Bioconversion of Tetracycline Antibiotics to Novel Glucoside Derivatives by Single-Vessel Multienzymatic Glycosylation.

The single-vessel multienzyme UDP-α-D-glucose recycling system was coupled with a forward glucosylation reaction to produce novel glucose moiety-conjugated derivatives of different tetracycline antibiotic analogs. Among five tetracycline analogs used for the reaction, four molecules (chlorotetracycline, doxytetracycline, meclotetracycline, and minotetracycline) were accepted by a glycosyltransferase enzyme, YjiC, from Bacillus licheniformis to produce glucoside derivatives. However, the enzyme was unable to conjugate sugar units to rolitetracycline. All glucosides of tetracycline derivatives were characterized by ultraviolet absorbance maxima, ultra-pressure liquid chromatography coupled with photodiode array, and high-resolution quadruple time-of-flight electrospray mass spectrometry analyses. These synthesized glucosides are novel tetracycline derivatives.

An Unprecedented Blue Chromophore Found in Nature using a "Chemistry First" and Molecular Networking Approach: Discovery of Dactylocyanines A-H.

Guided by a "chemistry first" approach using molecular networking, eight new bright-blue colored natural compounds, namely dactylocyanines A-H (3-10), were isolated from the Polynesian marine sponge Dactylospongia metachromia. Starting from ilimaquinone (1), an hemisynthetic phishing probe (2) was prepared for annotating and matching structurally related natural substances in D. metachromia crude extract network. This strategy allowed characterizing for the first time in Nature the blue zwitterionic quinonoid chromophore. The solvatochromic properties of the latter are reported.

Fluorescent lifetime imaging microscopy using Europium complexes improves atherosclerotic plaques discrimination.

The objective of this study is to characterize arterial tissue with and without atherosclerosis by fluorescence lifetime imaging microscopy (FLIM) using Europium Chlortetracycline complex (EuCTc) as fluorescent marker. For this study, twelve rabbits were randomly divided into a control group (CG) and an experimental group (EG), where they were fed a normal and hypercholesterolemic diet, respectively, and were treated for 60 days. Cryosections of the aortic arch specimens were cut in a vertical plane, mounted on glass slides, and stained with Europium (Eu), Chlortetracycline (CTc), Europium Chlortetracycline (EuCTc), and Europium Chlortetracycline Magnesium (EuCTcMg) solutions. FLIM images were obtained with excitation at 405 nm. The average autofluorescence lifetime within plaque depositions was ~1.36 ns. Reduced plaque autofluorescence lifetimes of 0.23 and 0.31 ns were observed on incubation with EuCTc and EuCTcMg respectively. It was observed a quenching of collagen, cholesterol and TG emission spectra increasing EuCTc concentration. The drastic reduction in fluorescence lifetimes is due to a resonant energy transfer between collagen, triglycerides, cholesterol and europium complexes, quenching fluorescence.

6-Iso-chlortetracycline or keto form of chlortetracycline? Need for clarification for relevant monitoring of chlortetracycline residues in food.

Chlortetracycline (CTC) is a broad-spectrum antibiotic used in veterinary medicine for pulmonary or digestive infections and having a regulatory maximum residue limit (MRL) necessitating an official analytical control method. The purpose of this study was to clarify the identification of different forms of CTC observed in standard solution, in spiked muscle samples and in naturally incurred muscle samples of pigs analysed by LC-MS/MS and to demonstrate the in vivo formation of 6-iso-chlortetracycline and 4-epi-6-iso-CTC as a metabolite of CTC and 4-epi-CTC in muscle. The six following forms were identified, all being isobaric with a protonated molecule at m/z 479 (precursor ion): the keto-enol forms of CTC and the keto-enol forms of 4-epi-chlortetracycline (4-epi-CTC), 6-iso-chlortetracycline (6-iso-CTC) and 4-epi-6-iso-chlortetracycline (4-epi-6-iso-CTC). The 6-iso-CTC and 4-epi-6-iso-CTC were observed only in incurred pig samples so were identified for the first time as metabolites of CTC and 4-epi-CTC. Identification of the different forms was obtained by comparing incurred muscle samples with standard solutions and with spiked samples. Then the differences between the features of the chromatograms obtained by LC-TQ-MS and the fragmentation study of the different forms of CTC obtained by LC-Q-TOF-MS helped us to support this identification. The extraction steps and the LC-MS/MS conditions developed to analyse muscle tissue samples are described. This clarification concerning the rigorous identification of chromatographic peaks allowed us to evaluate the relevance of our monitoring method with regard to the regulations in place in the European Union and could be of help to laboratories involved in official control of antibiotic residues in food of animal origin. Additional results are also presented highlighting the transformation of the CTC when prepared in a mixture with other antibiotics.

Heterologous expression and manipulation of three tetracycline biosynthetic pathways.

A very accommodating host: Three tetracycline biosynthetic pathways were overexpressed and manipulated in the heterologous host Streptomyces lividans K4-114. Through the inactivation of various genes and characterization of the resulting biosynthetic intermediates, new tetracycline-modifying enzymes were identified (see scheme).

Validation of Polo-like kinase 1 as a therapeutic target in pancreatic cancer cells.

Polo-like kinase 1 (PLK1) is a serine/threonine protein kinase and plays a critical role in mitosis. PLK1 has also been regarded as a valuable target for cancer treatment, and several PLK1 inhibitors are currently undergoing clinical investigations. In this study, our data show that the expression level of PLK1 is upregulated in human pancreatic cancer cells. Molecular modeling studies indicate that DMTC inhibits PLK1 activity through competitive displacement of ATP from its binding pocket. Our data further show that DMTC suppresses the proliferation of pancreatic cancer cells and induces the formation of multinucleated cells, ultimately resulting in apoptosis. In addition, combination index analysis demonstrates that DMTC acts synergistically with the chemotherapeutic drug gemcitabine in inhibiting the proliferation of pancreatic cancer cells. These results thus suggest a potential of using PLK1 inhibitors for the treatment of pancreatic cancer.

Stereoselective total syntheses of herbicidin†C and aureonuclemycin through late-stage glycosylation.

Better late than never! Two herbicidins, members of an important family of nucleoside antibiotics, have been synthesized for the first time. The route integrates a stereoselective C-glycosylation with several reagent-controlled stereoselective transformations and a surprisingly facile and highly diastereoselective late-stage N-glycosylation.

Interactions of tetracycline and its derivatives with DNA in vitro in presence of metal ions.

The interactions of calf thymus DNA with tetracycline (TC), 7-chlorotetracycline (CTC) and 6-dimethyl-7-chlorotetracycline (DMTC) were assessed employing spectrofluorometric and circular dichroism (CD) techniques. The Scatchard analysis revealed relatively lesser binding affinity of TC (Ka= 1.2 x 10(7) lmol(-1)) vis-a-vis CTC (Ka= 3.4 x 10(7) lmol(-1)) and DMTC (Ka= 3.0 x 10(7) lmol(-1)) with DNA. The data suggested both the intercalative and electrostatic nature of binding between the tetracyclines and DNA. The presence of Cu(II) augmented the interaction of tetracyclines with DNA, and resulted in red shift by 12 nm in CD spectra of tetracycline. The molar ellipticity (theta) also changed significantly for CTC and DMTC. The data unequivocally demonstrated the DNA binding potential of tetracyclines both in the presence and absence of Cu(II) ions in dark. The enhanced binding of tetracyclines in presence of Cu(II), ensuing conformational changes in DNA secondary structure to a varying extent, reflects differential reactivity of ligand chromophores.

Metabolism of chlortetracycline: drug accumulation and excretion in the hen's egg.

Chlortetracycline (CTC) is one of the few antibiotics that can be used without any withdrawal period in chickens laying eggs intended for human consumption. 6-Iso-CTC and 4-epi-6-iso-CTC have recently been identified as the principal metabolites of CTC in eggs. Although not covered by the European Union (EU) maximum residue limit (MRL) for CTC, these compounds, taken together, accumulate in the eggs of birds treated therapeutically with CTC to a mean concentration equivalent to more than twice the EU MRL (200 micrograms kg-1) in eggs. Plateau concentrations in eggs were achieved after approximately 3 d of medication. Following withdrawal of medication, mean egg concentrations of these compounds were maintained for 48 h, before falling below a level equivalent to the MRL after 5 d. Feeds containing typical sub-therapeutic contamination concentrations of CTC did not produce mean concentrations of 6-iso-CTC plus 4-epi-6-iso-CTC, combined, greater than 200 micrograms kg-1. It is not known whether these compounds are formed as a result of metabolism or of chemical degradation. However, analysis of ovules pre-lay showed that all of the CTC present in this matrix was in the form of 6-iso-CTC and 4-epi-6-iso-CTC, and not as the parent drug. Although microbiologically inactive, the toxicological properties of 6-iso-CTC and 4-epi-6-iso-CTC are not known.

Dactylocyclines, novel tetracycline derivatives produced by a Dactylosporangium sp. I. Taxonomy, production, isolation and biological activity.

A screen for antibiotics with activity against tetracycline-resistant microorganisms has led to the isolation of Dactylosporangium sp. (ATCC 53693), a producer of several novel tetracycline derivatives. The major fermentation products, dactylocyclines A and B, were purified and MIC values determined against tetracycline-resistant and tetracycline-sensitive Gram-positive bacteria. The dactylocyclines represent the first naturally occurring tetracycline C2 amides which lack cross resistance with tetracycline.

Dactylocyclines, novel tetracycline derivatives produced by a Dactylosporangium sp. II. Structure elucidation.

Fermentation of Dactylosporangium sp. (ATCC 53693) produces a mixture of tetracycline derivatives from which several related tetracycline glycosides, the dactylocyclines, were isolated and their structures determined. The most abundant glycoside in initial fermentations was found to be dactylocycline A. Each glycoside proved to be acid sensitive and readily hydrolyzed to a common aglycone, dactylocyclinone. While the aglycone was cross resistant with tetracycline, the dactylocyclines proved active against certain tetracycline-resistant organisms.

Dactylocyclines, novel tetracycline derivatives produced by a Dactylosporangium sp. III. Absolute stereochemistry of the dactylocyclines.

The dactylocycline antibiotics were found through circular dichroism measurements, NMR spectroscopy and chemical transformations to possess the usual tetracycline family absolute configuration at carbons 4, 4a, 5a and 12a. The absolute stereochemistry about the C-6 carbon, however, was the reverse of that found with previously investigated tetracyclines.

Quantitative analysis of chlortetracycline and related substances by high-performance liquid chromatography.

Isocratic high-performance liquid chromatography on Zorbax C8 7 microns allows quantitative determination of chlortetracycline, 4-epichlortetracycline, tetracycline, demethylchlortetracycline and isochlortetracycline using a mobile phase containing dimethylsulphoxide, 1 M perchloric acid and water (35:5:60). The minor impurities anhydrochlortetracycline and 4-epianhydrochlortetracycline, which are more strongly retained can be determined using a second isocratic system with a mobile phase containing more organic modifier. The method has been used for the comparison of official standards and for the analysis of a number of commercial samples.

A novel tetracycline from Actinomadura brunnea. Fermentation, isolation and structure elucidation.

A novel tetracycline antibiotic, Sch 33256, was isolated from a culture broth of a new species of Actinomadura. The antibiotic was isolated by solvent extraction, Sephadex G-25 column chromatography and crystallization. The structure was determined by comparison of the spectra with that of chlortetracycline. Spectroscopic analysis of the compound yielded 2'-N-methyl-8-methoxychlortetracycline as the proposed structure.

A new tetracycline antibiotic from a Dactylosporangium species. Fermentation, isolation and structure elucidation.

An actinomycete identified as a Dactylosporangium sp. produces a new tetracycline, 4a-hydroxy-8-methoxychlortetracycline (Sch 34164). The addition of magnesium ions to complex fermentation media increased the antibiotic titers. Sch 34164 was isolated by solvent extraction and Sephadex G-25 column chromatography. The novel structure was proposed based on spectroscopic analysis. The shift of C-4a (35 to 77 ppm) and C-8 (140 to 163 ppm) in the 13C NMR as compared to chlortetracycline was indicative of the novel hydroxyl and methoxy substituents, respectively.

Direct selection of a specifically blocked mutant of Actinomadura brunnea. Isolation of a third 8-methoxy substituted chlortetracycline.

Actinomadura brunnea produces 2'-N-methyl-8-methoxychlortetracycline. A derivative of this strain has been isolated that is specifically blocked in methylation of the 2'-amino position. This isolate was detected by screening approximately 30,000 colonies of a mutagenized population of Actinomadura brunnea using a direct soft agar overlay with an Escherichia coli indicator. The antibiotic produced by the blocked mutant has been identified as 8-methoxychlortetracycline (Sch 36969) based upon its biological activity, relative mobility on TLC and HPLC, and spectroscopic data.

In vitro and in vivo characterization of novel 8-methoxy derivatives of chlortetracycline.

The in vitro activities of three new 8-methoxychlortetracyclines, Sch 36969, 33256 and 34164 were compared to tetracycline, minocycline and doxycycline. Against aerobic Gram-negative rods Sch 36969 had a geometric mean MIC (GMM) of 4.2 micrograms/ml, about 8-fold more potent than Sch 33256, and similar to all the other compounds. Sch 36969 also had good activity against methicillin-resistant (GMM, 0.21 micrograms/ml) and -susceptible Staphylococci (GMM, 0.14 micrograms/ml), Streptococci (GMM, 0.06 micrograms/ml), and most anaerobic bacteria (GMM, less than 0.5 micrograms/ml). In general, Sch 36969 was similar to, or more potent than, all the other compounds tested. Serum levels of Sch 36969 in squirrel monkeys were 4-fold lower (AUC, 4.5 micrograms.hours/ml) than those of chlortetracycline (AUC, 16.1 micrograms.hours/ml). In mouse protection tests (PD50s) against various strains of bacteria, Sch 36969 was similar in activity to tetracycline, but up to 6-fold less active than chlortetracycline. The structure activity relationships for these new chlortetracyclines are described.

A double-blind trial of clomocycline in the treatment of persistent palmoplantar pustulosis.

Sixty patients with persistent palmoplantar pustulosis (PPP) were treated with clomocycline (Megaclor) in a double-blind, cross-over trial in an attempt to establish whether the condition responds to tetracycline. Each patient received 3 months each of clomocycline and placebo in random order. Forty patients completed the trial. Twenty-two failed to respond to either treatment, fifteen improved on clomocycline, two improved on placebo and one placebo and one improbed on both treatments. These results are highly significant (P = 0-003) and suggest that clomocycline may suppress pustulation in some patients. The twenty-two 'non-responders' were compared with the eighteen 'responders' for sex, age, length of history and associated psoriasis but no significant differences were found. Difficulties in assessment and the possiblity of improvement deing due to spontaneous remission are discussed. Further follow-up of both groups suggested that clomocycline used over long periods favourably influenced the course of the disease in the 'responder' group.