Thomas Yvorra

Anke Steinmetz

Pascal Retailleau

Olivier Lantz

Frédéric Schmidt
email: frederic.schmidt@curie.fr

Synthesis, biological evaluation and molecular modelling of new potent clickable analogues of 5-OP-RU for their use as chemical probes for the study of MAIT cell biology

Keywords: MAIT cells, ethinyl-5-OP-RU, MR1, TCR, immunology, molecular dynamics, ligand induced-fit

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

Introduction

In human, MAIT cells represent an abundant population of TCRαβ T cell lymphocytes with immediate effector functions and a unique specificity for a microbial compound [START_REF] Franciszkiewicz | MHC class I-related molecule, MR1, and mucosal-associated invariant T cells[END_REF][START_REF] Legoux | Unconventional or Preset αβ T Cells: Evolutionarily Conserved Tissue-Resident T Cells Recognizing Nonpeptidic Ligands[END_REF].

Because of their microbial specificity, MAIT cells have been studied during bacterial infections both in mice and humans and were found to be protective in several experimental infection models [START_REF] Salou | MAIT cells in infectious diseases[END_REF]. MAIT cells are able to sense viral infection (through cytokine stimulation) and indirectly protect host against viruses [START_REF] Ussher | MAIT cells and viruses[END_REF]. MAIT cells have also been implicated in cancer immunity and immune mediated diseases such as diabetes and autoimmune diseases, probably through their tissue repair functions [START_REF] Salou | A TCR-Dependent Tissue Repair Potential of MAIT Cells[END_REF][START_REF] Godfrey | The biology and functional importance of MAIT cells[END_REF].

In humans, MAIT cells are found mostly in liver, lungs, gastrointestinal tract and peripheral blood [START_REF] Kurioka | MAIT cells: new guardians of the liver[END_REF]. They express a semi-invariant T-cell receptor (TCR, TRAV1.2-TRAJ33/12/20 paired with a limited set of TCRβ chains) recognizing the microbial compound presented by the non-polymorphic and highly evolutionarily conserved MHCrelated protein 1 (MR1) [START_REF] Ussher | Mucosal-associated invariant T-cells: new players in anti-bacterial immunity[END_REF]. Unlike conventional T cells that recognize antigenic peptides, MAIT cells are activated by small organic molecules derived from an intermediate of the riboflavin (vitamin B2) biosynthesis pathway [START_REF] Kjer-Nielsen | MR1 presents microbial vitamin B metabolites to MAIT cells[END_REF]. These antigens are produced by most bacteria and by several yeasts but not by animal cells [START_REF] Mondot | MAIT, MR1, microbes and riboflavin: a paradigm for the co-evolution of invariant TCRs and restricting MHCI-like molecules?[END_REF]. The most potent stimulatory antigen is 5-(2-oxopropylideneamino)-6-(D-ribitylamino)uracil (5-OP-RU) [START_REF] Corbett | T-cell activation by transitory neo-antigens derived from distinct microbial pathways[END_REF]. It is formed in situ from condensation between the unstable 5-amino-6-(D-ribitylamino)uracil (5-A-RU) and methylglyoxal derived from the glycolysis pathway (Figure 1). 5-OP-RU is also an unstable molecule especially prone to hydrolysis and cyclization in an aqueous environment to give a stable but far less active ribityllumazine derivative [START_REF] Mak | Stabilizing short-lived Schiff base derivatives of 5-aminouracils that activate mucosal-associated invariant T cells[END_REF]. 5-OP-RU can be stabilized by binding to MR1 molecule, by the formation of a Schiff base between its α-iminocarbonyl moiety and lysine 43 (K43) of MR1. The complex formed with MR1 then interacts with the MAIT cell TCR leading to MAIT cell activation and secretion of pro-inflammatory cytokines such as INFγ and TNFα. MAIT cells can also kill bacterially infected cells through granzyme and perforin release [START_REF] Franciszkiewicz | MHC class I-related molecule, MR1, and mucosal-associated invariant T cells[END_REF].

Figure 1: 5-OP-RU biosynthesis and interactions with MR1 and the MAIT cell TCR. 5-A-RU reacts in situ with endogenous methylglyoxal yielding 5-OP-RU. The latter undergoes quick cyclization or binds covalently to MR1 stabilizing the structure and triggering the antigenic presentation to MAIT cells Despite growing interest in the study of MAIT cells over the past decades, many fundamental questions are still to be addressed about MAIT cell biology to better understand their protective role against diseases and to validate their use as therapeutic target for innovative immunotherapies. The role of MR1 for MAIT cell activation in physiological of pathological contexts and the nature of the cells able to present antigens to the MAIT cell TCR are key elements that have to be investigated. For this purpose, the development of tools allowing us to localize and track MR1 in vivo would be of great value. Thus, we developed ethinyl-functionalized analogues of 5-OP-RU that can be used through bio-orthogonal CuAAC reaction (copper(I)-catalyzed azide-alkyne cycloaddition also known as "click chemistry"). This compound will enable to visualize MR1 location and MAIT ligand dynamics in cells or in tissues during in vitro or in vivo experiments [START_REF] Cañeque | Visualizing biologically active small molecules in cells using click chemistry[END_REF].

Bio-orthogonal reactions refers to rapid and selective chemical reactions that can be done in biological systems under physiological conditions [START_REF] Sletten | Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality[END_REF]. CuAAC is the most widely used reaction for bioconjugation in biological systems especially because of its high efficiency and fast kinetics reported in physiological media. One major benefit of CuAAC is the small size of alkyne and azide groups disturbing as little as possible the interactions with the binding moiety. This motivated our choice to use CuAAC with 5-OP-RU, since we knew that the binding pocket formed between the MAIT cell TCR and MR1/5-OP-RU is narrow.

Herein we describe the design and chemical synthesis of the chemical probes followed by their biological assessment and in vitro validation of the click chemistry bioconjugation to MR1.

Results and discussion

Design of the ligands

In order to keep the maximum of interactions, we chose to introduce an alkyne functional group at the extremity of the ribityl tail, far from the α-iminocarbonyl moiety that covalently binds to MR1. We also wanted to keep all four hydroxyl groups of the ribityl tail to maximize the chances of maintaining the interaction with both TCR and MR1. In fact, the ribityl hydroxyl groups create a very important network of hydrogen bonds essential for the binding to both proteins [START_REF] Corbett | T-cell activation by transitory neo-antigens derived from distinct microbial pathways[END_REF]. Thus, 2'-OH group forms a critical hydrogen bond with Tyr95α of the TCR while 3'-OH is involved in interactions with MR1 and it also forms an intramolecular interaction with N1H of the uracil ring. The latter interaction is assumed to restrict movements of the ribityl tail allowing the molecule to fit perfectly inside the binding groove [START_REF] Ler | Computer Modelling and Synthesis of Deoxy and Monohydroxy Analogues of a Ribitylaminouracil Bacterial Metabolite that Potently Activates Human T Cells[END_REF]. It was recently shown that the modification of the ribityl chain by removing 2'-OH or 3'-OH groups was responsible for an important loss of potency [START_REF] Awad | The molecular basis underpinning the potency and specificity of MAIT cell antigens[END_REF]. In contrast, 5-OP-RU analogues lacking 4'-OH or 5'-OH groups kept a strong biological activity. Furthermore, the replacement of the D-ribityl chain by an D-allityl chain owning the same stereochemistry as 5-OP-RU but with a longer carbon chain did not lead to a dramatic loss of biological activity compared to 5-OP-RU [START_REF] Braganza | The effect of MR1 ligand glyco-analogues on mucosal-associated invariant T (MAIT) cell activation[END_REF]. All these results support the possibility of introducing a small additional moiety at the extremity of the ribityl tail without losing the affinity to both TCR and MR1.

Molecular modelling support for the design of derivatized 5-A-RU

Derivatization of ribityl hydroxyl groups by an ethinyl function as well as 5'-ethinylation were envisioned and evaluated by molecular docking. 4'-ethinyloxy-5-A-RU, 2'-ethinyloxy-5-A-RU, SRRR, and SRRS stereoisomers of 13 (13SRRR and 13SRRS, respectively) docked well to the MR1/TCR model prepared from the crystal structure of the ternary complex of 7hydroxy-6-methyl-8-D-ribityllumazine/MR1/TCR (PDB entry 4L4V [START_REF] Patel | Recognition of vitamin B metabolites by mucosalassociated invariant T cells[END_REF]). Comparison to the ternary complex of 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU) with MR1/TCR (PDB entry 4NQE [START_REF] Corbett | T-cell activation by transitory neo-antigens derived from distinct microbial pathways[END_REF]) showed perfect conservation of the 5-OE-RU binding mode by the uracil and ribityl moieties of 4'-, 2'-ethinyloxy-derivatives and 13SRRR in one of the three retained poses (Figure 2 and SM2 Figure 1). 13SRRS also conserved the binding mode of the uracil moiety, however the ribityl chain adapted its conformation to accommodate the rigid ethinyl group. The conformations of the three retained poses proposed different solutions to optimize hydrogen bonding to the protein. The conformation closest to the one of 5-OE-RU and forming a hydrogen bond by 3'-OH to the hydroxyl group of TCR Y95 G was retained for further studies (Figure 2). 5'-ethinyl and 4'-ethinyloxy derivatives pointed the ethinyl group to a solvent channel leading to bulk solvent whilst 5'R-ethinylation presented the advantage of preserving the entire potential of hydrogen bond formation of all hydroxyl groups in terms of donor or acceptor. Both 5'R-and 5'S-ethinyl ligand permitted excellent fitting of the reacted fluorophore probe to the solvent channel (vide infra). Thus, chemical synthesis of 5'-ethinyl derivatives was undertaken.

Figure 2:

Poses of 13 docked to MR1/TCR complex in comparison to the crystal structure of the ternary complex of 5-OE-RU; Top: close-ups of the ligand-binding site with 5'R and 5'S stereoisomers in A and B, respectively. Protein is depicted by ribbon diagrams with MR1 chain A, TCR chains G and H colored in olive, plum, and blue, respectively. Selected amino acids and ligands are represented as ball-and-sticks or tubes. Carbon atoms of 5-OE-RU, 5'R-and 5'S-ethinylated ligands are depicted in dark green, green, and bright green, oxygen, nitrogen, and hydrogen atoms in red, blue, and white, respectively. Bottom: 2-dimensional sketches of the chemical structure of 13.

Synthesis of ethinyl-5-OP-RU 14

One main challenge of the synthesis (Scheme 1) was the choice of protective groups for the ribityl chain. They were very important for purification purposes and for selective introduction of the alkyne function. Another crucial step was the coupling of the ribityl chain with uracil core (step xi) and we relied on the nitro group on the uracil ring to facilitate the coupling as previously described [START_REF] Li | Synthesis, stabilization, and characterization of the MR1 ligand precursor 5amino-6-D-ribitylaminouracil (5-A-RU)[END_REF]. An azido group was used as a stable precursor for an amine. Finally, we were aware that product 13 would probably be unstable due to sensitivity to oxidation like 5-A-RU [START_REF] Cushman | Design, Synthesis, and Evaluation of 9-d-Ribityl-1,3,7-trihydro-2,6,8-purinetrione, a Potent Inhibitor of Riboflavin Synthase and Lumazine Synthase[END_REF]. To overcome this issue, we synthetized the hydrochloride salt analogue because 5-A-RU was previously described stable in this form [START_REF] Li | Synthesis, stabilization, and characterization of the MR1 ligand precursor 5amino-6-D-ribitylaminouracil (5-A-RU)[END_REF][START_REF] Cushman | Design, Synthesis, and Evaluation of 9-d-Ribityl-1,3,7-trihydro-2,6,8-purinetrione, a Potent Inhibitor of Riboflavin Synthase and Lumazine Synthase[END_REF][START_REF] Cushman | Synthesis of epimeric 6,7bis(trifluoromethyl)-8-ribityllumazine hydrates. Stereoselective interaction with the light riboflavin synthase of Bacillus subtilis[END_REF].

The synthesis of 14 started from commercial D-ribose. Thioketal protection of the aldehyde followed by protection of the four hydroxyls with TBS triflate gave fully protected compound 3 [START_REF] Bender | A robust synthesis of 7,8-didemethyl-8-hydroxy-5deazariboflavin[END_REF]. Selective deprotection of the terminal hydroxyl function at this step with camphorsulfonic acid, p-toluenesulfonic acid, TFA/H2O or with TBAF was unsuccessful. A problem of reactivity was suspected due to the presence of the thioketal groups. Therefore, we decided to remove the thioketals first under oxidative conditions to give 4. The aldehyde was reduced by sodium borohydride and the resulting primary alcohol 5 was activated by mesylation to give 6. Reaction with sodium azide led to the stable azido 7. This time, selective deprotection of the primary alcohol was effective using camphorsulfonic acid to obtain 8 in a 19% yield together with the starting material which can be recovered. The resulting alcohol was quantitatively oxidized using Dess-Martin periodinane reagent to give 9.

TMS-acetylene addition on 9 was done with n-butyllithium leading to an expected mixture of two diastereomers of 10 but an unexpected migration of the neighbor TBS position (see section 1.4 for details). The synthesis was pursued with the mixture of the two diastereomers.

The azide group of 10 was reduced by Staudinger reaction to amine 11 which was coupled with 6-chloro-5-nitropyrimidine-2,4(1H,3H)-dione (purchased or synthetized following previously described protocol) [START_REF] Li | Synthesis, stabilization, and characterization of the MR1 ligand precursor 5amino-6-D-ribitylaminouracil (5-A-RU)[END_REF]. The TMS group was then removed using potassium methanolate and the resulting product 12 was converted to 13 by clean selective reduction of the aromatic nitro to amine with sodium dithionite followed by acid deprotection of the three remaining TBS groups. The mixture of diastereomers was isolated as hydrochloride salts which were found to be stable over several weeks upon storage at -20°C under inert atmosphere. In the presence of water, the salts were rapidly hydrolyzed leading to a quick degradation comparable to the one observed with 5-A-RU [START_REF] Cushman | Design, Synthesis, and Evaluation of 9-d-Ribityl-1,3,7-trihydro-2,6,8-purinetrione, a Potent Inhibitor of Riboflavin Synthase and Lumazine Synthase[END_REF]. Finally, product 14 was obtained from condensation reaction with methylglyoxal in DMSO during 48h to reach total conversion of 13 (reaction monitored by UPLC-MS). The solution was kept at -80°C and used in biology assays immediately after dilution in water because of its instability [START_REF] Mak | Stabilizing short-lived Schiff base derivatives of 5-aminouracils that activate mucosal-associated invariant T cells[END_REF].

Separation and identification of diastereomers 12a and 12b

The addition of TMS-acetylene on 9 gave a mixture of two diastereomers with a ratio of 95/5 according to HPLC analysis. This stereoselectivity is likely to be due to the presence of lithium ions in the mixture that would be chelated by the aldehyde and the oxygen of the neighboring TBS. Using a Cram chelation model allowed us to predict the absolute configuration of the two diastereomers. Thus, the major epimer should have R configuration and the minor S configuration. To prove this hypothesis, we were able to separate the two diastereomers of 12 by reverse phase HPLC to give 12a (major diastereomer) and 12b (minor diastereomer). We first tried to use Mosher esters method to determine the stereochemistry of the products, but no reaction occurred with Mosher's acid chloride derivatives, probably due to the bulkiness of the TBS groups. Finally, we managed to obtain crystals of 12a in a mixture of acetonitrile/water (Figure 3). X ray diffraction analysis of the crystal was done, and the data confirmed our initial hypothesis that the chiral center C5' was R. Unexpectedly, we also found that the TBS group had migrated from the 4'-OH to the 5'-OH during TMS-acetylene addition. To determine if the same migration occurred on compound 12b, we acetylated the free hydroxyl group and did 1 H NMR study that confirmed the same migration of the TBS position (clear increase of the chemical shift for the H4' NMR signal from 3.91 ppm to 5.13 ppm). These migrations did not change the synthetic scheme as all the TBS groups were removed later.

MRup-regulation assay

At steady state, MR1 resides in the endoplasmic reticulum (ER) of cells [START_REF] Mcwilliam | The intracellular pathway for the presentation of vitamin B-related antigens by the antigen-presenting molecule MR1[END_REF]. In the presence of vitamin B2 derivative antigens, the formed complex exits the ER and traffics through the secretory pathway to the cell membrane leading to antigenic presentation to MAIT cells. This induces an up-regulation of MR1 at the cell surface that can be measured by flow cytometry using an anti-MR1 antibody conjugated with fluorochrome. Thus, MR1 upregulation indirectly reflects the binding of the antigens to MR1.

The capacity of the two ethinyl-analogues of 5-OP-RU to up-regulate MR1 at the cell surface of WT3-m cells (embryonic murine fibroblast cells overexpressing MR1 [START_REF] Huang | MR1 antigen presentation to mucosal-associated invariant T cells was highly conserved in evolution[END_REF]) was assessed. Data are depicted both as percentage of MR1 positive cells (Figure 4A) and corresponding mean fluorescence intensity (Figure 4B) of PE-conjugated antibody anti-MR1.

The two diastereomers were responsible for a strong and similar MR1 up-regulation after 2 hours. The up-regulation was more pronounced than that obtained with positive control 5-OP-RU (synthetized following previously described protocol) [START_REF] Li | Synthesis, stabilization, and characterization of the MR1 ligand precursor 5amino-6-D-ribitylaminouracil (5-A-RU)[END_REF]. In agreement with a previous report [START_REF] Awad | The molecular basis underpinning the potency and specificity of MAIT cell antigens[END_REF], these data suggested that addition of an ethinyl substituent is well tolerated in binding and up-regulation of MR1. Most importantly, this result verified the mandatory high affinity binding of the ligands to MR1 for further validation of the probes. The two clickable ligands activated MAIT cells of double transgenic mice with activity close to 5-OP-RU (Figure 5A). In contrast, no biological activity was detected with splenocytes of single transgenic mice with 14a and 14b whereas 5-OP-RU strongly activated MAIT cells from this single transgenic mice (Figure 5B). These data suggested that activation of MAIT cells by compounds 14a and 14b is strongly dependent on the nature of the TCRβ chain expressed by MAIT cells.

An analogous specificity was previously described for diclofenac (DFC) and its metabolites. DFC was responsible for weak activation of Jurkat.MAIT A-F7 cell lines expressing TCR that uses TRBV6-1 but it did not activate Jurkat.MAIT-#6 and Jurkat.MAIT-C-F7 expressing TCR with TRBV6-4 and TRBV20, respectively [START_REF] Keller | Drugs and drug-like molecules can modulate the function of mucosal-associated invariant T cells[END_REF]. In contrast, the absolute configuration of its C6' stereocenter does not seem to have any influence on the biological activity the two molecules. As all published crystallographic models of MR1/TCR complexes lack a few N-or Cterminal residues as well as several short loops, we de novo modeled residues N1 H -G3 H , C-

termini V269 A -P270 A , D96 B -M99 B , P198 G -S203 G , E239 H -D245 H , loops L246 A - L253 A , R122 G -S130 G , N176 G -D179 G
, and F201 H -R210 H . Model completion issued a variety of alternative conformations for the missing residues of the 20 completed complexes (SM2). As these were bordering crystalline solvent channels, we assumed that conformational variability existed in these regions and accepted all proposed models for molecular dynamics simulations.

All simulations initiated with a standard pre-equilibration phase followed by 120 ns production phase as NPT ensemble at 300 K and 1 atm. Standard quality and structural analysis showed stable simulations in terms of potential and total energy, volume, temperature, pressure, and secondary structure [START_REF] Steinmetz | Data in Brief[END_REF]. In none of the simulations root mean square fluctuations (rmsf) of the protein chains exceeded 7.0 Å, thus illustrating moderate conformational changes [START_REF] Steinmetz | Data in Brief[END_REF] Rmsf of de novo modeled loops were observed at the same level as crystallographically observed loops. Detailed analysis and precise values of non-covalent intraligand and ligand-protein interactions are reported in Steinmetz & al [START_REF] Steinmetz | Data in Brief[END_REF] and supplementary materials SM2.

Altogether, the molecular dynamics simulations of natural and ethinylated ternary and binary ligand complexes as well as binary fluorophore-reacted complexes suggested uracil πstacking with Y7 A and hydrogen bonding with S24 A as canonical non-covalent interactions (SM2). Uracil and ribityl substructures could be relayed by either intraligand or R9 A mediated hydrogen bonding networks existing in varying constellations (SM2). Ligand interactions with TCR Y95 G leading to MAIT cell activation appeared resilient to alternative hydrogen bond patterns as we observed several alternative hydrogen bond partners of Y95 G in ternary complexes of 14a and both 5'S-ligands than crystallographically documented 2'-OH (SM2; [START_REF] Corbett | T-cell activation by transitory neo-antigens derived from distinct microbial pathways[END_REF]). Thus, 2'-OH hydrogen bonding with Y95 G did not appear obligatory for TCR-mediated MAIT cell activation.

The canonical ligand-protein interactions were complemented by a variety of numerous further interactions that led to rather stable placement of the ligands in the binding site (SM2).

Thus, the rmsf profiles of natural and 5'R-ethinylated ligands in ternary complexes were below 1.5 Å on average, whilst those of 5'S-ethinylated ligands were slightly higher at around 1.5 Å [START_REF] Steinmetz | Data in Brief[END_REF] Rmsd profiles of the latter were bimodal due to the exploration of the binding site by the ethinyl group, while the rmsd profiles of natural and 5'R-ethinylated ligands were unimodal due to more stable placement [START_REF] Steinmetz | Data in Brief[END_REF]. However, higher mobility of the ethinyl group of 5'S ligands did not appear to decrease MAIT cell activation as 14a and 14b were equally potent.

Crystal structure analysis suggested an interaction triad of MAIT cell activating ligands, TCR Y95 G , and MR1 Y152 A important for agonist activity, disruption of the interaction triad was associated with decreased agonist activity [START_REF] Awad | The molecular basis underpinning the potency and specificity of MAIT cell antigens[END_REF]. However, none of our molecular dynamics simulations of ternary complexes showed frequent or stable direct hydrogen bonding of natural or ethinylated ligands to Y152 A , at best it was observed as a transitory or water-mediated event ([26], SM2). Thus, our simulations did not reveal this triad as a dynamically stable network of strong MAIT cell activating ligands.

In binary MR1 complexes, the ribityl tail of natural ligands was solubilized in bulk solvent which was not observed for ethinylated or fluorophore-reacted binary complexes due to strong exclusion of hydrophobic and aromatic substructures forced by aqueous solvent.

Therefore, binary 5-OE-RU and 5-OP-RU complexes showed ligand rmsd values fluctuating between modes with lower and higher values while all other binary ligand rmsd profiles were unimodal with exception of the profile of fluorophore reacted 14 which documented slow evolution of one fluorophore placement to another [START_REF] Steinmetz | Data in Brief[END_REF]. In more detail, the molecular dynamics of covalently bound natural and 5'S-ethinylated ligands of binary complexes were reduced for the uracil moiety and more important for the ribityl substituent whilst those of 5'R-ethinylated ligands were equally low throughout their entire length (SM2). These observations suggested that stable occupation of the uracil site was necessary and sufficient for up-regulation of MR1 at the surface of WT3-m cells by natural, 5'S-and 5'R-ethinylated ligands while conformational dynamics of the ribityl chains did not perturb up-regulation.

This result agreed with MR1 up-regulation by a ribityl-less 5-OP-RU analogue [START_REF] Awad | The molecular basis underpinning the potency and specificity of MAIT cell antigens[END_REF].

Interpretation of studies on conformational flexibility of 5-OP-RU proposed that an intramolecular hydrogen bond between uracil N1 and ribityl 3'-OH restricts the conformation of the ribityl chain to facilitate formation of the hydrogen bond network in MR1/TCR ternary complexes [START_REF] Ler | Computer Modelling and Synthesis of Deoxy and Monohydroxy Analogues of a Ribitylaminouracil Bacterial Metabolite that Potently Activates Human T Cells[END_REF]. However, the torsional profiles of 3'-OH and 2'-OH differed in the simulations of ternary and binary 5-OE-RU and 5-OP-RU complexes presented here (SM2).

In the ternary complexes, 2'-OH/Y95 G and intramolecular N1/3'-OH hydrogen bonds accompanied dihedral sampling shifted away from the potential energy minimum to increased values [START_REF] Ayouchia | Understanding the mechanism and regioselectivity of the copper(I) catalyzed [3 + 2] cycloaddition reaction between azide and alkyne: a systematic DFT study[END_REF]. In the binary complexes, sampling centered symmetrically around the potential energy minimum of these dihedrals and the intraligand hydrogen bond was only transiently observed [START_REF] Ayouchia | Understanding the mechanism and regioselectivity of the copper(I) catalyzed [3 + 2] cycloaddition reaction between azide and alkyne: a systematic DFT study[END_REF]. Thus, the protein environment of the ternary complexes rather induced the conformations competent for efficient ligand-protein interactions at a small energetic cost of torsional sampling partly compensated by the intramolecular hydrogen bonds between uracil N1, 3'-OH, and 5'-OH. Thus, the entropic contribution of conformational dynamics of part of the ligand in solution or binary complexes to the thermodynamic balance of ternary ligand/MR1/TCR complex formation could be overcome in TCR binding by induced fit of the ribityl chain and its confinement to conformations competent to form favorable interactions in the ternary complexes.

Energetics and geometry of transition states and reaction intermediates of CuAAC were thoroughly studied by density functional theory (DFT) [START_REF] Ayouchia | Understanding the mechanism and regioselectivity of the copper(I) catalyzed [3 + 2] cycloaddition reaction between azide and alkyne: a systematic DFT study[END_REF]. σ-bound Cu(I)-acetylide π- Fluorescence quenching while labels are attached to their targets may hamper protein quantification or else impact interpretations of structural studies by FRET [START_REF] Lindhoud | Fluorescence of Alexa Fluor Dye Tracks Protein Folding[END_REF]. It arises by PET from an excited donor fluorophore to an acceptor group. This transfer of the excited state electron requires donor and acceptor moieties to be in ad minima transient van der Waals contact. Fluorescence of Alexa Fluor TM 488 dye could be quenched by tryptophan, tyrosine, methionine, and histidine [START_REF] Chen | Mechanisms of Quenching of Alexa Fluorophores by Natural Amino Acids[END_REF]. Quantum-mechanical optimization of ground and excited state geometries of the twice negatively charged 3,6-diamino-9-[2-carboxyphenyl]-4,5-disulfoxanthylium substructure of Alexa Fluor TM 488 evinced that the excited electron is predominantly localized on the diamino-disulfoxanthylium substructure as for related rhodamine fluorophores (SM2 Figure 3; [START_REF] Savarese | Fluorescence Lifetimes and Quantum Yields of Rhodamine Derivatives: New Insights from Theory and Experiment[END_REF]). Analysis of the protein-ligand interactions of both 5'R and 5'S fluorophore-reacted binary complexes suggested that partial quenching by PET from the diamino-disulfoxanthylium substructure to acceptor sidechains of MR1 could not be entirely excluded in spectroscopic or microscopic experiments (SM2).

Conclusion and perspectives

In this work, we described the synthesis of the first clickable MAIT cell agonists for its use through biorthogonal reactions. We managed to obtain a radiocrystallographic structure permitting us to assign unambiguously the stereochemistry of the generated stereocenters and to discover an unexpected migration of one TBS protecting group. The biological activity of the two molecules was confirmed with a strong MR1 up-regulation by both diastereomers reflecting a high affinity of the ligands for the protein complex. In situ click chemistry reaction in cells successfully revealed, under fluorescence microscopy, the clickable ligands selectively bound to MR1. Thus, we validated our CuAAC click chemistry strategy in vitro.

Interestingly, the two isolated diastereomers of 14 strongly activated double TCRαβ transgenic mice similarly to 5-OP-RU. However, no activation was detected with MAIT cells from single iVα19 transgenic mice underlying a structural specificity of the potent molecules towards the MAITs TCR. This discovery could bring additional information about the yet poorly understood basis of interaction to the TCRβ chain necessary for the recognition of vitamin B2 derivative antigens.

The broadly designed modeling approach by molecular dynamics simulations deepens the view on ligand-protein interactions of the natural, ethinylated, or fluorophore-reacted MAIT cell ligands in ternary and binary complexes. These observations fine-tune the appreciation of canonical and non-canonical non-covalent interactions and highlight the resilience of the interaction networks of strong MAIT cell agonists, particularly although not exclusively regarding hydrogen bonding with the sidechain of TCR Y95 G . The agonist binding pocket thus appears principally well druggable with the challenge of controlling the entropic contributions of flexible ligand substructures that are subject to induced-fit in ternary complex formation. To prepare the use of the novel tool compounds in quantitative or biophysical fluorescence studies we addressed the risk of fluorescence quenching by PET. The in silico analysis suggests that it is small, however, such a possibility cannot be entirely excluded and experimentalists should be aware of it.

Altogether, the new chemical probes open new avenues to study the mechanisms of action of MAIT cells in vitro and in vivo. They will further foster the discovery of new drugs in the fight against infectious diseases, cancer, or else conditions due to impaired immune processes. Propanethiol (14 mL, 2.2 eq, 0.15 mol) was added to a solution of D-ribose 1 (10 g, 1 eq, 0.067 mol) in 37% HCl (10 mL) at 0°C. The solution was stirred at room temperature during 1h30. Then, water (80 mL) was added and the solution was stirred for additional 1h. 3M

NaOH (50 mL) was added at 0°C. After a few minutes, a white precipitate appeared. It was filtrated and washed with hexane (3 x 50 mL) and water (3 x 50 mL) and dried under pressure to get the pure product as a white powder (14.50 g, 87%). 1 To 2 (1 g, 1 eq, 3.51 mmol) in DCM (8 mL) was added at 0°C 2,6-lutidine (2.58 mL, 5 eq, 17.6 mmol) and TBDMS triflate (3.63 mL, 4.5 eq, 16.0 mmol) dropwise at 0°C. The mixture was stirred at 0°C for 30 min and then at room temperature for 3h. After completion, water was added and the product was extracted by DCM. The organic layer was washed by CuSO4 10% aqueous solution to remove excess 2,6-lutidine. It was then dried over MgSO4 and concentrated under vacuum. The product was purified by column chromatography 100% hexane to give a colorless oil that solidified upon standing as a white powder (1.62 g, 54%). (m,4H), 1.01-0.89 (m, 42H), 0.28 (s, 3H), 0.21 (s, 3H), 0.17 (s, 3H), 0.14 (s, 3H), 0.12 (s, 3H), 0.10 (s, 3H), 0.05 (s, 6H). 13 C NMR (101 MHz, CDCl3) δ 78. 40, 77.96, 73.79, 64.26, 57.24, 35.23, 33.84, 26.60-26.31 (12C), 23.38, 22.98, 18.93, 18.70, 18.57, 18.51, 13.96, 13.76, -2.80, -3.27, -3.42, -4.12, -4.16, -5.14, -5. [START_REF] Cushman | Design, Synthesis, and Evaluation of 9-d-Ribityl-1,3,7-trihydro-2,6,8-purinetrione, a Potent Inhibitor of Riboflavin Synthase and Lumazine Synthase[END_REF]

(2C). HR-MS m/z

[M+Cl] -calculated for C35H80O4S2Si4: 775.4269, found: 775.4279

(2R,3R,4R)-2,3,4,5-tetrakis((tert-butyldimethylsilyl)oxy)pentanal (4)

Compound 4 was synthetized following literature modified synthesis protocol of a different molecule [START_REF] Bender | A robust synthesis of 7,8-didemethyl-8-hydroxy-5deazariboflavin[END_REF].

To 3 (1.0 g, 1 eq, 1.35 mmol) in a mixture of acetone/water (5/0.5 mL) was added at 0°C NaHCO3 (0.51 g, 4.5 eq, 6.08 mmol) and iodine (0.69 g, 2 eq, 2.70 mmol). The mixture was then stirred at room temperature for 16h. Additional NaHCO3 (0.11 g, 1 eq, 1.35 mmol) and iodine (1 eq, 0.34 g, 1.35 mmol) were added and the mixture was further stirred for 4h. A 10% Na2S2O3 aqueous solution was added to quench iodine and the product was extracted by DCM. The organic layer was dried over MgSO4 and concentrated under vacuum. The product was purified by column chromatography using a gradient 100% hexane to hexane/EtOAc 10/1 to get the product as a colorless oil (0.683 g, 83%). 1

(2S,3S,4R)-2,3,4,5-tetrakis((tert-butyldimethylsilyl)oxy)pentan-1-ol (5)

To 4 (290 mg, 1 eq, 0.48 mmol) in methanol (5 mL) was added at 0°C NaBH4 (27 mg, 1.5 eq, 0.48 mmol). The mixture was stirred at room temperature for 3h. The reaction was quenched by a saturated NH4Cl aqueous solution. The product was extracted by DCM and the organic layer was dried over MgSO4 and concentrated under vacuum. The product was purified by column chromatography hexane/EtOAc 10/1 and was obtained as a colorless oil (0.260 g, 90%). 1 H NMR (400 MHz, CDCl3) δ 3.90-3.83 (m, 3H), 3.72-3.66 (m, 3H), 3.57-3.53 (m, 1H), 2.04 (s, 1H), 0.92-0.90 (m, 36H), 0.12-0.05 (m, 24H). 13

(2S,3R,4R)-2,3,4,5-tetrakis((tert-butyldimethylsilyl)oxy)pentyl methanesulfonate (6)

To 5 (1.28 g, 1 eq, 2.1 mmol) in pyridine (5 mL) was added at 0°C methanesulfonyl chloride (0.2 mL, 1.2 eq, 2.5 mmol) dropwise. The mixture was stirred at room temperature for 3h.

After completion, pyridine was removed under vacuum. Water was added and the product was extracted by DCM. The organic layer was dried over MgSO4 and concentrated under vacuum. The product was purified by column chromatography hexane/EtOAc 10/0.5 and was isolated as a colorless oil (1.08 g, 75%). 1 H NMR (400 MHz, CDCl3) δ 4.50-4.46 (m, 1H), 4.17 To 6 (1.0 g, 1 eq, 1.46 mmol) in dry DMF (5 mL) was added sodium azide (0.14 g, 1.5 eq, 2.2 mmol) and the reaction was heated under reflux for 2h. DMF was then removed under vacuum, water was added and the product was extracted by DCM. The organic layer was dried over MgSO4 and concentrated under vacuum. The product was purified by column chromatography with hexane to get the product as a colorless oil (0.917 g, 99%). 1 3.34 (dd, 1H, J = 12.8 Hz, 6.3 Hz), 0.93-0.89 (m, 36H), 0.14-0.05 (m, 24H). 13

(2R,3S,4S)-5-azido-2,3,4-tris((tert-butyldimethylsilyl)oxy)pentan-1-ol (8)

Compound 8 was synthetized following literature modified synthesis protocol of different molecule [START_REF] Sun | The total synthesis of D-chalcose and its C-3 epimer[END_REF].

To 7 (367 mg, 1 eq, 0.58 mmol) in a 1:1 mixture of DCM (4 mL) and methanol (4 mL) was added (1R)-(-)-10-camphorsulfonic acid (13 mg, 0.1 eq, 0.06 mmol). The mixture was stirred at room temperature during 6h, then quenched by a 5% Na2CO3 aqueous solution and extracted by DCM. The combined organic layers were dried over MgSO4 and concentrated under vacuum. The product was purified by chromatography hexane/EtOAc 5%. The purification allowed to recover all the starting material that did not react in addition to the desired product obtained as a light yellow oil (0.058 g, 19%). 1 H NMR (400 MHz, CDCl3) δ 3.96-3.93 (m, 1H), 3.85 (dd, 1H, J = 5.9 Hz, 3.3 Hz), 3.73-3.70 (m, 2H), 3.62-3.58 (m, 1H), 3.42 (dd, 1H, J = 12.8 Hz, 3.6 Hz), 3.32 (dd, 1H, J = 12.8 Hz, 6.4 Hz), 1.76 (s, OH), 0.92-0.89 (m, 27H), 0.16-0.12 (m, 18H). 13

(2S,3S,4S)-5-azido-2,3,4-tris((tert-butyldimethylsilyl)oxy)pentanal (9)

To 8 (58 mg, 1 eq, 0.11 mmol) in dry DCM (1.5 mL) was added at 0°C NaHCO3 (37 mg, 4 eq, 0.45 mmol) and Dess-Martin periodinane (DMP) reagent (95 mg, 2 eq, 0.22 mmol). The reaction was stirred at room temperature for 30 min. Then, 10% Na2S2O3 aqueous solution was added followed by 5% Na2CO3 to quench the reaction. The product was extracted by DCM and the organic layer was dried over MgSO4 and concentrated under vacuum. The crude product was purified by chromatography hexane/EtOAc10/0.5 and was obtained as a colorless oil (0.041 g, 71%). 1 TMS-acetylene (0.049 mL, 3 eq, 0.58 mmol) was dissolved in dry THF (2 mL). The mixture was cooled at -78°C and n-butyllithium solution (2.5 M in hexane, 0.23 mL, 3 eq, 0.58 mmol) was added. The mixture was gradually warmed up to 0°C and further stirred for an additional 1h. The reaction mixture was again cooled to -78°C and 9 (1 eq, 0.100 g, 0.19 mmol) in THF (2 mL) was added dropwise. The mixture was gradually warmed-up to room temperature and stirred for 3h. After completion, the mixture was quenched by a saturated NH4Cl aqueous solution and the product was extracted by DCM. The organic layer was dried over MgSO4 and concentrated under vacuum. The product was purified by column chromatography hexane/EtOAc 10/0.5 and isolated as a mixture of two diastereomers (a fraction of the major diastereomer was isolated for NMR analysis). A light yellow oil was obtained (0.053 g, 45%). 1 H NMR (400 MHz, CDCl3) (major diastereomer) δ 4.55 (d, 1H, J = 3.7 Hz), 4.16-4.12 (m, 1H), 3.90 (dd, 1H, J = 6.6 Hz, 2.9 Hz), 3.75 (dd, 1H, J = 6.6 Hz, 3.8 Hz), 3.41 (dd, 1H, J = 12.8 Hz, 3.6 Hz), 3.34 (dd, 1H, J = 12.8 Hz, 6.9 Hz), 2.09 (s, OH), 0.95-0.88 (m, 27H), 0.18-0.12 (m, 27H). 13

(4R,5S,6S)-7-amino-4,5,6-tris((tert-butyldimethylsilyl)oxy)-1-(trimethylsilyl)hept-1-yn-3-ol (11)

To 10 (0.280 mg, 1 eq, 0.46 mmol) in a 5:1 mixture of THF/H2O (2.5 mL/0.5 mL) was added triphenylphosphine (1.19 g, 10 eq, 4.55 mmol). The mixture was heated under reflux during 2h. THF was removed under vacuum and water/DCM extraction was performed. The organic layer was dried over MgSO4 and concentrated under vacuum. The product was purified by column chromatography hexane/EtOAc 10/0.5 (triphenylphosphine excess removal) followed by hexane/EtOAc 5/5 and isolated as a mixture of the two diastereomers (a fraction of the major diastereomer was isolated for NMR analysis). A colorless oil was obtained (0.202 g, 75%). 1 To 11 (237 mg, 1 eq, 0.40 mmol) in dry DMF (3 mL) was added 6-chloro-5-nitrouracil (92 mg, 1.2 eq, 0.48 mmol) and triethylamine (5eq, 0.28 mL, 2.0 mmol). The mixture was heated at 50°C for 3h. DMF was removed under vacuum. Water was added and the product was extracted by EtOAc. The organic layer was dried over MgSO4 and concentrated under vacuum. TMS removal was directly done without further purification (120 mg of crude intermediate were obtained). The intermediate was dissolved in MeOH (2 mL) and K2CO3 (72 mg, 3 eq, 0.54 mmol) was added. The mixture was stirred at room temperature for 2h. Water was added to the mixture and the product was extracted by EtOAc. The organic layer was dried over MgSO4 and concentrated under vacuum. The product was purified by reverse phase HPLC using a gradient ACN/H2O 0.1% formic acid as following: 85% ACN-15% H2O for 1 min followed by gradually increase of ACN to 100% ACN after 20 min and 2 minutes 20 more of 100% ACN. The two diastereomers were isolated separately as light yellow powders (108 mg for the major one and 2.4 mg for the minor one, 41% yield). 1 H NMR (400 MHz, CDCl3) 12a δ 11.08 (s, 1H), 10.24 (brt, 1H, J = 6.8 Hz), 9.41 (s, 1H), 5.07 (s, 1H), 4.68 (dd, 1H, J = 3.6 Hz, 2.2 Hz), 4.39-4.37 (m, 1H), 4.21 (d, 1H, J = 4.9 Hz), 3.98 (t, 1H, J = 4.1 Hz), 3.88-3.81 (m, 1H), 3.75-3.69 (m, 1H), 2.52 (d, 1H, J = 2.2 Hz), 0.93-0.83 (m, 27H), 0.16-0.08 (m, 18H) 13 C NMR (101 MHz, CDCl3) 12a δ157. [START_REF] Patel | Recognition of vitamin B metabolites by mucosalassociated invariant T cells[END_REF] 12a (26 mg, 1 eq, 0.41 mmol) was heated to 90°C in a 10:1 mixture of methanol (3 mL) and water (0.3 mL). Then Na2S2O4 (58 mg, 8 eq, 0.33 mmol) was quickly added and the mixture was stirred under reflux for 15 minutes. Excess methanol was removed under vacuum and the product was extracted by EtOAc. The organic layer was dried over MgSO4 and concentrated under vacuum. The crude intermediate was dissolved in methanol (1 mL) and cooled to 0°C prior to addition of 37% HCl (1 mL). The mixture was then stirred at room temperature for 3 hours. The product was concentrated under vacuum, dissolved in few volumes of methanol and precipitated from EtOAc. The precipitate was filtered and washed with EtOAc. After deep drying under vacuum, the product was obtained as pink-red powder corresponding to the amine hydrochloride salt (7 mg, 58% yield). 1 [START_REF] Talukdar | O-Nucleoside, S-Nucleoside, and N-Nucleoside Probes of Lumazine Synthase and Riboflavin Synthase[END_REF]. 12b (7 mg, 1 eq, 0.011mmol) was heated to 90°C in a 10:1 mixture of methanol (1 mL) and water (0.1 mL). Then Na2S2O4 (15 mg, 8 eq, 0.09 mmol) was quickly added and the mixture was stirred under reflux for 15 minutes. Excess methanol was removed under vacuum and the product was extracted by EtOAc. The organic layer was dried over MgSO4 and concentrated under vacuum. The crude intermediate was dissolved in methanol (1 mL) and cooled to 0°C prior to addition of 37% HCl (1 mL). The mixture was then stirred at room temperature for 3 hours. The product was concentrated under vacuum, dissolved in few volumes of methanol and precipitated from EtOAc. The precipitate was filtered and washed with EtOAc. After deep drying under vacuum, the product was obtained as pink-red powder corresponding to the amine hydrochloride salt (2.5 mg, 75% yield). 1

Preparation of 14, 14a and 14b

Stock solution (1 mL) of freshly synthetized 13, 13a and 13b were prepared at 10 mM in DMSO. Methylglyoxal solution (40% in H2O) was diluted in DMSO to obtain a solution at 50 mM concentration and 1 mL of this solution was added to 1 mL of each 10 mM stock solution. The mixtures were allowed to react at room temperature for 48h to give 5 mM solutions of 14, 14a and 14b that were aliquoted and stored at -80°C. Completion of the reactions was controlled by UPLC-MS analysis of an aliquot using the following gradient conditions : A: water + 0.1% formic acid, B: CH3CN + 0.1% formic acid; gradient: 100% A to 100% B over 4 min. Corresponding chromatograms are displayed below:

X ray crystallography

12a was dissolved at room temperature in a 80/20 mixture of acetonitrile/water and was let at room temperature for solvent evaporation. After 2 days, small needles were obtained but necessarily brought to PROXIMA 2A (PX2A) beamline at the SOLEIL Synchrotron, Gif-sur-Yvette, Paris, France, for complete data collection at T = 100K. Crystal data, data collection and structure refinement details for 12a crystal model are available in the Supporting Information file but are summarized below.

CCDC 1990644 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Biology

2.2.1 Cell culture WT3 murine cell line (WT3-m) stably overexpressing MR1 was previously described [START_REF] Huang | MR1 antigen presentation to mucosal-associated invariant T cells was highly conserved in evolution[END_REF].

Culture was done in DMEM + GlutaMAX media supplemented with 10% FCS, penicillin and streptomycin, non-essential amino acids, HEPES, and sodium pyruvate (all from Gibco TM ).

Mice

The mice used in this study were described previously [START_REF] Martin | Stepwise Development of MAIT Cells in Mouse and Human[END_REF]. Briefly, iVα19-Tg animals were on a Cα-deficient B6 background to avoid endogenous Vα expression. All Vβ8-Tg mice were generated on the C57BL/6 background, and iVα19-Tg mice were backcrossed onto the C57BL/6 background for more than 10 generations. All mice were housed in our accredited specific pathogen-free colony and genotyped by PCR or FACS staining, as appropriate.

Animal experiments were done in accordance with the guidelines of the French Veterinary Department and the ethical committee of the Curie Institute.

MR1 up-regulation experiments

A suspension of WT3-m cells was prepared, and 100 000 cells were introduced into 96 wellsplates. The cells were incubated 3h at 37°C (5% CO2). The ligands to be tested were diluted to the desired concentrations in culture media and added into the wells. The plate was incubated for 2h at 37°C (5% CO2). Cells were detached using versene solution on ice for 10 minutes and further stained by anti-MR1-PE antibody. DAPI was used to exclude dead cells.

The cells were analyzed using CytoFLEX flow cytometer (Beckman Coulter). Analysis of the data was done with FlowJo software and graphs were made using Prism 8 software (GraphPad) with a two-way ANOVA test for the statistical analysis.

MAIT cell activation experiments

MAIT cell activation assay was performed as previously described [START_REF] Soudais | In Vitro and In Vivo Analysis of the Gram-Negative Bacteria-Derived Riboflavin Precursor Derivatives Activating Mouse MAIT Cells[END_REF]. A suspension of WT3-m cells was prepared, and 100 000 cells were introduced into 96 wells-plates. The cells were incubated 3h at 37°C (5% CO2). The ligands to be tested were diluted to the desired concentrations in culture media and added into the wells. The cells were incubated for 2h at 37°C (5% CO2) for APCs activation. Enriched MAIT cells suspension was obtained by mechanical disruption of splenocytes of transgenic mice (iVα19 simple transgenic or iVα19

Vβ8 double transgenic mice) on cell strainers followed by red blood cells lysis using a red blood lysis buffer. Then, 160 000 cells were added into the different wells. The cells were incubated overnight (16 hours). Cells were subsequently stained at 4°C in the dark by the following conjugated antibodies: Ab anti-TCRβ-APC, Ab anti-Vβ6-FITC, Ab anti-Vβ8-FITC, Ab anti-CD4-BV785, Ab anti-CD8-Alexa fluor 700, Ab anti-CD25-PE Texas red, Ab anti-CD69-PC7. DAPI was used to exclude dead cells. The cells were analyzed on CytoFLEX flow cytometer (Beckman Coulter). Activation of MAIT cells (Vβ6 + -Vβ8 + , CD4 -, CD8 +/- cells) was determined by the percentage of both CD69 and CD25 positive cells. Analysis of the data was done with FlowJo software and graphs were made using Prism 8 software (GraphPad) with a two-way ANOVA test for the statistical analysis.

Click chemistry experiments

Coverslips were incubated with a mixture of collagen (15 µg/mL) and fibronectine (1 µg/mL) for 1h and then 100 000 WT3-m or WT3-WT cells were added on the adherent coverslips.

The cells were incubated 16 hours at 37°C (5% CO2). Cells were subsequently treated with 5-OP-RU click mixture 14 or EdU used as positive control (10 µM final concentration for each product, 1 hour incubation at 37°C, 5% CO2). Coverslips were washed with PBS and cells were fixed using paraformaldehyde 2% in PBS during 12 minutes. Cells were then permeabilized using Triton X100 0.1% for 5 minutes. Click chemistry reaction was done following the supplier procedure (Click-it TM EdU cell proliferation kit for imaging Alexa

Fluor TM 488 dye supplied from ThermoFisher Scientific). After washing by PBS, coverslips were mounted on slides with Vectashield ® antifade mounting medium with DAPI (Vector laboratories). Fluorescent microscopy was performed with DeltaVision RT epifluorescence microscope. Images were acquired as z-stacks and deconvolution was done with SoftWorx software. Images were analyzed with ImageJ software (NIH).

Figure 3 :

 3 Figure 3: ORTEP view of one conformer of compound 12a with absolute configuration of ribose chain stereocenters. The thermal ellipsoids are drawn at the level of 30% probability

Figure 4 :

 4 Figure 4: MR1 up-regulation in the presence of 5-OP-RU, 14a or 14b. A: percentage of MR1 positive cells; B: MFI: mean fluorescence intensity of anti-MR1 PE conjugated antibody signal. Data displayed on the graphs are means ± SD of technical duplicates. (*) p ≤ 0.0332, (**) p ≤ 0.021, (***) p ≤ 0.0002, (****) p ≤ 0.0001 compared to 5-OP-RU

Figure 5 :

 5 Figure 5: MAIT cell activation by 5-OP-RU, 12a and 12b. A: iVα19 Vβ8 double transgenic mice; B: iVα19 single transgenic mice. Data displayed on the graphs are means ± SD of technical duplicates. (*) p ≤ 0.0332, (**) p ≤ 0.021, (***) p ≤ 0.0002, (****) p ≤ 0.0001 compared to 5-OP-RU

Figure 6 :

 6 Figure 6: Epifluorescence microscopy images showing 14 conjugated to AF488 (green) and DAPI (blue) in WT3-m cells or WT3-WT cells.

  complexing a second Cu(I) atom forms a non-planar reaction complex (RC) with methylazide that evolves over a planar transition state 1 (TS1) to a 6-membered Cu(I) containing planar cycle (intermediate complex; IC) that further brakes down via a non-planar transition state 2 (TS2) to the planar 5-membered Cu(I)-triazolyl pre-product (PP) releasing catalyst Cu(I) to form the final triazole product[START_REF] Ayouchia | Understanding the mechanism and regioselectivity of the copper(I) catalyzed [3 + 2] cycloaddition reaction between azide and alkyne: a systematic DFT study[END_REF]. To assess whether both stereoisomers of 14 could react with fluorescence label while bound to MR1 we analyzed radial distribution functions (rdf) of water molecules around the ethinyl or triazole groups of fluorophore-reacted 5'R and 5'S ligands. The analysis showed that roughly half of the volume of first and second hydration shells around the reactive or reacted group, respectively, was sheltered by MR1 in the molecular dynamics simulations of binary complexes ([START_REF] Steinmetz | Data in Brief[END_REF], SM2). The steric requirements of reaction intermediates and transition states of CuAAC were very small since the planes of TS1, IC, and PP including the coordinated water molecules fitted within circles of 6.0 Å radius, while RC and TS2 fitted within spheres of 6.0 Å. Thus, the simulations suggested that both stereoisomers of 14 could have participated in fluorescence labeling of WT3-m cells while adopting the modeled binding modes.

1 H

 1 NMR (400 MHz, CDCl3) δ 4.19 (ddd, 1H, 7.4 Hz, 3.9 Hz, 1.0 Hz), 4.10 (d, 1H, J = 1.4 Hz), 4.07 (dd, 1H, 8.5 Hz, 1.0 Hz), 3.90 (dd, 1H, J = 8.5 Hz, 1.4 Hz), 3.69 (dd, 1H, J = 10.5 Hz, 3.9 Hz), 3.58 (dd, 1H, J = 10.5 Hz, 7.4 Hz), 2.76-2.70 (m, 1H), 2.63-2.55 (m, 1H), 2.53-2.44 (m, 2H), 1.66-1.56

  H NMR (400 MHz, CDCl3) δ 9.64 (d, 1H, J = 1.6 Hz), 4.31 (dd, 1H, J = 3.4 Hz, 1.6 Hz), 4.01-4.00 (m, 1H), 3.89-3.85 (m, 1H), 3.71 (dd, 1H, J = 10.4 Hz, 5.4 Hz), 3.55 (dd, 1H, J = 10.4 Hz, 5.6 Hz), 0.91-0.86 (m, 36H), 0.10-0.05 (m, 24H). 13 C NMR (101 MHz, CDCl3) δ 202.25, 78.13, 77.82, 75.44, 64.81, 26.16-26.04 (12C), 18.49 (2C), 18.33 (2C), -4.13, -4.37 (4C), -4.80, -5.11, -5.18. MS m/z [M+Na] + calculated for C29H66O5Si4: 629.4, found: 631.3.

- 4 .

 4 25, -4.42 (2C), -4.73, -5.16, -5.25. MS m/z [M+H] + calculated for C29H68O5Si4: 609.4, found: 609.9.

  -4.13 (m, 2H), 3.92 (m, 1H), 3.74-3.70 (m, 1H), 3.62 (dd, 1H, J = 10.5 Hz, 6.0 Hz), 3.56 (dd, 1H, J = 10.5 Hz, 5.4 Hz), 2.96 (s, 3H), 0.91-0.89 (m, 36H), 0.14-0.05 (m, 24H).

13 C 7 2

 137 NMR (101 MHz, CDCl3) δ 77.16 (signal under solvent peak), 75.80, 72.40, 71.11, 64.46, 37.20, 26.17-26.09 (12C), 18.46-18.27 (4C), -4.17 to -4.44 (6C), -5.11 to -5.22 (2C). MS m/z [M+Na] + calculated for C30H70O7SSi4: 709.4, found: 709.

  H NMR (400 MHz, CDCl3) δ 3.98-3.93 (m, 1H), 3.88-3.86 (m, 1H), 3.78-3.74 (m, 1H), 3.64 (dd, 1H, J = 10.4 Hz, 5.2 Hz), 3.55 (dd, 1H, J = 10.4 Hz, 5.6 Hz), 3.49 (dd, 1H, J = 12.8 Hz, 2.5 Hz),

  H NMR (400 MHz, CDCl3) δ 9.61 (d, 1H, J = 1.5 Hz), 4.21-4.20 (m, 1H), 3.96-3.95 (m, 2H), 3.51-3.47 (m, 1H), 3.40-3.36 (m, 1H), 0.93-0.86 (m, 27H), 0.16-0.10 (m, 18H). 13 C NMR (101 MHz, CDCl3) δ 203.67, 78.64, 77.55, 71.72, 53.29, 26.10-26.01 (9C), 18.47, 18.36, 18.14, -4.03, -4.07, -4.32, -4.41, -4.67, -4.88. HR-MS m/z [M+NH4] + calculated for C23H51N3O4Si3: 535.3531, found 535.3540 2.1.2.9 (4R,5S,6S)-7-azido-4,5,6-tris((tert-butyldimethylsilyl)oxy)-1-(trimethylsilyl)hept-1-yn-3-ol (10) Compound 10 was synthetized following literature modified synthesis protocol of a different molecule [32].

  H NMR (400 MHz, CDCl3) (major diastereomer) δ 4.61 (d, 1H, J = 3.4 Hz), 4.12 (t, 1H, J = 5.8 Hz), 4.06 (dd, 1H, J = 7.0 Hz), 3.58 (dd, 1H, J = 7.0 Hz, 3.4 Hz), 2.90 (d, 2H, J = 13.1 Hz, 5.8 Hz), 2.78 (dd, 1H, J = 13.1 Hz, 5.8 Hz ), 2.42 (s, 2H), 0.92-0.89 (m, 27H), 0.16-0.11 (m, 27H). 13 C NMR (101 MHz, CDCl3) (major diastereoisomer) δ 103.99, 91.71, 77.56, 75.86, 74.75, 66.49, 42.73, 26.22-25.97 (9C), 18.46-18.37 (3C), -0.09 (3C), -3.52, -3.87, -4.50, -4.56, -4.74, -4.81. HR-MS m/z [M+H] + calculated for C28H63NO4Si4: 590.3907, found: ,3S,4R,5S)-2,3,4-tris((tert-butyldimethylsilyl)oxy)-5-hydroxyhept-6-yn-1yl)amino)pyrimidine-2,4(1H,3H)-dione (12b)

  Compound 13a was synthetized following literature modified synthesis protocol of a different molecule[START_REF] Talukdar | O-Nucleoside, S-Nucleoside, and N-Nucleoside Probes of Lumazine Synthase and Riboflavin Synthase[END_REF].

  H NMR (500 MHz, CD3OD) δ 4.61 (dd, 1H, J = 4.1 Hz, 2.2 Hz), 4.05-4.02 (m, 1H), 3.81 (dd, 1H, J = 7.5 Hz, 5.0 Hz), 3.70 (dd, 1H, J = 7.5 Hz, 4.1 Hz), 3.65-3.61 (m, 1H), 3.56 (dd, 1H, J = 14.7 Hz, 7.2 Hz), 2.86 (d, 1H, J = 2.2 Hz).

  H NMR (500 MHz, CD3OD) δ 4.58 (dd, 1H, J = 3.3 Hz, 2.3 Hz), 4.03-4.00 (m, 1H), 3.83 (dd, 1H, J = 7.3 Hz, 5.4 Hz), 3.75 (dd, 1H, J = 7.3 Hz, 3.4 Hz), 3.60-3.55 (m, 2H), 2.86 (d, 1H, J = 2.3 Hz).

  Crystal data: C29H56N4O8Si3, 0.5 (H2O), Mw = 682.34, monoclinic, P21, a = 13.108(1), b = 31.263(1), c = 19.120(1) Å, β= 90.008(6)°, V = 7835.3(8) Å 3 , Z = 8, ρcalcd = 1.156 g cm -3 , μ = 0.169 mm -1 , F(000) = 2048, colorless needle, 0.23 × 0.05 × 0.04 mm 3 , T = 100 K, synchrotron PX2A radiation, λ= 0.70848 Å, 97118 reflections, 17140 unique Laue group reflections, (97.1%) with overall Rsym = 0.090, 32863 point group reflections , Pseudomerohedral twin law ( 0 0, 0 0, 0 0 1), BASF = 0.646 (10), 1830 parameters, 218 restraints GOF = 1.027, wR2 = 0.1541, R = 0.0572 for 30820 reflections with I > 2σ(I), Flack parameter x = -0.08(6).

2. 1 . 5

 15 Molecular modellingMolecular modeling was carried out using Drug Discovery Suite by Schrödinger Inc. in versions 2016.u3 or 2020.u1 for model visualization, ligand design and preparation, protein structure preparation, homology modeling and loop refinement, energy minimizations, molecular dynamics simulations and analyses. More specifically the programs and tools Maestro, Protein Preparation Wizard, Ligprep, Epik, Glide, PRIME, Macromodel, and Desmond were used applying standard settings and procedures unless specified otherwise[START_REF] Talukdar | O-Nucleoside, S-Nucleoside, and N-Nucleoside Probes of Lumazine Synthase and Riboflavin Synthase[END_REF][START_REF] Jacobson | A hierarchical approach to all-atom protein loop prediction[END_REF][START_REF] Sastry | Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments[END_REF][START_REF] Greenwood | Towards the comprehensive, rapid, and accurate prediction of the favorable tautomeric states of drug-like molecules in aqueous solution[END_REF][START_REF] Halgren | Glide: A New Approach for Rapid, Accurate Docking and Scoring. 2. Enrichment Factors in Database Screening[END_REF][START_REF] Jacobson | On the Role of the Crystal Environment in Determining Protein Side-chain Conformations[END_REF]. Implicit or explicit SPC water models were applied with force field parameters OPLS3[START_REF] Harder | OPLS3: A Force Field Providing Broad Coverage of Drug-like Small Molecules and Proteins[END_REF].PDB entries 4L4V and 4NQE were prepared in the Protein Preparation Wizard by replacing missing side chains, commitment of alternative side chain conformations by visual inspection, optimization of hydrogen bonding networks with pKa calculation by Epik, and minimization of hydrogen atoms only. Missing loops were not modeled at this stage as these regions were at a distance of at least 24 Å from the ligand binding site. The MR1/TCR complex formed by chains A, B, G, and H of 4LV4 was prepared for docking by Glide. All water molecules were deleted prior to grid calculation by Glide. The coordinates of the grid center were visually optimized in the ligand binding site. Outer box dimensions were increased to 36 Å each and all relevant or potentially interesting hydrogen bond donors or acceptors were indicated for grid calculations. Subsets of anchoring points by hydrogen bonding were selected during subsequent standard precision docking. Models of both, SSRR and SSRS stereoisomers of 13 were prepared by ligprep for docking. 25 poses per ligand were retained for post-docking energy minimization and 3 final poses per ligand saved. Poses were selected by visual inspection that compared to ligand binding observed in 4NQE. Notably hydrogen bonding to Tyr95 G by 2'-or 3'-OH was an important criterium.Construction and preparation of models for MD simulations as well as setup, calculation, and analysis of MD are described in detail in an accompanying report which also gives access to raw and analyzed data (insert reference Steinmetz & al, 2020, Data in Brief, in press). Briefly, models of ternary TCR/MR1 and binary MR1 complexes with the natural ligands 5-OE-RU, 5-OP-RU, their ethinylated analogues in R and S configuration and the corresponding fluorophore-reacted analogues were derived from 4NQE and docked poses. Missing loops, Nand C-terminal residues were completed by homology modeling. The systems were preequilibrated according to standard procedure, simulated as NPT ensembles for 120 ns production phase at 300 k and 1 atm, and analyzed.Coordinates of Density Functional Theory (DFT) optimized intermediates and transition states of CuAAC were recovered from literature to estimate the volumetric requirements of the reaction when occurring at a protein-solvent interface[START_REF] Ayouchia | Understanding the mechanism and regioselectivity of the copper(I) catalyzed [3 + 2] cycloaddition reaction between azide and alkyne: a systematic DFT study[END_REF]. The twice negatively charged 3,6-Diamino-9-[2-carboxyphenyl]-4,5-disulfoxanthylium substructure of fluorophore labeled ligand was recovered, energy minimized to convergence in vacuo by Macromodel, and subsequently in vacuo optimized to convergence in Jaguar applying B3LYP-3D functional with Basis Set 6-311G, ** polarization and ++ diffuse basis functions by DFT at the ground state or by TDDFT at the first excited state to calculate multipole moments[START_REF] Chen | Mechanisms of Quenching of Alexa Fluorophores by Natural Amino Acids[END_REF][START_REF] Harder | OPLS3: A Force Field Providing Broad Coverage of Drug-like Small Molecules and Proteins[END_REF]. Both quantum-mechanically optimized geometries were input for TDDFT calculations of Natural Transition Orbital eigenvectors for visualization of excited electron localization at the first excited state as well as transition moments of excitation and emission at the same level of theory[START_REF] Savarese | Fluorescence Lifetimes and Quantum Yields of Rhodamine Derivatives: New Insights from Theory and Experiment[END_REF].

  C NMR (101 MHz, CDCl3) δ 77.01, 75.73, 73.04, 64.72, 63.37, 26.21-26.11 (12C), 18.53, 18.46, 18.36, 18.26, -3.87, -4.17,

  C NMR (101 MHz, CDCl3) δ 77.09, 75.46, 72.44, 64.58, 54.36, 26.22-26.17 (12C), 18.51, 18.47, 18.30, 18.21, -4.12, -4.17, -4.20, -4.36, -4.42, -4.45, -5.11, -5.23. HR-MS m/z [M+H] + calculated for

	C29H67N3O4Si4: 633.4287, found: 634.4285
	2.1.2.

  C NMR (101 MHz, CDCl3) (major diastereomer) δ 103.73, 91.90, 78.00, 76.21, 72.37, 65.90, 53.33, 26.21-26.15 (9C), 18.52, 18.44, 18.31, -0.08 (3C), -3.73, (2C), -4.05, -4.17, -4.57, -4.70. MS m/z [M+Na] + calculated for C28H61N3O4Si4: 638.4, found: 638.6 2.1.2.

-amino-6-(((2S,3S,4R,5S)-2,3,4,5-tetrahydroxyhept-6-yn-1- yl)amino)pyrimidine-2,4(1H,3H)-dione (13b)

  Compound 13b was synthetized following literature modified synthesis protocol of a different molecule

	72.96, 64.97, 46.24. HR-MS m/z [M-H] -calculated for C11H17ClN4O6: 335.0758, found
	335.0770
	2.1.2.12.1 5
	13 C NMR (126 MHz, CD3OD) δ 161.89, 151.65, 151.31, 83.45, 82.96, 76.46, 75.67, 73.61,
	21

Acknowledgements

The author want to thank Jean-Claude Florent and Bhanudas Dasari for chemical discussions.

Thomas Yvorra wants to thank the ANR ("In vivo analysis of MAIT cell functions in health and disease") for financial support and Aurélie Darbois Delahousse for training in biological experiments. We acknowledge the use of the synchrotron-radiation facility at the SOLEIL synchrotron, Gif-sur-Yvette, France under the BAG proposal 20191181. We also thank Dr Martin Savko and the staff of the PROXIMA 2A beamline at SOLEIL for assistance with data collection. We also want to thank the flow cytometry core of Institut Curie and the PICT-IBiSA at Pasteur Imaging Facility of Institut Curie, member of the France-BioImaging national research infrastructure.

Declaration of interests

None.