Chemical modification of neamine

The aminocyclitol antibiotic neamine has been modified by changing the configuration of one or two hydroxyl groups of the aminocyclitol moiety to elucidate the relationship between configuration and antimicrobial activity. 5-Epi-, 6-epi-, and 5,6-diepineamine have been prepared and their antimicrobial activity has been determined against several micro-organisms.     

Enzymatic synthesis of aminoglycoside antibiotics: novel adenosylmethionine:2-deoxystreptamine N-methyltransferase activities in hygromycin B- and spectinomycin-producing Streptomyces spp. and uses of the methylated products

Aminocyclitols structurally related to streptamine, a 1,3-diaminocyclitol, are common components of the RNA-binding aminoglycoside antibiotics. The respective aminocyclitol cores of hygromycin B and spectinomycin are N(3)-methyl-2-deoxy-D-streptamine and N(1),N(3)-dimethyl-2-epi-streptamine. Adenosyl[methyl-(14)C]methionine:2-deoxystreptamine N-methyltransferase activities were detected in extracts of early-stationary-phase mycelia of the hygromycin B producer Streptomyces hygroscopicus subsp. hygroscopicus ATCC 27438 and the spectinomycin producer Streptomyces flavopersicus ATCC 19756. Extracts of both strains methylated the N(1)- and N(3)-amino groups of 2-deoxystreptamine, streptamine, and 2-epi-streptamine; the N(1)-amino group of N(3)-methyl-2-deoxy-D-streptamine, and the N(3)-amino group of N(1)-ethyl-2-deoxy-D-streptamine, the semisynthetic aminocyclitol of netilmicin. The mono[(14)C]methyl derivatives of 2-deoxystreptamine, streptamine, and 2-epi-streptamine were excellent substrates for L-glutamine:aminocyclitol aminotransferase and thereby provided a sensitive assay for derepression of this key enzyme, a generic biosynthetic marker that we have shown to be the only enzyme common to the biosyntheses of all major aminoglycoside antibiotics. Other prospective uses for these methyl-labeled 2-deoxystreptamine analogs are also described.     

A new method for selective N-acylation of aminoglycoside antibiotics

Per-N-formylation of aminoglycoside (aminocyclitol) antibiotics followed by mild hydrolysis with aqueous ammonia gave mono-N-deformylated derivatives. Each positional isomer of the mono-N-deformylated derivatives thus obtained was separated by column chromatography on Amberlite CG-50 (NH₄⁺ ). Acylation of mono-N-deformylated derivatives gave the corresponding mono-N-acylated derivatives. The N-formyl groups of the mono-N-acylates were removed by the treatment with dilute aqueous hydrazine acetate, whereas the newly introduced N-acyl group was stable under these conditions. The 1-N-formyl group of the deoxystreptamine moiety of per-N-formylated aminoglycoside antibiotics containing neamine (or 3′-deoxyneamine) is more readily deformylated than the 3-N-formyl group. In this report, isolation and structural-elucidation studies, including ¹³C-n.m.r. spectral assignments, of positional isomers of tri-N-formyl derivatives of xylostasin (1), 3′-deoxyxylostasin (2), kanamycin A (3), and neamine (4) are described. This selective N-acylation provides a useful method for the preparation of 1-N-modified derivatives, and the synthesis of 3′-deoxybutirosin A (2f) from 2 is described in detail as an example.     

Preliminary studies on streptomutin A

Summary Supplementation of the culture medium with 2-deoxystreptidine allowsStreptomyces griseus MIT-5, an idiotrophic mutant, to produce an antibiotic which we have called streptomutin A. Degradation studies on the antibiotic showed that the aminocyclitol moiety is different from streptidine. Streptomutin A also is distinct from streptomycin in having a much different ratio of biological to chemical activity.This paper is Contribution No. 2746 from the Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, U.S.A.     

C7N氨基环醇家族的天然α-糖苷酶/淀粉酶抑制剂研究进展

C7N氨基环醇家族化合物是一类由放线菌产生的天然化合物,化学结构相对较新颖。其核心基团井岗胺赋予该家族化合物多种生物活性,其中最主要的是α-糖苷酶/淀粉酶抑制剂活性。该家族化合物在生物医学和农业领域中具有较大的应用价值。本文将结合作者的研究方向,综述已被报道的C7N家族α-糖苷酶/淀粉酶抑制剂的化学结构与生物活性。     

Determination of amikacin in human plasma by high-performance capillary electrophoresis with fluorescence detection

A selective and reproducible high-performance capillary electrophoretic (HPCE) method for the quantification of amikacin (AMK), an aminocyclitol antibiotic, in human plasma, has been developed for use in clinical laboratory tests. The method involves ultrafiltration (UF) of plasma before derivatization with the fluorescence derivatization reagent 1-methoxy-carbonylindolizine-3,5-dicarbaldehyde at room temperature for 15 min in the dark. An aliquot of the derivatives is directly introduced into the fused-silica capillary [75 cm (effective length)×50 μm I.D.] at the anode side by dynamic compression injection (50 hPa for 6 s). After electrophoresis with 40 mM SDS-20 mM phosphate-borate buffer (pH 7) in the micellar electrokinetic chromatography (MEKC) mode at 30 kV, the derivative had a retention time of 16.7 min and was detected by fluorescence intensity at 482 nm (with irradiation at 414 nm). The precision (n = 5) of the method is 4.08 and 1.59% (C.V.) at the 50 and 100 μg AMK/ml plasma levels, respectively. Linearity (r = 0.998) was established over the concentration range 5–100 mg of AMK/ml plasma and the detection limit (at a signal-to-noise ratio of 3) is 0.5 μg AMK/ml plasma. This assay method could potentially have wider application in the determination of other aminocyclitol antibiotics, such as arbekacin, dibekacin, kanamycin, in human plasma as well as of AMK.     

An Improved Synthesis of dl-Histidine

The 5-O-(2,6-diamino-2,6-dideoxy-α-d-glucopyranosyl)-2-deoxystreptamine derivative and its related compounds were synthesized by a modified Königs-Knorr condensation of 3,4-di-O-acetyl-2,6-dideoxy-2-(2′,4′-dinitroanilino)-6-phthalimido-α-d-glucopyranosyl bromide (I) with 4,6-di-O-acetyl-N,N′-dicarbobenzoxy-2-deoxystreptamine (V) and the corresponding streptamine (XI). The aglycons (V) and (XI) were prepared by selective acetylation of the aminocyclitol derivatives by taking advantage of the reactivity difference between the hydroxyl groups at C₅ and C₄ or C₆. The condensed products were converted to N-acetyl derivatives and were shown to have the α-configuration by PMR spectroscopy.     

Biochemical basis of resistance to hygromycin B in Streptomyces hygroscopicus--the producing organism

Hygromycin B, an aminocyclitol antibiotic that strongly inhibits both 70S and 80S ribosomes, is synthesized by Streptomyces hygroscopicus. Ribosomes from this Gram-positive mycelial bacterium are inhibited in vitro by the antibiotic. In contrast, the streptomycete is highly resistant to the drug in vivo since it possesses hygromycin B phosphotransferase activity. This enzyme has been shown by gel filtration to have a molecular weight of 42000, and to modify its antibiotic substrate to produce 7"-O-phosphoryl-hygromycin B which totally lacks biological activity both in vivo and in vitro.     

Chemistry and biology of trehazolins

Trehazolin (1) is a unique natural pseudodisaccharide possessing strong trehalase-specific inhibitory activity. To determine its argued correct stereochemistry, the syntheses of trehazolin (1), its components, the aglycon moiety, trehalamine (4) and its aminocyclitol hexaacetate (6), were accomplished from d-glucose using intramolecular [3+2] cycloaddition as the key step. In order to investigate the structure–activity relationships with regard to the stereochemistry of the aminocyclitol moiety and that of the anomeric position of trehazolin (1), trehalostatin (2) (trehazolin C-5 epimer), trehazolin β-anomer (32) and, trehazolin C-6 epimer (33) were all synthesized. In particular, with respect to the synthesis of trehazolin C-6 epimer (33), a tandem aldol–Wittig type reaction was developed as the key step to synthesize the highly functionalized 5-membered cyclitol. Moreover, on the basis of the outcome of these synthetic studies, a number of trehazolin-related compounds (49–52), modified at the terminal amino group of trehalamine (4), were synthesized to be evaluated as candidates directed to anti-NIDDM (non-insulin-dependent diabetes mellitus) drugs.     

Comparative study of the relationship between the blood concentration of gentamicin and streptomycin and their muscle-relaxing activity]

A mathematical model of relation between the miorelaxant effect value of aminocyclitol antibiotics and their blood levels is proposed. The kinetics of the reduction of the neuromuscle transmission damaged under the action of the drugs was studied in detail in acute experiments with cats treated with gentamycin or streptomycin administered intravenously. The changes in the effect value in time were satisfactorily described by the logistic function. Parallel determination of the blood levels of the antibiotics in the animals provided construction of an adequate model of their pharmacokinetics. Tabulation of the corresponding biexponential equations and logistic functions for the same time intervals during reduction of the neuromuscle transmission provided necessary information for plotting the "effect: concentration" curves. Comparison of the drug levels at which the effect of inhibition of the neuromuscle transmission was equal to 50% of the maximum one, revealed a statistically reliable difference in the miorelaxant activity of gentamycin and streptomycin. A high miorelaxant activity of gentamycin in comparison to streptomycin was also shown on comparison of the average "effect: concentration" curves plotted on the basis of tabulation of the general equations presenting a combination of the logistic and biexponential equations.     

Synthetic approaches to kasugamine

Reactions are described that lead to an immediate, chemical precursor of kasugamine the amino-sugar component of the aminocyclitol antibiotic kasugamycin. The key reaction involved the introduction of a bromine atom by a stereoselective ring-opening of a benzylidene acetal.     

Possible evolutionary relationships between streptomycin and bluensomycin biosynthetic pathways: detection of novel inositol kinase and O-carbamoyltransferase activities.

Bluensomycin (glebomycin) is an aminocyclitol antibiotic that differs structurally from dihydrostreptomycin in having bluensidine (1D-1-O-carbamoyl-3-guanidinodeoxy-scyllo-inositol) rather than streptidine (1,3-diguanidino-1,3-dideoxy-scyllo-inositol) as its aminocyclitol moiety. Extracts of the bluensomycin producer Streptomyces hygroscopicus form glebosus ATCC 14607 (S. glebosus) were found to have aminodeoxy-scyllo-inositol kinase activity but to lack 1D-1-guanidino-3-amino-1,3-dideoxy-scyllo-inositol kinase activity, showing for the first time that these two reactions in streptomycin producers must be catalyzed by different enzymes. S. glebosus extracts therefore possess the same five enzymes required for synthesis of guanidinodeoxy-scyllo-inositol from myo-inositol that are found in streptomycin producers but lack the next three of the four enzymes found in streptomycin producers that are required to synthesize the second guanidino group of streptidine-P. In place of a second guanidino group, S. glebosus extracts were found to catalyze a Mg2(+)-dependent carbamoylation of guanidinodeoxy-scyllo-inositol to form bluensidine, followed by a phosphorylation to form bluensidine-P. The novel carbamoyl-P:guanidinodeoxy-scyllo-inositol O-carbamoyltransferase and ATP:bluensidine phosphotransferase activities were not detected in streptomycin producers or in S. glebosus during its early rapid growth phase. Free bluensidine appears to be a normal intermediate in bluensomycin biosynthesis, in contrast to the case of streptomycin biosynthesis; in the latter, although exogenous streptidine can enter the pathway via streptidine-P, free streptidine is not an intermediate in the endogenous biosynthetic pathway. Comparison of the streptomycin and bluensomycin biosynthetic pathways provides a unique opportunity to evaluate those proposed mechanisms for the evolutionary acquisition of new biosynthetic capabilities that involve gene duplication and subsequent mutational changes in one member of the pair. In this model, there are at least five pairs of enzymes catalyzing analogous reactions that can be analyzed for homology at both the protein and DNA levels, including two putative pairs of inositol kinases detected in this study.     

Overexpression and mechanistic analysis of chromosomally encoded aminoglycoside 2'-N-acetyltransferase (AAC(2')-Ic) from Mycobacterium tuberculosis

The chromosomally encoded aminoglycoside N-acetyltransferase, AAC(2')-Ic, of Mycobacterium tuberculosis has a yet unidentified physiological function. The aac(2')-Ic gene was cloned and expressed in Escherichia coli, and AAC(2')-Ic was purified. Recombinant AAC(2')-Ic was a soluble protein of 20,000 Da and acetylated all aminoglycosides substrates tested in vitro, including therapeutically important antibiotics. Acetyl-CoA was the preferred acyl donor. The enzyme, in addition to acetylating aminoglycosides containing 2'-amino substituents, also acetylated kanamycin A and amikacin that contain a 2'-hydroxyl substituent, although with lower activity, indicating the capacity of the enzyme to perform both N-acetyl and O-acetyl transfer. The enzyme exhibited "substrate activation" with many aminoglycoside substrates while exhibiting Michaelis-Menten kinetics with others. Kinetic studies supported a random kinetic mechanism for AAC(2')-Ic. Comparison of the kinetic parameters of different aminoglycosides suggested that their hexopyranosyl residues and, to a lesser extent, the central aminocyclitol residue carry the major determinants of substrate affinity.     

Chemical modification of neamine.

The aminocyclitol antibiotic neamine has been modified chemically by removing one or two hydroxyl groups from the 2-deoxystreptamine moiety to give 5- and 6- deoxyneamines (5 and 10), as well as 5,6-dideoxyneamine (15). Their antimicrobial activities were determined against several microorganisms, including kanamycin-resistant strains.     

Synthesis and antibacterial activity of alkyl derivatives of the glycopeptide antibiotic A40926 and their amides

New derivatives of the glycopeptide antibiotic A40926 were synthesized and evaluated for antimicrobial activity against VRE. Deacylated A40926 was obtained by microbial transformation of the parent antibiotic with the use of Actinoplanes teichomyceticus ATCC 31121. Regioselective synthesis of alkylated derivatives of Deacyl A40926 was carried out using lipophilic aliphatic and aromatic halides or aldehydes. Further modification of the two carboxylic acids was performed to increase antibiotic activity. Poor antimicrobial activity was observed for the derivatives obtained by lipophilic mono- or dialkylation of the amino groups present on the molecule, while simultaneous condensation of both carboxylic groups, in hydrophobic derivatives, with dibasic amines led to a strong increase in antibiotic activity.     

Expression of prokaryotic genes for hygromycin B and G418 resistance as dominant-selection markers in mouse L cells

Novel bacterial resistance genes were cloned and expressed as dominant selection markers in mammalian cells. Escherichia coli genes coding for resistance to the aminocyclitol antibiotics hygromycin B (Hm) and G418 were cloned into the eukaryotic expression plasmid pSV5GPT [Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78 (1981) 2072–2076]. Mouse cells normally sensitive to 100 μg/ml Hm were transformed with these plasmids and selected in 200 μg/ml Hm. Transformants resistant to as much as l mg/ml Hm and 500 μg/ml G418 were isolated. Cell extracts contained an acetyltransferase activity capable of acetylating G418 and an Hm amino-cyclitol phosphotransferase activity. Plasmid DNA sequences were identified by Southern blot analysis of high M_r DNA isolated from transformed cells.     

Enzymatic modification of aminocyclitol antibiotics: Mutations affecting the expression of aminocyclitol acetyltransferase-3

Hydroxylamine treatment of plasmid DNA pLH1, a recombinant plasmid of colicin E1 and the gene for aminocyclitol acetyltransferase-3 (AAC-3), upon transformation provided a number of gentamicin-sensitive mutants. All gentamicin-sensitive strains lacked AAC-3 activity, and studies of partially defective and temperature-sensitive mutants have defined the role of the acetyltransferase in the determination of drug resistance. The results are consistent with the notion that modification of the entering drug per se, without interaction with membranes or transport systems, is responsible for the resistance phenotype, as was implied by the original finding of S. Okomoto and Y. Suzuki (Nature (London),208, 1301–1303, 1966), who demonstrated that dihydrostreptomycin and kanamycin were inactivated when incubated with extracts of cells containing plasmids.     

Acarbose, a pseudooligosaccharide, is transported but not metabolized by the maltose-maltodextrin system of Escherichia coli

The pseudooligosaccharide acarbose is a potent inhibitor of amylases, glucosidases, and cyclodextrin glycosyltransferase and is clinically used for the treatment of so-called type II or insulin-independent diabetes. The compound consists of an unsaturated aminocyclitol, a deoxyhexose, and a maltose. The unsaturated aminocyclitol moiety (also called valienamine) is primarily responsible for the inhibition of glucosidases. Due to its structural similarity to maltotetraose, we have investigated whether acarbose is recognized as a substrate by the maltose/maltodextrin system of Escherichia coli. Acarbose at millimolar concentrations specifically affected the growth of E. coli K-12 on maltose as the sole source of carbon and energy. Uptake of radiolabeled maltose was competitively inhibited by acarbose, with a Ki of 1.1 microM. Maltose-grown cells transported radiolabeled acarbose, indicating that the compound is recognized as a substrate. Studying the interaction of acarbose with purified maltoporin in black lipid membranes revealed that the kinetics of acarbose binding to LamB is asymmetric. The on-rate of acarbose is approximately 30 times lower when the molecule enters the pore from the extracellular side than when it enters from the periplasmic side. Acarbose could not be utilized as a carbon source since the compound alone was not a substrate of amylomaltase (MalQ) and was only poorly attacked by maltodextrin glucosidase (MalZ).     

A Rennin-like Enzyme from Penicillium expansum

dl-(1,3/2)-3-Acetamido-1,2-di-O-benzylcyclohex-4-enediol (IIIa) and dl-(1,3/4)-1-acetamido-3,4-di-O-benzylcyclohex-5-enediol (IIIb) were synthesized from dl-trans-1,2-di-O-benzylcyclohex-3-enediol (I) via the corresponding azide derivatives (IIa-b) prepared by bromination and subsequent treatment with sodium azide in N,N-dimethylformamide. Compounds (IIIa and IIIb) were converted into a variety of deoxyinosamine and deoxyinosadiamine derivatives via epoxides (VIII and IX) or by cis-hydroxylation with osmium tetroxide. Hexaacetyl-rac-inosamine-1 (XVIIIc) was synthesized from dl-(1,3,4/2,5)-3-acetamido-1,2-di-O-benzyl-5-bromocyclohexanetriol (VIa) via conduramine derivatives (XVIIa-c). Conformationai analysis of partially O-benzylated aminocyclitol derivatives were studied by means of NMR spectroscopy.