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.

Impact of stereochemistry on the biological activity of novel oleandomycin derivatives.

A set of 8-methylene-, 8-methyl-, and 8-methyl-9-dihydro-oleandomycin derivatives having different combinations of stereochemistries at positions C-8 and/or C-9 have been prepared in a chemoselective and stereoselective manner and tested in vitro for antibacterial activity and inhibition of IL-6 production. Configurations of the stereocenters at C-8 and C-9 were determined using 2D NMR techniques. We have shown that change of stereochemistry at these positions can exert a major influence on antibacterial activity as well as IL-6 inhibition, providing novel macrolide derivatives with diminished antibacterial and potent anti-inflammatory activity. In addition, the anti-inflammatory activity observed in vitro was confirmed in an in vivo model of lipopolysaccharide-induced inflammation.

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.

Engineered biosynthesis of a complete macrolactone in a heterologous host.

Macrocyclic polyketides have been subjects of great interest in synthetic and biosynthetic chemistry because of their structural complexity and medicinal activities. With expression of the entire 6-deoxyerythronolide B synthase (DEBS) (10,283 amino acids) in a heterologous host, substantial quantities of 6-deoxyerythronolide B (6dEB), the aglycone of the macrolide antibiotic erythromycin, and 8,8a-deoxyoleandolide, a 14-membered lactone ring identical to 6dEB except for a methyl group side chain in place of an ethyl unit, were synthesized in Streptomyces coelicolor. The biosynthetic strategy utilizes a genetic approach that facilitates rapid structural manipulation of DEBS or other modular polyketide synthases (PKSs), including those found in actinomycetes with poorly developed genetic methods. From a technological viewpoint, this approach should allow the rational design of biosynthetic products and may eventually lead to the generation of diverse polyketide libraries by means of combinatorial cloning of naturally occurring and mutant PKS modules.

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.

In vitro interaction of rat liver cytochromes P-450 with erythromycin, oleandomycin and erythralosamine derivatives. Importance of structural factors.

Several derivatives of the erythromycin, erythralosamine and oleandomycin series have been prepared. Their abilities to bind to rat liver microsomal cytochrome P-450 and to lead to the formation of stable 456 nm absorbing cytochrome P-450-metabolite complexes after their oxidative microsomal metabolism in vitro have been compared. The obtained data confirmed that cytochrome P-450 induced in rats either by macrolides or by 16 alpha-pregnenolone carbonitrile were the major isozymes involved in the binding of macrolides to liver microsomes and in metabolite-complex formation. They showed that (i) hydrophobicity was in general a beneficial factor for these two properties, (ii) the presence of a bulky substituent in position 3 of erythromycin dramatically decreased their affinity for these isozymes, and (iii) the simultaneous presence of bulky substituents in position 2' and 3 prevented iron-metabolite complex formation. These results led to the selection of two compounds, erythralosamine-2'-benzoate and erythralosamine-2',3-diacetate, which exhibited a particularly high affinity for macrolide inducible cytochrome P-450 and were very good precursors of cytochrome P-450-iron-metabolite complex formation.

[Oleandomycin and its natural and synthetic analogs. I. Dissociative ionization of oleandomycin and its derivatives]. / Oleandomycin, ego prirodnye i sinteticheskie analogi. I. Dissotsiativnaia ionizatsiia oleandomitsina i ego proizvodnykh.

Regularities of dissociative ionization of compounds belonging to the oleandomycin group were studied. Mass spectra of oleandomycin and some of its derivatives including anhydrooleandomycin, oleandomycin chlorhydrine, desoleandomycin, oleandomycin oxide, trimethylsilyl and acetyl derivatives were analyzed comparatively. Directions of disintegration with breakage of the glycoside bonds, macrolactone and carbon cycles were detected. The data are useful in structural analysis of not described oleandomycin-related compounds formed during biosynthesis and isolation of the main product.

[Oleandomycin and its natural and synthetic analogs. II. Structure and properties of oleandomycin B, a new compound of the oleandomycin group]. / Oleandomitsin, ego prirodnye i sinteticheskie analogi. II. Struktura i svoistva novogo soedineniia oleandomitsinovoi gruppy oleandomitsina B.

The revealed regularities of mass spectroscopic disintegration of oleandomycin and its derivatives made it possible to determine analytic criteria for identification of compounds related by their structure to oleandomycin. Analysis of the extracts from oleandomycin fermentation broth filtrates on the basis of the selected group of diagnostic ions showed that along with the main antibiotic there formed during the biosynthesis oleandomycin B, a structurally close minor component. The structure of the substance was assigned and its physico-chemical and biological properties were studied.

Synthesis and biological activities of 4"-deoxy-4"-sulfonamido-oleandomycin derivatives.

Chemical modification of the macrolide antibiotic oleandomycin (C-1) is described. Reductive amination of 11-acetyl-4"-deoxy-4"-oxo-oleandomycin (C-6) with ammonium acetate provides amino-oleandomycin derivative C-7 in which the 4"-amine is oriented in the axial configuration. The structure-activity relationship of a series of 4"-sulfonamide analogs prepared from amino-oleandomycin derivative C-7 is discussed. Noteworthy is the significant in vitro potency enhancement of the para-chlorobenzenesulfonamide analog C-12 over that of the parent oleandomycin. The absolute configuration of the 4"-amino-oleandomycin derivative C-7 was established through X-ray analysis of the para-iodobenzenesulfonamide analog C-14.

Evaluation of three 4"-deoxy-4"-sulfonamido-oleandomycin derivatives with erythromycin-like antibacterial potency.

Three derivatives of oleandomycin in which the C"-4 hydroxyl moiety was replaced for the first time with a nitrogen functionality have been compared with erythromycin base and oleandomycin base. The minimum inhibitory concentrations of these derivatives for 90% of a group of clinical isolates of Staphylococcus aureus were one-half to one-fourth those of erythromycin. The minimum inhibitory concentrations of the experimental macrolides for 50% of a group of S. aureus isolates resistant to greater than 12.5 micrograms of erythromycin per ml ranged from 0.2 to 0.39 micrograms/ml. The activities of these experimental compounds were equivalent to the activities of erythromycin against Staphylococcus epidermidis, Bacteroides fragilis, and Haemophilus influenzae isolates. In general, erythromycin was more active against Streptococcus species. Each experimental macrolide was superior to erythromycin in inhibiting RNA-directed, cell-free polypeptide synthesis. The three experimental compounds were markedly more active than erythromycin base after oral administration to mice infected with S. aureus. The 50% protective doses of the experimental compounds ranged from 27.4 to 45.7 mg/kg; that of erythromycin was approximately 100 mg/kg.

Ring contraction of oleandrose on the macrolide antibiotic oleandomycin with [(Methoxycarbonyl)sulfamoyl]triethylammonium hydroxide inner salt.

Ring contraction of the neutral oleandrose sugar in the 14-membered-ring macrolide antibiotic oleandomycin (2) has been accomplished using [(methoxycarbonyl)sulfamoyl]triethylammonium hydroxide inner salt (1). The product of this interesting rearrangement, after methanolic hydrolysis of the 2'-acetate, is the 11-acetyl-3-O-(3"-methoxy-4"-vinylfuranosyl)oleandomycin (12). The in vitro activity of furanoside 12 is only moderately less than that of 11-acetyloleandomycin (13).

A new semisynthetic macrolide antibiotic 3-O-oleandrosyl-5-O-desosaminylerythronolide A oxime.

A new antibiotic, 3-O-oleandrosyl-5-O-desosaminylerythronolide A oxime (3) was produced from erythronolide A oxime (1) by the oleandomycin-producing culture, Streptomyces antibioticus ATCC 11891. The structure of 3 was determined by degradative studies and confirmed by X-ray analysis. Compound 3 was found to be less active, but more stable to acid, then erythromycin A oxime.