Anhydrotetracycline

Under acidic conditions, dehydration to an anhydrotetracycline [20154-34-1] (8), C22H22N20y, occurs under basic ones, ring C opens to an isotetracycline [3811-31-2] (9), C22H24N20g. The anhydrotetracyclines, such as (8), appear to exhibit a mode of antibacterial action, but it is unlike that of tetracycline (24). Epimerization (23,25,26) at C-4 occurs in a variety of solvents within the pH range 2—6, particularly in acetic acid (25). A number of anions (27) facihtate this reaction. The reverse process, from 4-epitetracycline [79-85-6] C22H24N20g, to tetracycline, is promoted by chelation with ions such as calcium and magnesium (28). 

Anhydrotetracycline oxygenase from Streptomjces aureofaciens which cataly2es the conversion of anhydrotetracycline to dehydrotetracycline, has been isolated and characterized as a flavin-dependent oxygenase (83). It consists of two subunits of mol wt = 57, 500 based on SDS/polyacrylamide—gel electrophoresis. The cosynthetic factor 1 of Streptomjces aureofaciens involved in the reduction of 5a,lla-dehydrochlortetracycline to chlortetracycline, has been identified as 7,8-didemethyl-8-hydroxy-5-deazariboflavin. This work was aided by comparison of spectral data with that of an authentic sample obtained from the hydrolysis of coenzyme F-420 (84). 

Mass spectral studies, including low voltage techniques and accurate mass measurements, have been reported by Hoffman (40) on tetracycline and eight related compounds. As a result of good spectral correlations among these compounds, the major fragmentation processes are discussed in terms of two compounds in the series 5a,6-anhydrotetracycline and... 

Anhydro tetracycline oxygenase, 24 601 Anhydrotetracyclines, 24 594 Anhydrous acetic acid, extraction of, 10 786... 

MeOH-MeCN-DMF (45 49 6, v/v/v) with 25mM ammonium acetate, lOmM citric acid, and I 18 mM methanesulfonic acid added. Temperature 25°C. Voltage 30 kV resulting in a current of approximately 65 pA. Detection 254 nm. ATC anhydrotetracycline U unknown TC tetracycline DTC desmethyltetracycline EATC 4-epianhydrotetracycline ETC 4-epitetracycline. (Reprinted from reference 179, with permission.)... 

However, one should be aware of the fact that if a particular pharmaceutical is not detected in a WWTP effluent, this does not imply that it has been fully removed. On some occasions, it may have been degraded and give rise to unsuspecting or even more toxic compounds that will subsequently contaminate surface waters [30, 58-60]. As an example, anhydrotetracycline (ATC) and epianhydrote-tracycline (EATC) are tetracycline (TC) degradation products that cause Fanconi syndrome [61]. 

Stereospecific ene reaction. Singlet oxygen (meso-tetraphenylporphine) reacts with anhydrotetracycline (1) to give the hydroperoxide 2 in 97% yield. Hydrogenation of 2 provides the antibiotic tetracycline (3).2... 

Successful cell surface display of the protein can be verified by inducing gene expression via addition of anhydrotetracycline to the culture medium and immunofluorescence staining of the cells using an antibody directed against the protein to be displayed (Protocol 1). Analysis can be performed by fluorescence microscopy or flow cytometry. 

Anhydrotetracycline stock solution 2mgmL 1 anhydrotetracycline (Acros) in N, N-dimethy If ormamide. 

Gossen, M., and Bujard, H. (1993) Anhydrotetracycline, a novel effector for tetracycline controlled gene expression systems in eukaryotic cells. Nucleic Acids Res., 21, 4411—4412. 

They are approximately in agreement with values reported in literature, but the spectrum there was obtained with a HA-100 (23). The nmr shifts appear to be general regardless of solvent. Similar shifts are obtained in pyridine, trifluoroacetic acid and dimethyl sulfoxide. Particularly notable is the substantial downfield shift in the resoncance due to the C 4 proton when the tetracyclines epime-rize at C 4. This is especially convenient as both isomers are usually available and can be distinguished easily in this way. NMR was used to monitor the epimerization of TC since the dimethylamino resonance of TC and its C 4 epimer differ by 0.1 ppm (24). Formation of anhydrotetracycline could also be readily detected through NMR (23). 

As a viable synthetic relay from anhydrotetracycline (66) to tetracycline (68) has been reported by Wasserman and Scott via a two-step hydration at the 5a, 6-position (50), 66 was our first target. A reliable 12a-hydroxylation is required for the synthesis of 66, although evidence of such hydroxylation has been reported (27, 57). 

Hydrolysis of 65 to give the amide with concomitant removal of the N-Boc group was followed by JV-dimethylation and de-O-methylation to produce anhydrotetracycline (66). This was identical with a naturally derived sample in all respects. 

Anhydrotetracycline, dehydrotetracycline, and tetracycline were separated at 40°C on a Separon ODS glass-pack column (1 mm X 150 mm, 5 /urn). Mobile phase A was a 20 80 mixture of 20 mM EDTA (pH 6.4) and dimethyl-formamide. Solvent B was methanol. After 0.5 minute on solvent A, a 0.5-minute linear gradient from 0 to 50% B was started, with the higher concentration held for 2 minutes. This was followed by a return to the starting conditions with a 0.5-minute gradient. After a 1.5-minute delay, the next sample was injected. A diode-array detector was used, with peak areas integrated at 440, 400, and 360 nm for anhydrotetracycline, dehydrotetracycline, and tetracycline, respectively. 

The tetracycline dehydrogenase reaction was carried out under identical conditions except anhydrotetracycline was substituted by dehydrotetracycline, and tetracycline dehydrogenase was added. The reaction was often carried out using the anhydrotetracycline reaction mixture after a complete anhydrotetracycline to dehydrotetracycline conversion. 

Both reactions were linear over a wide range of amounts of enzyme added. The enzymes assayed were prepared from S. aureofaciens and were separated from each other by passing a cell-free extract through a phenyl-Sepharose CL-4B column. Tetracycline dehydrogenase activity was not retained on the column. Anhydrotetracycline oxygenase was liberated using a linear gradient of Lubrol. 

Acquired Lignac-De Toni-Fanconi syndrome, with polyuria, polydipsia, glycosuria, aminoaciduria, hyper-phosphaturia, and hypercalciuria, was described in a number of patients treated with outdated tetracycline formulations. The degeneration products responsible for the toxic action are probably epitetracycline, anhydro-4-epitetracycline, and anhydrotetracycline (117), as similar renal damage was produced in rats with anhydro-4-epite-tracycline (118). 

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