz, CDCl3) δ (ppm): 160. 5, 157.2, 149.5, 149.2, 147.4, 139.3, 135.3, 124.1, 116.0, 112.5, 110.1, 86.7, 81.0, 35.1, 31.8. ESI-HRMS: calcd for C17H16 I2NO2: 519.9186 (M+H), found 519.9117 (M+H). IR (ν, cm -1 ): 3066, 2955, 1600, 1568, 1532, 1483, 1426, 1411, 1334, 1274, 1249, 1176, 1126, 1065, 936, 854, 812, 760, 649.

Synthesis of HBO dye 1

Halogenated HBO dye 7 (0.5 mmol.), p-tolylacetylene (1.5 mmol.) and Pd(dppf)Cl2 (5 % molar) were dissolved in 25 mL of toluene/triethylamine (3/1 v:v). The resulting suspension was degassed with argon for 30 minutes before CuI (10 % molar) was added. The resulting mixture was stirred overnight at 90°C. The dark solution was taken up in dichloromethane, washed with water, dried (MgSO4) and concentrated in vacuo. The product was purified by silica gel chromatography (CH2Cl2/Pet. Ether 1:3 to 1:1) to afford clean HBO dyes 1 as a white powder. 37%. 1 H NMR (400MHz, CDCl3) δ (ppm): 11.33 (s, 1H, OH), 8.00 (dd, 1H, CH, 3 J = 7.9Hz, 4 J = 1.6Hz), 7.87 (s, 1H, CH), 7.52-7.57 (m, 2H, CH), 7.42-7.46 (m, 3H, CH), 7.16 (d, 2H, 3 J = 7.9Hz), 7.12 (d, 1H, 3 J = 7.9Hz), 7.00 (t, 1H, CH), 2.37 (s, 3H, CH3). 13 C NMR (100.5Hz, CDCl3) δ (ppm): 164.0, 159. 1, 149.1, 140.4, 138.8, 134.1, 131.7, 129.4, 129.4, 127.4, 122.5, 120.8, 120.2, 119.9, 117.7, 110.9, 110.5, 89.6, 88.3, 21.8. ESI-HRMS: calcd for C22H16NO2: 326.1176 (M+H), found 326.1163 (M+H). IR (ν, cm -1 ): 3027, 1631, 1586, 1513, 1486, 1464, 1428, 1296, 1252, 1224, 1192, 1053, 934, 809, 754, 719.

Synthesis of HBO dye 2

Halogenated HBO dye 9 (0.5 mmol.), p-tolylacetylene (1.5 mmol.) and Pd(PPh3)2Cl2 (5 % molar) were dissolved in 25 mL of toluene/triethylamine (3/1 v:v). The resulting suspension was degassed with argon for 30 minutes before CuI (10 % molar) was added. The resulting mixture was stirred overnight at 90°C. The dark solution was taken up in dichloromethane, washed with water, dried (MgSO4) and concentrated in vacuo. The product

Figure 1 .

 1 Figure 1.General structure of the ESIPT emitters HBO 1-6.

Scheme 1 .

 1 Scheme 1. Synthesis of HBO 1.

Figure 2 . 13 Scheme 3 .

 2133 Figure 2. Structure of dyes 3 and 4.13

Figure 3 .

 3 Figure 3. (a) Absorption spectra, (b) Emission spectra recorded in toluene at room temperature for HBO 1 (green), HBO 2 (navy blue), HBO 3 (pink), HBO 4 (light blue), HBO 5 (pink) and HBO 6 (red) and (c) Schematic representation of the value of the fluorescence quantum yield in toluene for each substitution position.

Figure 4 .

 4 Figure 4. (a) Structure of HBO 12, (b) UV-Visible (dashed) and emission (plain) spectra of HBO 12 in toluene (navy blue) and ethanol (pink) and (c) Photograph of HBO dye 12 in solution in toluene under irradiation by a bench UV lamp (λexc = 365 nm).

Figure 5 .

 5 Figure 5. (a) Absorption spectra and (b) emission spectra of HBO 6, recorded at room temperature in aerated solutions of toluene (navy blue), dichloromethane (light blue), THF (green), acetonitrile (orange), ethanol (purple) and DMF (red) and (c) Photographs of HBO 6 under irradiation with a bench UV lamp (λexc = 365 nm) in toluene, dichloromethane, THF, acetonitrile, ethanol and DMF (from left to right).

Figure 6 .

 6 Figure 6. Simplified energy diagram showing the K * emission (λem = 545-550 nm) stemming from the keto tautomer formed via ESIPT process and the D * emission (λem = 470-495 nm) originating from deprotonation of the enol tautomer in the ground state, occurring partially or fully in DMF, THF and CH3CN.

Figure 7 .

 7 Figure 7. (a) Overlay of absorption spectra of HBO dye 6 in acetonitrile recorded after successive addition of increasing equivalents of Me4NOH, (b) superposition of emission spectra, (c) plot of the ratio of intensity I(D * at 474 nm)/I(K * at 545 nm) vs. the number of added equivalents of Me4NOH and (d) Photographs of acetonitrile solutions of HBO dye 6 under irradiation (λexc = 365 nm) before addition of base, after addition of two, five and ten equivalents (from left to right).

Scheme 5 .

 5 Scheme 5. Synthesis of dye 13.

Figure 8 .

 8 Figure 8. (a) Emission spectra recorded in KBr pellets at room temperature for HBO 1 (green), HBO 2 (navy blue), HBO 3 (pink), HBO 4 (light blue), HBO 5 (purple) and HBO 6 (red) and (b) Photographs of HBO 6 as a powder under daylight (top) and under irradiation with a bench UV lamp (λexc = 365 nm) (bottom).

13 C

 13 Ether 1:2 leading to HBO 9 or 11 as a white powders. HBO dye 9. 57%. 1 H NMR (400MHz, CDCl3) δ (ppm): 12.60 (s, 1H, OH), 7.74-7.78 (m, 1H, CH), 7.68-7.72 (m, 1H, CH), 7.66 (dd, 1H, CH, 3 J = 7.6Hz, 4 J = 1.2Hz), 7.41-7.46 (m, 2H, CH), ), 7.12 (dd, 1H, CH, 3 J = 8.4Hz, 4 J = 1.2Hz), 7.03 (t, 1H, CH, 3 J = 8Hz). NMR (100.5Hz, CDCl3) δ (ppm): 161.4, 160.

Acknowledgments

J.M. and G.U. thank the Centre National de la Recherche Scientifique (CNRS), the Agence Nationale de la Recherche (ANR) (GeDeMi grant) and the SATT Conectus (Dualem maturation project) for funding. M.M. thanks the Région Alsace and Amoneta Diagnostics for funding of a Ph.D. grant.

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FULL PAPER

A series of ESIPT emitters based on ethynyl-extended HBO scaffolds has been synthesized. Their optical properties reveal increased quantum yields values in solution as compared to unsubstituted analogues while keeping strong fluorescence intensities in the solid-state. These scaffolds also allow the stabilization of an anionic species in selected cases, as evidenced by basic titrations. The nature of the excited-states was also rationalized using ab initio calculations.