Stefano Roelens

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.

Chiral Diaminopyrrolic Receptors for Selective Recognition of Mannosides, Part 1: Design, Synthesis, and Affinities of Second‐Generation Tripodal Receptors

A new generation of chiral tripodal receptors for recognition of carbohydrates, featuring trans-1,2-diaminocyclohexane as a key structural element, and their recognition properties toward a set of glycosides of biologically relevant monosaccharides is described. The introduction of a chelating diamino unit into the pyrrolic tripodal architecture markedly enhanced their binding abilities compared with the parent aminopyrrolic receptors previously reported by our group. In addition, the chirality of the structure had a clear impact on affinities, as well as on selectivities, displaying high enantiodiscrimination levels. These second-generation diaminopyrrolic tripodal receptors are highly selective for mannose among other monosaccharides, with two members of the family being selective for the α and the β anomers respectively. The measured affinities in acetonitrile, 83 μM of (S)-7 for the β mannoside and 127 μM of (R)-5 for the α mannoside, make them the most effective synthetic receptors for mannosides reported to date. The affinity assessment required a further evolution of the BC(0)(50) parameter, a previously developed binding descriptor, which in its ultimate formulation has now been extended to include, with no restrictions, complexes of any stoichiometry, and can thus be generally employed to rank affinity data from heterogeneous systems on a common scale.

A TRPA1 antagonist reverts oxaliplatin-induced neuropathic pain

Neuropathic pain (NeP) is generally considered an intractable problem, which becomes compelling in clinical practice when caused by highly effective chemotherapeutics, such as in the treatment of cancer with oxaliplatin (OXA) and related drugs. In the present work we describe a structurally new compound, ADM_09, which proved to effectively revert OXA-induced NeP in vivo in rats without eliciting the commonly observed negative side-effects. ADM_09 does not modify normal behavior in rats, does not show any toxicity toward astrocyte cell cultures, nor any significant cardiotoxicity. Patch-clamp recordings demonstrated that ADM_09 is an effective antagonist of the nociceptive sensor channel TRPA1, which persistently blocks mouse as well as human variants of TRPA1. A dual-binding mode of action has been proposed for ADM_09, in which a synergic combination of calcium-mediated binding of the carnosine residue and disulphide-bridge-forming of the lipoic acid residue accounts for the observed persistent blocking activity toward the TRPA1 channel.

Chiral Diaminopyrrolic Receptors for Selective Recognition of Mannosides, Part 2: A 3D View of the Recognition Modes by X‐ray, NMR Spectroscopy, and Molecular Modeling

The structural features of a representative set of five complexes of octyl α- and β-mannosides with some members of a new generation of chiral tripodal diaminopyrrolic receptors, namely, (R)-5 and (S)- and (R)-7, have been investigated in solution and in the solid state by a combined X-ray, NMR spectroscopy, and molecular modeling approach. In the solid state, the binding arms of the free receptors 7 delimit a cleft in which two solvent molecules are hydrogen bonded to the pyrrolic groups and to the benzenic scaffold. In a polar solvent (CD(3)CN), chemical shift and intermolecular NOE data, assisted by molecular modeling calculations, ascertained the binding modes of the interaction between the receptor and the glycoside for these complexes. Although a single binding mode was found to adequately describe the complex of the acyclic receptor 5 with the α-mannoside, for the complexes of the cyclic receptors 7 two different binding modes were required to simultaneously fit all the experimental data. In all cases, extensive binding through hydrogen bonding and CH-π interactions is responsible for the affinities measured in the same solvent. Furthermore, the binding modes closely account for the recognition preferences observed toward the anomeric glycosides and for the peculiar enantiodiscrimination properties exhibited by the chiral receptors.

Competition between gelation and crystallisation of a peculiar multicomponent liquid system based on ammonium salts

An exemplar competition between gelation and crystallisation phenomena was examined with an unusual synergistic multicomponent (organo)gelator solution (MGS), which consists of a well-defined methanolic solution of (1R,2R)-1,2-diaminocyclohexane L-tartrate containing 2.4 equiv. of concentrated hydrochloric acid. The optimal composition of the MGS was determined through meticulous solubility, gelation and structural studies, which support a transient gelation mechanism based on the kinetic self-assembly of the tartrate salt driven by hydrogen-bonding interactions, involving ammonium nitrogen donors and hydroxyl oxygen acceptors, and electrostatic interactions. The hydrochloric acid is involved in the solubilisation of the salt through an ionic dissociation-exchange process, which ends up with the formation–precipitation of (1R,2R)-1,2-diaminocyclohexane dihydrochloride. As a consequence, an irreversible destruction of the gel takes place, which indicates the metastable nature of this phase that cannot be accessed from the thermodynamically equilibrated state. Gelation of a variety of oxygenated and nitrogenated solvents with moderate polarity occurred efficiently using extremely low MGS concentrations at low temperatures, and the gel phase was confirmed by dynamic rheological measurements. Several features make the described MGS unique: (1) it is a multicomponent solution where each component and its stoichiometry play a key role in the reproducible formation and stabilization of the gels; (2) it is formed by simple, small, and commercially available chiral building blocks (dissolved in a well-defined solvent system), which are easily amenable for further modifications; (3) the gelation phenomenon takes place efficiently at low temperature upon warming up the isotropic solution, conversely to the typical gel preparation protocol; and (4) the formed organogels are not thermoreversible despite the non-covalent interactions that characterize the 3D-network.

A High-Affinity Carbohydrate- Containing Inhibitor of Matrix Metalloproteinases

Matrix metalloproteinases (MMPs) are a class of Zn-containing hydrolases secreted by living cells and which are specialized in endopeptidase activity. MMPs participate in various biological processes such as embryonic development, wound healing, nerve growth, and angiogenesis. Aberrant MMP activities, inducing excessive degradation of the extracellular matrix, are involved in the genesis of diseases such as cancer, rheumatoid arthritis, pulmonary emphysema, and skin ulceration. Since many of these pathologies may benefit from the control of MMP activity, the quest for suitable human MMP inhibitors (MMPIs) has been actively pursued for more than a decade. Many inhibitors endowed with high affinity but modest selectivity, based on a variety of molecular scaffolds, have been reported. Some of these inhibitors have entered into clinical trials for different indications, primarily cancer and arthritis. However, high affinity is often achieved by introducing lipophilic substituents on suitable binding scaffolds, thereby decreasing solubility in water and compromising oral bioavailability. Solubility in water is required for maintaining high drug levels in plasma, which is essential for treatments relying on oral administration. Only very few of the plethora of potentially useful MMPIs reported to date are water soluble. Doses of drug higher than those based on intrinsic efficacy must be administered because of limited bioavailability. High doses, in turn, exacerbate the adverse effects of modest selectivity by causing indiscriminate inhibition of other zinc endopeptidases. Furthermore, MMP-related pathologies are usually chronic and any plausible pharmacological scheme would require longterm treatment, during which time lipophilic drugs tend to accumulate in tissues and thereby enhance side effects. High lipophilicity also increases binding affinity to human serum albumin (HSA). Strong binding to HSA has an adverse effect on bioavailability by increasing the half-life in vivo and preventing the drug from reaching the target site. In the present communication we report a new high-affinity MMP inhibitor that addresses most of the fundamental issues discussed above, using a conceptually novel strategy whereby a glycosidic residue is introduced in the appropriate location of the molecule. The inhibitor is a prototype structure that opens the way to the design of a new class of highly effective MMPIs. N-Isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid (NNGH) is one of the most prominent representatives of a family of inhibitors possessing nanomolar affinity for several MMPs (Scheme 1). The NNGH family of inhibitors suffers from the major drawbacks discussed above, and is therefore inadequate for most applications. The recently published X-ray crystallographic structure of the NNGH–MMP-12 complex allowed us to ascertain that the interaction of the inhibitor with the active site of the enzyme involves binding of the hydroxamate moiety to the catalytic Zn ion and binding of the aromatic group to the S1’ subsite (MMP-12 is the enzyme implicated in the development of emphysema). The isopropyl group on the sulfonamide nitrogen atom points away from the shallow S2’ pocket and does not directly participate in binding. In an effort to overcome the limitations of NNGH, this structural information was used to prepare the new inhibitor 1, as depicted in Scheme 1, whereby the isopropyl group was replaced with a glucosylated N-hydroxyethyl chain. The leading concept in designing 1 was to replace a portion of the molecule not directly involved in binding with a watersoluble residue, linked through a spacer of appropriate length, in the hope that its inhibiting properties would not be affected. It must be emphasized that adding a carbohydrate residue to the inhibitor is unprecedented in the NNGH family. The diasteromerically pure b anomer of compound 1 was selectively obtained in good yield (26% over five steps) by reaction of trichloroacetimidate 2 with the hydroxyethyl sulfonamide 3 under Schmidt’s glycosylation conditions. Compound 3 was obtained by treatment of the corresponding sulfonamide of the glycine methylester 4 with ethylene oxide and methyl iodide. The synthesis, which produces the glycosidic b isoACHTUNGTRENNUNGmer exclusively, has been specifically devised as a general method easily amenable to the preparation of the desired derivatives (see below) on a multigram scale through the appropriately substituted imidate. As expected, compound 1 exhibited a marked increase in water solubility (>30 mm) compared with NNGH, which is essentially insoluble. Even more significantly, the sugar moiety of structure 1 is easily changed to adjust hydrophilicity. Indeed, the solubility of 1 can be scaled down in a stepwise manner by sequentially substituting non-hydrophilic substituents [a] Dr. V. Calderone, Dr. M. Fragai, Prof. C. Luchinat, Prof. C. Nativi Magnetic Resonance Center, University of Florence via Sacconi 6, Sesto Fiorentino 50019 (Italy) Fax: (+39)0554574271 E-mail : luchinat@cerm.unifi.it [b] Prof. C. Nativi, Dr. B. Richichi Department of Organic Chemistry, University of Florence via della Lastruccia 13, Sesto Fiorentino 50019 (Italy) Fax: (+39)0554573570 E-mail : cristina.nativi@unifi.it [c] Dr. V. Calderone Department of Chemistry, University of Siena, Siena (Italy) [d] Dr. M. Fragai, Prof. C. Luchinat Department of Agricultural Biotechnology, University of Florence, Florence (Italy) [e] Prof. C. Luchinat, Dr. B. Richichi ProtEra S.r.l. , Sesto Fiorentino 50019 (Italy) [f] Dr. S. Roelens CNR—Istituto di Chimica dei Composti Organometallici, Florence (Italy) Supporting information for this article is available on the WWW under http://www.chemmedchem.org or from the author: synthetic procedures and characterization details ; crystallographic and NMR spectroscopic data; fluorimetric details.

Aminopyrrolic synthetic receptors for monosaccharides: a class of carbohydrate-binding agents endowed with antibiotic activity versus pathogenic yeasts.

The biological activity of a set of structurally related aminopyrrolic synthetic receptors for monosaccharides has been tested versus yeast and yeast-like microorganisms and compared to their binding affinity toward mannosides. Antibiotic activity comparable to that of well-known polyene (amphotericin B) or azole (ketoconazole) drugs has been found for some members of the family, along with a general correlation with binding abilities. A systematic structure-activity-affinity investigation shed light on the structural and functional requirements necessary for antibiotic activity and identified the tripodal compound 1 as the most potent compound of the set. Together with toxicity tests and inhibitor localization experiments performed through fluorescence microscopy, these studies led to the characterization of a new class of carbohydrate binding agents possessing antibiotic activity, in which pyrrolic groups precisely structured on a tripodal architecture appear to be responsible for permeability through the cell wall of pathogens, as well as for antibiotic activity inside the cytoplasm.

Synthetic tripodal receptors for carbohydrates. Pyrrole, a hydrogen bonding partner for saccharidic hydroxyls.

The carbohydrate recognition properties of synthetic tripodal receptors relying on H-bonding interactions have highlighted the crucial role played by the functional groups matching saccharidic hydroxyls. Herein, pyrrole and pyridine, which emerged as two of the most effective H-bonding groups, were quantitatively compared through their isostructural substitution within the architecture of a shape-persistent bicyclic cage receptor. NMR and ITC binding studies gave for the pyrrolic receptor a 20-fold larger affinity toward octyl-β-d-glucopyranoside in CDCl(3), demonstrating the superior recognition properties of pyrrole under conditions in which differences would depend on the intrinsic binding ability of the two groups. The three-dimensional structures of the two glucoside complexes in solution were elucidated by combined NMR and molecular mechanics computational techniques, showing that the origin of the stability difference between the two closely similar complex structures resides in the ability of pyrrole to establish shorter/stronger H-bonds with the glucosidic ligand compared to pyridine.

13C NMR studies of D- and L-phenylalanine binding to cobalt (II) carboxypeptidase A

13C NMR T1 and T2 measurements have been performed on cobalt(II) substituted carboxypeptidase A in the presence of carboxylate-13C-enriched L- and D-phenylalanine. Upon binding to the cobalt enzyme, the longitudinal and transverse relaxation rates T1p-1 and T2p-1 of these inhibitors are enhanced significantly compared to the zinc enzyme, allowing both determination of an affinity constant for inhibitor binding, K, and calculation of the metal-13C carboxylate distances. The L-and D- Phe concentration dependence of T2p-1 yields affinity constants of 290 +/- 60M-1 and 670 +/- 90M-1. The distance measurements calculated for Co-13C from T1p-1 are 0.39 +/- 0.04 and 0.42 +/- 0.04 nm for L-Phe and D-Phe. Both values are too great for direct coordination of their carboxylate groups to the metal atom. Upon formation of their respective ternary enzyme.Phe.N3- complexes, the distances are essentially unaltered. In conjunction with electronic absorption studies on these complexes it can be concluded that N3-, but not the amino acid carboxylate, is bound to the metal.

Systematic Dissection of an Aminopyrrolic Cage Receptor for β‐Glucopyranosides Reveals the Essentials for Effective Recognition

A set of structures designed for the recognition of glucosides has been obtained by systematically destructuring a tripodal aminopyrrolic cage receptor that selectively recognizes octyl-β-D-glucopyranoside (OctβGlc). NMR spectroscopy and isothermal titration calorimetry binding measurements showed that cleavage of one pillar of the cage was beneficial to the binding properties of the receptor, as long as two residual amino groups of the cleaved pillar were present. Removal of these two residual amino groups produced a dramatic loss of affinity for OctβGlc of the resulting monocyclic analogue of the parent cage receptor. A significant improvement in the binding ability was achieved by replacing one pillar with two aminopyrrolic hydrogen-bonding arms, despite the loss of a preorganized structure. In contrast to the cage receptor, recognition of OctβGlc was observed, even in a competitive medium (30 % DMF in chloroform). Structural studies in solution, carried out through NMR spectroscopy and molecular modeling calculations, led to the elucidation of the 3D binding modes of the side-armed monocyclic receptors; this highlighted the key role of the amino groups and demonstrated the occurrence of a rotaxane-like complex, which featured the octyl chain of the glucoside threaded through the macrocyclic ring.