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Ansamitocins are potent antitumor agents produced by Actinosynnema pretiosum. As deduced from their structures, an N-methylation on the amide bond is required among the various modifications. The encoded protein by asm10 belongs to SAM-dependent methyltransferase family. Through gene inactivation and complementation, asm10 was proved to be responsible for the N-methylation of ansamitocins. Asm10 is 33.0 kDa in size and present as monomer as determined by gel filtration. Using N-desmethyl-ansamitocin P-3 as substrate, the optimal temperature and pH were determined to be 32 °C and 10.0 respectively for Asm10 catalysis. Asm10 also showed broad substrate flexibility toward other N-desmethyl ansamycins and synthetic indolin-2-ones. Through site-directed mutagenesis, Asp154 and Leu155 of Asm10 were confirmed to be essential for its catalysis possibly thorough the binding of SAM. The characterization of this unique N-methyltransferase enriched the toolbox for engineering N-methylated derivatives from both natural and synthetic compounds, which will allow modification of known potential drugs.

The conformational properties of a series of pyridoxal amino acid Schiff's bases have been calculated. The calculations were performed using the program CAMSEQ which employs a set of empirical potential energy functions. The cndo/2 charge distributions are also given in this preliminary report. The locations of the local energy minima are consistent with Dunathan's hypothesis that the course of enzymatic reaction is related to the conformation of the pyridoxal phosphate Schiff's base. Calculated results are also in agreement with experiment.

Maytansine, a potent clinically evaluated plant-derived anti-tumor drug, and its microbial counterpart, ansamitocin P-3, showed a substantially higher cytoxicity than many other anti-tumor drugs. Owing to a shortage of material and lack of sufficiently sensitive analytical methods at the time, no metabolism studies were apparently carried out in conjunction with the initial preclinical and clinical studies on maytansine, but some products of decomposition during the period of storage of the formulated drug were reported. In the current study, the in vitro metabolism of maytansine and ansamitocin P-3 was studied after incubation with rat and human liver microsomes in the presence of NADPH, and with rat and human plasma and whole blood, using liquid chromatography/multi-stage mass spectrometry. Unchanged ansamitocin P-3 and 11 metabolites and unchanged maytansine and seven metabolites were profiled and the structures of some metabolites were tentatively assigned based on their multi-stage electrospray ion-trap mass fragmentation data and in some cases accurate mass measurement. The major pathway of ansamitocin P-3 metabolism in human liver microsomes appears to be demethylation at C-10. Oxidation and sequential oxidation/demethylation also occurred, although to a lesser extent. However, the major pathway of maytansine metabolism in human liver microsomes is N-demethylation of the methylamide of the ester moiety. Several minor pathways including O/N-demethylation, oxidation and hydrolysis of the ester bond were also observed. There were no differences in maytansine metabolism between rat and human liver microsomes; however, the rate of metabolism of ansamitocin P-3 was different in rat and human liver microsomes. About 20% of ansamitocin P-3 was converted to its metabolites in rat liver microsomes and about 70% in human liver microsomes under the same conditions. Additionally, 10-O-demethylated ansamitocin P-3 was also detected in the urine after i.v. bolus administration of ansamitocin P-3 to Sprague-Dawley male rats. No metabolites were detected following incubation of maytansine and ansamitocin P-3 with human and rat whole blood and plasma.

Echinosporin (1), a known antibiotic with a unique tricyclic acetal-lactone structure, is produced by Streptomyces erythraeus (strain Tu 4015), together with the novel 7-deoxyechinosporin (2) as minor compound, which was fully characterized. The biosynthesis of 1 was established by feeding experiments with 13C-labelled precursors. The results revealed that 1 was formed by the shikimate pathway, with chorismate as a biosynthetic intermediate. The proposed mechanism for the conversion of chorismate into 1 represents a new branch of the shikimate pathway, producing a nonaromatic metabolite. In addition, the influence of aromatic amino acids and of glyphosate, an inhibitor in the shikimate pathway of plants, was investigated. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

Alkylation-amination of the enamine 2 in the presence of ethyl α,α-bis(dibromomethyl)acetate, triethyl-amine, and methylamine lead to the construction of the aza-transposed ergoline 3. Sequential reduction, hydrolysis, reesterification, and indolization of 3, produced three diastereomers of 6. The structure of these three diastereomers was assigned on the basis of nmr and ir spectral analysis to be (α-cis) syn, (β-cis) anti, and (α-trans) syn. The isomer (β-cis) anti was reduced with lithium aluminum hydride to the corresponding alcohol.

The thiostrepton-resistance gene encoding the 23S rRNA A1067 methyltransferase from Streptomyces azureus has been overexpressed in Escherichia coli using a T7-RNA-polymerase-dependent expression vector. The protein was efficiently expressed at levels up to 20% of total soluble protein and purified to near homogeneity. Kinetic parameters for S-adenosyl-L-methionine (Km = 0.1 mM) and an RNA fragment containing nucleotides 1029-1122 of the 23S ribosomal RNA from E. coli (Km = 0.001 mM) were determined. S-Adenosyl-L-homocysteine showed competitive product inhibition (Ki = 0.013 mM). Binding of either thiostrepton or protein L11 inhibited methylation. RNA sequence variants of the RNA fragment with mutations in nucleotides 1051-1108 were tested as substrates for the methylase. The experimental data indicate that methylation is dependent on the secondary structure of the hairpin including nucleotide A1067 and the exact sequence U(1066)-A(1067)-G(1068)-A(1069)-A(1070) of the single strand.

Interspecies cloning of antibiotic biosynthesis genes in streptomycetes can be used to generate new hybrid natural product structures; the application of this approach to polyketide and its extension to thiopeptide antibiotics are summarized.

The mode of C-ring formation in ergot alkaloid biosynthesis was probed by synthesizing two potential intermediates, compounds 6 and 7, in deuterated form from the prenylated indole 8. Both compounds were incorporated into the ergot alkaloid elymoclavine by washed mycelia of Claviceps sp., strain SD 58, but only the formation of 7, and not 6, could he demonstrated in the cultures. Hence it is proposed that only 7 is an intermediate in ergot alkaloid biosynthesis, whereas 6 is not on the pathway but can he converted into 7 when added to the cultures

The formation of the cyclohexanecarboxylic acid moiety in the biosynthesis of ansatrienin (mycotrienin) has been studied. [sup 13]C- and [sup 2]H-labeled samples of shikimic acid were used to probe the stereochemistry of processing the cyclohexane ring of shikimic acid and to establish the fate of all the precursor hydrogens in this transformation. A sample of [2-[sup 13]C]shikimic acid was fed to Streptomyces collinus Tu 1982, and [sup 13]C in the resulting ansatrienin was found to reside exclusively at C-36. The l-cyclohexenecarboxylic acid accompanying the cyclohexanecarboxylic acid in the hydrolysis of the biosynthetic sample of ansatrienin carried the [sup 13]C label not at C-2 but at C-6. Samples of [2-[sup 2]H]-, [3-[sup 2]H]-, [4-[sup 2]H], [2,5-[sup 2]H[sub 2]]-, [2,3,4,5-[sup 2]H[sub 4]]-, and [6-[sup 2]H[sub 1]]shikimic acid were fed to S. collinus. Deuterium from C-2, C-3, C-4, and C-5 was effectively incorporated and occupied the 36R (axial), 35R (equatorial), 34E (equatorial), and 33R (axial) positions, respectively, in the resulting ansatrienin A. However, absolutely no deuterium from C-6 of shikimic acid was incorporated. Potential intermediates specifically labeled with [sup 13]C and [sup 2]H were used to further delineate the pathway. Combined, the results from these studies define the pathway by which shikimic acidmore » is converted into cyclohexanecarboxylic acid. 1,4-Conjugate elimination of the hydroxy group at C-3 and a proton from C-6 of shikimic acid gives rise to a cross-conjugated dihydroxy diene, which undergoes reduction of the double bond conjugated to the carbonyl group. Another 1,4-elimination involving the C-4 hydroxy group and the proton at C-1 gives a 5-hydroxy 1,3-diene. Reduction to 5-hydroxycyclohex-1-enecarboxylic acid proceeds either directly or via the [Delta][sup 2] isomer. Another reduction gives the hydroxy acid, which undergoes dehydration involving a nonacidic proton. 52 refs., 3 figs., 3 tabs.« less

Sulfur plays a critical role in many biochemical reactions both as a nucleophile and a nucleofuge and, in the form of the sulfhydryl-disulfide pair, as a redox system. It is involved in stabling pr...

The biosynthesis of the highly modified thiopeptide antibiotic, nosiheptide (1), was studied by feeding radioactive and stable-isotope-labeled precursors to cultures of the producing organism, Streptomyces actuosus. The stable isotope enrichments and/or coupling patterns in the isolated 1 were analyzed by NMR spectroscopy using various 1D and 2D NMR techniques. The results complete the confirmation of the amino acid components of the antibiotic and shed light on a number of mechanistic and stereochemical aspects of its assembly from these basic building blocks. The dehydroalanine and butyrine moieties are formed by an anti elimination of water from serine and threonine, respectively, the thiazole rings from cysteine residues with loss of the pro-3R hydrogen in the oxidation step, and the hydroxypyridine moiety from two intact serine residues, situated nine amino acids apart in the peptide chain, and the carboxyl group of an adjacent cysteine. According to {sup 15}N studies which included the complete assignment of the {sup 15}N NMR resonances of 1, the carboxy-terminal amide nitrogen of 1 originates from the amino group of serine, suggesting that the precursor peptide giving rise to 1 carries an additional carboxyl-terminal serine residue which, except for its nitrogen, is removed during processing. 64 refs., 7 figs.,more » 3 tabs.« less

The biosynthesis of the antibiotic reductiomycin (I) in Streptomyces xanthochromogenus was investigated by feeding experiments with radioactive and stable isotope-labeled precursors. NMR and mass spectroscopic analyses of the labeled I samples revealed that the acetoxy group comes from acetate, the 2-amino-3-hydroxycyclopent-2-enone moiety arises by a novel intramolecular cyclization of 5-aminolevulinic acid (ALA), and the dihydrofuranylacrylic moiety is formed by aromatic ring cleavage of a symmetrical product of the shikimate pathway. Both 4-hydroxy-[7-[sup 13]C]benzoic acid and 4-hydroxy-[7-[sup 13]C]benzaldehyde label 1 very efficiently, and deuterium from various positions in these precursors is incorporated into the predicted positions in the dihydrofuranylacrylic acid moiety of 1. The results are interpreted in terms of a dioxygenase mechanism for the ring cleavage reaction and pyridoxal phosphate catalysis for the ALA cyclization. 42 refs., 6 figs., 5 tabs.

The synthesis of 4-amino-3,4-dideoxy- d -arabino-heptulosonic acid 7-phosphate (15), the first committed intermediate in a new branch of the shikimate pathway of aromatic biosynthesis, is reported from 2-deoxy- d -glucose in 14 steps in 10–12% overall yield.

The biosynthesis of the plant antitumor agent, taxol, was studied by feeding radioactive or stable isotope-labeled precursors to cut stems or to inner bark tissue of Taxus brevifolia. The labeled taxol was purified to radiochemical purity and subjected to chemical degradation or was analyzed by electrospray tandem mass spectrometry. It was demonstrated that in the plant taxol is synthesized from baccatin-111, containing the fully functionalized diterpene moiety, and a precursor of the phenylpropanoid side chain. The latter arises from phenylalanine not via cinnamic acid, but via B-phenylalanine and phenylisoserine. Benzoylation of the side chain occurs only after its attachment to the diterpene moiety. The benzoate moiety is also formed from phenylalanine via B-phenylalanine and phenylisoserine, not via cinnamic acid. Our studies focused initially on the origin of the phenylpropanoid side chain and its mode of attachment to the diterpene moiety. Feeding experiments with radiolabeled precursors were carried out with cut twigs of Taxus brevifolia to which the radioactive material was administered through the cut stem in a small volume of water. Extensive purification of the resulting taxol, first by repeated HPLC and then by co-crystallization with non-labeled carrier material was found necessary to achieve constant specific radioactivity or, in other cases, to remove traces of radioactive impurities. Under these conditions incorporation of (7-14C)benzoic acid, as its N-acetylcysteamine thioester (0.09%), and ( 13-3Hlbaccatin-III (0.12%) was obtained. Degradation of the radioactive taxol from the benzoate feeding experiment showed that 88% of the radioactivity was recovered in the side chain fragment and only 1 1 % in the baccatin-111, suggesting that under the conditions of the experiment relatively little of the diterpene moiety was synthesized de novo. Upon degradation of the taxol from the baccatin-I11 feeding experiments, all the radioactivity was recovered in the baccatin-111 moiety, none in the side chain fragment.

Incubation of (R,S)-DL-phenyl[2-3H]lactic acid with Datura stramonium generates hyoscyamine 2 with the tritium isotope located at C-3′ of the tropic acid ester moiety; the C-3′ hydroxymethyl group of 2 is converted into a chiral methyl group and is oxidised to generate chiral sodium acetate, with the (R) configuration (96% ee); the tritium is therefore located at the 3′-pro-S site of 2; it follows that the 3′-pro-R hydrogen is introduced with inversion of configuration by the mutase operating during hyoscyamine biosynthesis.

Alkylation-amination of the enamine 2 in the presence of ethyl α,α-bis(dibromomethyl)acetate, triethyl-amine, and methylamine lead to the construction of the aza-transposed ergoline 3. Sequential reduction, hydrolysis, reesterification, and indolization of 3, produced three diastereomers of 6. The structure of these three diastereomers was assigned on the basis of nmr and ir spectral analysis to be (α-cis) syn, (β-cis) anti, and (α-trans) syn. The isomer (β-cis) anti was reduced with lithium aluminum hydride to the corresponding alcohol.

Five new type II manumycins, containing the hydroxyquinol mC7N unit, asukamycins A-II, B-II, C-II, D-II, E-II, were discovered in cultures of Streptomyces nodosus ssp. asukaensis. The biosynthetic origin of the type II manumycins from the type I compounds, containing an epoxyquinol mC7N unit, was deduced from the time course of production and proven by preparing [7'-13C]asukamycin A and demonstrating its incorporation into asukamycin A-II.

The chemical synthesis of 99% enriched D(-)-[1,7-13C2]shikimic acid from [1,2-13C2]acetate in 25% overall yield is described. A two-carbon phosphonate synthon was prepared from [1,2-13C2]acetate and used in the shikimate synthesis of Fleet and coworkers.

Die Feinheiten der chemischen Synthesen, die von lebenden Zellen beim Aufbau komplexer organischer Naturstoffe ausgefuhrt werden, lassen sich durch Markierung mit stabilen Isotopen in Verbindung mit hochentwickelten 2D- und Mehrquanten-NMR-Analysenmethoden detailliert untersuchen. Mechanistische Einzelheiten werden durch Aufklarung der „kryptischen” Stereochemie biosynthetischer Reaktionen mit Hilfe stereospezifischer Isotopenmarkierung enthullt. Die Analyse blockierter Mutanten und das Klonieren von Biosynthesegenen bieten neue Moglichkeiten fur die Untersuchung von Biosynthesewegen und fur die Ausnutzung der chemischen Fahigkeiten lebender Organismen.

Nitrosourea derivatives of ergolines have been synthesized for the purpose of obtaining agents with both prolactin-and tumor-inhibitory activity. Two derivatives of 8-amino-6-methylergoline (3), 8-[3-(2-chloroethyl)-3-nitrosoureido]-1-nitroso-6-methylergoline (5c) and 8-[3-2-chloroethyl)-3-nitrosoureido]-6-methylergoline (5a), have been prepared. In addition, nitroso (7) and chloroethylcarbamyl (8) derivatives of elymoclavine (6) are reported. Compounds 5a and 5c have activity against L1210 leukemia in mice but only moderate prolactin-inhibiting activity. The chloroethylcarbamyl derivative 8 of elymoclavine is a potent prolacting inhibitor.

Die Modifizierung von Peptid-Strukturelementen von Ergot-Alkaloiden verlauft unter Umwandlung einer α-Aminosaureeinheit in die entsprechende α-Hydroxy-a-aminosaureeinheit.

A partir d'indolecarbaldehyde-5, via l'action du [methyl trichloroethoxycarbonyl] amino malonate de dimethyle sur le [t-butyldimethylsiloxymethyl-3' hydroxy-3' butene-1' yl]-4 dimethylaminomethyl-3 indole, synthese de [dihydroxy-3',4' methyl-3' butene-1' yl]-4 N-methyl indolyl-3alanine. L'etude montre ensuite qu'aucun des diastereoisomeres de ce compose n'est un precurseur d'alcaloides de l'ergot