- Heterologous Catalysis of the Final Steps of Tetracycline Biosynthesis by Saccharomyces cerevisiae
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Developing treatments for antibiotic resistant bacterial infections is among the highest priority public health challenges worldwide. Tetracyclines, one of the most important classes of antibiotics, have fallen prey to antibiotic resistance, necessitating the generation of new analogs. Many tetracycline analogs have been accessed through both total synthesis and semisynthesis, but key C-ring tetracycline analogs remain inaccessible. New methods are needed to unlock access to these analogs, and heterologous biosynthesis in a tractable host such as Saccharomyces cerevisiae is a candidate method. C-ring analog biosynthesis can mimic nature's biosynthesis of tetracyclines from anhydrotetracyclines, but challenges exist, including the absence of the unique cofactor F420 in common heterologous hosts. Toward this goal, this paper describes the biosynthesis of tetracycline from anhydrotetracycline in S. cerevisiae heterologously expressing three enzymes from three bacterial hosts: the anhydrotetracycline hydroxylase OxyS, the dehydrotetracycline reductase CtcM, and the F420 reductase FNO. This biosynthesis of tetracycline is enabled by OxyS performing just one hydroxylation step in S. cerevisiae despite its previous characterization as a double hydroxylase. This single hydroxylation enabled us to purify and structurally characterize a hypothetical intermediate in oxytetracycline biosynthesis that can explain structural differences between oxytetracycline and chlortetracycline. We show that Fo, a synthetically accessible derivative of cofactor F420, can replace F420 in tetracycline biosynthesis. Critically, the use of S. cerevisiae for the final steps of tetracycline biosynthesis described herein sets the stage to achieve a total biosynthesis of tetracycline as well as novel tetracycline analogs in S. cerevisiae with the potential to combat antibiotic-resistant bacteria.
- Herbst, Ehud,Lee, Arden,Tang, Yi,Snyder, Scott A.,Cornish, Virginia W.
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- X-ray crystallography of tetracycline, doxycycline and sancycline
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The molecular and crystal structures of α-6-deoxy-β-5- oxytetracycline (doxycycline) hydrochloride, of tetracycline hydrochloride and tetracycline hexahydrate were re-determined. The crystal structures of 6-deoxy-6-dimethyl-tetracycline (sancycline) hydrochloride were, for the first time, determined by single crystal X-ray diffraction technique. All crystals studied exhibit the orthorhombic space group P21212 1 with 4 molecules per unit cell. The starting material tetracycline (TC) hydrochloride crystallizes from aqueous solution, independently of the pH of the mother liquor, as hexahydrate complex of the zwitterion with the same molecular structure (a = 9.585(3) A, b = 12.112(3) A, c = 21.671(6) A). From methanolic solution, tetracycline hydrochloride crystallizes as hydrochloride (a = 11.001(3) A, b = 12.852(4) A, c = 15.795(3) A). Doxycycline hydrochloride crystallizes from acidic aqueous solution as dihydrate complex (a = 11.115(4) A, b = 12.768(4) A, c = 16.921(5) A). In both hydrochloride crystals, the amide group is protonated and oriented such that an intramolecular hydrogen bond is formed between the amide oxygen and O3. In the tetracycline-hexahydrate crystal, the amide group of the zwitterion is rotated by about 180 (vs the cation) with a hydrogen bond being made by one hydrogen of its amino group and O3-. Sancycline hydrochloride crystallized from an acidified solvent mixture (water and methanol) adopts a geometry close to that of the tetracycline cation, e.g., with a hydrogen bond between the protonated amide oxygen and O3 (a = 6.8944(4) A, b = 16.815(2) A, c = 18.190(2) A). But the dimethyl ammonium group of sancycline hydrochloride is disordered with respect to its orientation. In the majority fraction (65.9 %), the proton at N4 is directed towards O3, which has a water molecule next to it. In the minority fraction (34.1 %), the proton at N4 points away from O3 and has a methanol molecule in its neighborhood. The bond length variations in ring A of all compounds studied do not reflect the location of single and double bonds in any of the classical mesomeric structures. By comparison with calculated Wiberg-bond orders we show that this apparent discrepancy is not due to the existence of different tautomers in the crystals as discussed previously but merely a consequence of the π-conjugation in one tautomer. Graphical Abstract: Although the packing of the three title compounds in the respective crystals is different, the geometries of the molecular framework are nearly the same as the one shown for the sancycline cation. A comparison of experimentally determined bond lengths with calculated Wiberg-Bond orders provides evidence that only one tautomer is present in the crystal.[Figure not available: see fulltext.].
- Heinemann, Frank W.,Leypold, Clemens F.,Roman, Cyprian R.,Schmitt, Matthias O.,Schneider, Siegfried
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- METHODS FOR ENGINEERING THERAPEUTICS AND USES THEREOF
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The disclosed subject matter provides for genetically modified cells, e.g, fungal cells, that autonomously generates and/or secretes one or more therapeutic molecules, e.g, therapeutic peptides, therapeutic proteins or small therapeutic molecules, in situ. In certain embodiments, the present disclosure provides genetically-engineered fungal cells that generate and secrete tetracycline and analogues thereof.
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Page/Page column 148
(2021/05/15)
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- Pharmaceutical Composition for the Eradication of Helicobacter Pylori and Preparation Method Thereof
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The present invention relates to a pharmaceutical composition and its preparation method for the eradication of Helicobacter pylorif in the forms of effervescent tablet, suspension or powder. The pharmaceutical composition comprises an effective dose of β-lactam antibiotic, an effective dose of macrolide antibiotic, an effective dose of antacid such as proton pump inhibitor and H2 blocker, and a pharmaceutical acceptable carrier. An effective dose of alkaline substance such as carbonate or bicarbonate can be added to increase the pH of the stomach when the PPI antacid is used, which can protect the degradation of acid-labile antibiotics or PPI to further increase the bioavailability of the pharmaceutical composition for the purpose of Helicobacter pylori eradication.
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- Technology for the Preparation of Microparticles
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Microspheres are produced by contacting a solution of a macromolecule or small molecule in a solvent with an antisolvent and a counterion, and chilling the solution. The microspheres are useful for preparing pharmaceuticals, nutraceuticals, cosmetic products and the like of defined dimensions.
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- SYNTHESIS OF TETRACYCLINES AND ANALOGUES THEREOF
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The tetracycline class of antibiotics has played a major role in the treatment of infectious diseases for the past 50 years. However, the increased use of the tetracyclines in human and veterinary medicine has led to resistance among many organisms previously susceptible to tetracycline antibiotics. The modular synthesis of tetracyclines and tetracycline analogs described provides an efficient and enantioselective route to a variety of tetracycline analogs and polycyclines previously inaccessible via earlier tetracycline syntheses and semi-synthetic methods. These analogs may be used as anti-microbial agents or anti-proliferative agents in the treatment of diseases of humans or other animals.
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Page/Page column 106-107
(2008/06/13)
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- Synthesis of (-)-tetracycline
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We describe a convergent, enantioselective synthesis of (-)-tetracycline (1) from benzoic acid (17 steps, 1.1% yield). Benzoic acid was transformed into the AB precursor 2 in 10 steps (11% yield), as previously described, and the latter compound was activated toward Diels-Alder cycloaddition by the introduction of an α-phenylthio group (two steps, 66% yield). Heating of the resulting α-(phenylthio)enone (3) with the triethylsilyloxybenzocyclobutene derivative 4 at 85 °C gave the endo-Diels Alder adduct 5 in 64% yield. Deprotection and oxidation of the latter intermediate gave the 2-(phenylthio)-1,3-diketone 7, which was oxidized with m-chloroperoxybenzoic acid in the presence of trifluoroacetic acid. The sulfoxide intermediate(s) formed eliminated upon warming to 35 °C to give the anyhydrotetracycline derivative 8. Intermediate 8 underwent spontaneous autoxidation at 23 °C to form the hydroperoxide keto-9 stereoselectively. Without isolation, hydrogenolysis of 9 in the presence of palladium black gave (-)-tetracycline (42% yield from 7), indistinguishable from an authentic sample. Copyright
- Charest, Mark G.,Siegel, Dionicio R.,Myers, Andrew G.
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p. 8292 - 8293
(2007/10/03)
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- Fast rolitetracycline stability determination by spectrophotometer Variable-temperature kinetic experiments
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The pseudo-first order rate constant for the hydrolysis of rotitetracycline as a function of temperature was obtained by performing variable-temperature kinetic experiments. The results are in agreement with those obtained at constant-parameter kinetics but saving experimental time and chemicals.
- Alibrandi,Coppolino,Micali,Villari
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- Compositions and methods for treating hemorrhagic virus infections and other disorders
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Cytokine-receptor and cytokine antagonist-enriched blood-dervided compositions and methods of preparing and using the compositions are provided. Also provided are compositions and methods for the treatment or prevention of disorders, especially acute inflammatory disorders involving pathological responses of the immune system, such as viral hemorrhagic diseases, sepsis, rheumatoid arthritis and other autoimmune disorders, acute cardiovascular events, flare-ups and acute phases of multiple sclerosis, wasting disorders and other disorders involving deleterious expression of cytokines and other factors, including tumor necrosis factor (TNF) and interleukin-1 (IL-1) are provided.
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- The first total synthesis of natural (-)-tetracycline
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Natural (-)-tetracycline has been stereoselectively synthesized from D-glucosamine through [4 + 2] cycloaddition and Michael-Dieckmann type reaction.
- Tatsuta, Kuniaki,Yoshimoto, Takuji,Gunji, Hiroki,Okado, Yoshiya,Takahashi, Masaaki
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p. 646 - 647
(2007/10/03)
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- Pharmaceutical controlled-release composition with bioadhesive properties
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A pharmaceutical composition for the controlled release of medicinal drugs, which has the property of adhering to biologic tissues is described. The characteristic features of the composition are a plurality of small-size units capable of ensuring a gradual release of the active ingredient they contain the units being coated with a bioadhesive polymer layer. The composition makes it possible to keep the release controlling function separate from the function providing bioadhesion and can be adapted inter alia to oral, ocular, rectal, vaginal, nasal or periodontal administrations. An advantageous process for making the composition is also disclosed.
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