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metals in all biological systems (Stamler, 1994). The S-nitrosothiol (SNO) and metal nitrosyl (MNO) products and Nikolaus J. Sucher ‡ § have intrinsic reactivities that enable local action. The * Departments of Medicine, Cell Biology, NO group is transferred by covalent exchange mecha- † Chemistry and Biochemistry nisms including thiol/nitrosothiol (RSNO/R'S Ϫ ⇔ R'SNO/ Duke University RS Ϫ ) or nitrosothiol/metal (SNO/M ⇔ MNO/S) exchange. Durham, North CarolinaThis mode of intra-and intercellular communication has ‡ Laboratory of Cellular and Molecular Neuroscience advantages over free diffusion and is better established Children's Hospital in vivo (Stamler et al., 1992a;Jia et al., 1996). Harvard Medical SchoolNO also exploits reactions with O 2 /O 2 Ϫ to complete the Boston, Massachusetts armamentarium of species responsible for "NO" effects (Table 1; Stamler et al., 1992b). This is well exemplified Nitric oxide (NO) is a signaling molecule that has capin the immune system in work by DeGroote and Fang tured our imagination. According to the common view, (DeGroote et al., 1996). These researchers found that NO diffuses over a large sphere of influence, moving bacterial virulence is conferred by a gene that protects freely through membranes of target cells to raise levels against the lethal effects of SNOs produced by the (muof cGMP. In the brain, NO influences synaptic plasticity, rine) host, whereas NO is harmless against the same apoptosis, neuronal development, and even complex bacteria. Molecular recognition of the SNO onslaught behavioral responses. This image has been reinforced and the activation of bacterial resistance is achieved by by observations in the cardiovascular and immune sys-S-nitrosylation of proteins involved in defense (Hauslatems, for example, the relaxation of blood vessels by den et al., 1996). Thus, in this system, S-nitrosylation is cGMP, and the killing of tumor cells and bacteria by the the signal and the regulator of the response. macrophage NO synthase (NOS).A similar NO signal is used by mammalian cells. Images can also be misleading. First, a wide sphere For example, the endothelium-derived relaxation factor of NO diffusion implies that it travels down concentration (EDRF)/NO-mediated relaxation of blood vessels occurs gradients that are established by extracellular sinks partly by direct activation of a potassium channel (Lancaster, 1994;Stamler, 1996). The problem with this through reactions of EDRF with critical thiols (Bolotina picture is that NO cannot achieve local action: it would et al., 1994). Likewise in the heart, SNO and peroxynitrite be leaving cells more rapidly than it reacts within. Sec-(OONO Ϫ ) directly activate calcium channels by a redox ond, we have come to appreciate that many NO signals mechanism that opposes the effects of cGMP (Campbell are cGMP independent. These pathways, typically et al., 1996). Ion channel activation may also account grouped under the broad heading of "redox", have not for NO/SNO-mediated relaxations of third to fourth order been i...

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The inhibition of protein−carbohydrate interaction provides a powerful therapeutic strategy for the treatment of myriad human diseases. To date, application of such approaches have been frustrated by the inherent low affinity of carbohydrate ligands for their protein receptors. Because lectins typically exist in multimeric assemblies, a variety of polyvalent saccharide ligands have been prepared in the search for high affinity. The cluster glycoside effect, or the observation of high affinity derived from multivalency in oligosaccharide ligands, apparently represents the best strategy for overcoming the “weak binding” problem. Here we report the synthesis of a series of multivalent dendritic saccharides and a biophysical evaluation of their interaction with the plant lectin concanavalin A. Although a 30-fold enhancement in affinity on a valence-corrected basis is observed by agglutination assay, calorimetric titration of soluble protein with a range of multivalent ligands reveals no enhancement in binding free energies. Rather, IC50 values from agglutination measurements correlate well with entropies of binding. This observation suggests that hemagglutination measures a phenomenon distinct from binding that is typified by a large favorable entropy and an unfavorable enthalpy:  this process is almost certainly aggregation. Supporting this assertion, we report crystal structures of multivalent ligands cross-linking concanavalin A dimers. To the best of our knowledge, these structures are the first reported of their kind. Our results indicate that hemagglutination assays evaluate the ability of ligands to inhibit the formation of cross-linked lattices, a process only tangentially related to reversible ligand binding. Cluster glycoside effects observed in agglutination assays must, therefore, be viewed with caution. Such effects may or may not be relevant to the design of therapeutically useful saccharides.