Structure Elucidation of Macrolide Antibiotics Using MSn Analysis and Deuterium Labelling.

The 14- and 16-membered macrolide antibiotics are an important structural class. Ubiquitously produced by a number of bacterial strains, namely actinomycetes, purification and structure elucidation of the wide array of analogs is challenging, both for discovery efforts and methodologies to monitor for byproducts, metabolites, and contaminants. Collision-induced dissociation mass spectrometry offers an attractive solution, enabling characterization of mixtures, and providing a wealth of structural information. However, interpretation of these spectra can be difficult. We present a study of 14- and 16-membered macrolide antibiotics, including MSn analysis for unprecedented depth of coverage, and complimentary analysis with D2O and H218O labeling to elucidate fragmentation mechanisms. These analyses contrast the behaviors of varying classes of macrolides and highlight how analogues can be identified in relation to similar structures, which will provide utility for future studies of novel macrolides, as well as impurities, metabolites, and degradation products of pharmaceuticals. Graphical Abstract.

Regiodivergent Glycosylations of 6-Deoxy-erythronolide B and Oleandomycin-Derived Macrolactones Enabled by Chiral Acid Catalysis.

This work describes the first example of using chiral catalysts to control site-selectivity for the glycosylations of complex polyols such as 6-deoxyerythronolide B and oleandomycin-derived macrolactones. The regiodivergent introduction of sugars at the C3, C5, and C11 positions of macrolactones was achieved by selecting appropriate chiral acids as catalysts or through introduction of stoichiometric boronic acid-based additives. BINOL-based chiral phosphoric acids (CPAs) were used to catalyze highly selective glycosylations at the C5 positions of macrolactones (up to 99:1 rr), whereas the use of SPINOL-based CPAs resulted in selectivity switch and glycosylation of the C3 alcohol (up to 91:9 rr). Additionally, the C11 position of macrolactones was selectively functionalized through traceless protection of the C3/C5 diol with boronic acids prior to glycosylation. Investigation of the reaction mechanism for the CPA-controlled glycosylations revealed the involvement of covalently linked anomeric phosphates rather than oxocarbenium ion pairs as the reactive intermediates.

Deconstruction-reconstruction strategy for accessing valuable polyketides. Preparation of the C15-C24 stereopentad of discodermolide.

An advanced, known intermediate for discodermolide synthesis was prepared by an efficient sequence from the readily available fermentation product oleandomycin. The scheme makes use of a new method for the direct cleavage of aminoglycosides, a critical double-bond isomerization, and a selective protection of two of three hydroxyl groups in a modified oleandolide. This synthesis illustrates a new strategy, "deconstruction-reconstruction", for accessing stereochemically complex polyketide building blocks.

Systematic approach to understanding macrolide-ribosome interactions: NMR and modeling studies of oleandomycin and its derivatives.

The three-dimensional structures of oleandomycin (1) and its derivatives oleandomycin-9-oxime (2) and 10,11-anhydrooleandomycin (3) were determined in different solvents by the combined use of NMR and molecular modeling methods. The experimental NMR data were compared with the results of molecular modeling and known crystal structures of the related molecules. It was shown that the dominant conformation of the lactone ring is the folded-out conformation with some amounts of the folded-in one depending on the solvent and temperature, while desosamine and cladinose sugars adopt the usual chair conformations. Modeling calculations provided evidence for conformational changes in the upper lactone region as well. Saturation transfer difference (STD) NMR experiments have provided information on the binding epitopes of 1-3 in complexes with E. coli ribosomes. The obtained molecular surfaces in close contact with ribosomes were compared with recently available 3D structures of the related macrolide-ribosome complexes, and the observed differences were discussed. The knowledge gained from this study can serve as a platform for the design of novel macrolides with an improved biological profile.

Identification and antibacterial activity of a new oleandomycin derivative from Streptomyces antibioticus.

During the study on the oleandomycin production, we purified a new oleandomycin derivative having a macrolactone of which biosynthesis does not follow the genetic architecture of the oleandomycin PKS. The molecular formula for the compound was suggested as C35H59NO11 on the basis of the analysis of NMR and HRMS data (m/z 670.4185, Delta-1.9mmu, calcd for C35H60NO11). 13C NMR assignments and analysis of COSY, HMBC and HMQC data suggested that the compound differs from oleandomycin by formation of the olefinic functionality resulting from the dehydration of a hydroxy group in oleandomycin. The new oleandomycin derivative has antibacterial activities similar to those of oleandomycin agaisnt Enterococcus faecalis, Bacillus subtilis and Staphylococcus aureus.

Conformational analysis of oleandomycin and its 8-methylene-9-oxime derivative by NMR and molecular modelling.

Conformations of the 14-membered macrolide antibiotic oleandomycin and its 8-methylene-9-oxime derivative were determined in various solvents. The experimental NMR data--coupling constants and NOE contacts--were compared with the results of molecular modelling--molecular mechanics calculations and molecular dynamics simulations. The conformational changes, on the right-hand side of the 14-membered ring, affected mostly the 3JH2,H3 values and NOE crosspeaks H3 or H4 to H11. Oleandomycin was found to be present predominantly in the C3-C5 folded-in conformations in DMSO-d6 solution, whereas in buffered D2O, acetone-d6 and CDCl3, there was a mixture of folded-in and folded-out conformational families. The predominant conformation of the 8-methylene-oleandomycin-9-oxime derivative in solution was a folded-out one although different amounts of folded-in conformation were also present depending on the solvent. Oleandrose and desosamine sugar moieties adopted the usual and expected chair conformation. The conformation around the glycosidic bonds, governing the relative orientation of sugars vs. the lactone ring, showed a certain flexibility within two conformationally close families. We believe that by combining the experimental NMR data and the molecular modelling techniques, as reported in this paper, we have made significant progress in understanding the conformational behaviour and properties of macrolides. Our belief is based on our own current studies on oleandomycins as well as on the previously reported results and best practices concerning other macrolides. A rational for macrolide conformational studies and advances in methodology has been suggested accordingly.

Comparative study of coulometric and amperometric detection for the determination of macrolides in human urine using high-performance liquid chromatography.

A high-performance liquid chromatography (HPLC) method using chromatographic conditions optimised in a previous work was applied for the separation of three macrolide antibiotics roxithromycin (Rox), oleandomycin (Ole) and rosamicin (Ros) and further determination of two of them, roxithromycin (Rox) and oleandomycin (Ole), in human urine samples. A comparative study of the behaviour of these macrolides under the two types of electrochemical detection (EC) widely coupled with HPLC, that is coulometric (EC-C) and amperometric (EC-A), was carried out by applying the same multiresidue method. From the assays performed using both detectors the comparison was made taking relevant criteria such as detection limits, linearity, recovery and precision values into account. As a result of this comparison, the coulometric detector appears slightly more suitable than the amperometric one for macrolide analysis.

Inversion of the anomeric configuration of the transferred sugar during inactivation of the macrolide antibiotic oleandomycin catalyzed by a macrolide glycosyltransferase.

Macrolides are a group of antibiotics structurally characterized by a macrocyclic lactone to which one or several deoxy-sugar moieties are attached. The sugar moieties are transferred to the different aglycones by glycosyltransferases (GTF). The OleI GTF of an oleandomycin producer, Streptomyces antibioticus, catalyzes the inactivation of this macrolide by glycosylation. The product of this reaction was isolated and its structure elucidated. The donor substrate of the reaction was UDP-alpha-D-glucose, but the reaction product showed a beta-glycosidic linkage. The inversion of the anomeric configuration of the transferred sugar and other data about the kinetics of the reaction and primary structure analysis of several GTFs are compatible with a reaction mechanism involving a single nucleophilic substitution at the sugar anomeric carbon in the catalytic center of the enzyme.

Separation and determination of the macrolide antibiotics (erythromycin, spiramycin and oleandomycin) by capillary electrophoresis coupled with fast reductive voltammetric detection.

Separation and determination of erythromycin, spiramycin and oleandomycin by capillary zone electrophoresis coupled with fast reductive voltammetric detection using an Hg-film electrode was investigated in a simple aqueous phosphate buffer system. The influence of pH, concentration of phosphate, applied voltage, capillary length and dimension on the separation was examined and optimized. The entire separation of erythromycin, spiramycin, and oleandomycin was achieved in a 0.2 mol/L phosphate buffer system without organic modifiers. The electrochemical detection parameters, such as electrode material, applied waveform, scan rate, preconcentration potentials and preconcentration times, were investigated and discussed. This approach provides high separation efficiency and high sensitivity for all compounds, with detection limits (3 x peak-to-peak baseline noise) of 7.5 x 10(-8) mol/L for spiramycin, and 3 x 10(-7) mol/ L for erythromycin and oleandomycin. The calibration plot of peak areas for each separated peak vs. concentration of analyte was found to be linear over three orders of magnitude.

[Determination of 1H- or 13C NMR spectra of oleandomycin (OL), the esterolitic cleavage compound of OL and OL 2'-phosphate using two-dimensional methods in D2O solution].

All signals of 1H- and 13C NMR spectra of oleandomycin and the esterolitic cleavage compound of oleandomycin, and all signals of 1H-NMR spectra of oleandomycin 2'-phosphate were determined using two-dimensional methods as 1H-1H and 13C-1H COSY NMR, and DEPT NMR in D2O solution. The two modified products of oleandomycin were prepared by two strains of Escherichia coli highly resistant to erythromycin. These results are basically useful in determination of the structure of novel metabolites of oleandomycin inactivated by bacterial action.

New oleandomycin 9-oximes. Synthesis, characterization and biological activity.

A series of the novel oleandomycin 9-oximes has been prepared and characterized by spectroscopic data and X-ray analysis. The antibacterial in vitro activities of the oximes (6-10) were compared with that of oleandomycin (1). Among the novel derivatives the most active compound was 8(R)-methyloleandomycin-9-oxime (9) in contrast ot its 8(S)-isomer (10) which possessed only low potency. Some preliminary pharmacokinetic data of 9 confirmed its activity. Compound 9 has been advanced to further biological study.

Application of nuclear magnetic resonance spectrometry to measure the activity of bacterial macrolide esterase.

A new method of measuring the activity of macrolide antibiotic esterase in whole bacteria and in crude enzyme extracts was determined through the application of nuclear magnetic resonance spectrometry. The structure of the enzymatically esterolytic cleavage compound of oleandomycin was clearly shown to be the cyclic hemiacetal between the C-9 keto and the hydroxy group of C-13 in the open macrolide nucleus by physicochemical techniques.