2 Potent Immunostimulator Agents: Total Synthesis and Biological 3 Evaluation in Human Invariant Natural Killer T Cells and Mice 4 Julie Hunault,† Mette Diswall,‡ Jean-Ced́ric Frison,† Virginie Blot,† Jeźabel Rocher,‡ 5 Sev́erine Marionneau-Lambot, Thibauld Oullier, Jean-Yves Douillard, Steṕhane Guillarme, 6 Christine Saluzzo, Gilles Dujardin, Denis Jacquemin,† Jeŕo ̂me Graton,† Jean-Yves Le Questel,† 7 Michel Evain, Jacques Lebreton,† Didier Dubreuil,† Jacques Le Pendu,*,‡ and Muriel Pipelier*,† 8 †Universite ́ de Nantes, CNRS, Chimie et Interdisciplinarite:́ Synthes̀e, Analyse, Modeĺisation (CEISAM), UMR CNRS 6230, 9 Faculte ́ des Sciences et des Techniques, 2 rue de la Houssinier̀e, BP 92208, 44322 Nantes Cedex 3, France 10 ‡INSERM, U892, Universite ́ de Nantes, IRT UN, 8 Quai Moncousu, 44007 Nantes Cedex 1, France 11 Plateforme in vivo du Canceŕopôle Grand Ouest, 8 Quai Moncousu, 44093 Nantes Cedex, France 12 Centre Rene ́ Gauducheau, Centre Reǵional de Lutte Contre le Cancer Nantes-Atlantique, 44805 Saint-Herblain Cedex, France 13 Universite ́ du Maine, CNRS, Unite ́ de Chimie Organique Molećulaire et Macromolećulaire, (UCO2M), UMR CNRS 6011, 14 Faculte ́ des Sciences et Techniques, Universite ́ du Maine, Avenue Olivier Messiaen, 72085 Le Mans Cedex 9, France 15 Universite ́ de Nantes, Nantes Atlantique Universite,́ CNRS, Faculte ́ des Sciences et des Techniques, Institut des Mateŕiaux Jean 16 Rouxel, UMR CNRS 6502, 2 rue de la Houssinier̀e, BP 92208, 44322 Nantes Cedex 3, France

We propose here the synthesis and biological evaluation of 3,4-dideoxy-GalCer derivatives. The absence of the 3- and 4-hydroxyls on the sphingoid base is combined with the introduction of mono or difluoro substituent at C3 (analogues 8 and 9, respectively) to evaluate their effect on the stability of the ternary CD1d/GalCer/TCR complex which strongly modulate the immune responses. Biological evaluations were performed in vitro on human cells and in vivo in mice and results discussed with support of modeling studies. The fluoro 3,4-dideoxy-GalCer analogues appears as partial agonists compared to KRN7000 for iNKT cell activation, inducing T(H)1 or T(H)2 biases that strongly depend of the mode of antigen presentation, including human vs mouse differences. We evidenced that if a sole fluorine atom is not able to balance the loss of the 3-OH, the presence of a difluorine group at C3 of the sphingosine can significantly restore human iNKT activation.

A set of pyrimidine nucleosides fused with a 4'-C,3'-O-propylene bridge was successfully synthesised in 12 steps from 1,2:5,6-di-O-isopropylidene-α-d-glucofuranose, an inexpensive starting material, based on a ring-closing metathesis (RCM) reaction followed by Vorbrüggen-type nucleobase coupling. Antiviral and cytotoxicity activities of the targeted modified nucleosides, as well as their phosphoramidate prodrugs, are described.

4-Deoxy-alpha-GalCer analogues are considered weaker agonists than KRN7000 for the stimulation of human iNKT cells, but this remains strongly debated. In this work, we described a strategy toward 4-deoxy-alpha-GalCers with, as a key step, a metathesis reaction allowing sphingosine modifications from a single ethylenic alpha-galactoside precursor. The 4-deoxy-KRN7000 derivative 2, described here, induced potent cytokinic responses, comparable to those of KRN7000, both from human iNKT cells in vitro and from their murine counterpart in vivo.