Timothé Vucko

Nicolas Pétry

François Dehez

Alexandrine Lambert

Antonio Monari

Cécile Lakomy

Patrick Lacolley

Véronique Regnault

Charlotte Collet

Gilles Karcher

Nadia Pellegrini- Moïse

Sandrine Lamandé-Langle
email: sandrine.langle@univ-lorraine.fr

C-glyco"RGD" as αIIbβ3 and αvβ integrin ligands for imaging applications: synthesis, in vitro evaluation and molecular modeling

Keywords:

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

Introduction

Angiogenesis is a fundamental process involved in tumor growth, malignant transformation of tumors and metastasis. 1,[START_REF] Ribatti | History of research on angiogenesis[END_REF]3 Integrins are heterodimeric cell-adhesion proteins involved in angiogenesis signaling pathways and overexpressed in many angiogenic processes. These receptors mediate the attachment of cells to the extracellular matrix and are crucial for cell-to-cell interactions. [4][5][6] Integrins are composed of two non-covalently associated subunits: an α domain and a β domain, whose combination determines signaling properties and ligand binding affinity. 4,6,7 During angiogenesis, αv integrins are overexpressed on the surface of endothelial cells in angiogenic vessels to promote their growth and survival. The most extensively studied integrin is the αvβ3 integrin, which represents a highly specific biomarker, in particular to monitor tumor progression. [8][START_REF] Demircioglu | αvβ3 Integrin and tumour blood vessels-learning from the past to shape the future[END_REF][START_REF] Nieberler | Exploring the Role of RGD-Recognizing Integrins in Cancer[END_REF] The αIIbβ3 integrin lying across the plasma membrane of human platelets is also widely studied. Its physiological function is intimately linked to the process of platelet activation and thrombosis. 6,[START_REF] Fong | Directly Activating the Integrin αIIbβ3 Initiates Outside-In Signaling by Causing αIIbβ3 Clustering[END_REF][START_REF] Deckmyn | Inhibitors of the Interactions Between Collagen and Its Receptors on Platelets[END_REF] Therefore, ---instance, c(RGDfK) and c(RGDfC) cyclopeptides are often used as both lysine and cysteine lateral chains are efficient anchors while such modifications do not generally significantly affect the binding affinity of the native peptide. [START_REF] Kapp | A Comprehensive Evaluation of the Activity and Selectivity Profile of Ligands for RGD-binding Integrins[END_REF] The inherent physico-chemical properties of peptides impair their biodistribution, [START_REF] Nguyen | Peptide Chemistry and Drug De-sign[END_REF] limiting their direct use in the cellular context. The introduction of a carbohydrate moiety on bioactive peptides has been shown to reduce significantly their lipophilicity and consequently enhancing their bioavailability, pharmacokinetics, and in vivo clearance properties, which is of great interest for therapeutic and diagnostic applications. [START_REF] Moradi | Glycosylation, an effective synthetic strategy to improve the bioavailability of therapeutic peptides[END_REF][START_REF] Haubner | Galacto-RGD: synthesis, radiolabeling, metabolic stability, and radiation dose estimates[END_REF] Moreover, it has been demonstrated that the inclusion of a carbohydrate moiety enhances the capacity of the conjugate peptide to form intermolecular interactions, inducing enriched functionalities such as the ability to modulate peptide structure and cell-cell recognition in multicellular systems. [START_REF] Pratta | Synthetic glycopeptides and glycoproteins as tools for biology[END_REF][START_REF] Grogan | Homogeneous Glycopeptides and Glycoproteins for Biological Investigation[END_REF] Notably, the glycosylation of RGD containing peptides has proven useful in particular to target tumor cells and for imaging of tumor angiogenesis. [START_REF] Haubner | Galacto-RGD: synthesis, radiolabeling, metabolic stability, and radiation dose estimates[END_REF][START_REF] Chapleur | Toward imaging glycotools by click coupling in Click Chem-istry in Glycoscience: New Developments and strategies[END_REF][START_REF] Maschauer | Fglyco-RGD peptides for PET imaging of integrin expression: efficient radiosynthesis by click chemistry and modulation of biodistribution by glycosylation[END_REF][START_REF] Gaertner | Radiolabelled RGD peptides for imaging and therapy[END_REF][START_REF] Haubner | Noninvasive Imaging of avb3 Integrin Expression Using 18F-labeled RGD-containing Glycopeptide and Positron Emission Tomography[END_REF][START_REF] Beer | PET Imaging of αvβ3 Expression in Cancer Patients in Methods in Molecular Biology[END_REF][31][START_REF] Kuijpers | Preparation and evaluation of glycosylated arginine-glycineasparate (RGD) derivatives for integrin targeting[END_REF][START_REF] Maschauer | Labeling and Glycosylation of Peptides Using Click Chemistry: A General Approach to 18F-Glycopeptides as Effective Imaging Probes for Positron Emission Tomography[END_REF] The applicability of cyclic RGD-glycoconjugates in PET imaging has been recently investigated. Prante and coworkers synthetized glyco"RGD" by forming either a C-N or a C-S bond directly on the pseudo-anomeric center. [START_REF] Maschauer | Fglyco-RGD peptides for PET imaging of integrin expression: efficient radiosynthesis by click chemistry and modulation of biodistribution by glycosylation[END_REF]31,[START_REF] Maschauer | Labeling and Glycosylation of Peptides Using Click Chemistry: A General Approach to 18F-Glycopeptides as Effective Imaging Probes for Positron Emission Tomography[END_REF] Haubner's group developed c(RGDfK) conjugated on position 6 of a sugar amino acid. [START_REF] Haubner | Galacto-RGD: synthesis, radiolabeling, metabolic stability, and radiation dose estimates[END_REF][START_REF] Gaertner | Radiolabelled RGD peptides for imaging and therapy[END_REF][START_REF] Haubner | Noninvasive Imaging of avb3 Integrin Expression Using 18F-labeled RGD-containing Glycopeptide and Positron Emission Tomography[END_REF] We prepared glyco"RGD" where an O-glycosidic moiety was grafted on the RGD peptide by means of a spacer arm. 19,[START_REF] Collet | Development of 6-[18F]fluoro carbohydrate-based pros-thetic groups and their conjugation to peptides via click chemis-try[END_REF][START_REF] Vala | Neoglycopeptides through direct functionalization of cysteine[END_REF][START_REF] Chapleur | Pro-cess for the preparation of 18F-radiolabeled triazolyl-linked glycopeptides as radiotracers via "click" chemistry[END_REF] In the perspective of developing more performant and selective diagnostic tools for PET imaging, the use of RGD derivatives conjugated with a C-glycosidic moiety appears as an appealing strategy. Some O-glycoside RGD derivatives previously developed 19,[START_REF] Collet | Development of 6-[18F]fluoro carbohydrate-based pros-thetic groups and their conjugation to peptides via click chemis-try[END_REF] are recognized by glycosidases, eventually hydrolyzing their O-glycosidic bond and limiting thereby their stability in vivo. Interestingly, C-glycosyl compounds, which is a subject of interest in our group for several years, [START_REF] Pellegrini-Moïse | Exo-glycals as useful tools for anomeric functionalization of sug-ars[END_REF][START_REF] Richard | Base-and radical-mediated regio-and stereoselective additions of thiols, thio-sugars and thiol-containing peptides on trisubstituted activated exo-glycals[END_REF][START_REF] Tran Thien | Tetrasubstituted C-Glycosylidenes and C-Glycosyl Compounds from Di-and Monobromo-Substituted exo-Glycals[END_REF][START_REF] Richard | Carbohydrate-based peptidomimetics targeting neuropilin-1: Synthesis, molecular docking study and in vitro biological activities[END_REF] are resistant to acidic and enzymatic hydrolysis thanks to their robust C-C link in anomeric position, conferring them a high in vivo stability. [START_REF] Leclerc | Fluoro-C-glycosides and fluoro-carbasugars, hydrolytically stable and synthetically challenging glycomimetics[END_REF][START_REF] Beau | C-Glycosyl analogs of oligosaccharides and glycosyl amino acids in Glycoscience[END_REF] Copper-catalyzed azide-alkyne cycloaddition (CuAAC) is an ideal tool for conjugating a sugar moiety to RGD peptides. [START_REF] Collet | Development of 6-[18F]fluoro carbohydrate-based pros-thetic groups and their conjugation to peptides via click chemis-try[END_REF][START_REF] Li | Development and Applications of the Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) as a Bioorthogonal Reaction[END_REF][START_REF] Meyer | Click Chemistry and Radiochemistry: The First 10 Years[END_REF] It produces a stable triazolyl group which could further increase the peptide binding affinity for integrins. [START_REF] Kuijpers | Preparation and evaluation of glycosylated arginine-glycineasparate (RGD) derivatives for integrin targeting[END_REF][START_REF] Bock | 1,2,3-Triazoles as peptide bond isosteres: synthesis and biological evaluation of cyclotetrapeptide mimics[END_REF] In this contribution, we designed a new type of glyco"RGD" integrating a stable and flexible C-glycosidic bond (Fig 1). A 19/18 F-fluorine atom was introduced on the sugar moiety, allowing the future use of these glycosidic compounds as diagnostic tools for PET imaging. Cyclic and linear glyco"RGD" derivatives were synthetized and their ability to target integrins was evaluated in vitro. The experimental results were supplemented by moleculardynamics simulations, offering an atomistic description of the binding patterns of C-glyco"RGD" and αvβ3 integrin.

Results

Synthesis of C-glyco"RGD".

The synthesis of new C-glyco"RGD" derivatives by CuAAC requires that one partner bears an azido group and the other a propargyl function. Based on our expertise in the synthesis of Oglycopeptides by CuAAC, 19,[START_REF] Vala | Neoglycopeptides through direct functionalization of cysteine[END_REF][START_REF] Chapleur | Pro-cess for the preparation of 18F-radiolabeled triazolyl-linked glycopeptides as radiotracers via "click" chemistry[END_REF] we introduced propargyl and azide moieties on peptide and sugar groups respectively. Indeed, propargyl bromide is easily and quickly introduced on the thiol group of cysteine-containing peptides by a standard alkylation reaction in aqueous medium. 19,[START_REF] Vala | Neoglycopeptides through direct functionalization of cysteine[END_REF][START_REF] Lo Conte | Photoinduced Addition of Glycosyl Thiols to Alkynyl Peptides: Use of Free-Radical Thiol-Yne Coupling for Post-Translational Double-Glycosylation of Peptides[END_REF] C-glycosides containing an azido propyl arm on the anomeric carbon were chosen as sugar derivative to take advantage of the flexibility given by the three carbons arm between the carbohydrate and the triazole of the final C-glyco"RGD" conjugate. In this study, we focused on a glucopyranose in both α and β configurations after introduction of the flexible arm (Scheme 1).

Bromo derivatives 2 (β configuration) and 3 (α configuration) were prepared according to known methods [START_REF] Petry | Synthesis and revised stereo-chemistry assignment of C-allyl glucopyranosides and derivatives[END_REF] (Scheme 1). Briefly, 1-O-acetyl-2,3,4,6-tetra-O-benzyl-D-glucopyranose 1 [START_REF] Han | Concise and scalable synthesis of aspalathin, a powerful plasma sugar-lowering natural product[END_REF][START_REF] Kulkarni | Efficient Synthesis of a C-Analogue of the Immunogenic Bacterial Glycolipid BbGL2[END_REF] was stereoselectively converted in the corresponding βor α-Callylglucopyranoside. [START_REF] Thota | Synthesis of β-C-galactosyl D-and L-alanines[END_REF][START_REF] Lewis | Highly stereose-lective approaches to alpha-and beta-C-glycopyranosides[END_REF][START_REF] Hosomi | Stereoselective synthesis of 3-(d-glycopyranosyl)propenes by use of allylsilanes[END_REF][START_REF] Hosomi | Highly stereoselective callylation of glycopyranosides with allylsilanes catalyzed by silyl triflate or iodosilane[END_REF] The alkenes were treated with 9-Borabicyclo(3.3.1)nonane (9-BBN) [START_REF] Martin | Synthesis of Chiral Spiroacetals from Carbohydrates[END_REF][START_REF] Petry | Synthesis and revised stereo-chemistry assignment of C-allyl glucopyranosides and derivatives[END_REF] and the resulting alcohols were converted in bromide derivatives 2 and 3. [START_REF] Petry | Synthesis and revised stereo-chemistry assignment of C-allyl glucopyranosides and derivatives[END_REF] Analogous pathways were then carried out on both anomers to provide the targeted azidopropyl-C-glycosides 14 and 15 (Scheme 1). The removal of the benzyl ether protecting groups by hydrogenolysis re-quired a meticulous optimization due to the presence of a bromide atom. We observed previously that catalytic hydrogenolysis under H2 atmosphere (33 psi) with Pd(OH)2/C (20% w/w) in a THF/H2O (5/1 v/v) mixture during 13 hours gave the best results for both β and α anomers. [START_REF] Vucko | Valueadded carbohydrate building blocks by regioselective O-alkylation of C-glucosyl compounds[END_REF] The fully deprotected derivatives 4 and 5 were thus obtained in 95 and 93% yields respectively while the formation of debrominated side-product was limited. Compounds 4 and 5 were reacted with sodium azide in an acetone/water (5/1 v/v) mixture at reflux during 12 hours affording compounds 6 and 7. [START_REF] Zhang | Livertargeted antiviral peptide nanocomplexes as potential anti-HCV therapeutics[END_REF] In a one-pot procedure, selective protection of the primary alcohol of 6 and 7 with a trityl group using trityl chloride and a catalytic amount of 4dimethylaminopyridine (DMAP) followed by peracetylation produced intermediates 8 and 9 in 62 and 75% yields respectively. Compounds 10 and 11 were obtained after selective deprotection of the primary hydroxyl with trifluoroacetic acid (TFA) in dichloromethane at 0°C during 10 minutes in 73 and 75% yields respectively. [START_REF] Miyagawa | Selective deprotection of trityl group on carbohydrate by microflow reaction inhibiting migration of acetyl group[END_REF] In view of potential PET applications, [START_REF] Meyer | Click Chemistry and Radiochemistry: The First 10 Years[END_REF][START_REF] Kuhnast | The challenge of labeling macromolecules with fluorine-18: three decades of research[END_REF] C-glycosyl derivatives were fluorinated at position 9 (numbering in Scheme 1) standing as a non-radioactive reference of prosthetic groups to be coupled to peptides. Indeed, in the perspective of fluorine-18 labeling, this position is the most reactive and ideally suited for introducing a fluorine atom via nucleophilic substitution. The reaction was carried out with (diethylamino)sulfur trifluoride (DAST) as a fluorinating agent in diglyme at 110°C to provide the corresponding 6-fluoro-C-glycosides 12 and 13. 19 F-NMR of fluoro-compounds showed only one doublet of triplet, corresponding to the fluorine atom coupling with H9 and H8, observed around -231.4 ± 0.2 ppm with coupling constants around 47 and 21 Hz (JF,H9 and JF,H8 respectively). Zemplén deprotection (Na 0 , MeOH) was then carried out leading to 14 (β-anomer) and 15 (α-anomer) in quantitative yields. Next, we introduced a triple bond on peptide derivatives. The S-propargylation of cysteine-containing RGD peptides was carried out by taking advantage of the high nucleophilicity of the thiol group of the cysteine. It has already been demonstrated that this simple, efficient and robust method 19,[START_REF] Vala | Neoglycopeptides through direct functionalization of cysteine[END_REF][START_REF] Lo Conte | Photoinduced Addition of Glycosyl Thiols to Alkynyl Peptides: Use of Free-Radical Thiol-Yne Coupling for Post-Translational Double-Glycosylation of Peptides[END_REF] enables one-step propargylation of non-protected peptides. Two commercially available RGD peptides containing a cysteine residue were investigated, one linear tetrapeptide i.e. RGDC 16 and one cyclic pentapeptide i.e. c(RGDfC) 17. Regioselective S-propargylation of the cysteine residue was carried out according to a described method, 19,[START_REF] Vala | Neoglycopeptides through direct functionalization of cysteine[END_REF][START_REF] Lo Conte | Photoinduced Addition of Glycosyl Thiols to Alkynyl Peptides: Use of Free-Radical Thiol-Yne Coupling for Post-Translational Double-Glycosylation of Peptides[END_REF] giving the S-propargyl-RGD derivatives 18 and 19 in 85 and 82% yields respectively (Scheme 2). Coupling of S-propargylated peptides 18 and 19 with 6-fluoro-C-glycosides 14 and 15 was performed via CuAAC. This rapid and usually side-product-free reaction proceeded efficiently under very mild conditions in water using copper (II) acetate (10 mol%) and sodium ascorbate (20 mol%) as reducing agent (Scheme 3). 19 Interestingly, the reaction medium turned pale green upon addition of copper acetate to the azide and alkyne mixture and became blue within 2 hours. TLC monitoring showed no unreacted C-glycoside and completion of the reaction. The workup consisted of removal of copper ions with a chelating resin (Chelex®) followed by freeze-drying. Linear and cyclic C-glyco"RGD" derivatives 20-23 were obtained in good yields (65-74%) after purification by steric exclusion gel filtration. As expected with these experimental conditions, the cycloaddition was regioselective and afforded 1,4disubstituted-2,3,4-triazole as the only product in all cases (Scheme 3). High resolution mass spectrometry and 1D and 2D NMR data confirmed the structure of the cycloadducts. S-propargyl-cysteine RGD derivatives reacted efficiently with azido-fluoro-C-glycosides in CuAAC click coupling whatever the anomeric configuration and the peptide type i.e. linear or cyclic. The reaction proceeds well in water in short reaction time. Beyond CuAAC advantages (vide supra), the incorporation of a triazole moiety in a peptide or glycopeptide is of particular interest in terms of electronic properties and potential additional (intra)molecular interactions. [START_REF] Kuijpers | Preparation and evaluation of glycosylated arginine-glycineasparate (RGD) derivatives for integrin targeting[END_REF][START_REF] Bock | 1,2,3-Triazoles as peptide bond isosteres: synthesis and biological evaluation of cyclotetrapeptide mimics[END_REF] Scheme 3. Synthesis of C-glyco"RGD" derivatives 20-23. Reagents and conditions: (i) H2O, Cu(OAc)2, Na Ascorbate, 25°C, 2h, 20 (74%), 21 (71%), 22 (69%), 23 (65%).

In vitro evaluation.

The in vitro biological properties of the new linear and cyclic C-glyco"RGD" derivatives were evaluated on human platelets and human vascular smooth muscle cells (VSMCs) selected for their expression of αIIbβ3 and αvβ3 respectively. 8,[START_REF] Deckmyn | Inhibitors of the Interactions Between Collagen and Its Receptors on Platelets[END_REF][START_REF] Kapp | A Comprehensive Evaluation of the Activity and Selectivity Profile of Ligands for RGD-binding Integrins[END_REF][START_REF] Gaertner | Radiolabelled RGD peptides for imaging and therapy[END_REF][START_REF] Chen | Clinical Application of Radiolabeled RGD Peptides for PET Imaging of Integrin αvβ3[END_REF][19][START_REF] Zhou | Radiolabeled cyclic RGD peptides as radiotracers for imaging tumors and thrombosis by SPECT[END_REF] Additionally, to assess the influence of the saccharide moiety, the biological effects of the corresponding reference peptides, i.e. RGDC and c(RGDfC), were also measured.

The ability of the four RGD-conjugates 20-23 to inhibit ADPinduced platelet aggregation was first evaluated. Platelet aggregation was triggered by 2.5 µM ADP in human citrated platelet-rich plasma (PRP adjusted to 300×109 platelets/L) after performing or not a preincubation with reference peptides RGDC 16, c(RGDfC) 17 and C-glyco"RGD" 20-23 at concentrations varying from 12.5 to 200 µM (Fig 2). All peptides dosedependently inhibited aggregation of PRP induced by 2.5 µM ADP. For all tested concentrations, linear C-glyco-RGDC 20 and 21 exerted a more potent inhibitory effect than RGDC and c(RGDfC) reference peptides whereas cyclic C-glyco-c(RGDfC) 22 and 23 had only a slight effect. IC50 values are listed in Table 1. As described in the literature, [START_REF] Kapp | A Comprehensive Evaluation of the Activity and Selectivity Profile of Ligands for RGD-binding Integrins[END_REF] the IC50 value was higher for the linear RGDC than for the cyclic c(RGDfC) reference peptides (compare entries 1 and 2). This is however not the case for the Cglyco"RGD" conjugates. Indeed, it appears that linear C-glyco-RGDC 20 and 21 are more effective inhibitors (IC50 of 100 and 65 µM respectively, entries 3 and 4) compared to both RGD references (IC50 223 and 139 µM). Conversely, cyclic C-glycoc(RGDfC) 22 and 23 display very modest inhibitory activities (IC50 >250 µM, entries 5 and 6). These findings are in line with the binding properties of O-glyco"RGD" previously synthesized in our group. The in vitro stability of C-glyco"RGD" was determined using a routine protocol notably used in molecular imaging. [START_REF] Maschauer | Fglyco-RGD peptides for PET imaging of integrin expression: efficient radiosynthesis by click chemistry and modulation of biodistribution by glycosylation[END_REF] The stability of these conjugates was determined in human serum in vitro at 37°C by HPLC monitoring. HPLC analysis of acetonitrile serum extracts at different incubation time points during the incubation period of 120 minutes demonstrating the in vitro stability of those tools built with a stable C-glycoside.

Molecular-dynamics simulations.

We built molecular models of a linear and a cyclic Cglyco"RGD", using compounds 20 and 22 respectively. The initial 3D structures were first built using the Molefacture plugin of VMD [START_REF] Humphrey | VMD: visual molecular dynamics[END_REF] and subsequently solvated in a bath of explicit water. To rationalize the biological profile of C-glyco-c(RGDfC) 22 toward αvβ3, a molecular assay of the integrin in complex with the cyclic c(RGDfC) was further built. We started from the crystal structure of the extracellular segment of integrin αvβ3 in complex with cyclic peptide c(RGDfV) (PDB:1L5G). [START_REF] Xiong | Crystal structure of the extracellular segment of integrin alpha Vbeta3 in complex with an Arg-Gly-Asp ligand[END_REF] For the protein, missing residues were not incorporated, similarly to the work of Xiong et al. [START_REF] Gilad | Synthesis, biological studies and molecular dynamics of new anticancer RGD-based peptide conjugates for targeted drug delivery[END_REF] Two molecular assays were constructed using either the crystal RGD peptide or the cyclic C-glyco-c(RGDfC) 22. The complex involving the latter ligand was generated by aligning its c(RGDfC) moiety onto the RGD structure of the peptide in the crystal. Finally, a solution of 150 mM NaCl was added to simulation boxes (Fig 4).

Discussion

The development of high affinity and selective ligands toward integrins for in vivo applications like molecular imaging remains a daunting challenge. RGD binding regions are very similar in most integrins, it is thus not easy to design ligands exhibiting simultaneously high selectivity and high affinity for distinct subtypes. Nevertheless, this is an essential requirement for the use of bioactive molecules in molecular imaging. [START_REF] Kapp | A Comprehensive Evaluation of the Activity and Selectivity Profile of Ligands for RGD-binding Integrins[END_REF][START_REF] Kuhnast | The challenge of labeling macromolecules with fluorine-18: three decades of research[END_REF] Of equal importance is bioavailability, especially in the context of PET imaging relying on radioisotopes of short half-life. In this contribution, we have designed original linear and cyclic Cglyco"RGD" tools which possess a carbohydrate moiety bearing a fluorine atom, making them suitable for future applications in PET imaging. The radiosynthesis of these conjugates was performed to produce four [ 18 F]fluoro-C-glyco"RGD" ready to be investigated in PET imaging. The introduction of a three-carbon triazolyl linker provides a rather high flexibility to the synthetized ligands. Molecular simulations revealed that, in solution, the carbohydrate group explores an ensemble of conformations irrespective of the topology (cyclic or linear) of the RGD peptides, however subtle differences in the flexibility of the linear and cyclic conjugates may be highlighted. Such a conformational dynamic is anticipated to modulate their level of affinity and selectivity towards αIIbβ3 and αvβ3 integrins. Of particular importance, the introduction of a C-C bond to link the C-glycoside prosthetic group increases significantly the resistance to proteolytic degradation compared to O-glycoside derivatives that were developed previously. 19,[START_REF] Collet | Development of 6-[18F]fluoro carbohydrate-based pros-thetic groups and their conjugation to peptides via click chemis-try[END_REF] The stability of the latter compounds was shown to be limited to one hour in vitro, a time range suboptimal for PET imaging. At variance, C-glycoside derivatives remain stable for two hours in human serum, making them perfect candidates towards in vivo imaging application.

Testing the efficacy of integrin ligands in the cellular context is a difficult endeavor. There is no consensual protocol to measure unambiguously the binding affinity of RGD-ligands towards integrins, making the comparison of the ensemble of published data hazardous. [START_REF] Kapp | A Comprehensive Evaluation of the Activity and Selectivity Profile of Ligands for RGD-binding Integrins[END_REF] In an effort to reconcile the various binding studies, Kessler et al. have evaluated systematically the affinity of a wide range of ligands towards integrins using a unique solid phase binding assay. [START_REF] Kapp | A Comprehensive Evaluation of the Activity and Selectivity Profile of Ligands for RGD-binding Integrins[END_REF] Here, the biological effect on αIIbβ3 and αvβ3 integrins were examined using functional tests. While the binding affinity of RGD peptide derivatives can be accurately measured using a solid phase binding assay, their ability to modulate biological functions has to be also assessed to evaluate their relevance as diagnostic tools. Previous studies with integrin antagonists and knockout mice have clearly demonstrated that αIIbβ3, the most abundant integrin expressed on platelets and whose expression is restricted to cells of the megakaryocyte lineage, is a crucial adhesive receptor for platelet aggregation. [START_REF] Versteeg | New fundamentals in hemostasis[END_REF] A key feature of vascular cells is their adhesive status which is determined by a repertoire of adhesion receptors. In the vascular wall the αv subunit pairs with β1, β3, β5, β6 and β8 that play different roles in attachment and migration, but the αv subunit only pairs with the β3 subunit on the surface of VSMCs [START_REF] Lacolley | Vascular Smooth Muscle Cell and Arterial Stiffening: Relevance in Development, Ageing and Disease[END_REF] and only αvβ3 interacts with all major matrix ligands. [START_REF] Moiseeva | Adhesion receptors of vascular smooth muscle cells and their functions[END_REF] The first identified function of αvβ3 was its capacity to mediate attachment of cells including VSMCs to vitronectin. Therefore, inhibition of platelet aggregation and inhibition of VSMC adhesion to vitronectin are highly relevant assays to identify RGD derivatives that can be further used as functional probes.

The addition of a C-glycoside moiety on either a linear or a cyclic RGD scaffolds affects their binding to αIIbβ3 and αvβ3 integrins in a different manner. While cyclic ligands 22 and 23, demonstrate promising affinity for αvβ3, it was not the case towards αIIbβ3, in sharp contrast with the affinity reported for standard cyclic-RDGs. [START_REF] Kapp | A Comprehensive Evaluation of the Activity and Selectivity Profile of Ligands for RGD-binding Integrins[END_REF] Interestingly enough, linear conjugates 20 and 21 displayed only weak affinity to αvβ3, following the trend observed for non-functionalized linear RGDs, but their inhibitory affinities toward αIIbβ3 is highly enhanced by the addition of a carbohydrate group and the chemical linker. The peculiar biological profile of C-glyco"RGD" can be rationalized by analyzing their structural dynamics and the topology of the binding pockets of αvβ3 and αIIbβ3. For the latter integrin the binding site appears more ensconced compare to that of αvβ3, as revealed by of X-Ray crystallography (PDB:1LG5 and PDB:3ZE2) (see Figure S20 for a side-by-side comparison of the two binding sites). Moleculardynamics simulation clearly shows that the linear peptide is much more flexible than its cyclic homolog, allowing to adapt to the somewhat buried cavity of αIIbβ3, whereas the more rigid cyclic compound is anticipated to be sterically hindered preventing its binding to this integrin. However, the relative rigidity of the cyclic C-glyco-c(RGDfC) does not impair its recognition by the flatter binding site of αvβ3. Molecular-dynamics simulation performed on the most potent compound 22 show that the RGD/integrin interface remains essentially the same for 22 and native c(RGDfC) 17. The in-silico experiments further highlight that the glyco-moiety of compound 22 interacts transiently with the βA domain of the integrin, most often with charged residues K125 and D126, rationalizing possibly the higher affinity of the conjugated compared to its native analog.

Conclusion

Four new 6-fluoro-C-glyco"RGD" tools aimed at targeting integrins for molecular imaging purposes have been synthesized and radiosynthesized by conjugating RGD peptide derivatives with 6-fluorosugars via a triazolyl moiety. The design of all the derivatives has exploited the robustness and hydrophilicity of Cglycosides to enhance their possible efficiency, while a threecarbon flexible spacer was selected in order to minimize the steric hindrance. Remarkably, distinct affinity profiles towards αIIbβ3 and αvβ3 integrins can be achieved by modulating the RGD conjugates topology. Indeed, the linear C-glyco-RGDC 20 and 21 showed a notable effect for αIIbβ3 while cyclic C-glyco-c(RGDfC) 22 and 23 preferentially bind to αvβ3 with an IC50 of 4.4 and 9.3 µM respectively. Important effects on cell adhesions have been highlighted. Molecular modeling and simulation rationalized the distinct biological properties of C-glyco"RGD" and in particular the preference of cyclic C-glyco-c(RGDfC) 22 in the targeting αvβ3 integrin due to a combined effect of the ligand flexibility and of the integrin binding pocket hindrance. The achievement of this distinct affinity profile is clearly of crucial results that will show all its importance for in vivo applications. Our results allow us to propose the use of the linear [ [START_REF] Chen | Clinical Application of Radiolabeled RGD Peptides for PET Imaging of Integrin αvβ3[END_REF]

Experimental section

Materials and methods

Solvents and liquid reagents were purified and dried according to recommended procedures. Thin layer chromatography (TLC) analyses were performed using standard procedures on Kieselgel 60F254 plates (Merck, Kenilworth, NJ, USA). Compounds were visualized using UV light (254 nm) and a methanolic solution of sulfuric acid and charred. Column chromatography was performed on silica gel SI 60 (63-200 µm) (Merck). Purification of glycoconjugates was achieved by gel filtration on LH20 using water or water/methanol mixture as eluent. Melting points were determined with a Tottoli apparatus and are uncorrected. FTIR spectra were recorded on a Shimadzu IRAffinity-1, ATR PIKE Technologies model GladiAT (cm-1). Optical rotations were measured on an Anton-Paar MCP 300 polarimeter. Analytical High performance liquid chromatography (HPLC) analyses were run on a Waters system (2695eb pump, auto sampler injector, 2998 PDA detector, 2424 ELSD detector, and NaI detector from Berthold [Bad Wildbad, Germany]) controlled by the Empower Software (Orlando, FL, USA). Analyses were performed on a Vydac 218TP C18 (5 μm, 250 × 4.6 mm) from Grace or Alltima C8 (5 μm, 150 × 4.6 mm) from Grace with ACN/H2O/TFA mixture (proportions given in brackets) at 1 mL/min. Integrated HPLC system on AllInOne synthesizer was equipped with a radioactivity detector, Smartline UV detector 200 (Knauer, Berlin, Germany) at 254 nm and a HPLC pump P4.1.5 (Knauer, Berlin, Germany). Semipreparative HPLC were performed on an Alltima C8 endcapped (5µm, 150×10 mm) from Grace (HPLC n°1) and on a Vydac 218TP C18 (5 µm, 250x10mm) from Grace (HPLC n°2) with ACN/H2O or ACN/H2O/TFA mixture (proportions given in brackets) at 4.5 or 3 mL/min respectively. 1H, 13C, and 19F NMR spectra were recorded on a Bruker spectrometer DPX250 (250 MHz, 62.9 MHz and 235 MHz, respectively) or Avance III 400 (400 MHz and 100.6 MHz, respectively). For complete assignment of 1H and 13C signals, two-dimensional 1H, 1H COSY and 1H, 13C correlation spectra were recorded. Chemical shifts (δ) are given in parts per million relative to the solvent residual peak. For clarity, atom numbering starts from the end of the anomeric arm rather than resulting from the IUPAC naming of compounds (cf fig S1). The following abbreviations are used for multiplicity of NMR signals: s = singlet, d = doublet, t = triplet, q = quadruplet, m = multiplet, b = broad signal and app = apparent multiplicity. The given J values refer to apparent multiplicities and do not represent the true coupling constants. Mass and high resolution mass spectrometry spectra were recorded on a Bruker microTOFQ apparatus. No-carrier-added fluoride-18 was produced via the [START_REF] Chen | Clinical Application of Radiolabeled RGD Peptides for PET Imaging of Integrin αvβ3[END_REF] O (p,n) [START_REF] Chen | Clinical Application of Radiolabeled RGD Peptides for PET Imaging of Integrin αvβ3[END_REF] F nuclear reaction on a PET Trace cyclotron (GE). The bombardment was performed at 10 µA during 5 minutes to provide about 5 GBq of fluoride-18 delivered as a solution in [START_REF] Chen | Clinical Application of Radiolabeled RGD Peptides for PET Imaging of Integrin αvβ3[END_REF] O-enriched water (1.6 mL). Radiosynthesis was carried out on an AIO module from Trasis®.

Experimental procedure and physico-chemical characterization

Compounds 2, 3 47 4, 6 [START_REF] Vucko | Valueadded carbohydrate building blocks by regioselective O-alkylation of C-glucosyl compounds[END_REF] and 18, 19 19 were prepared according to previously described methods.

Complete experimental details and physico-chemical characterization of new compounds, biological assays and molecular-dynamics simulation protocols are described in the supporting information part.

. 2 . 1 . G e n e r a l m e t h o d f o r t h e s y n t h e s i s o f f l u o r o -Cg l y c o " R G D "

To a solution of S-propargyl derivative 18 and 19 (0.02 mmol) and azido derivatives 14 or 15 (0.02 mmol) diluted in water (0.4mL), was added at room temperature sodium ascorbate (0.004 mmol) in water (0.4 mL) and a solution of Cu(OAc)2 (0.002 mmol) in water (0.045 mL). The solution turned immediately pale green. The reaction was stopped when the intense blue color reappeared (2h of stirring). Chelex ® resin (150 mg) was then added to the blue solution and the suspension was stirred until the solution turned colorless. The resin was filtered off and the resulting solution was freeze-dried. Purification was achieved on Sephadex LH20 resin. Elution with water provided pure fluoro C-glycopeptides 20, 21, 22 or 23. For c(RGDfC) 19, 0.005 mmol of peptide was introduced, proportion of other reactive was respected.

. 2 . 2 . L -C y s t e i n e , L -a r g i n y l g l y c y l -L -α -a s p a r t y l -S -[ [ 1 -[ 1 -(4 , 8 -a n h y d r o --f l u o r o -, 1 , 2 , 3 -t r i d e o x y -Dg l y c e r o -D -g u l o -n o n i t o l )] -1 H -1 , 2 , 3 -t r i a z o l -4y l] m e t h y l] (2 0 )

Yield 74%. White foam.

[α]D: + 4.9 (c 0.05; H2O).

Fig 1 .

 1 Fig 1. Schematic topology of C-glyco"RGD" compounds.

Scheme 1 .

 1 Scheme 1. 6-fluoro-C-glycosides synthesis. Reagents and conditions: (i) Pd(OH)2/C (20% w/w), THF/H2O (5/1 v/v), H2, 33 psi, 13h, 4 (95%) , 5 (93%); (ii) NaN3, acetone/water (5/1 v/v), reflux, 12h, 6 (95%), 7 (96%); (iii) 1) TrCl, DMAP, pyridine, 48h, 45°C. 2) Ac2O, 0°C to rt, 2h, 8 (62%), 9 (75%); (iv) TFA (20% v) in CH2Cl2, 0°C, 10 min, 10 (73%), 11 (75%); (v) DAST, diglyme, 110°C, 8 min, 12 (61%), 13 (52%); (vi) Na 0 , MeOH, 25°C, 1h, quantitative.

Scheme 2 .

 2 Scheme 2. Propargylation of RGD peptides. Reagents and conditions: (i) propargyl bromide (1.1 eq.), NH4OH, H2O, MeOH, 25°C, 3h, 18 (85%), 19 (82%).

Scheme 4 .

 4 Scheme 4. Radiosynthesis of [ 18 F]F-C-glyco"RGD" derivatives [ 18 F]20-[ 18 F]23. Reagents and conditions: (i) 1) K[ 18 F]F-K222, CH3CN, 95°C, 5 min, decay-corrected radiochemical yields (48 to 69%). 2) HPLC purification. 3) NaOH, 30 °C, 9 min then HCl. (ii) Cu(OAc)2, Na ascorbate, H2O, 55°C, 25 min, (40 to 65 %).

  [START_REF] Chen | Clinical Application of Radiolabeled RGD Peptides for PET Imaging of Integrin αvβ3[END_REF] F-radiosynthesis of [ 18 F]F-C-glyco"RGD" [ 18 F]20-[ 18 F]23 was then investigated (Scheme 4). Precursors of radiolabeling 24 and 25 were easily obtained starting from 10 or 11 by reacting triflic anhydride with pyridine in 80% and 45% yield respectively.[START_REF] Chen | Clinical Application of Radiolabeled RGD Peptides for PET Imaging of Integrin αvβ3[END_REF] F-radiolabeling of 24 and 25 was carried out on an AllInOne (AIO) synthesizer with classical method of radiofluorination using the K[ 18 F]F-K222 complex in acetonitrile at 95°C during 5 minutes. Radiolabelled mixture was then purified on a silica SPE cartridge following by semi-preparative HPLC. [ 18 F]14 or [ 18 F]15 was obtained after saponification with NaOH 1M of the collected product. After neutralization by HCl 1M, the CuAAC coupling was then implemented to afford [ 18 F]20-[ 18 F]23. The reaction proceeded with propargylated peptide (18 or 19), Cu(OAc)2 and sodium ascorbate in water at 55°C during 25 min. Pre-purification of 18 F-labeled C-glyco"RGD" can be operated by simple filtration on a cartridge loaded with Chelex® 100 resin to remove most of the unreacted prosthetic group and copper. Semi-preparative HPLC of [ 18 F]F-C-glyco"RGD" was then performed. Identity of [ 18 F]20-[ 18 F]23 was confirmed with analytical HPLC by a second injection with the authentic non-radioactive standard (identical tR). [ 18 F]20-[ 18 F]23 were radiosynthesized with satisfactory decay corrected radiochemical yields (2-4%) considering the complexity of the radiosynthesis.

Fig 2 .

 2 Fig 2. Effect of C-glyco"RGD" 20-23 and reference peptides on human platelet aggregation in response to ADP. The results are the means ± SEM of 4 to 5 independent experiments with PRP from different donors.

Fig 3 .

 3 Fig 3. a) Effect of C-glyco"RGD" 20-23 and reference peptides on human VSMC adhesion to immobilized vitronectin. The results are the means ± SEM of 4 to 5 independent experiments. b) Zoom for 0-100 µM peptide concentration.

Fig 4 .

 4 Fig 4. Initial simulation box for the cyclic C-glyco"RGD" 22 in complex with the extracellular segment of integrin αvβ3 (PDB:1L5G). Chlorine, sodium and magnesium ions are depicted by violet, grey and green van der Waals spheres respectively. Water molecules are represented by the cyan molecular surface. Orange and ice blue cartoons stand for the integrin propeller and βA domains respectively. The RGD ligand is represented by van der Waals sphere colored by atom name (oxygen: red, hydrogen: white, sulfur: yellow, nitrogen: blue and carbon: cyan) The conformational dynamics of the linear and the cyclic Cglyco"RGD" 20 and 22 differs markedly in solution. The cycle formed by the c(RGDfC) backbone is essentially rigid along the MD trajectory while the spacer arm carrying the carbohydrate group remains predominantly in an extended conformation, preventing intramolecular interactions. Consequently, the glyco moiety moves in a well-defined volume opposite to the RGD sequence and extending from the arginine to the phenylalanine side chains (Fig 5). At variance, the linear C-glyco-RGDC appears more flexible navigating between various conformations mainly characterized by the formation of intramolecular interactions. The most frequent conformation observed during the simulation involves an electrostatic interaction between the arginine side chain and the terminal carboxylate of the cysteine residue, resulting in a cyclic-like arrangement of the RGD sequence. The space-volume being accessed by the carbohydrate group is larger than in the case of the cyclic ligand (Fig 5), suggesting a higher plasticity.

Fig 5 .

 5 Fig 5. Representative snapshots of (top) cyclic C-glyco-c(RGDfC) 22 and (bottom) linear C-glyco-RGDC 20 extracted from the 500 ns-long moleculardynamics simulations of the free ligands in water. The RGD ligand is represented by licorices colored by atom name. Semi-transparent white surface represents an isosurface of the mass-weighted atomic density of the carbohydrate moiety computed over the MD trajectory with the VolMap plugin of VMD.Binding of compound 22 (IC50 = 4.4 µM) and reference c(RGDfC) 17 (IC50 = 2.8 µM) to the extracellular domain of αvβ3 integrin was investigated using MD simulations (Fig6). The RGD/integrin interface observed all along the 500 ns-long trajectories remains essentially the same for both peptides. More specifically, the aspartic acid of the ligand interacts tightly with a magnesium ion, the latter being strongly associated to S121, S123 and E220. It further forms a hydrogen bond with the backbone of Y122. The arginine is forming a robust salt-bridge with D218 irrespective of the interacting compound. The in-silico experiments further highlighted that the glyco-moiety of compound 22 interacts transiently with the βA domain of the integrin, most often with charged residues K125 and D126.

Fig 6 .

 6 Fig 6. Representative snapshots of the binding interface of αvβ3 integrin and (top) cyclic c(RGDfC) and (bottom) cyclic C-glyco-c(RGDfC) 22 extracted from the 500 ns-long molecular-dynamics simulations. Integrin propeller and βA domains are depicted by orange and ice blue surfaces respectively. Magnesium ions are represented by green van der Waals spheres. Integrin amino acids interacting with the peptide are colored by residue type (polar: green, acidic: red and basic: blue). The RGD ligand is represented by licorices colored by atom name (hydrogens are omitted for clarity).

  F]F-C-glyco-RGDC [ 18 F]20 and [ 18 F]21 for thrombosis imaging, while the cyclic [ 18 F]F-C-glycoc(RGDfC) [ 18 F]22 and [ 18 F]23 could be promising tools for cancer diagnosis.

  1 H NMR (400 MHz, D2O): δ 1.40-1.50 (m, 1H, H3a), 1.70-1.88 (m, 3H, H2a and CH2), 1.96-2.22 (m, 4H, H2b, H3b and CH2), 2.60-2.75 (m, 1H, ½CH2), 2.77-3.06 (m, 3H, ½CH2 and CH2), 3.15-3.35 (m, 4H, H4, H5 and CH2), 3.44-3.57 (m, 3H, H6, H7 and H8), 3.83-4.20 (m, 4H, 2 CH2), 4.39 (bs, 1H, CH), 4.45-4.70 (m, 5H, H1, H9 and 2 CH), 8.06 (bs, 1H, Htriazole). 13 C NMR (100.6 MHz, D2O): δ 23.4 (CH2), 25.4 (C2 and CH2), 27.7 (C3), 28.0 (CH2), 33.3 (CH2), 40.4 (2 CH2), 42.5 (CH2), 50.3 (C1), 51.6 (CH), 52.8 (CH), 54.3 (CH), 68.6 (d, JC,F = 7.0 Hz, C7), 73.3 (C5), 77.2 (C6), 78.0 (d, JC,F = 17.0 Hz, C8), 78.9 (C4), 82.3 (d, JC,F = 168.0 Hz, C9), 125.2 (CHtriazole), 140.8 (Ctriazole), 156.8 (C=N), 170.1 (C=O), 170.7 (2

Table 1

 1 Inhibition of ADP-induced platelet aggregation and VSMC adhesion to

vitronectin Entry Peptide IC50 (µM)

			Platelet	VSMC
			aggregation	adhesion
	1	RGDC 16	223 ± 11	> 2000
	2	c(RGDfC) 17	139 ± 6	2.8 ± 0.7
	3	20	100 ± 5	> 2000
	4	21	65 ± 7	> 2000
	5	22	> 250	4.4 ± 0.6
	6	23	> 250	9.3 ± 0.6

Acknowledgment

This work was supported by Institut Jean Barriol and the Investments for the Future program under grant agreement No ANR-15-RHU-0004. We acknowledge The Ministère de l'Enseignement Supérieur et de la Recherche for PhD funding to T. Vucko. We acknowledge the State-Region Plan "Technological Innovations, Modeling and Personalized Medical Support" (IT2MP) and the European Regional Development Funds (ERDF) for generous support. We thank S. Adach, F. Lachaud and P. Ponce for technical assistance.

Graphical Abstract

To create your abstract, type over the instructions in the template box below. Fonts or abstract dimensions should not be changed or altered. Leave this area blank for abstract info.

C-glyco"RGD" as αIIbβ3 and αvβ3 integrin ligands for imaging applications: synthesis, in vitro evaluation and molecular modeling