DOI: 10.1002/chem.201200887 Strong Aphicidal Activity of GlcNAcACHTUNGTRENNUNG(b1!4)Glc Disaccharides: Synthesis, Physiological Effects, and Chitinase Inhibition Christophe Dussouy, [a, b] Laurent Bultel, [a] Julien Saguez, [b] Anas Cherqui, [b] Mounia Khelifa, [b] Eric Grand, [a] Philippe Giordanengo,* [b] and JosØ Kovensky* [a] Introduction In most insect species, chitin is the major component of the peritrophic membrane, [1, 2] a non-cellular barrier formed by the digestive epithelium to compartmentalize digestive en- zymes and protect epithelial cells from disruption and the penetration of parasites. [3, 4] The turnover of the peritrophic membrane involves chitinases, which represent a digestive target in the strategy of chitinase inhibitors. The hemipteran Myzus persicae (Aphididae) is one of the most polyphagous insects worldwide, as it successfully develops on hundreds of plant species. M. persicae, as all homoptera, is deprived of a peritrophic membrane, and no chitinase activity has so far been described in the aphid midgut. Thus, aphids could be considered as non-digestive target pests in the context of chitinase-inhibitor use. Aphids are hemimetabolous and lar- viparous insects, that is, the offspring develop in the genital tracts of adults. During their embryonic development, the growth of larvae is influenced by the physiology of the fun- datrix, and their integument chitinases, involved in cuticle remodelling, are possible targets. All these characteristics make M. persicae an interesting model to study chitinase-in- hibitor effects. We recently demonstrated the potential use of chitinase inhibitors as an aphid-management tool. [5] Differential aphi- cidal effects on M. persicae have been induced by allosami- din, a pseudotrisaccharide that is known to inhibit competi- tively chitinases [6, 7] by mimicking chitin. Blattner et al., [8, 9] synthesized and tested a set of allosami- din analogues to evaluate the structural requirements for chitinase inhibition. For a Chironomus tentans (Diptera, Chironomidae) enzyme, one N-acetylallosamine residue can be omitted without impairment of enzyme inhibition and glucosamine can replace allosamine in inhibitory activity without any negative effect. Similar requirements have been reported for Tineola bisselliella (Lepidoptera, Tineidae) and Lucilia cuprina (Diptera, Calliphoridae) larvae mortality, [8] and these results showed that the b-1,4 linkage between the sugar (or pseudosugar) moieties is necessary for biological activity. Moreover, these authors also demonstrated a direct correlation between larval mortality and chitinase inhibition. These findings suggest that b-1,4-gluco disaccharides could be appropriate candidates for insect-chitinase inhibition. We report herein: 1) The synthesis of four GlcNAcACHTUNGTRENNUNG(b1!4)Glc disaccharides. Our synthetic approach involved the introduction of a sulfate group at C6 to investigate the possible addition- al cooperative interactions with the protein. Sulfated oli- gosaccharides display a wide spectrum of biological ac- tivities, mainly through the interaction of sulfate groups with side chains of basic residues (i.e., arginine (Arg), lysine (Lys)) of proteins. [10] The role of a 2-O-acetyl group on the glucose unit (as a N-acetylglucosamine mimic) was also addressed, thus giving rise to four ana- logues: non-sulfated 2-O-acetyl 1, 6-sulfate 2-O-acetyl 2, non-sulfated 2-OH 3, and 6-sulfate 2-OH 4 (Scheme 1). Keywords: carbohydrates · chiti- nases · glycosylation · inhibitors · insect Abstract: The synthesis of four GlcNAcACHTUNGTRENNUNG(b1!4)Glc disaccharides containing 2-O- acetyl and/or 6-sulfate groups was performed in high yields with total 1,2-trans ste- reoselectivity. These disaccharides were evaluated as candidates for insect chiti- nase inhibition and aphicidal activity. All the compounds prepared displayed phys- iological effects on M. persicae aphids; however, the inhibition of chitinases of dif- ferent sources (bacteria, fungus, and aphid) followed different patterns according to subtle structural characteristics. [a] Dr. C. Dussouy, Dr. L. Bultel, Dr. E. Grand, Prof. J. Kovensky Laboratoire des Glucides CNRS FRE 3517 Institut de Chimie de Picardie UniversitØ de Picardie Jules Verne 33 Rue Saint-Leu, 80039 Amiens Cedex 1 (France) Fax: (+ 33) 322827567 E-mail: jose.kovensky@u-picardie.fr [b] Dr. C. Dussouy, Dr. J. Saguez, Dr. A. Cherqui, Dr. M. Khelifa, Prof. P. Giordanengo Laboratoire dEcologie Dynamique des Systmes AnthropisØs JE 2532 UniversitØ de Picardie Jules Verne 33 Rue Saint-Leu, 80039 Amiens Cedex 1 (France) Fax: (+ 33) 322827879 E-mail: philippe.giordanengo@u-picardie.fr Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201200887. Chem. Eur. J. 2012, 00,0–0  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim These are not the final page numbers! ÞÞ &1& FULL PAPER