Kanamycin A 3 -phosphate

When a large amount of kanamycin was inactivated in a reaction mixture, besides kanamydn A 3 -phosphate, another product, which showed similar analytical values but which was less basic than kanamycin A 3 -phosphate, was obtained. This compound was not affected by alkaline phosphatase and phosphate diesterase. However, when kanamycin A was inactivated in dilute solution, the production of this unidentified compound was negligible. 

The reaction mixture is heated at 80°C for 5 min to cease the reaction, followed by centrifugation. The supernatant is run onto a column of 100 parts by volume of cation-exchange resin [Amberlite IRC-50, NH4+-form]. The column is washed with water, and then eluted with 1 N-aqueous ammonia to give fractions which contain kanamycin B-3 -phosphate. The fractions are collected and concentrated under reduced pressure, and then the concentrate is run onto a column of 100 parts by volume of cation-exchange resin [carboxy-methyl Sephadex C-25, NH4+-form]. The column is washed with water, and eluted with 0.2 N-aqueous ammonia to give fractions which contain kanamycin B-3 -phosphate. The fractions are collected, concentrated and lyophilized, whereby 4.5 parts of kanamycin B-3 -phosphate. 

ATP -I- kanamycin = ADP + kanamycin 3 -phosphate (<3> isoenzyme APH3 -Illa follows a Theorell-Chance mechanism [7])... 

Lividomycins lack a 3 -hydroxyl group that could undergo the reaction of a kanamycin-neomycin phosphate transferase, but the 5"-hydroxyl group is phosphorylated by kanamycin-neomycin phosphate transferase... 

Mobashery and co-workers also reported the synthesis and inhibitory apph-cation of 2 -N02 derivatives of neamine and kanamycin (Scheme 4.28). Using the metal-chelating method, the 2 -NH2 was selectively unmasked and then oxidized into 2 -N02, which will increase the acidity of 2 -H. Upon phosphorylation at the 3 -0H, elimination of phosphate will lead to the formation of a nitroalkene intermediate, 198, that can function as a Michael acceptor and... 

Because H. Umezawa and coworkers had found that an enzyme named phosphate transferase I (which is produced by some strains of resistant Escherichia coli) phosphorylates not only the 3 -hydroxyl group of kanamycins but also the 5"-hydroxyl group of ribostamycin and livi-domycin A, 5"-deoxy derivatives of ribostamycin and lividomydns were prepared. 3, 4, 5"-Trideoxyribostamycin (197) was prepared from the... 

Independently, Umezawa and coworkers and Brzezinska and Davies" found another enzyme, designated kanamycin phosphate transferase II or kanamycin-neomydn phosphate transferase II, in E. coli canning R factor, and it was found by the former authors that this enz)one also catalyzes the transfer of a phosphate group from adenosine 5 -triphos-phate to the 3 -hydroxyl group of the kanamycins, paromamine, neamine, neomycin, ribostamydn, and the butirosins. Therefore, kanamycin-neomycin phosphate transferase II differs from I in the following points II phosphorylates butirosin, but not lividomydn, whereas I phospho-rylates the latter, but not the former. 

D-glucopyranosyl)-2-deoxystreptamine, neamine, paromamine, neomycin, paromomycin, ribostamycin, butirosin A, and butirosin B. Thus, the 3-amino-3-deoxy-D-gIucose moiety in the kanamycins and the d-ribose moiety in the butirosins are not involved in the reaction. Similar structural requirements for inhibitors of kanamycin-neomycin phosphate transferase of Pseudomonas aeruginosa, as later described (see p. 201), were also observed for kanamycin-neomycin phosphate transferase II. Phosphorylation of butirosin A was competitively inhibited by one tenth the concentration of 3, 4 -dideoxykanamycin B. 

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