Synthesis of an Amphiphilic Tetraantennary Mannosyl Conjugate and Incorporation Into Liposome Carriers Socorro Espuelas, Philippe Haller, Francis Schuber and Benoı ˆ t Frisch* Laboratoire de Chimie Bioorganique, UMR 7514 CNRS/ULP, Universite ´ Louis Pasteur, Faculte ´ de Pharmacie, 74 route du Rhin, 67400-Illkirch, France Received 24 February 2003; revised 7 April 2003; accepted 30 April 2003 Abstract—We have synthesized a novel conjugate (Man 4 K 3 DOG) composed of a tetramannosyl head group connected, via a polyethylene glycol spacer, to a lipid moiety. This amphiphilic molecule was easily incorporated into the bilayers of liposomes. As expected from the clustering effect, such multivalent mannose residues when exposed on the surface of the vesicles showed much higher binding affinity for Concanavalin A than their monomannosyl analogue. Mannosylated liposomes prepared with the tetra- valent antenna could be promising carriers for e.g., loading dendritic cells with antigens for vaccination purposes. # 2003 Elsevier Ltd. All rights reserved. The human mannose receptor (MR) is predominantly present on macrophages and dendritic cells. 1 It binds carbohydrates present on the surface of microorganisms contributing to their clearance and takes part to the innate immunity. 2 The mannose receptor also strongly enhances effector immune functions through efficient antigen (ag) uptake and delivery to MHC class II molecules. 3 It has been demonstrated that the interac- tions of MR with carbohydrates are usually of low affi- nity unless these ligands are organized as multivalent clusters in specific arrangements. 4 In the search of effective MR ligands, several groups have synthesized multivalent glycopeptide mimics and demonstrated that the affinity of MR was enhanced for branched struc- tures with valencies increasing from 2 up to 8 mannose residues. 5 Disparity of results about optimal valency was attributed to other factors affecting binding affinity and specificity, such as charge or ligand geometry. 6 Liposomes have been described as tools for the efficient presentation of ligands to cell surface receptors, which require multi- valent contacts. Moreover, ag associated to such vesicles were also shown to be more efficiently captured and pre- sented by antigen presenting cells than free ag in solution. 7 Thus, in the present work, we have synthesized two novel neutral mannosyl amphiphilic compounds (Schemes 1 and 2). They have a hydrophilic head group, with one or four mannose residue(s), linked via a poly- ethyleneglycol spacer arm to a lipid moiety that allows their insertion into the bilayers of liposomes. Several considerations have guided our approach: (i) A tetra- mannosyl cluster was considered to represent a good compromise between its affinity for the human MR and synthetic accessibility; 5 (ii) These ligands when exposed at the surface of vesicles could in principle engage in multiple interactions, and thus dramatically increase their apparent affinity for the cells expressing MRs; (iii) The lengths of the spacer arms between the a-d-man- nose groups and the scaffold, and between the ligand and the anchoring moiety, were chosen to provide a good flexibility and accessibility of the liposome-asso- ciated ligands to the MR carbohydrate recognition domains; (iv) The bilayer anchoring moiety contained oleyl chains to ensure to these lipids a low phase transi- tion temperature and a good miscibility with the lipo- somal phospholipids. The feasibility of preparing liposomes with variable amounts of these neoglycolipids was assessed. The accessibility of the mannose residues on the surface of the vesicles and their ability to engage into multivalent interactions with the lectin concanavalin A (Con A) was also evaluated. Synthesis of the Amphiphilic Tetramannosyl Conjugate Man 4 K 3 DOG 8 The synthesis of Man 4 K 3 DOG (Scheme 1) first required the production of lipid 3. Compound 1 was synthesized 0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0960-894X(03)00472-4 Bioorganic & Medicinal Chemistry Letters 13 (2003) 2557–2560 *Corresponding author. Tel.: +33-9024-4168; fax: +33-9024-4306; e-mail: frisch@pharma.u-strasbg.fr