Franck Talmont
email: franck.talmont@ipbs.fr

Remi Veneziano

Gilles Dietrich

Lionel Moul

Catherine Mollereau

Jean-Marie Zajac

Pharmacological insight into the activation of the human neuropeptide FF2 receptor

Keywords:

The neuropeptide FF2 (NPFF2) receptor, predominantly expressed in the central nervous system, plays an important role in the modulation of sensory input and opioid analgesia, as well as in locomotion, feeding, intestinal motility, reward, and the control of obesity. The NPFF2 receptor belongs to the RFamide peptide receptor family and to the G protein coupled receptor (GPCR) super family, but contrary to many other class A GPCRs, no 3D structure has been solved. Thus, it is essential to perform mutagenesis to gain information on the fine functioning of the NPFF2 receptor. In this study, we examined the role of aspartic acid (D) from the " D/ERY/F " motif found in the second intracellular loop (ICL2) and the role of the C-terminal end of the receptor in ligand binding and signal transduction. We found that mutation D3.49A does not impair binding capacities but inhibits G protein activation as well as adenylyl cyclase regulation. Truncation of the C terminal part of the receptor has different effects depending on the position of truncation. When truncation was realized downstream of the putative acylation site, ligand binding and signal transduction capabilities were not lost, contrary to total deletion of the C terminus, which totally impairs the activity of the receptor.

Introduction

The neuropeptide FF2 (NPFF2) receptor belongs to the RFamide peptide receptor family and to the G protein coupled receptor (GPCR) super family [START_REF] Vass | Chemical diversity in the g protein-coupled receptor superfamily[END_REF]. The NPFF2 receptor and its closest homologue (50 % homology), the neuropeptide FF1 (NPFF1) receptor, are activated by the neuropeptide FF (NPFF; FLFQPQRF-NH2) [START_REF] Mollereau | Pharmacological characterization of human NPFF(1) and NPFF(2) receptors expressed in CHO cells by using NPY Y(1) receptor antagonists[END_REF]. This peptide and other related peptides are generated from two precursors, pro-NPFFA and pro-NPFFB [START_REF] Mouledous | Opioid-modulating properties of the neuropeptide FF system[END_REF]. The NPFF system is known as an anti-opioid effector since NPFF and NPFF-like peptides can oppose some of the acute effects of opioids [START_REF] Mouledous | Opioid-modulating properties of the neuropeptide FF system[END_REF]. Just like the NPFF peptide, the NPFF2 receptor is predominantly expressed in the central nervous system in accordance with its role in the modulation of sensory input and opioid analgesia. In addition to their implications in pain perception and opioid modulation, NPFF peptides and receptors have demonstrated roles in locomotion, feeding, intestinal motility, reward, and the control of obesity [START_REF] Jhamandas | Role of neuropeptide FF in central cardiovascular and neuroendocrine regulation[END_REF][START_REF] Lin | Neuropeptide FF modulates neuroendocrine and energy homeostasis through hypothalamic signaling[END_REF][START_REF] Panula | A mammalian neuropeptide with multiple functions[END_REF]. Since the first X-ray crystal structure determination of bovine rhodopsin [START_REF] Palczewski | Crystal structure of rhodopsin: a G protein-coupled receptor[END_REF], an increasing number of 3D structures of GPCR have been solved [START_REF] Thal | Recent advances in the determination of G protein-coupled receptor structures[END_REF]. Together with site-directed mutagenesis and domain deletion, techniques for the determination of 3D structure are powerful methods for providing insight into how a GPCR works. Nevertheless, to provide relevant structure -function information, these techniques must be combined and interact closely. Until recently, no 3D structure has been solved for the NPFF2 receptor, it is thus essential to perform mutagenesis to gain information on its fine functioning. Since this receptor belongs to the class A GPCR family [START_REF] Isberg | Generic GPCR residue numbers -aligning topology maps while minding the gaps[END_REF], it shares structural domains, amino acids, and functional similarities with other class A receptors. These receptors have N-and C-terminal tails in their extracellular and intracellular compartments respectively. They are constituted with seven α -helices crossing the membrane and with a putative eighth helix located immediately after the seventh transmembrane helical domain (Fig. 1). However, there is limited information regarding the structure -function of the NPFF2 receptor [START_REF] Findeisen | Structure-activity studies of RFamide peptides reveal subtype-selective activation of neuropeptide FF1 and FF2 receptors[END_REF][START_REF] Findeisen | Selective mode of action of guanidine-containing non-peptides at human NPFF receptors[END_REF] performed by site-directed mutagenesis. Here, we present the pharmacological profile of the NPFF2 receptor mutated at the aspartic acid (D) site in the D/ERY/F " motif in the second intracellular loop (ICL2), known to be involved in GPCR activation [START_REF] Li | Constitutive activation of the mu opioid receptor by mutation of D3.49(164), but not D3.32 (147): D3.49(164) is critical for stabilization of the inactive form of the receptor and for its expression[END_REF][START_REF] Rasmussen | Mutation of a highly conserved aspartic acid in the beta2 adrenergic receptor: constitutive activation, structural instability, and conformational rearrangement of transmembrane segment 6[END_REF][START_REF] Surratt | G protein-coupled receptor structural motifs: relevance to the opioid receptors[END_REF][START_REF] Weis | The molecular basis of g protein-coupled receptor activation[END_REF]. We also investigated the effect of truncation of the C-terminal end, a domain involved in receptor signaling [START_REF] Deng | Role for the C-terminus in agonist-induced mu opioid receptor phosphorylation and desensitization[END_REF][START_REF] Okura | Agonist-specific down-regulation of the human delta-opioid receptor[END_REF][START_REF] Pankevych | Truncation of the A1 adenosine receptor reveals distinct roles of the membrane-proximal carboxyl terminus in receptor folding and G protein coupling[END_REF]. enzymes, the Δ C63-hNPFF2 receptor and the Δ C80-hNPFF2 receptor were inserted into the bicistronic pEFIN3 vector [START_REF] Kotani | Functional characterization of a human receptor for neuropeptide FF and related peptides[END_REF]. The sequences were confirmed by DNA sequencing.

Cell expression and transfection

Chinese hamster ovary (CHO) cell line was stably transfected with the mutant receptor constructs using fugene 6 according to the manufacturer ' s instructions (Roche Applied Science, France • C, samples were rapidly filtered on Whatman GF/B filters presoaked in 0.3 % polyethylenimine. The filters were rinsed 3 times with 4 mL of icecold buffer containing 0.1 % bovine serum albumin, and the bound radioactivity was counted in a liquid scintillation spectrophotometric counter (Packard).

Assay for intracellular 3 ′ , 5 ′ -cyclic adenosine monophosphate

An assay for intracellular 3 ′ , 5 ′ -cyclic adenosine monophosphate (cAMP) was performed as previously described [START_REF] Mollereau | Pharmacological characterization of human NPFF(1) and NPFF(2) receptors expressed in CHO cells by using NPY Y(1) receptor antagonists[END_REF]. In this procedure,

[35S] GTP γ S binding assay

The assay buffer consisted of 20 mM HEPES (pH 7.

Western blot analysis

Proteins from CHO membranes were separated according to their size by 10 % sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and transferred onto an immun-blot polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA). The receptors were probed with hNPFF2-primed mouse immune sera (1:1000) [START_REF] Talmont | Denatured G-protein coupled receptors as immunogens to generate highly specific antibodies[END_REF].

Bound immunoglobulin G (IgG) was revealed using horseradish peroxidase-labeled sheep anti-mouse IgG antibodies diluted at 1:10000 (Jackson Immunoresearch Lab, West Grove, PA).

Analysis of the data

Non-linear regression analyses of the data were performed using Prism 4.0 software (GraphPad Inc., USA).

Results

We constructed three mutants of the hNPFF2 receptor (Fig. 1) and analyzed the effects of each mutant on ligand interaction and signal transduction. The first mutant (D3.49A-hNPFF2 receptor) was created by changing the amino acid aspartic acid (D), located at the interface between transmembrane helix three (TM3) and intracellular loop (ICL2), to alanine (A). This mutant was designated using the Ballesteros and Weinstein nomenclature [START_REF] Ballesteros | Integrated methods for the construction of threedimensional models and computational probing of structure-function relations in G protein-coupled receptors[END_REF] and belongs to the highly conserved E/DRY/F motif of class A GPCRs. The two other mutants were C-terminal end deleted, with one next to the interface between transmembrane helix seven (TM7) and the C terminus ( Δ C80-hNPFF2 receptor) and the second downstream to a putative palmitoylation site ( Δ C63-hNPFF2 receptor).

Binding of the agonist [3H]-EYF

Saturation binding curves were established with a membrane fraction using the tritiated agonist compound [3H]-EYF [START_REF] Talmont | Characterization of two novel tritiated radioligands for labelling Neuropeptide FF (NPFF(1) and NPFF (2)) receptors[END_REF] (Fig. 2). The results obtained for the mutants were compared with the wild type receptor (Table 1). A slight decrease in affinity was observed for the Δ C63-hNPFF2 and D3.49A-hNPFF2 receptors mutants compared to the wild type, but KD values were all in the nM range. The Δ C80-hNPFF2 receptor mutant was unable to bind the specific agonist ligand EYF. The clone that express Δ C80-hNPFF2 receptor was resistant to G418 and the expression of the mutant was confirmed by Western blot (data not show).

Competition binding assays

Competition experiments (Table 2, Fig. 3) were then performed to compare the pharmacological binding profile of wild type and mutant hNPFF2 receptors. All peptides used in this study belong to the RF-amide peptide family [5,[START_REF] Mouledous | Opioid-modulating properties of the neuropeptide FF system[END_REF]. They are all agonists to NPFF receptors except RF9 that is an antagonist [START_REF] Simonin | RF9, a potent and selective neuropeptide FF receptor antagonist, prevents opioid-induced tolerance associated with hyperalgesia[END_REF]. Two peptides precursors have been the Δ C63-hNPFF2 receptor, whereas there was a four-fold increase for the wild type receptor.

Ligand-induced cAMP accumulation

As for the stimulation of [35S]-GTP γ S binding, the Δ C63-hNPFF2

receptor was the only mutant able to mediate the inhibition of the forskolin-induced accumulation of cAMP by the agonists (Fig. 5). The rank orders of ligand potencies in wild type hNPFF2 and mutant Δ C63-hNPFF2 receptors were very similar (NPAF ≥ SQA-NPFF > 1DMe > NPVF > NPSF >> RF9 for wild type and SQA-NPFF ≥ NPAF > 1DMe > NPSF > NPVF >> RF9 for Δ C63-hNPFF2 receptor). However, the potencies decreased 10-fold in compared to the wild type receptor (Table 4). Concerning these receptors, the inhibition constants (Ki) were related to the functional potencies (EC50) for nearly all peptides tested, indicating that the agonist activity reflected the binding affinity.

Discussion

In the present study, mutants of the hNPFF2 receptor (Fig. 1) were generated and transfected in CHO cells. The mutants were analyzed for their ability to interact with ligands specific for the human NPFF2 receptor and for their capacity to activate signal transduction cascades.

For the first mutant D3.49A-hNPFF2 receptor, the aspartic acid at the absorption spectra, but it failed to stimulate the G protein [START_REF] Franke | Rhodopsin mutants that bind but fail to activate transducin[END_REF]. In the human β 2 adrenergic receptor, it was suggested that D3.49 (Asp-130) is the receptor between its active and inactive state, and, thus, it may govern the spatial disposition of TMs 3 and 6 [START_REF] Rasmussen | Mutation of a highly conserved aspartic acid in the beta2 adrenergic receptor: constitutive activation, structural instability, and conformational rearrangement of transmembrane segment 6[END_REF]. In the hNPFF2 receptor, intramolecular interactions between the DRF amino acids cluster and an amino acid located in the TM6 helix remain to be verified.

Thus, replacing the charged aspartic acid residue located in the highly conserved D/ERY/F motif with the non-polar amino acid alanine has a slight effect on the ligand binding affinities. In fact, the interaction between hNPFF2 receptor and specific ligands is located in a binding pocket narrowed down to the upper part of transmembrane helices 5, 6, and 7 and the extracellular loop 2 [START_REF] Findeisen | Selective mode of action of guanidine-containing non-peptides at human NPFF receptors[END_REF]. The Beck-Sickinger group performed an important work [START_REF] Findeisen | Selective mode of action of guanidine-containing non-peptides at human NPFF receptors[END_REF] to investigate this binding pocket of NPFF receptors after mutagenesis of key residues.

However, this D3.59A amino acid modification has a huge effect on the ability of the receptor to transduce a signal after ligand interaction, suggesting the involvement of this amino acid in a conformational structure with G proteins. It seems possible that the loss of function of the mutant D3.49A-hNPFF2 receptor and its higher affinity for the antagonist RF9 reflects an inactive and uncoupled intermediary conformation of the receptor. The C-terminal end of class A GPCRs interacts with G proteins and interferes with signal transduction when modified. The conformational structure of the C-terminus of a GPCR, its ability to interact with G proteins, and its role in signal transduction depends on its composition in amino acids (e.g., phosphorylation sites [START_REF] Bray | Identification and functional characterization of the phosphorylation sites of the neuropeptide FF2 receptor[END_REF]) (Fig. 1), and the occurrence of an amphipathic eighth α -helix [START_REF] Sato | Functional role of the C-Terminal amphipathic Helix 8 of olfactory receptors and other g protein-coupled receptors[END_REF].

In the mutant Δ C63-hNPFF2, deletion of 63 amino acids from the C-terminal end of the hNPFF2 receptor, which was realized downstream of the helix8, had a slight effect on the binding of [3H]-EYF (Fig. 2, Table 1) and on the ability of the ligand to compete with [3H]-EYF (Fig. 3, Table 2). The decrease in stimulation of [35S]-GTP γ S binding with 1DMe at high and low NaCl concentrations (Fig. 4, Table 3) was in accordance with the decrease in 1DMe binding affinity. Nevertheless, we can observe a more significant decrease in potency and relative efficacy of [35S]-GTP γ S stimulation by 1DMe (Table 3) for the Δ C63 mutant relative to wild type at low salt than in high salt. This effect has already been observed for partial agonist compared to full agonist [START_REF] Selley | Effects of sodium on agonist efficacy for G-protein activation in mu-opioid receptor-transfected CHO cells and rat thalamus[END_REF][START_REF] Williams | Somatostatin5 receptormediated [35S]guanosine-5 ' -O-(3-thio)triphosphate binding: agonist potencies and[END_REF]. The effect of the deletion of the C-terminal part of the receptor seems to be comparable to that observed for partial agonists in other systems. The cAMP signal transduction pathway that follows G protein activation was more affected (Fig. 5, Table 4), and EC50 values increase by a factor 10 -30, depending on the ligand considered. In the hNPFF2 receptor, eight residues can be phosphorylated at the C-terminal part, either in the basal state or after agonist treatment [START_REF] Bray | Identification and functional characterization of the phosphorylation sites of the neuropeptide FF2 receptor[END_REF], and as for the results obtained for the deletion in the Δ C63-hNPFF2 receptor mutant, the removal of individual conserved phosphorylation sites does not strongly affect NPFF2 receptor signaling [START_REF] Bray | Identification and functional characterization of the phosphorylation sites of the neuropeptide FF2 receptor[END_REF]. Truncation of the C-terminal parts of human mu opioid receptor ( Δ C45hMOR) [START_REF] Deng | Role for the C-terminus in agonist-induced mu opioid receptor phosphorylation and desensitization[END_REF] and mouse delta opioid

2 .

 2 Materials and methods 2.1. Materials NPFF-related peptides were synthesized in-house using manual solid phase synthesis with Fmoc-amino acid chemistry. Peptides used were: NPSF: SLAAPQRF-NH2, NPFF: FLFQPQRF-NH2, NPAF: NPAFLFQPQRF-NH2, NPVF: VPNLPQRF-NH2, 1DMe: ([D.Tyr1, (NMe) Phe3] NPFF), SQA-NPFF: SQAFLFQPQRF-NH2. The NPFF antagonist RF9 [25] was synthesized at Universit ´ e Louis Pasteur, Illkirch, France. [3H]-EYF, 72 Ci/mmol (2.66 T Bq/mmol), was custom made by RC TRITEC AG (Teufen, Switzerland) by hydrogenation of the EYWS (3 -4 dehydro)LAAPQRFa precursor with 99 % tritium gas[START_REF] Talmont | Characterization of two novel tritiated radioligands for labelling Neuropeptide FF (NPFF(1) and NPFF (2)) receptors[END_REF].2.2. Construction of human neuropeptide FF2 receptor mutantsMutagenesis was performed on the human NPFF2 receptor cloned in pBluescript II (SK) + . The D3.49A mutation was realized by polymerase chain reaction using the primers 5 ′ TGCAATTGCTGTAGCTAGGTTCCAGTGTGT3 ′ and 5 ′ TTTCCTGATTTGGCCAGTCTTCCCG3 ′ to amplify a 233 bp double strand cDNA. This fragment was cloned in a TOPO TA cloning vector (Invitrogen), cut with MfeI and BbsI enzymes and inserted at the same sites in the human NPFF2 receptor pBluescript II (SK) + . After digestion with BamHI and XbaI, the D3.49A hNPFF2 receptor sequence was inserted into the bicistronic pEFIN3 vector[START_REF] Kotani | Functional characterization of a human receptor for neuropeptide FF and related peptides[END_REF]. For construction of the deleted C′ ( Δ C63-hNPFF2 receptor) and 5 ′ TCTAGATTAGCGGAAATTCTCGTT3 ′ ( Δ C80-hNPFF2 receptor). The double strand cDNAs (445bp for the Δ C63-NPFF2 receptor and 394 bp for the Δ C80-NPFF2 receptor) were cloned into a TOPO TA cloning vector, cut with pBluescript II (SK) + vector. After digestion with BamHI and XbaI restriction

  200,000 recombinant cells were incubated for one hour at 37 • C under an atmosphere of 5% CO2 with 0.6 mCi [3H] adenine (26 Ci/mmol, GE Healthcare) in Ham ' s F12 medium. cAMP production was stimulated by 5 μ M Forskolin (Sigma, France) for 10 min at 37 • C in 200 μ L HEPES ([4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid])-buffered Krebs -Ringer solution in the presence of 0.1 mM of the phosphodiesterase inhibitors IBMX (Sigma, France) and Ro-20 1724 (Fisher, France). The desired concentration of the tested ligands were added and the reaction was stopped by the addition of 20 μ L of HCl (2.2 N) and rapid mixing. The [3H] cAMP content of each tube was isolated by chromatography on acidic alumina columns (Sigma, France) and counted in a liquid scintillation analyzer (Packard).

1 %

 1 4), 20 mM or 150 mM NaCl, 3 mM MgCl2, 1 μ M GDP (guanosine-5 ′ -diphosphate) and 0.BSA (bovine serum albumin). Membranes were incubated in polypropylene tubes at 30 • C for 60 min in 500 μ L of buffer containing 0.05 nM [35S]GTP γ S. The reaction was stopped by rapid filtration, and the radioactivity was detected and counted in a Packard liquid scintillation analyzer.

3 . 3 .

 33 identified in mammals. NPFF, 1DMe which is a stable NPFF analogue, SQA-NPFF, NPSF and NPAF are pro-NPFFA-derived peptides whereas NPVP is a pro-NPFFB-derived peptide. The utilization of these peptides allow to make pharmacological profiles on NPFF receptors and mutants due to their different selectivity on NPFF type 1 and 2 receptors[5,[START_REF] Findeisen | Structure-activity studies of RFamide peptides reveal subtype-selective activation of neuropeptide FF1 and FF2 receptors[END_REF][START_REF] Mollereau | Pharmacological characterization of human NPFF(1) and NPFF(2) receptors expressed in CHO cells by using NPY Y(1) receptor antagonists[END_REF].For the D3.49A-hNPFF2 receptor, the rank order of the potency of the inhibition of specific [3H]-EYF binding by seven ligands was SQA-NPFF ≥ 1DMe ≥ NPAF > NPFF > NPSF > RF9 ≥ NPVF. For the Δ C63-hNPFF2 receptor, the order was SQA-NPFF > NPAF ≥ NPFF ≥ 1DMe > NPVF > NPSF > RF9. The Ki values measured for the D3.49A-hNPFF2 receptor were very similar to those of the wild type receptor, except for RF9, which was four-fold more potent for the mutant. For the mutant Δ C63-hNPFF2 receptor, the tested ligand was globally as potent as the wild type receptor. Binding of [35S]-GTP γ S to the G proteins in wild type and mutantChinese hamster ovary (CHO)-NPFF2 receptor membranesStimulation of [35S] GTP γ S binding by 1DMe was assessed, and EC50and Emax parameters were determined from the concentration -response curves (Table3, Fig.4). Experiments were performed under low(20 mM) and high sodium (150 mM) conditions. High Na + concentration is suspected to unmask the constitutive activity of receptors[START_REF] Gouarderes | Functional differences between NPFF1 and NPFF2 receptor coupling: high intrinsic activities of RFamide-related peptides on stimulation of [35S]GTPgammaS binding[END_REF]. No stimulation was observed for the Δ C80-hNPFF2 receptor, in accordance with the absence of binding, and, unexpectedly, for the D3.49A-hNPFF2 receptor mutant. A comparison of the EC50 values of hNPFF2 and Δ C63-hNPFF2 receptors revealed a decrease in potency for the mutant at low Na + (EC50, 17-fold higher) and at high Na + concentrations (EC50, 3.5-fold higher). The rightward shift of the curve induced by high NaCl concentrations was less pronounced in the Δ C63-hNPFF2 receptor (8-fold) compared to that of the wild type receptor (40-fold). The Emax between the low and high NaCl conditions increased by a factor two for

ICL2

  glutamic acid/aspartic acid-arginine-tyrosine/phenylalanine (D/ ERY/F) motif was mutated to alanine and the two other mutants were Cterminal truncated forms of the hNPFF2 receptor. The ICL2 " D/ERY/F " motif is a highly conserved motif among GPCRs[START_REF] Surratt | G protein-coupled receptor structural motifs: relevance to the opioid receptors[END_REF]. It is located at the cytoplasmic interface between TM3 and ICL2. This motif is usually known as a DRY motif (position 3.49 -3.51 in the Ballesteros nomenclature). The mutant D3.49A-hNPFF2 receptor displays a 3-fold decrease in [3H]-EYF affinity compared to the wild type. Competition studies against the tritiated ligand [3H]-EYF have shown no change in affinity for various specific ligands, except for the antagonist RF9, whose affinity increased by a factor of four. In contrast, the signal transduction pathway was deeply impaired since neither G-protein coupling nor adenylate cyclase activation were observed for this mutant. An ionic lock was detected in the rhodopsin crystal structure where arginine (3.50) is engaged in a double salt bridge with glutamic acid (3.49) and another glutamic acid (6.30) positioned on helix six [19]. It was proposed that breaking these interactions might have important consequences in the activation of rhodopsin. The mutation of aspartic/glutamic acid (3.49) in class A GPCRs has various effects on ligand binding and signal transduction. The aspartic acid to asparagine, lysine, or arginine mutations (D3.49 N, D3.49 K, D3.49R) in the adenosine A3 receptor and expressed in COS-7 cells have no impact on agonist binding affinity, cAMP production, and phospholipase C activation [3]. On the contrary, the human D3.49 CXCR1 mutants (D3.49 N and D.3.49 V) expressed in HEK 293 cells were not able to bind specific ligands and did not retain G protein coupling and agonist-induced responses [10]. In rhodopsin, the ionically charged pair E3.49/R3.50 found in the DRY motif was mutated to R3.49/E3.50, and this mutant normally binds 11-cis-retinal to yield pigments with native rhodopsin

  receptor ( Δ C31mMOR)[34] expressed in CHO cells, downstream of the putative palmitoylation site (thus defining a 4th ICL), has no impact on the binding affinity of specific ligands and on the inhibition of forskolin-stimulated cAMP accumulation after exposure to the agonist. Our results obtained for the Δ C63-hNPFF2 mutant receptor prompted us to analyze a mutant with a total C-terminal amino acid truncation, namely the Δ C80-hNPFF2 receptor mutant. Contrary to the Δ C63-hNPFF2 receptor mutant, no binding was observed in the [3H-EYF] saturation test. No positive results were obtained in the stimulation of [35S] GTP γ S binding and cAMP accumulation tests, despite the detection of the protein by Western blot (data not shown). The absence of crystallographic data for the NPFF2 receptor does not allow for clear positioning of the structural domains that compose the protein e.g., TM7 or helix eight. However, the position of the Δ C80 truncation could be at the level of the putative helix eight, which would indicate an essential role of this domain in receptor binding and signaling. The specific amino acid composition of the C-terminus is not the only actor that trigger receptor activity. Post-translational modification such as palmitoylation, O-glycosylation or phosphorylation are also important. Moreover, the activity of GPCR depend on the type of G-proteins or β -arrestins and channels, which may vary in cells and tissues.In conclusion, the replacement of aspartic acid 3.49 with alanine from the highly conserved ICL2 " D/ERY/F " motif does not impair the binding capacities of receptor-specific ligands. On the other hand, this amino acid is essential with regard to signal transduction since the activation of G proteins by the ligand as well as the capacity to inhibit the activity of adenylyl cyclase were totally damaged. Truncation of the C-terminal part of the receptor has different effects depending on the position of the truncation. When the truncation was realized downstream of the putative acylation site ( Δ C63-hNPFF2 receptor), ligand binding and signal transduction capabilities were not lost, although they were less efficient than for the entire receptor. On the contrary, total deletion of the C-terminus ( Δ C80-hNPFF2 receptor) totally impairs the activity of the receptor. This suggests that this domain, comprising the putative eighth α -helix and acylation site, is essential for the correct folding of the receptor.

Fig. 1 .Fig. 2 .

 12 Fig. 1. Serpentine model representative of the human neuropeptide FF2 receptor. (From the GPCRDB site [11]. The arrows indicate mutants. ECL, extracellular loop; ICL, intracellular loop; N-term, Nterminal end; C-term, C-terminal end.

Fig. 3 .

 3 Fig. 3. Competition studies of [ 3 H]-EYF (1 -2 nM) with increasing concentrations of NPFF receptor ligands (1DMe, NPFF, SQA-NPFF, NPSF, NPVF, NPAF, RF9) on D3.49A-hNPFF 2 and Δ C63-hNPFF 2 receptors. The curves are representative of at least three experiments performed in duplicate. Ki (nM) results are presented as bar graphs.

Fig. 4 .

 4 Fig. 4. [ 35 S]-GTP γ S binding induced by 1DMe on the wild type and mutant Δ C63-hNPFF 2 receptor membranes was realized with 1 μ M guanosine-5 ′ -diphosphate (GDP), various concentrations of 1DMe and 0.1 nM [ 35 S]-GTP γ S. With 10 mM NaCl or 150 mM NaCl. The curves are representative of at least three experiments performed in duplicate. Inset: EC 50 (nM) and Emax (%) values.

Fig. 5 .

 5 Fig. 5. Inhibition by various ligands of the forskolin-induced accumulation of 3 ′ , 5 ′ -cyclic adenosine monophosphate (cAMP) in intact recombinant Chinese hamster ovary (CHO) cells that express the mutant Δ C63-hNPFF2 receptor. The curves are representative of at least three experiments performed in duplicate. EC 50 (nM) results are presented as bar graphs. WT h-NPFF 2 values are from [16,29].

Table 2

 2 Ki values determined by displacement.

	Ki values determined by displacement of [ 3 H]-EYF (1 -2 nM) by a series of NPFF
	receptor ligands. Shown data are mean Ki ± standard error of the mean from
	three or more independent experiments. * results from [20]. ND: not
	determined.
		WT *	ΔC63	D3.49A
		Ki nM	Ki nM	Ki nM
	1DMe	0.38 ± 0.04	1.18 ± 0.36	0.17 ± 0.053
	NPFF	0.63 ± 0.05	0.83 ± 0.34	0.73 ± 0.014
	NPSF	19 ± 5	57.9 ± 17.3	13.8 ± 1.3

Table 4

 4 Inhibition of forkolin-induced cAMP accumulation.Inhibition of forkolin-induced cAMP accumulation. Shown data are mean EC 50 ± standard error of the mean from three or more independent experiments.

*

WT hNPFF2 EC50 (nM) ΔC63-hNPFF2 EC50 (nM) 1DMe 2.7 ± 0.5 36.9 ± 7.1

			20 mM NaCl		150 mM NaCl
			EC 50 (nM)	Emax %	EC 50 (nM)	Emax %
	hNPFF 2 receptor	0.3 ± 0.2 116 ± 4	12 ± 3	458 ± 46
	Δ C63-hNPFF 2 receptor	5 ± 2 122 ± 2	41 ± 4	233 ± 5
	NPSF	222 ± 26	2276 ± 792
	SQA-NPFF 0.56 ± 0.05	6.19 ± 0.84
	NPVF	133 ± 11	4111 ± 842
	NPAF	0.53 ± 0.03	7.31 ± 1.75
	RF9	> 1000		> 1000

Acknowledgement

The authors acknowledge financial support from the Centre National de la Recherche Scientifique (CNRS).


Rostom Ahmed-Belkacem

Priscila Sutto Ortiz

Etienne Decroly

Jean-Jacques Vasseur

Françoise Debart

Synthesis of adenine dinucleosides 2',5'-bridged by sulfur-containing linkers as bisubstrate SAM analogues for viral RNA 2'-O-methyltransferases

Viral RNA 2'-O-methyltransferases play a crucial role for luring the host cell innate antiviral response during a viral infection by catalyzing either the methylation of the 5'-end RNA cap-structure at 2'-OH of nucleoside N1 or by inducing internal 2'-O-methylation of adenosines within RNA sequence using S-adenosyl-L-methionine (SAM) as the methyl donor. Our goal is to synthetized bisubstrate SAM analogues mimicking the transition state of the 2'-O-methylation of the RNA in order to block viral 2'-O-methyltransferases and struggle against emerging viruses. Here we designed and synthesized five dinucleosides by connecting a 5'-thioadenosine representing the SAM to the 2'-OH of another adenosine unit mimicking the RNA substrate, via various sized sulfur-containing linkers such as alkylthioether linkers, sulfoxide or sulfone derivatives, or a disulfide bond.

Introduction

Emerging RNA viruses (eg., Dengue, Zika, SARS, MERS, Ebola viruses) are important human pathogens causing substantial health and economic burden. [1] Their spreading is, among others, linked to their rapid evolution combined with their capacity to escape antiviral response by hiding their RNA from detection by antiviral sensors or restriction factors. [2] The viral replication/transcription complex contains enzymes essential for virus replication, which are involved in RNA synthesis (polymerase) and RNA capping. The cap structure, consisting of a guanosine linked by a 5'-5'-triphosphate bridge to the 5'-end of messenger RNAs, protects viral RNA from degradation by cellular nucleases. Particularly, our research aims at studying and targeting viral RNA Methyltransferases (MTases) which play a crucial role by catalyzing the methylation of the RNA cap-structure using S-adenosyl-L-methionine (SAM) as the methyl donor. Viral N7-MTase methylates the cap at the nitrogen in position N7 of guanosine in order to allow RNA translation into viral proteins. [3] The cap structure is also often methylated at the 2'-O-position of the N1 residue (adenosine or guanosine) by 2'-O-MTase. Moreover, internal 2'-Omethylations of viral RNA have been demonstrated with Sudan ebolavirus, Dengue and Zika viruses, and HIV. [START_REF]5'-bis-O-(tert-butyldimethylsilyl)-S-(2'-O-ethyladenosyl)-5'-thioadenosine 14 To a suspension of 8 (135 mg, 0.42 mmol, 1.00 eq) in MeOH (4 mL) was added a solution of KOH (47 mg, 0.83 mmol, 2.00 eq) in MeOH (2 mL). After stirring for 20 minutes at 0°C, a solution of 13 (346 mg, 0.50 mmol, 1.20 eq) in CH2Cl2 (4 mL) was added and the reaction mixture was stirred for additional 2 h at room temperature. The solvents were removed under vacuum, and the resulting paste was purified by chromatography (dry-load) with a 0-15% linear gradient MeOH in CH2Cl2 yielding to 14 as a white solid (63 mg, 0.078 mmol, 19%). Rf 0.67[END_REF] These 2'-O-methylations were recently evidenced as self-markers, hiding viral RNA from detection by RIG-like receptors [2c, 5] and limiting the restriction of viral replication by IFIT1/3 molecules. [START_REF]HRMS (ESI+): m/z calcd for C34H57N10O7SSi2[END_REF] It is now currently admitted that these key enzymes are potent antiviral targets, as their inhibition will both unmask the viral RNA to the innate immunity and limit the virus replication. Smallmolecule RNA MTase inhibitors (Sinefungin, 5'-methylthioadenosine (MTA), SAM or S-adenosyl-Lhomocysteine (SAH) have already been described but these SAM analogs show inadequate selectivity due to the high homology of SAM binding domain of the different RNA MTases. [7] To overcome this lack of selectivity, we propose to develop another approach with bisubstrate nucleosidic analogues as 2'-O-MTase inhibitors by mimicking the transition state of the 2'-O-methylation of the RNA cap structure (Scheme 1). [7] Scheme 1. The 2'-O-methyltransferase reaction on the cap structure of a mRNA with its transition state intermediate These analogues consist of a SAM analogue without the amino acid side chain, covalently bound to the 2'-OH of an adenosine unit via various sized linkers containing one or two sulfur atoms. In this bisubstrate approach, the SAM analogue has been designed to accommodate the SAM binding pocket of the 2'-O-MTase and the adenosine unit represents the 5'-end nucleoside (N1) of mRNA to fit in the RNA binding pocket (Figure 1). In the same way, several bisubstrates for diverse methyltransferases (DNA MTases, [8] catechol MTase, [9] protein MTases, [10] nicotinamide MTase [11] …) have been previously reported in the literature. Nevertheless, it is noteworthy some relevant examples of the use of bisubstrate nucleosidic analogues for the study of nucleic acids methyltransferases. Particularly, P. Arimondo developed transition state analogues of DNA methylation based on the coupling of cytosine analogues to adenosine to give 5methylcytosine-adenosine compounds. [12] Moreover, the first bisubstrates targeting RNA methyltransferases have been described in 1986 and one compound designed with the SAM moiety linked to the C6 of a guanine derivative demonstrated an inhibitory activity against vaccinia RNA N7guanine MTase for the N7-methylation of the 5'-cap structure. [13] More recently, in the context of deciphering the roles of N6m-A RNA modifications and consequently exploring the functions of N6-A RNA MTases, SAM-adenosine conjugates mimicking the transition state of methylation at N6 were synthesized by connecting a SAM analogue to the N6-position of an adenosine unit via alkyl and urea linkers. [14] The binding of these bisubstrate analogues for Ribosomal RNA large subunit MTase J (RlmJ) has been studied and they were shown to be useful as starting scaffolds for inhibitor design against m6A RNA MTases. [15] Beside these few examples, none other RNA MTases have been targeted by bisubstrate analogues.

In this work, we report on the synthesis of five S-adenosyl-5'-thioadenosine conjugates as bisubstrate analogues for the study of RNA 2'-O-methyltransferases. All these compounds were designed with a 5'-thioadenosine linked to the 2'-OH of an adenosine unit through alkyl linkers of various length (methyl or ethyl) and/or different oxidation degrees of the sulfur atom. Indeed, we first focused on the sulfidecontaining linkers (S-linkers) that represents the most fitting motives to mimic the SAM structure closely in comparison to other linkages with diverse heteroatoms in place of S. Moreover, S-linkers are attractive as they are stable and non-hydrolysable. This was previously shown with the synthesis of several 2'dialkyl S-linked dinucleosides which were incorporated into oligonucleotide analogues for the study of their hybridization properties in antisense purposes. [16] Furthermore, the corresponding sulfoxides and sulfones are of interest for chemists as they result from simple thioether oxidation and these groups are commonly found in nature as well as in the structure of some active drugs (Disulone ® , Modiodal ® ). The presence of sulfoxide and sulfone was also noted in aminoglycoside-Coenzyme A bisubstrates targeting aminoglycoside N-6'-acetyltransferase. [17] Recently, two SAM structural analogues, a sulfoxide and a sulfone derived from SAH have been synthesized as substrates for the study of their reductive cleavage by radical SAM enzymes. [18] In addition, it should be mentioned that a cytosine or a thymine dinucleoside 3',5'-bridged by a sulfone-containing linker has been described in the literature for the synthesis of stable sulfonyl-containing antisense oligonucleotides. [19] Inspired by this work, the S-linked dinucleoside 1 has been oxidized in sulfoxide (SO, 2) or sulfone (SO2, 3) since these sulfur functional groups might improve chemical stability and solubility in water of the bisubstrates (Figure 1). Likewise, the disulfide bridge represents an attractive functional group to design another S-linker. The disulfide bonds are widely found in natural biological systems and play a central role in protein stability, they are able to undergo disulfide-exchange reactions with thiols over a broad range of pH. Interestingly, some disulfide dinucleosides as nicotinamide adenine dinucleotide (NAD) mimics were reported to inhibit several NAD kinases. [20] In similar way, we linked two adenosines via an alkyl disulfide bond to yield the 2',5'-disulfanyl dinucleoside 5.

Results and Discussion

Synthesis of adenine dinucleoside 1 with a methylthioether linker. Two strategies have been tested to obtain the dinucleoside 1 with a methylthioether linker between both adenosines A1 and A2 (Figure 1). The first one has consisted in the coupling of 2'-O-acetylthiomethyl-N 6 -phenoxyacetyl adenosine previously described by our group [21] , bearing a pro-nucleophile site at 2'-position, and the commercially available 5'-O-tosyl adenosine or 5'-Cl-adenosine, both bearing an electrophile site at 5'-position. A basic medium (7M NH3/MeOH or BuNH2/THF) released a nucleophilic thiolate at 2'-position of A1 prone to react with A2. Nevertheless, the dinucleoside coupling was unsuccessful due to the too fast degradation of the 2'-thiohemiacetal species into adenosine. In the second strategy, the reactivity centers were reversed in the synthons A1 and A2 with a thioacetyl group as the pro-nucleophile site at 5'position of A2 and a 2'-chloromethyl group as electrophile in A1. The coupling was similarly achieved in basic medium to generate the nucleophilic thiolate in A2, capable to attack the chloromethyl group.

Following this strategy, we first synthesized the 2'-chloromethyl derivative 6 from the commercial 3',5'-O-tetraisopropyldisiloxane (TIPDS) N 6 -phenoxyacetyl adenosine via a 2'-O-methylthiomethyl derivative (Pummerer rearrangement) upon a described procedure (Scheme 2). [21] This compound 6 remains stable for 2 h at room temperature therefore requires a rapid utilization in the next coupling with a 5'-thiolate. In parallel, the introduction of the thioacetyl group at 5'-position of 2',3'isopropylideneadenosine was achieved by a Mitsonubu reaction with 98% yield to give compound 7 which was subsequently 2',3'-deprotected in acidic conditions affording 5'-thioacetyl adenosine 8. [22] Then, several basic conditions (7M NH3 / MeOH; nBuNH2 in THF; NaOMe / MeOH; KOH / MeOH) have been screened to give the thiolate derivatives of 7 or 8, and to compare the coupling efficiency with 6. It is worth mentioning that the 5'-thioacetyl adenosine 8 was totally converted into its thiolate derivative within short reaction times from 10 min with KOH / MeOH to 30 min with other basic conditions whereas the 5'-thiolate of 2',3'-protected adenosine 7 was only formed at 50% within 2 h with NH3 / MeOH or 16 h with NaOMe or KOH in MeOH. Consequently, the coupling was more efficient with 5'-thiolate from 8 than from 7. The 5',3'-TIPDS dinucleoside 10 was obtained with 61% yield from the coupling of potassium thiolate salt of 8 with the chloromethyl nucleoside 6 in a mixture dichloromethane / methanol after 2 h reaction at room temperature. The last step was the removal of the TIPDS group with a fluoride ions treatment for 2 h at 50°C to release the S-(2'-O-methyladenosyl) 5'thioadenosine 1 with 89% yield and high purity after purification by C18-reversed-phase silica gel chromatography.

S A O O O O A Pac O O O O Si Si O Cl 6 A O O O O Si Si O S A O O O S A O OH OH O A O O O O Si Si O S A O OH OH 5' 5' 7 8 N N N N NH 2 O O OH HO S N N N N NH 2 O OH OH 1 Et 3 N.3HF / THF A1 A2 A2 9 10 + K - S A O O O + K - S A O OH OH KOH / MeOH 0°C, 10 min KOH / MeOH 0°C, 10 min CH 2 Cl 2 / MeOH 2 h, r.t. 50°C, 2 h A1 A2 A Pac O O O O Si Si O SO 2 Cl 2 DCM 25°C, 2 h A Pac O OH O O Si Si O Ac 2O, AcOH DMSO 25°C, 24 h puis 50°C, 4 h 67% S S A O O O O 5' 7 A2 HCCOH, H 2 O 25°C, 44 h, 99% Scheme 2. Synthesis of S-(2'-O-methyladenosyl) 5'-thioadenosine 1. A = adenine. A Pac = N 6 - phenoxyacetyl adenine
Synthesis of adenine dinucleosides 2 and 3 with sulfoxide-or sulfone-containing linkers. To extend the series of thioether-linked dinucleoside, sulfoxide-or sulfone-containing linkers were evidently designed. The ease of preparation of oxidized sulfides and the potential increase of affinity for enzymes by allowing two extra H-bonds between the oxygen of S=O and these two hydrogen bonds prompted us to oxidize the sulfur atom of dinucleoside 1 into sulfoxide 2 or sulfone 3 derivatives (Scheme 3). Selective oxidation of the sulfide can be performed with several oxidizing agents such as mchloroperbenzoic acid (m-CPBA), sodium periodate, potassium hydrogen persulfate (oxone ® ).

However, m-CPBA was not soluble in the THF/MeOH/H2O solution mixture required for dissolution of the substrate 1 and sodium periodate led to the oxidizing cleavage of the cis-diol-containing adenosine as a side reaction. Finally, oxone ® was selected for its ease of use, stability, non-toxicity and solubility in the reaction mixture. The reaction with 3',5'-TIPDS dinucleoside 10 was complete after 3 h at 0°C after addition of three equivalents of oxidant in three portions. HPLC analysis of the crude material exhibited two peaks with a 68/32 ratio corresponding to the sulfoxide 11 and the sulfone 12, respectively. Dinucleosides 11 and 12 were isolated after purification with 30% and 21% yield, respectively. Then, 11 and 12 were deprotected with a fluoride treatment to give the sulfoxide dinucleoside 2 with 38% yield and the sulfone dinucleoside 3 with 32% yield after purification by C18 reversed-phase chromatography. HPLC analysis of 2 exhibits two peaks with 74/26 ratio corresponding to the two (R) and (S) diastereoisomers. No attempt was made to determine the absolute stereochemistry at the sulfur atom. The sulfoxide-linked dinucleoside 2 will be first evaluated as a diastereoisomeric mixture in the inhibition assays. Synthesis of adenine dinucleoside 4 with an ethylthioether linker. The dinucleoside 4 with a longer ethylthioether linker than the one of 1 was synthesized by coupling the 2'-O-modified adenosine 13 with an electrophile site at 2'-position and the 5'-thiolate derivative from 8. Thus, the 2'-O-(tosylethyl) adenosine 13 was prepared in four steps from adenosine following a described procedure for the three first steps (Scheme 4). [23] N The first step has consisted in introducing a methylester group preferentially at 2'-position whereas the 3'-OH and 5'-OH were unprotected. The reaction was conducted in the presence of NaH and the 2'-O-(methoxycarbonylmethyl) adenosine was the main compound isolated with satisfactory 42% yield. The 3'-and 5'-isomers were also formed at a lower extent. The next step was the masking of 3'-OH and 5'-OH by tert-butyldimethylsilyl (TBS) groups with 83% yield. The reduction of the ester function in the presence of LiAlH4 gave the 5',3'-O-TBS 2'-O-(2-hydroxyethyl) adenosine quantitatively. Unlike reported work described the alcohol activation with a mesylate group, [23] in our case the mesylate derivative was unstable and the coupling between both adenosines did not succeed. In contrast, the tosylate derivative 13 obtained with 86% yield was stable and was reacted with the 5'-potassium thiolate adenosine from 8 to give the 2',5'-ethylthioether-linked dinucleoside 14 (19%). [24] This low yield would have been improved by addition of 18-crown-6 ether to increase the reactivity of the thiolate nevertheless preliminary assays had shown that the separation by gel chromatography of 14 from 18-C-6 ether could not be achieved. Finally, an Et3N. 3HF treatment was applied to remove the TBS groups from 14 to afford the dinucleoside 4 in 44% yield after purification.

Synthesis of adenine dinucleoside with a disulfide linker 5. To generate the disulfide bridge between two adenosines, a thiol-disulfide exchange reaction has been intended between a thiolate derivative at 2'-position of A1 and a 5'-disulfanylnitropyridine adenosine A2 in basic conditions with release of 2thio-5-nitropyridine. In a first attempt, the coupling was performed between one equivalent of 2'-Omethylthioacetyl N 6 -Pac-adenosine 16 [21] (at 58 mM concentration) obtained from the chloromethyl derivative 6, and an excess (1.2 eq) of 5'-disulfanylnitropyridine adenosine 18 prepared from 5'acetylthioadenosine 8 (Scheme 5). [22a] The reaction was carried out at 0°C in the presence of 7M ammonia in methanol and after 15 min, three peaks were noticed in the reverse-phase HPLC chromatogram with a 35/45/20 ratio (Figure S14). These three major peaks have been assigned to three dinucleosides with the linker attached at different positions in the adenosines (Scheme S1). The main peak corresponds to the desired dinucleoside 5 with a 2',5'-disulfide linker. The other peaks were assigned to the symmetrical dinucleosides with 2',2'-or 5',5'-disulfide-linker resulting from the coupling of synthon 16 or synthon 8 with themselves, respectively. Next, the conditions of the coupling reaction have been optimized to obtain the dinucleoside 5 with an improved yield. Either a higher (91 mM) or a lower (23 mM) concentration for 16 were tested and it was shown that a diluted solution of 16 at 23 mM was favorable to a high proportion (73%) of 5 in the crude mixture. In contrast, increasing the amount of 18 up to 3 equivalents rather promoted the formation of the 5',5'-disulfide link dinucleoside. However, even though in the optimized conditions, the dinucleoside 5 was the major compound in the mixture, we were not able to isolate 5 with high purity and in sufficient amount due to a delicate separation of the three dinucleosides which may be explained by the similarity of their structure. To improve this separation, we introduced the lipophilic dimethoxytrityl (DMTr) group at 5'-position of 16 to give 5'-O-DMTr 2'-O-SAc adenosine 17. This nucleoside 17 used at 23 mM concentration was reacted with 1.8 equivalent of 18 in 7M NH3/MeOH to give the three dinucleosides as previously. However, the ratio was different and the benefit of the lipophilicity of DMTr group was crucial for the separation since the dinucleoside with 2',2'-disulfide linker is DMTr-protected at both 5'-positions (Rt 15.66), the dinucleoside with 5',5'-disulfide linker is the most polar with both 5'-OH (Rt 4.29) and the dinucleoside 19 has only one DMTr group at 5'position of A1 (Rt 10.68) (Figure 2). It is noteworthy that to get rid of the remaining excess of 5'nitropyridinyl disulfide adenosine 18 by reverse-phase chromatography the use of a 50 mM triethylammonium acetate buffer pH7 instead of water as eluent was recommended to avoid contamination of all the fractions by the nitropyridine derivative. Indeed, the pyridine moiety exists under protonated/deprotonated equilibrium in water and 18 spreads out all over the column chromatography. After purification, HPLC analysis showed that the isolated compound 19 was 75% pure and 5'-O-DMTr adenosine was characterized as the main contaminant resulting from the total deprotection of 17 in basic medium during the coupling reaction. Finally, dinucleoside 19 was engaged in the last deprotection step to remove the DMTr group with 80% acetic acid in water to give the disulfanyl-linked dinucleoside 5 that was isolated with high purity after purification by C18-chromatography in 15% yield over 2 steps (coupling and deprotection).

Conclusion

In this paper, we report the synthesis of five adenine dinucleosides with S-linkers as bisubstrate SAM analogues for 2'-O-methyltransferases that catalyze the 2'-O-methylation of the 5' cap of viral mRNA or at internal positions within RNA sequence. These bisubstrates contain a 5'-thioadenosine mimicking the SAM adenosine and attached to the 2'-position of another adenosine through an alkyl S-linker, mimicking the 5'-end of RNA substrate. Such analogues were designed as mimics of the transition state of the 2'-O-methylation of RNA with both partners of the reaction. The evaluation of the bisubstrate analogues as inhibitors of various 2'-O-MTases of emerging viruses (Zika, Dengue, Ebola, SARS, MERS) is currently in progress. Moreover, the S-linked adenine dinucleosides are valuable tools to start structural studies on viral MTases before further studies with short RNAs incorporating the bisubstrate molecules at 5'-end or at internal positions. Rationally, the prospects of this work stand the synthesis of new bisubstrates with other heteroatom-containing linkers therefore the synthesis of analogues with amine-type linkages (OCH2CH2NR2) is ongoing.

Experimental section

General Methods. DIEA was distilled over calcium hydride. All dry solvents and reagents were purchased from commercial suppliers and were used without further purification. Thinlayer chromatography (TLC) analyses were carried out on silica plate 60 F254. Purifications by column chromatography were performed using Biotage Isolera 1 system with Column Flash Pure from Buchi. NMR experiments were recorded on Bruker 400, 500 or 600 spectrometers at 20°C. HRMS analyses were obtained with electrospray ionization (ESI) in positive mode on a Q-TOF Micromass spectrometer. Analytical HPLC was performed on a UHPLC Thermoscientific Ultimate 3000 system equipped with a LPG-3400RS pump, a DAD 3000 detector and an WPS-3000TBRS Autosampler, Column Oven TCC-3000SD. Dinucleosides 1-5 were analyzed by RP-HPLC (Macherey Nagel Nucleodur C18 3 µm, 4.6 x 75 mm). The following HPLC solvent systems were used: 1% CH3CN in 12.5 mM TEAAc (buffer A), 80% CH3CN in 12.5 mM TEEAc (buffer B). Flow rate was 1 mL/min. UV detection was performed at 260 nm. Lyophilized compounds 1-5 were stored at -20°C for several months without any degradation.

S-(3',5'-(tetraisopropyldisiloxane-1,3-diyl)-2'-O-methyladenosyl)-5'-thioadenosine 10

To a solution of 3',5'-O-tetraisopropyldisiloxane 2'-O-methylthiomethyl N 6 -phenoxyacetyl adenosine [21] (0.50 g, 0.71 mmol, 1.00 eq) in anhydrous CH2Cl2 (2.50 mL) was added 1.0 M sulfuryl chloride (SO2Cl2) in dichloromethane (1.10 mL, 1.06 mmol, 1.50 eq) diluted in anhydrous CH2Cl2 (2.50 mL) in a dropping funnel. The reaction mixture was stirred for 2 h at room temperature under argon.

The solvent was removed under vacuum and the crude mixture of 6 was diluted in anhydrous CH2Cl2 (3 mL). In parallel, 5'-thioacetyl adenosine 8 (0.39 mg, 1.20 mmol, 1.70 eq) suspended in MeOH (2 mL) in a round flask was treated with a solution of potassium hydroxide KOH (0.14 g, 2.49 mmol, 3.50 eq) in MeOH (4 mL). The reaction mixture was stirred for 30 min at 0°C under argon. The chloromethyl derivative 6 was directly added to the solution containing the potassium thiolate of 8 and the reaction mixture was stirred for 2 h at room temperature under argon. The solvents were removed under vacuum, and the resulting residue was purified by silica gel chromatography (dry-loading) with a 0-15% MeOH linear gradient in CH2Cl2 to give the dinucleoside 10 as a white solid (0.35 g, 0.435 mmol, 61%). Rf = 0.66 (MeOH/CH2Cl2 15:85);

S-(2'-O-methyladenosyl)-5'-thioadenosine 1

To a solution of 10 (0.10 g, 0.12 mmol, 1.00 eq) in anhydrous THF (6 mL) was added 1M Et3N-3HF solution in THF (60 µL, 3.72 mmol, 3.00 eq). After stirring for 2 h at 50°C, the reaction mixture was treated with 2 M triethylammonium acetate buffer (pH 7). The solvents were removed under vacuum then water (10 mL) and CH2Cl2 (10 mL) were added. The aqueous layer was extracted three times with CH2Cl2 and once with Et2O and was evaporated under vacuum. The resulting residue was purified by chromatography on reversed-phase silica gel column C18 (4 g, 40 µm) with a 0-25% acetonitrile linear gradient in TEAAc buffer 50 mM, pH 7. The fractions containing the pure compound were pooled, concentrated and lyophilized to give 1 as a white powder (62 mg, 110 µmol, 89%) with 99% purity determined by HPLC analysis at 260 nm. Compound 10 (0.93 mg, 1.16 mmol, 1.00 eq) was suspended in a mixture of THF (7 mL), MeOH (7 mL) and water (1.80 mL). After sonication, NaHCO3 (0.378 g, 4.52 mmol, 3.90 eq) and oxone ® (0.177 g, 1.16 mmol, 1.00 eq) were added. After 1 h stirring at room temperature under argon, an additional equivalent of oxone ® was added and the reaction mixture was stirred for an additional hour. Another additional equivalent of oxone ® was added and the reaction mixture was stirred again for an additional hour. The solution was then quenched with saturated aqueous NaHCO3 (10 mL). The aqueous layer was extracted with AcOEt (6 x 50 mL) and the combined organic extracts were washed with saturated aqueous NaCl (3 x 50 mL), dried over Na2SO4 and concentrated under vacuum. The residue was purified via chromatography (dry-loading). Compound 12 was first eluted with 14% MeOH in AcOEt and isolated as a white solid (0.20 g, 0.239 mmol, 21%). Compound 11 was isolated as a white solid (0.288 g, 0.351 mmol, 30%) after elution with 18% MeOH in AcOEt.

S-(3',5'-(tetraisopropyldisiloxane

(11) Rf = 0.30 (MeOH/CH2Cl2 15:85). 1 H-NMR (600 MHz, DMSO-d6) δ = 8.33 ;8.31 ;8.19 ;8.18 ;8.14 ;8.13 ;8.04 ;8.04 ;7.33 ;7.28 ;5.90 ;5.59 ;5.56 ;5.45 ;5.09 ;5.06 ;4.90 ;4.82 ;3.92 ;152.57 ;152.46 ;149.21 ;149.17 ;148.49 ;148.47 ;140.19 ;139.85 ;139.79 ;119.34 ;119.32 ;119.28 ;88.32 ;88.01 ;87.68 ;87.45 ;86.45 ;85.88 ;83.57 ;80.35 ;80.28 ;79.18 ;78.97 ;78.75 ;77.33 ;76.62 ;73.31 ;73.23 ;72.77 ;72.74 ;69.97 ;69.91 ;60.11 ;51.22 ;49.25 ;

S-(2'-O-methyladenosyl)-5'-sulfoxide adenosine 2

To a solution of 11 (0.10 g, 0.12 mmol, 1.00 eq) in anhydrous THF (6 mL) was added Et3N-3HF (60 µL, 3.66 mmol, 3.00 eq). After 2 h stirring at 25°C, the reaction mixture was treated with 2 M triethylammonium acetate buffer (pH 7). The solvents were removed under vacuum then water (10 mL) and CH2Cl2 (10 mL) were added. The aqueous layer was extracted three times with CH2Cl2 and once with Et2O and was evaporated under vacuum. The resulting residue was purified by chromatography on a C18 reversed-phase silica gel column (4 g, 40 µm) with a 0-25% acetonitrile linear gradient in 50 mM TEAAc buffer, pH 7. The fractions containing the pure compound were pooled, concentrated and lyophilized to give 2 as a white powder (27 mg, 47 µmol, 39%) with 98% purity determined by HPLC analysis at 260 nm. 1 H-NMR (600 MHz, DMSO-d6) δ = 8.35 ;8.33 ;8.32 ;8.14 ;8.13 ;7.34 ;7.30 ;6.10 ;6.07 ;5.90 ;5.59 ;5.57 ;5.54 ;5.47 ;5.44 ;5.39 ;4.85 ;4.82 ;4.72 ;4.68 ;4.61 ;4.40 ;4.36 ;4.25 ;3.99 ;3.66 ;3.30 ;3.18 ;156.56 ;153.13 ;153.03 ;153.00 ;149.77 ;149.67 ;149.45 ;140.57 ;140.35 ;140.18 ;140.13 ;119.74 ;119.68 ;88.55 ;88.32 ;86.53 ;86.36 ;86.29 ;85.45 ;83.78 ;83.39 ;78.47 ;77.92 ;73.61 ;73.45 ;73.24 ;73.01 ;69.53 ;69.42 ;61.73 ;51.29 ;50.44. HRMS (ESI+): m/z calcd for C21H27N10O8S: 579.17286, found: 579.17224.

S-(2'-O-methyladenosyl)-5'-sulfone adenosine 3

To a solution of 12 (0.10 g, 0.12 mmol, 1.00 eq) in anhydrous THF (6 mL) was added Et3N-3HF (60 µL, 3.59 mmol, 3.00 eq). After 2 h stirring at 25°C, the reaction mixture was treated with 2 M triethylammonium acetate buffer (pH 7). The solvents were removed under vacuum then water (10 mL) and CH2Cl2 (10 mL) were added. The aqueous layer was extracted three times with CH2Cl2 and once with Et2O and was evaporated under vacuum. The resulting residue was purified by chromatography on a C18 reversed-phase silica gel column (4 g, 40 µm) with a 0-25% acetonitrile linear gradient in 50 mM TEAAc buffer, pH 7. The fractions containing the pure compound were pooled, concentrated and lyophilized to give 3 as a white powder (23 mg, 39 µmol, 32%) with 98% purity determined by HPLC analysis at 260 nm.

3',5'-bis-O-(tert-butyldimethylsilyl)-2'-O-(2-toluene-sulfonylethyl) adenosine 13

To a solution of 3',5'-bis-O-(tert-butyldimethylsilyl)-2'-O-(hydroxyethyl) adenosine [23] (1.23 g, 2.34 mmol, 1.00 eq) in anhydrous CH2Cl2 (7 mL) was added successively 4-dimethylaminopyridine (29 mg, 0.23 mmol, 0.10 eq), Et3N (0.70 mL, 4.92 mmol, 2.10 eq) and 4-toluenesulfonyl chloride (0.893 g, 4.68 mmol, 2.00 eq). After stirring for 4 hours at 0°C under argon, the solution was diluted with CH2Cl2 (30 mL) and washed with saturated aqueous NaHCO3. The aqueous layer was extracted with CH2Cl2 (3 x 40 mL) and the combined organic extracts were washed with saturated aqueous NaCl (60 mL), dried over Na2SO4 and concentrated under vacuum. The residue was purified by chromatography with a linear gradient 0-10% MeOH in CH2Cl2 yielding to 13 as a white solid (1.40 g, 2.02 mmol, 86%). Rf 0.71 (MeOH/CH2Cl2 5:95).

Figure 1 .

 1 Figure 1. Rationale for designing a library of diverse bisubstrate SAM analogues for RNA 2'-O-methyltransferases

Scheme 3 .

 3 Scheme 3. Synthesis of S-(2'-O-methyladenosyl) 5'-sulfoxide adenosine 2 and S-(2'-O-methyladenosyl) 5'-sulfone adenosine 3

Scheme 4 .

 4 Scheme 4. Synthesis of S-(2'-O-ethyladenosyl)-5'-thioadenosine 4

  Scheme 5. Synthesis of S-(2'-O-methylthioadenosinyl)-5'-thioadenosine 5

Figure 2 .

 2 Figure 2. Reverse-phase HPLC profile of the crude mixture after the coupling reaction between 17 (23 mM) and 18 (1.80 eq)

  1 H-NMR (400 MHz, DMSO-d6) δ = 8.30 (s, 1H, A2H8) ; 8.16 (s, 1H, A1H8) ; 8.13 (s, 1H, A2H2) ; 8.06 (s, 1H, A1H2) ; 7.33 (s, 2H, A1NH2) ; 7.26 (s, 2H, A2NH2) ; 5.99 (s, 1H, A1H1') ; 5.85 (d, J = 5.7 Hz, 1H, A2H1') ; 5.50 (d, J = 6.0 Hz, 1H, A2OH2') ; 5.29 (d, J = 5.1 Hz, 1H, A2OH3') ; 5.04 (m, 1H, A1H3') ; 5.03 (d, J = 11.7 Hz, 1H, OCH2S) ; 4.93 (d, J = 11.6 Hz, 1H, OCH2S) ; 4.81 (d, J = 5.1 Hz, 1H, A1H2') ; 4.77 (q, J = 5.7 Hz, J = 11.1 Hz, 1H, A2H2') ; 4.14 (q, J = 4.9 Hz, J = 9.0 Hz, 1H, A2H3') ; 4.02-4.07 (m, 2H, A2H4', A1H5 x ) ; 3.90-3.99 (m, 2H, A1H4', A1H5 x ); 2.87 -3.06 (m, 2H, A2H5', A2H5'') ; 0.96 -1.03 (m, 28H, HTIPDS).

  1 H-NMR (600 MHz, DMSO-d6) δ = 8.35 (s, 1H, A2H8) ; 8.31 (s, 1H, A1H8) ; 8.16 (s, 1H, A2H2) ; 8.14 (s, 1H, A1H2) ; 7.42 -7.23 (m, 4H, A1NH2, A2NH2) ; 6.08 (d, J = 4.9 Hz, 1H, A1H1') ; 5.91 (d, J = 5.3 Hz, 1H, A2H1') ; 5.62 (d, J = 5.8 Hz, 1H, A2OH2') ; 5.49 (d, J = 5.2 Hz, 1H, A2OH3') ; 5.38 (d, J = 5.3 Hz, 1H, A1OH3') ; 5.35 (dd, J = 6.5, 4.7 Hz, 1H, A1OH5') ; 4.90 -4.83 (m, 1H, A1H2') ; 4.75 (d, J = 12.6 Hz, 1H, OCH2SO2) ; 4.71 -4.63 (m, 2H, OCH2SO2, A2H2') ; 4.46 -4.40 (m, 1H, A1H3') ; 4.28 -4.23 (m, 1H, A2H4') ; 4.21 -4.16 (m, 1H, A2H3') ; 3.95 -3.89 (m, 1H, A1H4') ; 3.73 -3.68 (m, 1H, A2H5') ; 3.68 -3.65 (m, 1H, A1H5') ; 3.56 -3.49 (m, 1H, A1H5'') ; 3.46 -3.39 (m, 1H, A2H5'').

  1H, A1OH5') ; 5.28 (d, J = 5.3 Hz, 1H, A1OH3') ; 5.24 (d, J = 5.3 Hz, 1H, A2OH3') ; 4.81 (d, J = 11.7 Hz, 1H, OCH2S) ; 4.76 (t, J = 5.3 Hz, 1H, A1H2') ; 4.69 (d, J = 11.7 Hz, 1H, OCH2S) ; 4.67 (q, J = 5.8 Hz, J = 11.2 Hz, 1H, A2H2') ; 4.35 (m, 1H, A1H3') ; 4.03 (m, 1H, A2H3') ; 3.98 (q, J = 3.4 Hz, J = 6.9

	Hz, 1H, A1H4') ; 3.94 (m, 1H, A2H4') ; 3.69 & 3.56 (2m, 2H, A1H5' & A1H5'') ; 2.73 (dd, J = 1.4 Hz, J =
	6.7 Hz, 2H, A2H5'& A2H5''). 13 C-NMR (150 MHz, DMSO-d6) δ = 156.14 & 156.05 (A1C6 & A2C6) ;
	152.7 & 152.5 (A1C2 & A2C2) ; 149.4 (A2C4) ; 148.9 (A1C4) ; 139.7 (A2C8) ; 139.6 (A1C8) ; 119.3 (A2C5)
	; 119.1 (A1C5) ; 87.3 (A2C1') ; 86.3 (A1C4') ; 86.1 (A1C1') ; 83.6 (A2C4') ; 78.2 (A1C2') ; 72.7 (OCH2S) ;
	72.6 (A2C2') ; 72.5 (A2C3') ; 68.9 (A1C3') ; 61.4 (A1C5') ; 32.2 (A2C5'). HRMS (ESI+): m/z calcd for
	C21H27N10O7S [M+H] + : 563.1785, found: 563.1786.

1

H-NMR (600 MHz, DMSO-d6) δ = 8.35 (s, 1H, A1H8) ; 8.29 (s, 1H, A2H8) ; 8.13 & 8.14 (2s, 2H, A1H2 & A2H2) ; 7.33 (s, 2H, A2NH2) ; 7.28 (s, 2H, A1NH2) ; 6.03 (d, J = 5.9 Hz, 1H, A1H1') ; 5.83 (d, J = 5.9 Hz, 1H, A2H1') ; 5.47 (d, J = 6.

1 Hz, 1H, A2OH2') ; 5.45 (m,

Page No. -Page No. Synthesis of adenine dinucleosides 2',5'-bridged by sulfur-containing linkers as bisubstrate SAM analogues for viral RNA 2'-O-methyltransferases

  1 H-NMR (500 MHz, CDCl3) δ = 8.30 (s, 1H, H2) ; 8.15 (s, 1H, H8) ; 7.75 (d, J = 8.3 Hz, 2H, Hortho Ts) ; 7.29 (d, J = 8.0 Hz, 2H, Hmeta Ts) ; 6.02 (d, J = 3.4 Hz, 1H, H1') ; 5.74 (s, 2H, NH2) ; Rostom Ahmed-Belkacem, 1 Priscila Sutto Ortiz, 2 Etienne Decroly, 2 Jean-Jacques Vasseur 1 and Françoise Debart1,*

	FULL PAPER
	A nucleosidic bisubstrate approach	RNA methyltransferases
	is developed to target 2'-O-RNA
	methyltransferases that catalyse
	the methylation of the 2'-OH of the
	cap nucleoside N1 or internal
	adenosines of RNA substrate using
	S-adenosyl methionine as the
	methyl donor. Bisubstrate
	analogues have been designed as
	a mimic of the transition state of the
	2'-O-methylation on RNA with two
	adenosines connected by various
	sulphur atom-containing linkers.

Acknowledgments

Rostom Ahmed-Belkacem thanks the University of Montpellier for financial support.

Keywords

Bisubstrate; RNA 2'-O-methylation; transition states; viral RNA methyltransferases; Nucleosides

S-(2'-O-ethyladenosyl)-5'-thioadenosine 4

To a solution of 14 (63 mg, 0.078 mmol, 1.00 eq) in anhydrous THF (3.7 mL) was added 1M Et3N-3HF solution in THF (50 µL, 0.31 mmol, 4.00 eq). After stirring for 3 h at 50°C, the reaction mixture was treated with 2M triethylammonium acetate buffer (pH 7). The solvents were removed under vacuum before water (10 mL) and CH2Cl2 (10 mL) were added. The aqueous layer was extracted three times with CH2Cl2 and once with Et2O. The solvent was removed under vacuum and the resulting paste was purified by chromatography on a reversed-phase C18 silica gel column (4 g, 40 µm) with a 0-25% linear gradient of acetonitrile in 50 mM TEAAc buffer, pH 7 followed by concentration and lyophilization of pure fractions to give 4 as a white powder (20 mg, 0.035 mmol, 44%) with 99% purity determined by HPLC analysis at 260 nm. 1 H-NMR (600 MHz, DMSO-d6) δ = 8.37 (s, 1H, A1H8) ; 8.33 (s, 1H, A2H8) ; 8.14 (2s, 2H, A1H2 & A2H2) ; 7.34 & 7.28 (2 br s, 4H, A1NH2 & A2NH2); 6.00 (d, J = 5.9 Hz, 1H, A1H1') ; 5.87 (d, J = 5.7 Hz, 1H, A2H1') ; 5.49 (d, J = 6.1 Hz, 1H, A2OH2') ; 5.38 (dd, J = 6.9, 4.8 Hz, 1H, A1OH5') ; 5.29 (d, J = 5.1 Hz, 1H, A2OH3') ; 5.17 (d, J = 5.2 Hz, 1H, A1OH3') ; 4.71 (q, J = 5. 6 Hz, 1H, A2H2') ; 4.50 (m, 1H, A1H2') ; 4.32 (m, 1H, A1H3') ; 4.10 (m, 1H, A2H3') ; 3.98 (m, 2H, A1H4' & A2H4') ; 3.72 -3.65, 3.55 -3.52 (m, 4H, A1H5', A1H5'', 2'OCH2) ; 2.90 -2.79 (m, 2H, A2H5', A2H5'') ; 2.65 (m, 2H, CH2S). 13

S-(5'-O-(4,4'-dimethoxytrityl-2'-O-methylthioadenosyl)-5'-thioadenosine 19

To a solution of 17 [21] (202 mg, 0.26 mmol, 1.00 eq) in MeOH (3.5 mL) was added 18 [22a] (200 mg, 0.46 mmol, 1.80 eq) and an ammonia solution (7 M in MeOH) (7.5 mL). After stirring for 20 min at -10°C under argon, the solvents were removed and the resulting residue was purified by chromatography (dryload) on reversed-phase silica gel column C18 with a 20-70% linear gradient of acetonitrile in 50 mM TEAAc buffer, pH 7. The fractions containing 19 with more than 75% purity were pooled and concentrated to dryness. Traces of TEAAc salts were removed by several coevaporations with water and acetonitrile to give compound 19 (92 mg, 0.102 mmol, 40% corrected yield) with 75% purity determined by HPLC analysis at 260 nm. The main contaminant was the 5'-O-DMTr adenosine. Full characterization of 19 was performed with a 99% pure fraction isolated after purification.

S-(2'-O-methylthioadenosyl)-5'-thioadenosine 5

The mixture of 19 and the by-product 5'-O-DMTr adenosine was treated with a solution of 80% acetic acid in water (8.76 mL) and stirred for 15 min at room temperature. The mixture solution was washed with CHCl3 (10 x 5 mL) then Et2O (1 x 10 mL). The solvent was removed under vacuum and the resulting residue was purified by chromatography on a reversed-phase silica gel column C18 (4 g, 40 µm) with a 0-25% linear gradient of acetonitrile in TEAAc buffer 50 mM, pH 7. The fractions containing the pure compound was pooled, concentrated and lyophilized to give 5 as a white powder (23 mg, 38.6 µmol, 15% over two steps) with 99% purity determined by HPLC analysis at 260 nm. 1 H-NMR (400 MHz, 1,4-Dioxane-d8) δ = 8.19 (s, 1H, A1H2) ; 8.18 (s, 1H, A2H2) ; 7.98 (s, 1H, A1H8) ; 7.97 (s, 1H, A2H8) ; 6.62 (br s, 2H, NH2) ; 6.50 (br s, 2H, NH2) ; 6.00-5.95 (m, 2H, A1H1', A1OH5') ; 5.86 (d, J = 4.3 Hz, A2H1') ; 4.99 (m, 1H, A1H2') ; 4.82 (s, 2H, OCH2S) ; 4.80 (m, 2H, A2H2', A2OH2') ; 4.44 (m, 2H, A2OH3', A2H3') ; 4.30 (m, 1H, A2H3') ; 4.12 (m, 2H, A1H4', A2H4') ; 3.99 (d, J = 4.2 Hz, A1OH3') ; 3.84-3.59 (m, 2H, A1H5', A1H5'') ; 3.00 (dd, J = 1.7 Hz, J = 5.9 Hz, 2H, A2H5', A2H5'').