Kanamycin preparation

Antibiotic for SD-citrate-CAA and SG-citrate-CAA cultures. Use 5() ig/mL kanamycin. Prepare 50mg/mL stock by dissolving 500mg kanamycin sulfate (Sigma cat. no. K4000) in lOmL ddH20. Filter sterilize. Aliquot and store at -20°C. 

Stock solutions of antibiotics Kanamycin at 100 mglml Dissolve 3.2 g in 25 ml H2O note that most commercial kanamycin preparations have a purity of approximately 80%. G418 at SO mglml Dissolve 1.25 g in 25 ml water. Hygromycin at 10 mglml Dilute from commercial stock solution into water. All solutions are filter-sterilized into 5-ml aliquots and stored at -20°C. Cefotaxime... 

Preparation of 1-[L-l-)-y-Bemyloxycarbonylamino-a-Hydroxybutyryl]-6 -Carbobenzoxy-kanamycin A A solution of 1.6 grams (4.6 mmol) of L-(-)-7-benzyloxycarbonylamino-... 

Preparation of the Monosulfate Salt of 1-[L-(-l-y-Amino-a-HydroxybutyrylJKanamycin A One mol of 1-[L-(-)-7-amino-o-hydroxybutyryl] kanamycin A is dissolved in 1 to 3 liters of water. The solution is filtered to remove any undissolved solids. To the chilled and stirred solution is added one mol of sulfuric acid dissolved in 500 ml of water. The mixture is allowed to stir for 30 minutes, following which cold ethanol Is added to the mixture till precipitation occurs. The solids are collected by filtration and are determined to be the desired monosulfate salt. 

The next problem for H. Umezawa was to use his findings to design new kanamycin derivatives effective against resistant bacteria. The synthetic work was undertaken in cooperation with his brother. Prof Sumio Umezawa of Keio University, and one of the writers (T. Tsuchiya). The first useful derivatives active against resistant bacteria, namely, 3, 4 -dideoxy-kanamycin B (dibekacin) and 3 -deoxykanamycin A, were prepared in 1971. These were also found active against Pseudomonas known to have intrinsic resistance. These results supported the truth of H. Umezawa s theory. In the synthesis of dibekacin, the Tipson-Cohen method for introducing unsaturation, developed by one of the writers (D. Horton, 1966) for pyranoside... 

Preparation of several fluorinated analogs of kanamycin A (673) were reported. 6"-Deoxy-6"-fluorokanamycin A (674) was prepared through... 

Dideoxy-2 -fluorokanamycin A (Ref. 170), a kanamycin derivative in which the OH-2 or NH2-2 of kanamycin A and B, respectively, is replaced by a fluorine atom, was prepared by condensation of 6-azido-4-0-benzoyl-2,3,6-trideoxy-2-fluoro-a-D-n77ohexopyranosyl bromide, derived from 179 (see Section 11,2), with the pseudodisaccharide component of kanamycin. The compound showed mediocre antibacterial activity however, on attachment of an (5)-4-amino-2-hydroxybutanoyl residue, as in amikacin, to the NHj-l group, the derivative showed considerably enhanced activity. ... 

Coupling of affinity molecules to surfaces also can be enhanced by the use of discrete PEG linkers. Nishimura et al. (2005) modified an amino surface with a NHS-PEG -maleimide crosslinker to create a hydrophilic self-assembled monolayer (SAM) surface that was thiol reactive for the conjugation of sulfhydryl-modified RNAs. This array then was used to investigate the binding specificity of synthetic kanamycins with selected RNA sequences to prove the specific interaction of ribosomal RNA with this molecule. The PEG linkers on surfaces provide lower nonspecific binding character than alkyl linkers, when preparing SAM surfaces for affinity interactions. 

Vergnes D, Moatti N, Monrozies X, Lazorthes F, Enjalbert L Pre-operative colonic preparation using kanamycin and metronidazole Qualitative and quantitative effects on the bacterial flora of the intestine. J Antimicrob Che-mother 1980 6 709-716. 

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). ... 

Recently, Hanessian has reported methods for direct modification of tobramycin. Tobramycin (or nebramycin) differs from kanamycin B only in its 3 -deoxygenation, which makes tobramycin immune from the modification catalyzed by APH(3 ). A library of tobramycin analogs bearing various functionalities at 0-5 was prepared (Scheme 4.23). In general, these tobramycin analogs were less active than tobramycin (Table 4.15). However, compounds 157 and 159 showed activity against P. aeruginosa (ATCC 27853) (MIC = 12.5 fig/mL). 

Kanamycin stock solution is prepared by dissolving at 50 mg/ ml in water, and is filter-sterilized. Stock solution should be kept at -20°C. 

X TTliquid medium. Prepared as described above. Ampicil-lin or kanamycin should be added at 50 pg/ml if needed. 

Colonies are examined with the CloneChecker System. Plasmid sizes are analyzed by cutting with Pmel according to the supplier s recommendations. Clones with a plasmid of an appropriate size are selected, and the corresponding suspensions that are prepared for analysis with the CloneChecker System are respectively inoculated to 600 pL of LB medium containing 50 pg/mL of kanamycin in a 96-well format and cultured at 37°C overnight. 

Three to several colonies for each sample are inoculated into the same 600 pL of LB medium containing 50 pg/mL of kanamycin and cultured at 37°C overnight. For glycerol stock, 50 pL of the culture is added to 25 pL of Cell Stock Buffer in a 96-well plate. The remaining suspensions are applied to the plasmid preparation with the Wizard SV 96 Plasmid DNA Purification System see Note 6). One microliter of the resultant plasmid solution is directly applied to agarose gel electrophoresis for estimation of the size of plasmid in a form of covalently closed circular. Similarly, another 1 pL of the plasmid solution is treated with S fi/Pmel and analyzed by agarose gel electrophoresis for an insert size check. This plasmid solution set is the final product and is reserved in a freezer. 

Clones with ampicillin resistance but without kanamycin resistance are examined with the CloneChecker System. By this antibiotic selection, the undesired vector heterodimer is removed. If both of the clones are false, additional clones are analyzed using the same protocol. When appropriate clones are obtained for almost all of the genes, they are inoculated from the ampicillin plates to prepare plasmids in a 96-well format by using the Wizard SV 96 Plasmid DNA Purification System see Note 6). 

Preparation of l-[L-(-)-y-Amino-a-Hydroxybutyryl] Kanamycin A The crude product l-[L-(-)-y-benzyloxycarbonylamino-a-hydroxybutyryl]-6 -carbobenzoxykanamycin A was dissolved in 40 ml of 50% aqueous dioxane and a small amount of insoluble material was removed by filtration. To the filtrate was added 0.8 ml of glacial acetic acid and 1 gram of 10% palladium-on-charcoal and the mixture was hydrogenated at room temperature for 24 hours in a Parr hydrogenation apparatus. The reaction mixture was filtered to remove the palladium catalyst and the filtrate was evaporated to dryness in vacuum. 

Ampicillin (100 mg/ml) prepared in sterile distilled water—Sigma catalog A-9518 Kanamycin (100 mg/ml) prepared in sterile distilled water—Sigma catalog K-4000... 

---