Analysis of Major Components of Bacitracin, Colistin and Virginiamycin in Feed Using Matrix Solid-phase Dispersion Extraction by Liquid Chromatography-electrospray Ionization Tandem Mass Spectrometry.

A quantitative LC-MS/MS method has been developed for simultaneous determination of bacitracin A, bacitracin B, colistin A, colistin B and virginiamycin M1 in feed. This rapid simple and effective extraction method was based on matrix solid-phase dispersion. Qualitative and quantitative analyses were performed by LC-ESI-MS/MS. CCß of polypeptide antibiotics upon the method ranged from 9.6 to 15.8 µg kg-1 and 19.4 to 27.5 µg kg-1, respectively. The limit of quantification of polypeptide antibiotics was 25 µg kg-1 in feed samples. The recoveries of polypeptide antibiotics spiked in feed samples at a concentration range of 25-100 µg kg-1 were found above 75.9-87.9% with relative standard deviations within days less than 15.7% and between days less than 20.6%. This rapid and reliable method can be used to efficiently separate, characterize and quantify the residues of polypeptide antibiotics in feed with advantages of simple pretreatment and environmental friendly.

The conformation of acetylated virginiamycin M1 and virginiamycin M1 in explicit solvents.

The three-dimensional structure of acetylated virginiamycin M(1) (acetylated VM1) in chloroform and in a water/acetonitrile mixture (83:17 v/v) have been established through 2D high resolution NMR experiments and molecular dynamics modeling and the results compared with the conformation of the antibiotic VM1 in the same and other solvents. The results indicated that acetylation of the C-14 OH group of VM1 caused it to rotate about 90 degrees from the position it assumed in non-acetylated VM1. The conformation of both VM1 and acetylated VM1 appear to flatten in moving from a nonpolar to polar solvent. However, the acetylated form has a more hydrophobic nature. The acetylated VM1 in chloroform and in water/acetonitrile solution had a similar configuration to that of VM1 bound to 50S ribosomes and to the Vat(D) active sites as previously determined by X-ray crystallography. Docking studies of VM1 to the 50S ribosomal binding site and the Vat(D) gave conformations very similar to those derived from X-ray crystallographic studies. The docking studies with acetylated VM1 suggested the possibility of a hydrogen bond from the acetyl carbonyl group oxygen of acetylated VM1 to the 2' hydroxyl group of ribose of adenosine 2538 at the ribosomal VM1 binding site. No hydrogen bonds between acetylated VM1 and the Vat(D) active sites were found; the loss of this binding interaction partly accounts for the release of the product from the active site.

Structures of five antibiotics bound at the peptidyl transferase center of the large ribosomal subunit.

Structures of anisomycin, chloramphenicol, sparsomycin, blasticidin S, and virginiamycin M bound to the large ribosomal subunit of Haloarcula marismortui have been determined at 3.0A resolution. Most of these antibiotics bind to sites that overlap those of either peptidyl-tRNA or aminoacyl-tRNA, consistent with their functioning as competitive inhibitors of peptide bond formation. Two hydrophobic crevices, one at the peptidyl transferase center and the other at the entrance to the peptide exit tunnel play roles in binding these antibiotics. Midway between these crevices, nucleotide A2103 of H.marismortui (2062 Escherichia coli) varies in its conformation and thereby contacts antibiotics bound at either crevice. The aromatic ring of anisomycin binds to the active-site hydrophobic crevice, as does the aromatic ring of puromycin, while the aromatic ring of chloramphenicol binds to the exit tunnel hydrophobic crevice. Sparsomycin contacts primarily a P-site bound substrate, but also extends into the active-site hydrophobic crevice. Virginiamycin M occupies portions of both the A and P-site, and induces a conformational change in the ribosome. Blasticidin S base-pairs with the P-loop and thereby mimics C74 and C75 of a P-site bound tRNA.

A novel route to the vinyl sulfide nine-membered macrocycle moiety of Griseoviridin.

The synthetic potentialities of cerium(III) chloride are demonstrated by the synthesis of a nine-membered ring heterocycle component of Griseoviridin (3) in optically active form. The key step involves the stereospecific formation of the alpha-carbalkoxy alkenyl sulfide moiety using a combination system of cerium(III) chloride heptahydrate and sodium iodide.

[The influence of steric crowding on the electrochemical reduction of amide groups in a pyridylcarboxamide seriesapplication to rote ction of amines in peptide synthesis]. / Incidence de l'encombrement stérique sur la réduction électrochimique du groupement amide en série pyridylcarboxamide: application à la protection des amines en synthèse peptidique.

To gain a better understanding of the effect exerted by the 3-hydroxypicolinoyl residue on the antibiotic activity of Pristinamycin IA, the C-N bond of picolinamide was cleaved electrochemically. A mechanistic study demonstrated that the presence of the peptidic macrolactone M markedly modified the expected cathodic behavior of pyridylcarboxamides. In order to assess the influence of steric crowding exerted by M on this original behavior, we look for models using two different approachs. First, tertiary pyridylcarboxamides were used to increase steric hindrance at the amide nitrogen position; second, M was opened by ammonolysis to decrease steric crowding at the amide nitrogen position. The electrochemical behavior of the selected compounds is presented in the first and the second parts of this study. Determination of pyridine nitrogen basicity in an N-substituted-3-methoxypicolinamide series is treated in the third part as a useful probe to evaluate the intensity of steric crowding at the amide nitrogen position. Finally, in the last part of this work, we propose the use of the picolinoyl residue (C6H4N-CO-ou Pic) as a protecting group for amines in peptide synthesis.

Quinupristin (RP 57669): a new tool to investigate ribosome-group B streptogramin interactions.

Streptogramin antibiotics consist of two types of molecules, group A and group B. The group B molecule quinupristin (RP 57669) and the group A molecule dalfopristin (RP 54476) constitute the first water-soluble semisynthetic streptogramin, quinupristin/dalfopristin (RP 59500). When group B molecules bind to 50S subunits or to tightly coupled ribosomes, there is an increase in their fluorescence intensity, which is proportional to the concentration of the antibiotic-ribosome complex formed. We found here that the background fluorescence of unbound quinupristin is 10-fold lower than that of unbound virginiamycin S, a natural group B molecule often used experimentally. The association constants were found (i) to be similar for the binding of the two group B molecules to tightly coupled 70S ribosomes in the absence of the group A molecules (quinupristin: 3.5 x 10(7) M(-1); virginiamycin S: 2.8 x 10(7) M(-1)) and (ii) to similarly increase about 20-fold in the presence of the corresponding group A molecule (quinupristin + dalfopristin: 69 x 10(7) M(-1); virginiamycin S + virginiamycin M: 60 x 10(7) M(-1)). Similar results were obtained with 50S ribosomal subunits. Additionally, we provide evidence that the failure of the group B molecules to inhibit poly(Phe) synthesis is due to the displacement of the group B molecule during poly(Phe) polymerization on the ribosome, indicating that the artificial poly(Phe) peptide competes with the binding of the group B molecule.

Recent developments in streptogramin research.

The streptogramins are a class of antibiotics remarkable for their antibacterial activity and their unique mechanism of action. These antibiotics are produced naturally, but the therapeutic use of the natural compounds is limited because they do not dissolve in water. New semisynthetic derivatives, in particular the injectable streptogramin quinupristin/dalfopristin, offer promise for treating the rising number of infections that are caused by multiply resistant bacteria. The streptogramins consist of two structurally unrelated compounds, group A and group B. The group A compounds are polyunsaturated macrolactones: the group B compounds are cyclic hexadepsipeptides. Modifications of the group B components have been mainly performed on the 3-hydroxypicolinoyl, the 4-dimethylaminophenylalanine and the 4-oxo pipecolinic residues. Semi-synthesis on this third residue led to the water-soluble derivative quinupristin. Water-soluble group A derivatives were obtained by Michael addition of aminothiols to the dehydroproline ring of pristinamycin IIA. Followed by oxidation of the intermediate sulfide into the sulfone derivatives (i.e., dalfopristin). Water-soluble derivatives (both group A and group B) can now be obtained at the industrial scale. Modified group B compounds are now also being produced by mutasynthesis, via disruption of the papA gene. Mutasynthesis has proved particularly useful for producing PIB, the group B component of the oral streptogramin RPR 106972. The streptogramins inhibit bacterial growth by disrupting the translation of mRNA into protein. Both the group A and group B compounds bind to the peptidyltransferase domain of the bacterial ribosome. The group A compounds interfere with the elongation of the polypeptide chain by preventing the binding of aa-tRNA to the ribosome and the formation of peptide bonds, while the B compounds stimulate the dissociation of the peptidyl-tRNA and may also interfere with the release of the completed polypeptide by blocking its access to the channel through which it normally leaves the ribosome. The synergy between the group A and group B compounds appears to result from an enhanced affinity of the group B compounds for the ribosome. Apparently, the group A compound induces a conformational change such that B compound binds with greater affinity. The natural streptogramins are produced as mixtures of the group A and B compounds, the combination of which is a more potent antibacterial agent than either type of compound alone. Whereas the type A or type B compound alone has, in vitro and in animal models of infection, a moderate bacteriostatic activity, the combination of the two has strong bacteriostatic activity and often bactericidal activity. Minimal inhibitory concentrations of quinupristin/dalfopristin range from 0.20 to 1 mg/l for Streptococcus pneumonae, from 0.25 to 2 mg/l for Staphylococcus aureus and from 0.50 to 4 for Enterococcus faecium, the principal target organisms of this drug. Quinupristin/dalfopristin also has activity against mycoplasmas, Neisseria gonorrhoeae, Haemophilus influenz, Legionella spp. and Moraxella catarrhalis. Bacteria develop resistance to the streptogramms by ribosomal modification, by producing inactivating enzymes, or by causing an efflux of the antibiotic. Dimethylation of an adenine residue in rRNA, a reaction that is catalyzed by a methylase encoded by the erm gene class, affects the binding of group B compounds (as well as the macrolides and lincosamides; hence, MLSB resistance), but group A and B compounds usually maintain their synergy and their bactericidal effect against MLSB-resistant strains. erm genes are widespread both geographically and throughout numerous bacterial genera. Several types of enzymes (acetyltransferases, hydrolases) have been identified that inactivate the group A or the group B compounds. Genes involved in streptogramin efflux have so far been found only in staphylococci, particularly in coagulase-negative species

Unusual transformation of the 3-hydroxy-picolinoyl residue of pristinamycin IA.

Pristinamycin IA was modified in a two-step procedure to give original derivatives possessing a tricyclic nucleus (8a, 8b, 8c) or a substituted pyrrole ring (10a, 10b) in place of the natural exocyclic 3-hydroxy-picolinoyl residue. This transformation involved firstly preparation of pyridinium betaines 5 from pristinamycin IA and secondly a 1-3 dipolar cycloaddition between 5 and N-substituted maleimides or diethyl acetylenedicarboxylate. The compounds obtained were evaluated as antibacterial agents alone and in association with pristinamycin IIA.