Simple Synthesis of Nodulation‐Factor Analogues Exhibiting High Affinity towards a Specific Binding Protein

Abstract

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.

Lipo-Chitin Oligosaccharides, Plant Symbiosis Signalling Molecules That Modulate Mammalian Angiogenesis In Vitro

“…The compounds used were fully synthesised (compounds 11, 13–16), or partially synthesised after first purifying precursors from the supernatants of Escherichia coli harbouring rhizobial LCO genes ( i.e. for compounds 1–3, 5–10, 22, 23) [40] , [41] , isolated directly from the supernatants of Escherichia coli harbouring rhizobial LCO genes (compounds 4, 11) [40] , [41] , obtained from commercial sources (compounds 12, 17–21) or isolated directly from rhizobial secretions (compound 24; Fig. 1 ; Methods).…”

“…S2 and Methods S1 in S1 File ). All other compounds were synthesised as described [40] , [58] . Compound 11 was also derived independently from the supernatant of transgenic E. coli .…”

“…LCO and LCO-like compounds were generated in transgenic E. coli [39] , [40] , [41] , [42] , [43] , [44] , [45] , [46] , [47] unless stated otherwise in Fig. 1 or synthesised as stated below.…”

From Chitin to Bioactive Chitooligosaccharides and Conjugates: Access to Lipochitooligosaccharides and the TMG‐chitotriomycin

“…For the initial use of these NHTFA building blocks, we prepared glycosyl acceptors, which are useful in the synthesis of the lipochitooligosaccharides5 or analogues,5c, 10c, 19 with a free hydroxy group at C4 of the reducing unit. Standard anomeric acetate cleavage,20 silylation,21 deacetylation, benzylidene formation, acetylation, and reductive opening of the acetal in CH 2 Cl 2 with triethylsilane and trifluoroacetic acid22 gave acceptors 11 and 12 in respective overall yields of 57 and 53 % from 6 and 7 (Scheme ).…”

Lipo-Chitooligosaccharidic Nodulation Factors: Synthesis and Agricultural Perspectives

“…Thus, using the cellular approach reported above, we have synthesized a series of diverse mimics of the Nod factor of S. meliloti, the microsymbiont of alfalfa (Scheme 3). [24] ample of a non-conventional construction of a linear oligosaccharide starting from the non-reducing end (Box 2, Scheme 1). [9] The first b-glycosylation reaction was secured by a participating benzyloxycarbonyl carbamate group at C(2) of donor 5.…”