Aminoglycosides methods

Urease assay. When Proteus mirabilis grows in a urea-containing medium it hydrolyses the urea to ammonia and consequently raises the pH of the medium. This production of urease is inhibited by aminoglycoside antibiotics (inhibitors of protein synthesis Chapter 8). In practice, it is difficult to obtain reliable results by this method. 

Luciferase assay. In this technique, firefly luciferase is used to measure small amounts of adenosine triphosphate (ATP) in a bacterial culture, ATP levels being reduced by the inhibitory action of aminoglycoside antibiotics. This method may find more application in the future as more active and reliable luciferase preparations become available. 

Breeze A.S. Simpson A.M. (1982) An improved method using acetyl-coenzyme A regeneration for the enzymic inactivation of aminoglycosides prior to sterility testing. JApplBacteriol, 53, 277-284. 

These methods complement other approaches [159] to the preparation of 2-deoxy-2-aminoglycosides and serve to further emphasize the rich diversity of products available from the glycals. 

Wienen, R, and Holzgrabe, U. (2003). A new micellar electrokinetic capillary chromatography method for separation of the components of the aminoglycoside antibiotics. Electrophoresis 24, 2948-2957. 

The 3D structures of several aminoglycoside-enzyme complexes have been described recently by X-ray methods [35,36]. Interestingly, in some cases, the 3D structure of the antibiotic in the enzyme binding site significantly... 

Although a number of chromatographic methods have been reported for determinations of aminoglycoside antibiotics in blood serum and urine, the application of chromatographic methods to residue analysis has been very limited. Shaikh et al (93) recently described an HPLC method for neomycin in animal tissue, and Lachatre et al (94) described a method for nine aminoglycosides in plasma, urine, and renal cortex tissue. Both procedures use post column derivatization with a-pthalaldehyde and fluorescence detection. 

Attaching fnnctionalities at the N-1 position of the 2-deoxystreptamine among kanamycin or neomycin class antibiotics, is one of the other most effective methods of reviving the activity against aminoglycoside resistant bacteria. This strategy has led to the development of semisynthetic amikacin that has an (5 )-4-amino-2-hydroxybutyryl (AHB) group at N-1 position. 

Metal-chelating method has also been commonly employed for the regios-elective incorporation of AHB at N-1 of kanamycin class aminoglycoside (Scheme 4.20). Other kanamycin derivatives have been prepared in similar fashion (Figure 4.10). ... 

Aminoglycosides remain clinically important antibiotics. NMR provided the initial breakthrough in structural understanding of aminoglycoside action on the ribosome, and it remains a powerful tool for the biophysical characterization of drug-RNA interaction. The combined use of NMR, X-ray crystallography, thermodynamic and functional assays, and computational methods is needed to drive forward the development of new aminoglycosides with improved clinical properties. The rich data described above, combined with the application of new synthetic methods, bode well for the future. 

In the example of the aminoglycoside/ A site complexes, different crystallization solutions were prepared to test various glycerol/MPD ratios 5, 2, 1, 0.75, 0.67, and 0.5 (Table 14.2). All trials are performed at the optimal temperature of 37°C using the vapour diffusion method in the hanging drop set-up 1 xl RNA-antibiotic complex solution was added to 1 xl crystallization solution and equilibrated over a 40% MPD reservoir. 

In Australia, the general antimicrobial screen is performed on kidney and is able to detect -lactam, aminoglycoside, tetracycline, and macrolide antimicrobials and to identify the class of antimicrobial compound present. Where the screen test identifies a class of compounds, confirmation and quantitation are done by the specific HPLC or gas chromatographic (GC) method appropriate for the class of antimicrobial. 

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