Does size matter? - Comparing pyranoses with septanoses as ligands of the bacterial lectin FimH.

The pharmacological modulation of disease-relevant carbohydrate-protein interactions represents an underexplored area of medicinal chemistry. One particular challenge in the design of glycomimetic compounds is the inherent instability of the glycosidic bond toward enzymatic cleavage. This problem has traditionally been approached by employing S-, N-, or C-glycosides with reduced susceptibility toward glycosidases. The application of ring-extended glycomimetics is an innovative approach to circumvent this issue. On the example of the bacterial adhesin FimH, it was explored how design principles from pyranose glycomimetics transfer to analogous septanose structures. A series of ring-extended FimH antagonists exhibiting the well-proven pharmacophore necessary for targeting the tyrosine-gate of FimH was synthesized. The resulting septanoses were evaluated for their affinity to the conformationally rigid isolated lectin domain of FimH (FimHLD), as well as a structurally flexible full-length FimH (FimHFL) construct. Some elements of potent mannoside-based FimH antagonists could be successfully transferred to septanose-based ligands, ultimately resulting in a 32-fold increase in binding affinity. Interestingly, the canonical ca. 100-fold loss of binding affinity between FimHLD and FimHFL is partly mitigated by the more flexible septanose antagonists, hinting at potentially differing interaction features of the flexible glycomimetics with intermediately populated states during the conformational transition of FimHFL.

Characterization and Fate of a Septanosyl Ferrier Cation in the Gas and Solution Phases.

Ferrier reactions follow a mechanistic pathway whereby Lewis acid activation of a cyclic enol ether facilitates departure of an allylic leaving group to form a glycosyl Ferrier cation. Attack on the Ferrier cation provides a new acetal linkage concurrent with the transposition of the alkene moiety. The idiosyncratic outcomes of Ferrier reactions of seven-membered ring carbohydrate-based oxepines prompted an investigation of its corresponding septanosyl Ferrier cation. Experiments that characterized the ion, including gas-phase cryogenic IR spectroscopy matched with density functional theory-calculated spectra of candidate cation structures, as well as product analysis from solution-phase Ferrier reactions, are reported here. Results from both approaches revealed an inclination of the seven-membered ring cation to contract to five-membered ring structures. Gas-phase IR spectra matched best to calculated spectra of structures in which five-membered dioxolenium formation opened the oxepine ring. In the solution phase, an attack on the ion by water led to an acyclic enal that cyclized to a C-methylene-aldehydo arabinofuranoside species. Attack by allyl trimethylsilane, on the other hand, was diastereoselective and yielded a C-allyl septanoside.

Synthesis and Characterization of Preacinetobactin and 5-Phenyl Preacinetobactin.

We report the first total synthesis of 5-phenyl preacinetobactin and its characterization. The route was developed for the synthesis of preacinetobactin, the siderophore critical to the Gram-negative pathogen A. baumannii. It leverages a C5-substituted benzaldehyde as a key starting material and should enable the synthesis of similar analogs. 5-Phenyl preacinetobactin binds iron in a manner analogous to the natural siderophore, but it did not rescue growth in a strain of A. baumannii unable to produce preacinetobactin.

Synthesis of a Diacetonide-Protected, Mannose-Based Oxepine: Configurational Control of Anomeric Acetate Activation.

Carbohydrate-based oxepines are seven-membered-ring oxacycles containing an enol ether moiety. These compounds have been used as intermediates in the preparation of septanose carbohydrates by functionalization through their double bond. Reported here is a new synthesis of a carbohydrate based oxepine that uses 2,3;4,6-di-O-acetonide mannose as a key starting material. The oxepine is an important precursor used in the synthesis of septanose glycomimetics of mannopyranosides. The central feature of the synthesis is a two-step sequence that converts a septanose 1,2-di-O-acetate to the septanosyl bromide and onward to the oxepine via a reductive elimination.

Characterization of the low energy conformations and differential reactivities of D-glucose and D-mannose based oxepines.

Carbohydrate-based oxepines are valuable intermediates for the synthesis of septanose carbohydrates. Here we report the characterization of the preferred conformations of D-glucose and D-mannose based oxepines 1 and 2 using computational chemistry and NMR spectroscopy. Monte Carlo conformational searches on 1 and 2 were performed, followed by DFT optimization and single-point energy calculations on the low energy conformations of each oxepine. Coupling constants were computed for all unique conformations at a B3LYP/6-31G(d,p)u+1s level of theory and weighted based on a Boltzmann distribution. These values were then compared to the experimental values collected using 3JH,H values collected from 1H NMR spectra. Information from the MC/DFT approach was then used in a least squares method that correlated DFT calculated and observed 3JH,H coupling constants. The conformations of 1 and 2 are largely governed by a combination of the rigidifying enol ether element in combination with the reduction of unfavorable interactions. The vinylogous anomeric effect (VAE) emerged as a consequence, rather than a driver of conformations. Oxepine 1 showed greater reactivity in Ferrier rearrangement reactions relative to oxepine 2, in line with its greater %VAE.

Indolyl Septanoside Synthesis for In Vivo Screening of Bacterial Septanoside Hydrolases.

Building-up and breaking-down of carbohydrates are processes common to all forms of life. Glycoside hydrolases are a broad class of enzymes that play a central role in the cleavage of glycosidic bonds, which is fundamental to carbohydrate degradation. The large majority of substrates are five- and six-membered ring glycosides. Our interest in seven-membered ring septanose sugars has inspired the development of a way to search for septanoside hydrolase activity. Described here is a strategy for the discovery of septanoside hydrolases that uses synthetic indolyl septanosides as chromogenic substrates. Access to these tool compounds was enabled by a route where septanosyl halides act as glycosyl donors for the synthesis of the indolyl septanosides. The screening strategy leverages the known dimerization of 3-hydroxy-indoles to make colored dyes, as occurs when the ß-galactosidase substrate X-Gal is hydrolyzed. Because screens in bacterial cells would enable searches in organisms that utilize heptoses or from metagenomics libraries, we also demonstrate that septanosides are capable of entering E. coli cells through the use of a BODIPY-labeled septanoside. The modularity of the indolyl septanoside synthesis should allow the screening of a variety of substrates that mimic natural structures via this general approach.

Exploring the physicochemical and antiproliferative properties of biaryl-linked [13]-macrodilactones.

A macrocyclic motif fosters productive protein-small molecule interactions. There are numerous examples of both natural product and designed, synthetic macrocycles that modulate the immune system, slow microbial infection, or kill eukaryotic cells. Reported here are the synthesis, physicochemical characterization, and antiproliferative activity of a group of [13]-macrodilactones decorated with a pendant biaryl moiety. Biaryl analogs were prepared by Suzuki reactions conducted on a common intermediate that contained a bromophenyl unit alpha to one of the carbonyls of the [13]-macrodilactone. Principal component analysis placed the new compounds in physicochemical context relative to a variety of pharmaceuticals and natural products. Modest inhibition of proliferation was observed in ASZ cells, a murine basal cell carcinoma line. This work underscores the value of an approach toward the identification of bioactive compounds that places the evaluation of physicochemical parameters early in the search process.

Structures of three disubstituted [13]-macro-di-lac-tones reveal effects of substitution on macrocycle conformation.

The synthesis and crystal structures of three new disubstituted [13]-macro-di-lactones, namely, trans-4,8-dimethyl-1,10-dioxa-cyclo-tridec-5-ene-2,9-dione, C13H20O4, I, cis-4-(4-bromo-phen-yl)-13-methyl-1,10-dioxa-cyclo-tridec-5-ene-2,9-dione C18H21BrO4, II, and trans-11-methyl-4-phenyl-1,10-dioxa-cyclo-tridec-5-ene-2,9-dione, C18H22O4, III, are reported and their conformations are put in the context of other [13]-macrodilactone structures reported previously. Together, they show that the number, location, and relative disposition of groups attached at the termini of planar units of the [13]-macrodilactones subtly influence their aspect ratios.

Post-Glycosylation Diversification (PGD): An Approach for Assembling Collections of Glycosylated Small Molecules.

Many small molecule natural products with antibiotic and antiproliferative activity are adorned with a carbohydrate residue as part of their molecular structure. The carbohydrate moiety can act to mediate key interactions with the target, attenuate physicochemical properties, or both. Facile incorporation of a carbohydrate group on de novo small molecules would enable these valuable properties to be leveraged in the evaluation of focused compound libraries. While there is no universal way to incorporate a sugar on small molecule libraries, techniques such as glycorandomization and neoglycorandomization have made signification headway toward this goal. Here, we report a new approach for the synthesis of glycosylated small molecule libraries. It puts the glycosylation early in the synthesis of library compounds. Functionalized aglycones subsequently participate in chemoselective diversification reactions distal to the carbohydrate. As a proof-of-concept, we prepared several desosaminyl glycosides from only a few starting glycosides, using click cycloadditions, acylations, and Suzuki couplings as diversification reactions. New compounds were then characterized for their inhibition of bacterial protein translation, bacterial growth, and in a T-cell activation assay.

The price of flexibility - a case study on septanoses as pyranose mimetics.

Seven-membered ring mimetics of mannose were studied as ligands for the mannose-specific bacterial lectin FimH, which plays an essential role in the first step of urinary tract infections (UTI). A competitive binding assay and isothermal titration calorimetry (ITC) experiments indicated an approximately ten-fold lower affinity for the seven-membered ring mannose mimetic 2-O-n-heptyl-1,6-anhydro-d-glycero-d-galactitol (7) compared to n-heptyl α-d-mannopyranoside (2), resulting exclusively from a loss of conformational entropy. Investigations by solution NMR, X-ray crystallography, and molecular modeling revealed that 7 establishes a superimposable H-bond network compared to mannoside 2, but at the price of a high entropic penalty due to the loss of its pronounced conformational flexibility. These results underscore the importance of having access to the complete thermodynamic profile of a molecular interaction to "rescue" ligands from entropic penalties with an otherwise perfect fit to the protein binding site.

Epitope-Resolved Detection of Peanut-Specific IgE Antibodies by Surface Plasmon Resonance Imaging.

Peanut allergy can be life-threatening and is mediated by allergen-specific immunoglobulin E (IgE) antibodies. Investigation of IgE antibody binding to allergenic epitopes can identify specific interactions underlying the allergic response. Here, we report a surface plasmon resonance imaging (SPRi) immunoassay for differentiating IgE antibodies by epitope-resolved detection. IgE antibodies were first captured by magnetic beads bearing IgE ϵ-chain-specific antibodies and then introduced into an SPRi array immobilized with epitopes from the major peanut allergen glycoprotein Arachis hypogaea h2 (Ara h2). Differential epitope responses were achieved by establishing a binding environment that minimized cross-reactivity while maximizing analytical sensitivity. IgE antibody binding to each Ara h2 epitope was distinguished and quantified from patient serum samples (10 µL each) in a 45 min assay. Excellent correlation of Ara h2-specific IgE values was found between ImmunoCAP assays and the new SPRi method.

Macrocycles All Aflutter: Substitution at an Allylic Center Reveals the Conformational Dynamics of [13]-Macrodilactones.

The shapes adopted by large-ring macrocyclic compounds play a role in their reactivity and their ability to be bound by biomolecules. We investigated the synthesis, conformational analysis, and properties of a specific family of [13]-macrodilactones as models of natural-product macrocycles. The features of our macrodilactones enabled us to study the relationship between stereogenic centers and planar chirality through the modular synthesis of new members of this family of macrocycles. Here we report on insights gained from a new [13]-macrodilactone that is substituted at a position adjacent to the alkene in the molecule. Analysis of the compound, in comparison to an α-substituted regioisomer, by using X-ray crystallography, NMR coupling constants, and reaction-product characterization in concert with computational chemistry, revealed that the alkene unit is dynamic. That is, the data support a model in which the alkene in our [13]-macrodilactones oscillates between two conformations. A difference in reactivity of one conformation compared to the other leads to manifestation of this dynamic behavior. The results underscore the local conformational dynamics observed in some natural-product macrocycles, which could have implications for biomolecule binding.

Unequivocal determination of complex molecular structures using anisotropic NMR measurements.

Assignment of complex molecular structures from nuclear magnetic resonance (NMR) data can be prone to interpretational mistakes. Residual dipolar couplings and residual chemical shift anisotropy provide a spatial view of the relative orientations between bonds and chemical shielding tensors, respectively, regardless of separation. Consequently, these data constitute a reliable reporter of global structural validity. Anisotropic NMR parameters can be used to evaluate investigators' structure proposals or structures generated by computer-assisted structure elucidation. Application of the method to several complex structure assignment problems shows promising results that signal a potential paradigm shift from conventional NMR data interpretation, which may be of particular utility for compounds not amenable to x-ray crystallography.

Synthesis and structure of a carbohydrate-fused [15]-macrodilactone.

The design, synthesis and structural characterization of a new α-d-glucose fused [15]-macrodilactone is reported. The macrolide was synthesized by a route involving sequential acylations of glucose at the C4' and C6' hydroxyl groups followed by an intramolecular Stille reaction previously established for other [15]-macrodilactones. Analysis of the X-ray crystallographic structure of the macrolide revealed a unique conformation of this macrocycle that differs from earlier models for [13]- and [15]-macrodilactones. Organizing the three planar units and the pyranose moiety into a macrocyclic ring resulted in a cup-shaped structure with planar chirality. Further, the gt conformation of the exocyclic hydroxymethyl group in the glucose unit was found to be crucial for controlling the planar chirality and, hence, governing the molecular shape and overall topology of the compound.

A practical and scalable synthesis of carbohydrate based oxepines.

An efficient, seven-step synthesis of carbohydrate based oxepines is reported using per-O-acetyl septanoses as key intermediates. The scope of the synthesis was evaluated by varying both the pyranose starting materials and protecting groups incorporated into the oxepine products. The practicality of the method make it amenable to scale up as demonstrated by the gram-scale synthesis of the d-glucose derived oxepine.

Rules of Macrocycle Topology: A [13]-Macrodilactone Case Study.

Shape is an inherent trait of a molecule that dictates how it interacts with other molecules, either in binding events or intermolecular reactions. Large-ring macrocyclic compounds in particular leverage their shape when they are selectively bound by biomolecules and also when they exhibit macrocyclic diastereoselectivity. Nonetheless, rules that link structural parameters to the conformation of a macrocycle are still rudimentary. Here we use a structural investigation of a family of [13]-macrodilactones as a case study to develop rules that can be applied generally to macrocycles of different sizes and with a variety of functionality. A characteristic "ribbon" shape is adopted by the [13]-macrodilactones in the absence of stereogenic centres, which exhibits planar chirality. When one stereogenic centre at key positions on the backbone is incorporated into the structure, the planar chirality is dictated by the configuration of the centre. In cases where two stereogenic centres are present, their relationships can either reinforce the characteristic ribbon shape or induce alternative shapes to be adopted. The rules established in the case study are then applied to the analysis of a structure of the natural product migrastatin. They lay the groundwork for the development of models to understand macrocycle-biomolecule interactions and for the preparation of macrocycles with designed properties and activities.

Turning Spiroketals Inside Out: A Rearrangement Triggered by an Enol Ether Epoxidation.

Invited for this month's cover picture is the group of Professor Mark Peczuh at the University of Connecticut. The cover picture compares the rearrangement of a small molecule to the process of turning a stuffed animal inside out. The recycled, inside-out stuffed animals are both artistic and philosophically provocative. They capture the essence of the rearrangement reaction because the compounds themselves turn inside out over the course of the reaction, extending the diversity of products that can arise from simple starting materials. Small molecules often have functional groups with latent reactivity; under the appropriate conditions, those groups can react with other compounds (e.g., reagents) and also with other groups in the same molecule in an intramolecular reaction. The research team found that the epoxidation of some highly functionalized spiroketal compounds promoted rearrangements of their structures that turned them inside out. Some of the features of the products led them to use X-ray crystallography or a combination of computer-assisted structure elucidation, computation, and a new version of the 1,1-ADEQUATE NMR experiment to determine their structures. For more details, see the Communication on p. 577 ff.

Turning Spiroketals Inside Out: A Rearrangement Triggered by an Enol Ether Epoxidation.

Spiroketals organize small molecule structures into well-defined, three-dimensional configurations that make them good ligands of proteins. We recently discovered a tandem cycloisomerization-dimerization reaction of alkynyl hemiketals that delivered polycyclic, enol-ether-containing spiroketals. Here we describe rearrangements of those compounds, triggered by epoxidation of their enol ethers that completely remodel their structures, essentially turning them "inside out". Due to the high level of substitution on the carbon skeletons of the substrates and products, characterization resorted to X-ray crystallography and advanced computation and NMR techniques to solve the structures of representative compounds. In particular, a new proton-detected ADEQUATE NMR experiment (1,1-HD-ADEQUATE) enabled the unequivocal assignment of the carbon skeleton of one of the new compounds. Solution of the structures of the representative compounds allowed for the assignment of product structures for the other compounds in two separate series. Both the rearrangement and the methods used for structural determination of the products are valuable tools for the preparation of characterization of new small molecule compounds.

Synthesis, structure and reactivity of [15]-macrodilactones.

A strategy for utilizing parameters such as the ring size, planar units and the connections between them, and the location of asymmetric centers has been applied to the design and synthesis of a new class of 15-membered macrocycles. The interplay between three planar units in combination with a hinge atom and a stereogenic center, introduces a planar chirality that defines the molecular topology of these [15]-macrodilactones.

Stereogenic α-carbons determine the shape and topology of [13]-macrodilactones.

The synthesis and characterization of new [13]-macrodilactones substituted at stereogenic centers α- to the carbonyl are reported. When one center is substituted, it directs the topology of the macrocycle; when two centers are substituted, both the shape and the topology are influenced. The findings indicate that the number and configuration of α-centers fine-tune macrocyclic structure.