Chemoenzymatic acylation of aminoglycoside antibiotics.

The chemoenzymatic installation of the clinically valuable (S)-4-amino-2-hydroxybutyryl side chain onto a number of 2-deoxystreptamine-containing aminoglycosides is described using the purified Bacillus circulans biosynthetic enzymes BtrH and BtrG in combination with a synthetic acyl-SNAC surrogate substrate.

Aph(3')-IIc, an aminoglycoside resistance determinant from Stenotrophomonas maltophilia.

We report the characterization of an intrinsic, chromosomally carried aph(3')-IIc gene from Stenotrophomonas maltophilia clinical isolate K279a, encoding an aminoglycoside phosphotransferase enzyme that significantly increases MICs of kanamycin, neomycin, butirosin, and paromomycin when expressed in Escherichia coli. Disruption of aph(3')-IIc in K279a results in decreased MICs of these drugs.

Significance of the 20-kDa subunit of heterodimeric 2-deoxy-scyllo-inosose synthase for the biosynthesis of butirosin antibiotics in Bacillus circulans.

A gene (btrC2) encoding the 20-kDa subunit of 2-deoxy-scyllo-inosose (DOI) synthase, a key enzyme in the biosynthesis of 2-deoxystreptamine, was identified from the butirosin-producer Bacillus circulans by reverse genetics. The deduced amino acid sequence of BtrC2 closely resembled that of YaaE of B. subtilis, but the function of the latter has not been known to date. Instead, BtrC2 appeared to show sequence similarity to a certain extent with HisH of B. subtilis, an amidotransferase subunit of imidazole glycerol phosphate synthase. Disruption of btrC2 reduced the growth rate compared with the wild type, and simultaneously antibiotic producing activity was lost. Addition of NH4Cl to the medium complemented only the growth rate of the disruptant, and both the growth rate and antibiotic production were restored by addition of yeast extract. In addition, a heterologous co-expression system of btrC2 with btrC was constructed in Escherichia coli. The simultaneously over-expressed BtrC2 and BtrC constituted a heterodimer, the biochemical features of which resembled those of DOI synthase from B. circulans more than those of the recombinant homodimeric BtrC. Despite the similarity of BtrC2 to HisH the heterodimer showed neither aminotransfer nor amidotransfer activity for 2-deoxy-scyllo-inosose as a substrate. All the observations suggest that BtrC2 is involved not only in the secondary metabolism, but also in the primary metabolism in B. circulans. The function of BtrC2 in the butirosin biosynthesis appears to be indirect, and may be involved in stabilization of DOI synthase and in regulation of its enzyme activity.

Kinetic isotope effect and reaction mechanism of 2-deoxy-scyllo-inosose synthase derived from butirosin-producing Bacillus circulans.

The mechanism of 2-deoxy-scyllo-inosose synthase reaction, a carbocycle formation step from D-glucose-6-phosphate in the biosynthesis of the 2-deoxystreptamine aglycon of clinically important aminocyclitol antibiotics, was investigated with a partially purified enzyme from butirosin-producing Bacillus circulans SANK 72073, Nonlabeled and double-labeled D-[4-2H, 3-15O]glucose-6-phosphate were used for cross-over experiment, and the oxime-TMS ether derivative of the 2-deoxy-scyllo-inosose product was analyzed by GC-MS. The deuterium label at C-4 of the substrate appeared to be retained at C-6 of the inosose product without scrambling of the double-labeled isotopes. Since the transient reduction of NAD+ cofactor was proved to be essential in the 2-deoxy-scyllo-inosose reaction, the hydride abstraction and returning appeared to take place within the same glucose molecule. The observed kinetic isotope effect was estimated to be kH/kD = 2.4. These results strongly suggest that this carbocycle formation is catalyzed by a single 2-deoxy-scyllo-inosose synthase enzyme with catalytic requirement of NAD+, the mechanism of which appears to be resembled closely to the 2-deoxy-scyllo-inosose synthase in the Streptomyces fradiae.

Synthesis and activity of butirosin derivatives with 5''-amidino and 5''-guanidino substituents.

The preparation and antibacterial activity of the 5''-guanidino (6) and 5''-amidino (7) derivatives of 4'-deoxybutirosin A (1) as well as the 5''-guanidino derivative (8) of butirosin A are described. The key intermediates, tetra-N-benzyloxycarbonyl-5''-azido derivatives were selectively reduced with NiCl2-NaBH4 to give the corresponding 5'-amino derivatives. Subsequent guanidination or amidination followed by deblocking afforded the final compounds 6, 7 and 8. The 5''-guanidino derivatives (6 and 8) were more active against Gram-positive and Gram-negative bacteria than the corresponding 5''-hydroxy derivatives (1 and butirosin A). Compound 6 was also active against a variety of methicillin-resistant Staphylococcus aureus (MRSA).

Deoxy derivatives of butirosin A and 5"-amino-5"-deoxybutirosin A, aminoglycoside antibiotics resistant to bacterial 3'-phosphorylative enzymatic inactivation. Synthesis and NMR studies.

3'-Deoxybutirosin A (4), 5"-amino-3', 5"-dideoxybutirosin A (6), and 5"-amino-4', 5"-dideoxybutirosin A (7) were prepared by deoxygenation of the appropriate hydroxyl in suitably protected derivatives of butirosin A, using sequentially trifluoromethylsulfonylation, displacement with benzenethiolate, and hydrogenolysis. The structures of the compounds were confirmed by NMR spectroscopy, using 13C NMR and 1H NMR at up to 600 MHz. The compounds are broad-spectrum antibiotics active against resistant microorganisms which inactivate butirosin and related aminoglycosides by 3'-phosphorylation.

Susceptibility to butirosin and neomycin B in Bacillus circulans, the butirosin-producing organism.

Butirosin, an aminoglycoside antibiotic, is produced by Bacillus circulans B-3312. Experiments using recombined ribosomal and supernatant fractions from this strain and from B. megaterium KM have shown that the ribosome of both are sensitive to butirosin. The aminoglycoside 3'-phosphotransferase present in B. circulans modifies butirosin and neomycin in vitro but confers resistance only to the former in vivo. The phosphotransferase does not modifya detectable amount of extracellular butirosin while mediating resistance to the antibiotic. In vitro, however, the enzyme appears to protect against inhibition by butirosin by inactivating the bulk of the antibiotic in the system. An extrachromosomal element of unknown function has been detected in B. circulans.

1-N-Alkyl analogs of butirosin.

Butirosin (Ia), 5''-amino-5''-deoxy-(Ic), 3',4'-dideoxy-(Ie), and 5''-amino-3',4',5''-trideoxy-butirosin A (If) were converted into the corresponding 1'''-deoxo derivatives, Ib, Id, Ig, and Ih by borane reduction. In addition, xylostasin (IIIa) was converted into its 1-N-ethyl derivative (IIIb) by reductive ethylation. Their antibacterial activities were discussed.

Mutational biosynthesis of butirosin analogs. I. Conversion of neamine analogs into butirosin analogs by mutants of Bacillus circulans.

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.

Mutational biosynthesis of butirosin analogs. II. 3', 4'-Dideoxy-6'-N-methylbutirosins, new semisynthetic aminoglycosides.

A pair of new butirosin analogs was isolated from the fermentation broth obtained by cultivating a neamine-negative mutant of the butirosin-producing organism Bacillus circulans in the medium supplemented with 6'-N-methylgentamine C1a. These antibiotics were characterized and elucidated as 3', 4'-dideoxy-6'-N-methylbutirosins A and B (DMB-A & DMB-B), by chemical and spectroscopic studies. DMB-A and DMB-B exhibited broad-spectrum antibacterial activities with in vitro potency similar to or slightly less than that for the butirosin A, with the exception of strains of Pseudomonas aeruginosa and Serratia marcescens against which they exhibited activities equal to or slightly greater than that for butirosin A. As expected, they exhibited stronger activities against butirosin-resistant organisms which contain acetylating enzymes AAC(6')-I and AAC(6')-IV, and phosphorylating enzyme APH(3')-II. They were also active against some of the clinical isolates resistant to butirosins, dibekacin and/or gentamicin. The acute intravenous toxicity in mice of the DMB complex (B:70 APPROXIMATELY 80%) was somewhat less than that of the butirosin A.

Mutational biosynthesis of butirosin analogs. III. 6'-N-methylbutirosins and 3', 4'-dideoxy-6'-c-methylbutirosins, new semisynthetic aminoglycosides.

Two pairs of butirosin analogs were isolated from the fermentation broths obtained by cultivating a neamine-negative mutant of the butirosin-producing organism Bacillus circulans in the medium supplemented with 6'-N-methylneamine and gentamine C2, respectively. These amtibiotics were characterized as 6'-N-mentylbutirosins A and B (NMB-A & NMB-B), and 3', 4'-dideoxy-6'-C-methylbutirosins A and B (DCB-A & DCB-B), respectively, by chemical and spectroscopic studies. NMB-A and NMB-B exhibited broad-spectrum antibacterial activities with in vitro potency similar to or slightly less than that for butirosin A, but with greater activities against butirosin-resistant strain which contains acetylating enzyme AAC-(6')-I. The activities of NMB-A and NMB-B against Pseudomonas aeruginosa strains were relatively weak. DCB-B had broad-spectrum activity, and exhibited greatly improved activity against butirosin-resistant organisms which contain acetylating enzymes AAC(6')-I and AAC(6')-IV, and phosphorylating enzyme APH(3')-II. These new butirosin analogs were also active against some of the clinical isolates resistant to butirosins, dibekacin and/or gentamicin.

Synthesis of 5''-amino-3,5''-dideoxybutirosin A.

The titled compound was prepared by condensation of 3'-deoxyparomamine derivative (5) with 2,3-O-bis(p-nitrobenzoyl)-5-O-tosyl-D-xylofuranosyl bromide followed by 1-N-acylation with the active ester of (S)-4-benzyloxycarbonylamino-2-hydroxybutyric acid. The compound was slightly more active than 3'-deoxybutirosin A against Pseudomonas.

Mistranslation in a eucaryotic organism.

Previous work from our laboratory has demonstrated that a subclass of the aminoglycoside antibiotics, those containing the drug fragment paromamine, stimulates mistranslation in cell-free protein-synthesizing systems derived from eucaryotic cells. We report here experiments which show that the ciliate Tetrahymena thermophila (formerly T. pyriformis, syngen 1) is sensitive to the paromamine-containing aminoglycoside antibiotics. The drugs are active with respect to growth inhibition, inhibition of protein synthesis in the whole organism, inhibition of protein synthesis in vitro and the stimulation of mistranslation in cell-free protein-synthesizing systems. Because of their misreading properties, these drugs may be useful in isolating and propagating strains carrying mutations which can be translationally suppressed (that is, missense and nonsense mutations).

A new methods for the 3'-deoxygenation of butirosins A and B.

An aziridine ring-formation involving the reaction of adjacent amino and alcohol groups with triphenylphosphine, carbon tetrachloride, and triethylamine was applied at the 2' and 3' positions of butirosin A (1a) and B (1b). The amino groups at the 2' position of 1a and 1b were p-methoxybenzylated to increase the nucleophilicity of the nitrogen atom and to avoid the formation of a P-N linkage, and the N-p-methoxybenzyl derivatives were converted into the aziridine derivatives, which were then subjected to hydrogenolysis and removal of the protecting groups to give 3'-deoxybutirosin A (7a) and B (7b), respectively. This new method is compared with the conventional N,O-protecting method that involves several complex steps.

Effect of combinations of penicillin and aminoglycosides on Streptococcus faecalis: a comparative study of seven aminoglycoside antibiotics.

The 50% inhibitory concentrations (IC50) of benzylpenicillin, streptomycin, sisomicin, gentamicin, tobramycin, kanamycin, amikacin, and butirosin were determined for 58 clinical isolates of Streptococcus faecalis, 28 of which were recovered from cultures of blood samples from patients with endocarditis. The IC50 of streptomycin was less than 100 microng/ml for 42 strains, 192-10,000 microng/ml for eight, and larger than or equal to 10,000 micron/ml for eight. One isolate that was highly resistant to streptomycin was also highly resistant to kanamycin and butirosin. Extraordinarily high resistance to the other aminoglycosides was not observed. The bactericidal effects of combinations of penicillin and aminoglycosides were studied in 20 strains of S. faecalis that represented different levels of resistance to streptomycin. Significant enhancement of the effect of the combination of penicillin and streptomycin was found only in strains with an IC50 of smaller than or equal to 190 microng/ml. Combinations of penicillin and sisomicin, gentamicin, or tobramycin were effective even against strains that were highly resistant to streptomycin and kanamycin.

Amikacin, an aminoglycoside with marked activity against antibiotic-resistant clinical isolates.

A total of 319 clinical isolates known to be resistant to one or more aminoglycoside antibiotics were tested for their susceptibility to 10 aminoglycosides. The percentages of isolates found by an agar dilution method to be susceptible were: amikacin, 83.7%; tobramycin, 41.4%; butirosin A, 33.2%; dideoxykanamycin B, 32.6%; gentamicin C, 27.3%; lividomycin A, 17.6%; neomycin B, 10.7%; paromomycin, 10.3%; kanamycin A, 10.0%; and ribostamycin, 7.2%. The effectiveness of the antibiotics was related to their degree of resistance to bacterial enzymes; e.g., of the nine enzymes known to inactivate antibiotics containing 2-deoxystreptamine, amikacin was affected by one enzyme, tobramycin by five, and gentamicin and kanamycin by six. Examination of cell-free extracts from the 52 strains resistant to amikacin revealed that only four contained the amikacin-inactivating enzyme aminoglycoside-6'-acetyltransferase, a finding indicating that this mechanism of resistance is rare. Other experiments suggest that most amikacin-resistant strains, which are almost invariably resistant to all aminoglycosides, lack the ability to accumulate effectively either amikacin or presumably the other antibiotics intracellularly.