Chiara Venturi

Binding of Ionic Species: A General Approach To Measuring Binding Constants and Assessing Affinities

Bound together: The association of receptors with ionic species cannot be assimilated to the binding of neutral guests. When dealing with salts, both ion pairing and binding to the free and the ion-paired ionic guest determine the actual association pattern (see figure). The general issue of measuring association constants and assessing affinities for ions is addressed and validated in two cases of anion binding.A general approach to the largely underestimated issue of measuring binding constants and assessing affinities in the binding of ionic species is described. The approach is based on a rigorous, nongraphical determination of binding constants in multiequilibrium systems by nonlinear regression of chemical shift data from NMR titrations and on the use of the BC(50) descriptor for assessing affinities and ranking the binding ability of receptors on a common scale. The approach has been validated with two tripodal anion-binding receptors, namely, a ureidic (1) and a pyrrolic (2) receptor, binding to tetramethylammonium chloride in CDCl(3)/CD(3)CN (80:20). A set of five and six formation constants could be measured for 1 and 2, respectively, including, in addition to the ion pair, complexes of the free and the ion-paired anion. The BC(50) values calculated from the measured constants allowed a quantitative assessment of each receptors binding affinity towards the chloride anion, the pyrrolic receptor showing a 15-fold larger affinity over the ureidic receptor, a figure that quantifies the improvement obtained by replacing the amido-pyrrolic for ureidic binding groups on the tripodal scaffold of the receptor. The results have shown that, in contrast to common practice, neither of the two systems could be appropriately described by a 1:1 association with the anion only, but required the ion-pairing and ion-pair binding equilibria to be taken into account because these contribute substantially to the complexation process. The BC(50) descriptor has also been shown to be a useful and general tool for the assessment of affinities of systems involving ionic species. The required extension of the BC(50) binding descriptor to include the treatment of ion-binding has been described in detail.

α‐O‐Linked Glycopeptide Mimetics: Synthesis, Conformation Analysis, and Interactions with Viscumin, a Galactoside‐Binding Model Lectin

Efficient cycloaddition of a silylidene-protected galactal with a suitable heterodiene yielded the basis for a facile diastereoselective route to a glycopeptide-mimetic scaffold. Its carbohydrate part was further extended by beta1-3-linked galactosylation. The pyranose rings retain their (4)C(1) chair conformation, as shown by molecular modeling and NMR spectroscopy, and the typical exo-anomeric geometry was observed for the disaccharide. The expected bioactivity was ascertained by saturation-transfer-difference NMR spectroscopy by using the galactoside-specific plant toxin viscumin as a model lectin. The experimental part was complemented by molecular docking. The described synthetic route and the strategic combination of computational and experimental techniques to reveal conformational properties and bioactivity establish the prepared alpha-O-linked glycopeptide mimetics as promising candidates for further exploitation of this scaffold to give O-glycans for lectin blocking and vaccination.

Design In Silico, Synthesis and Binding Evaluation of a Carbohydrate-Based Scaffold for Structurally Novel Inhibitors of Matrix Metalloproteinases

The matrix metalloproteinases (MMPs) are members of a continuously growing family of Zn-dependent endopeptidases that function extracellularly. This clan of enzymes, specialized in endopeptidase activity, is involved in both normal and pathological tissue remodeling. Activation and over-expression of MMPs seem to be connected with pathological conditions such as arthritis, cardiovascular diseases, multiple sclerosis, and cancer-cell metastasis. Since preclinical studies clearly showed that the inhibition of MMPs would be therapeutic for such diseases, the generation of effective and selective inhibitors has become an extremely attractive goal. Early approaches to the identification of potential MMP inhibitors (i.e. substrate-based design of peptidomimetics and random screening of natural product or compound libraries) have recently been replaced by structure–activity relationship (SAR) studies. Deeper insights into enzyme–ligand interactions have been possible from SAR studies, and structure elucidation through X-ray and NMR spectroscopy of MMP catalytic domain/inhibitor complexes showed that the interaction of the inhibitor with zinc in the active site is very important in determining biological potency. Beside the active-site zinc(ii) ion, all MMPs are characterized by a hydrophobic cavity, conventionally designated the S1’ pocket, which offers the greatest opportunity for selective-inhibitor design because there is considerable variation between the MMPs in its dimensions and the residues that line the pocket. Thus peptidomimetics that incorporate a zinc ligand and S1’ side chain represent one of the most common classes of MMP inhibitors (i.e. Marimastat, Batimastat), while another class of zinc ligand compounds are sulfonamide-based inhibitors such as NNGH or AG3340.

Characterisation of the MMP-12-elastin adduct.

MMP-12 (matrix metalloproteinase 12)is an important protein of the MMP family. Its substrate is elastin, which is composed of a cross-linked insoluble network of polypeptide chains of tropoelastin of MW 65 kDa each. Insoluble elastin is responsible for keeping some extracellular connective tissues elastic. The full-length MMP-12 is made up by a catalytic (CAT) domain and a hemopexin-like (HPX) domain. The two domains have relatively large conformational freedom, as also recently found in MMP-1 and MMP-9. The role of the HPX domain is not certain, as the CAT domain is always active by itself, even if it may have a smaller turnover for some substrates. Experimental structures and models, representative of the binding mode of peptide substrates on protein active site are already present in the Protein Data Bank. However, the interaction of native elastin with full length MMP-12 and its isolated domains had not been structurally investigated so far. Here we report a NMR study of MMP-12 with elastin. MMP-12, with the catalytic zinc replaced by cadmium 15] and with the catalytically competent Glu219 replaced by alanine, is inactive and allows the investigation of its interaction with elastin without cleavage of the substrate. Insoluble elastin can be made soluble by limited proteolysis that provides a mixture of cross-linked polymers of several tens of kDa or more. The soluble mixture from bovine neck ligament (elastin hereafter) is the closest viable model to native elastin. This protein shares 76 % identity (excluding long insertions/deletions) with the human ortholog. The assignment of the C-, Hand N-labelled full-length MMP-12 protein is available. In the presence of elastin some H-N HSQC signals are shifted, as reported in Figure 1 A. Shifted signals are observed both in the CAT and HPX domains; in the former these are more numerous and a decrease in intensity of several signals is also observed. The spectra of the isolated CAT domain in the presence of elastin undergo similar shifts and intensity decreases as those observed for the fulllength protein (Figure 1 B) and the isolated HPX domain also maintains similar shifts (Figure 1 C). Comparison of Figures 1 B and 1C with 1A shows that both interactions are clearly operative separately, although an overall reinforcement of the interaction of the CAT domain in the fulllength protein may be noted, possibly brought about by a cooperative behaviour of the HPX domain. A comparison of the chemical shift changes with their signs in the fulllength protein and in the isolated domains is reported in Supporting Information (see Figure S1). The pattern of residues shifted upon addition of elastin is largely similar in the full length protein and in the isolated domains, but is more pronounced in the former (Figure S2). The overall pattern is consistent with a simultaneous interaction of both domains with the elastin polymer, also in view of the recently reported conformational flexibility of the two domains. While this finding suggests that the HPX domain may have a role in elastin recognition, it is clear that the isolated CAT domain is essentially able to recognise its natural substrate by itself. By analyzing in more detail the pattern of shifted NH nuclei in the isolated CAT domain, it is immediately apparent that the affected nuclei all belong to the active crevice and to the internal basal layer of the catalytic domain of MMP-12 (Figure 2). These results demonstrate the occur[a] Prof. I. Bertini, Dr. M. Fragai, Prof. C. Luchinat, Dr. M. Melikian, Dr. C. Venturi Magnetic Resonance Center (CERM), University of Florence Via L. Sacconi 6, 50019 Sesto Fiorentino (Italy) Fax: (+39) 0554574271 E-mail : bertini@cerm.unifi.it [b] Prof. I. Bertini Department of Chemistry, University of Florence Via della Lastruccia 3, 50019 Sesto Fiorentino (Italy) [c] Dr. M. Fragai, Prof. C. Luchinat Department of Agricultural Biotechnology, University of Florence Via Maragliano 75-77, 50144 Florence (Italy) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200901009.

Stereoselective Synthesis of 1,3-Disaccharides through Diels-Alder Reactions: Part 2[ 1 ]: Convenient Protecting Groups for Heterodienes and Conformational Evaluations

Improved synthesis of 1,3-disaccharides, obtained diastereomerically pure by [4+2] hetero cycloadditions between glycals and α -thiono-β -keto-δ -lactones, has been reported. The choice of appropriate protecting groups for β -keto-δ -lactones, stable under cycloaddition conditions employed, sensibly shortened the synthesis of heterodienic precursors. The first example of a β -oxy-imino-δ -lactone synthesized from β -keto-δ -lactones was also reported. Molecular modeling and conformational evaluations about the cycloaddition features allowed tentatively rationalizing the experimental results.