Rhizobial glycolipids (lipochitooligosaccharides (LCOs), also called nodulation (Nod) factors) are important signaling molecules involved in the establishment of symbiosis with legumes.[1] This association results in the formation of rhizobia-infected nodules on plant roots in which atmospheric nitrogen is reduced to ammonia for the benefit of the plant. These nodulation factors represent a large family of lipooligosaccharides consisting of a chitin fragment (three-five Nacetyl-d-glucosamine units) with an N-acyl chain attached to the nonreducing unit and a variety of additional substituents.[1a] The variations are characteristic of each bacterial strain and define the high degree of specificity in the legumerhizobia interaction. One of the most studied models is the symbiosis of Sinorhizobium meliloti and alfalfa (Medicago sativa) in which the major bacterial signal molecule 1 (Scheme 1) contains four d-glucosamine residues with a 6-O-acetyl group and an N-(2E,9Z)-hexadecadienoyl side chain at the nonreducing residue and an essential 6-O-sulfate group at the reducing unit which is the main determinant of the host specificity in M. sativa.[2]Although highly active, signal molecule 2 is approximately ten times less active than the acetylated derivative 1 in morphogenic activity. However, since it is more stable, 2 is more suitable for biological studies. Structure-function relationship studies have shown that the correct nature of the lipid chain, in totally synthetic molecules, [3] is essential for triggering an optimal morphogenic activity in M. sativa roots.[4] Moreover, the length and the structure of the lipid chain play a role in the high-affinity binding of LCOs to a Nod factor binding site (NFBS2) characterized in cell suspension cultures of legumes. [5][6][7] As a step forward in the search for modified molecules for the study of the Nod factor perception mechanisms by the plant, we now report a simple synthesis of analogues by using metabolically engineered bacterial cells, a method that avoids the complexity of total synthetic approaches from N-acetyl-dglucosamine monomers. [3,8] We further demonstrate that the incorporation of a substituted benzamide (see B in Scheme 2) provides new analogues able to interact with a putative LCO receptor at nanomolar concentrations.The aromatic motif was selected because a) it leads to benzamides that are potentially more stable chemically and metabolically than the natural conjugated amides, b) it makes it possible to introduce a photoactivatable group at a strategic position, and c) it is readily prepared from commercial precursors. Although replacement of unsaturated bonds in lipids by aromatic derivatives is frequently used to produce molecular probes and agonists or antagonists of bioactive molecules, this substitution combining an aromatic motif and Scheme 1. Structures of the major nodulation factors produced by Sinorhizobium meliloti.Scheme 2. An alkoxybenzamide (X = O) or benzylamine (X = H 2 ) structure B can mimic the natural a,b-unsaturated amide A.

Nod factors are lipochitoligosaccharides originally produced by the soil bacteria Rhizobia that are involved in the symbiotic process with leguminous plants. Some synthetic analogs of the Nod factors present a strong biological activity, and the conformational behavior of these molecules is of interest for structure/function studies. Nod factor analogs containing an insertion of a phenyl group in the acyl chain at the oligosaccharidic non-reducing end were previously synthesized (Grenouillat N, Vauzeilles B, Bono J-J, Samain E, Beau J-M. 2004. Simple synthesis of nodulation-factor analogues exhibiting high affinity towards a specific binding protein. Angew Chem Int Ed Engl. 43:4644). Conformational studies of natural compounds and synthetic analogs have been performed combining molecular dynamics simulations in explicit water and NMR. Data revealed that the glycosidic head group can adopt only restricted conformations, whereas chemical modifications of the lipid chains, highly flexible in a water environment, influence the global shape of the molecules. Collected structural data could be used in the future to rationalize and understand their biological activity and affinity toward a putative receptor.