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The syntheses of 4′-deoxy-4′-fluoro neamine and 4′-deoxy-4′-fluoro 4′-epi neamine from the readily available neamine and paromamine are described. Different approaches for the introduction of fluorine such as the use of DAST, sulfonic esters displacement, and epoxide ring opening by fluoride anion were studied. Examples of anchimeric assistance and ring contraction in the aminoglycoside series are highlighted.

Enzymatic activity of a glycosyltransferase KanM2 encoded in the kanamycin biosynthetic gene cluster

Natural aminoglycoside antibiotics recognize an internal loop of bacterial ribosomal-decoding-site RNA by binding to the deep groove of the RNA structure. We have designed, synthesized, and tested RNA-targeted paromamine derivatives that exploit additional interactions on the shallow groove face of the decoding-site RNA. An in vitro transcription-translation assay of a series of 6'-derivatives showed the 6'-position to be very sensitive to substitution. This result suggests that the group at the 6'-position plays a pivotal role in RNA target recognition.

Based on the published NMR structure of the antibiotic paromomycin complexed with the A-site of Escherichia coli 16S ribosomal RNA, a set of C-5 functionalized analogs of paromamine was designed and synthesized, incorporating cyclic and acyclic appendages and terminating with one or more ammonium groups.

Natural aminoglycoside antibiotics recognize an internal loop of bacterial ribosomal-decoding-site RNA by binding to the deep groove of the RNA structure. We have designed, synthesized, and tested RNA-targeted paromamine derivatives that exploit additional interactions on the shallow groove face of the decoding-site RNA. An in vitro transcription-translation assay of a series of 6'-derivatives showed the 6'-position to be very sensitive to substitution. This result suggests that the group at the 6'-position plays a pivotal role in RNA target recognition.

By N-methyl-N'-nitro-N-nitrosoguanidine treatment, neamine-negative mutants which required neamine for biosynthesis of butirosins were obtained from a butirosin-producing organism Bacillus circulans. These mutants also produced butirosins from paromamine and could be divided into two types I and II. Mutants of type I could not produce butirosins from 2-deoxystreptamine, whereas those of type II could. Two typical mutants MCRL 5003 (type I) and MCRL 5004 (type II) could produce butirosin analogs, 3', 4'-dideoxybutirosins, 6'-N-methylbutirosins, 3', 4'-dideoxy-6'-N-methylbutirosins and 3', 4'-dideoxy-6'-C-methyl-butirosins from neamine analogs, gentamine Cla, 6'-N-methylneamine, 6'-N-methylgentamine Cla and gentamine C2, respectively.

Based on the knowledge that certain bacterial strains can produce enzymes that are capable of phosphorylating the 3′-hydroxyl group of a series of aminoglycoside antibiotics, the pseudodisaccharide 3′-epiparomamine was prepared through a series of high yieldingsteps, starting from paromamine. Application of selective halogenation and subsequent animation reactions allowed the efficient transformation of 3′-epiparomamine to 3′-epineamine. The two compounds were not substrates for the inactivating enzymes, however, they are also much weaker antibiotics compared to the parent compounds paromamine and neamine. Model synthetic studies in the 2-amino-2-deoxy-D-ghicose series are also reported.

By use of a decarboxylation reaction with lead tetraacetate and a deacetoxylation reaction with sodium borohydride as key-reactions, paromamine ( 12 and tri-N-benzyloxycarbonylparomamine ( 12a ) were synthesized from D-glucosamine.

Paromamine has been synthesized by condensing the N,N′-dinitrophenyl derivative of deoxystreptamine with the N-dinitrophenyl derivative of acetobromoglucosamine in nitromethane in the presence of mercuric salts, and by then removing the protecting groups by treatment with methanolic ammonia and hydrolysis with Dowex 1X2. The synthetic sample was shown to be identical with natural paromamine by a comparison of their infrared spectra and by microbiological assay. The identity was also proved by the determination of the Δ[M]Cu Am values of the tri-N-acetate of the synthetic and natural specimens by the copper-complex methods.

Streptomyces kanamyceticus produces 14C-kanamycin and other labeled kanamycin-related compounds in the presence of 14C-glucose or 14C-glucosamine. One of the latter is paromamine. In order to find a condition to obtain high incorporation rate of radioactive precursors, the culture phase for the addition of labeled compounds was studied. The maximal incorporation rate was obtained by addition of 14C-glucosamine in the later phase of the culture and the specific activity of resultant kanamycin A was found to be much higher than in the case of the addition of 14C-glucose. In some experiments one third of radioactivity of the added 14C-glucosamine was taken into kanamycin, and the distribution was limited only in 6-amino-6-deoxy-D-glucose moiety of kanamycin A when 14C-glucosamine was added. In contrast, when 1JC-glucose was added, approximately equal distribution of radioactivity was observed in each of three moieties of kanamycin A molecule.

4-O-(3-O-Benzyl-2-carbobenzoxyamino-4,6-O-isopropylidene-2-deoxy-α-D-glucopyranosyl)-N,N′-dicarbobenzoxy-2-deoxystreptamine has been prepared from paromamine and condensed with 3-acetamido-2,4,6-tri-O-benzyl-3-deoxy-α-D-glucopyranosyl chloride by a modified Koenigs-Knorr reaction. The identity of the synthetic α,α-diglycoside with a substance of the same structure derived from natural kanamycin C was shown. Removal of the masking groups gave the product identical with natural kanamycin C.

In a previous paper1) we reported that Bacillus circulans, strain No. YQW-B6, produced a complex of aminoglycoside antibiotics designated Bu-1709, of which the two major components, A1 and A2, were identified as butirosins A and B2). Two additional components, E1 and E2, have now been isolated and characterized. They have antibacterial spectra similar to the butirosins but with substantially less intrinsic activity. Bu-1709 E1 and E2 contain the paromamine moiety in place of the neamine moiety of the butirosins.

The structures of seldomycin factors 1 and 2 have been determined by consideration of chemical degradation and spectral properties. Factor 1, also known as XK-88-1, is shown to be 6-O-(2-amino-2-deoxy-alpha-D-xylopyranosyl) paromamine (1) and factor 2, also known as XK-88-2, is shown to be 4'-deoxy-neamine (2). Mass spectral evidence has been obtained that suggests the most probable structure for seldomycin factor 3, also known as XK-88-3, is 6'-amino-6'-deoxyseldomycin factor 1 (12).

Based on the knowledge that certain bacterial strains can produce enzymes that are capable of phosphorylating the 3′-hydroxyl group of a series of aminoglycoside antibiotics, the pseudodisaccharide 3′-epiparomamine was prepared through a series of high yieldingsteps, starting from paromamine. Application of selective halogenation and subsequent animation reactions allowed the efficient transformation of 3′-epiparomamine to 3′-epineamine. The two compounds were not substrates for the inactivating enzymes, however, they are also much weaker antibiotics compared to the parent compounds paromamine and neamine. Model synthetic studies in the 2-amino-2-deoxy-D-ghicose series are also reported.

A fraction enriched in the minor components of the kanamycin complex was prepared from a commercial sample. The enriched fraction was subjected to Amberlite CG-50 and Dowex 1-X2 column chromatography. Two minor components were isolated in addition to the known A, B, and C components. These congeners were identified as paromamine and 6- o -(3-amino-3-deoxy-α- d -glucopyranosyl)- deoxystreptamine. The identification was based on degradation and mass spectral studies.

Kanamycin and paromamine were inactivated in the presence of ATP and Mg++ by an enzyme solution obtained from E. coli carrying R factor. The inactivated kanamycin was isolated and it was found that the hydroxyl group on C-3 of 6-amino-6-deoxy-D-glucose moiety was phosphorylated. In the inactivated paromamine, the hydroxyl group on C-3 of D-glucosamine moiety was phosphorylated. Besides the inactivated kanamycin above described, another inactivated kanamycin containing phosphate was isolated, though it is not certain that this is the product of enzymatic inactivation.

The synthesis of an N-protected 2′-amino-2′-deoxy-1′-O-(2-deoxystreptamin-4-yl)-α-D-threo-D-gluco-octo-1′,5′:4′,8′-dipyranose in nine steps from paromamine is described; the key steps are the conversion of paromamine into a protected octuronic acid derivative, followed by its 8′,4′-lactonization upon removal of the O-protecting groups, and partial reduction of the resulting ‘trans-decalone-like’ lactone.