Ulrich Girreser

Biosynthesis of the orthosomycin antibiotic avilamycin A: deductions from the molecular analysis of the avi biosynthetic gene cluster of Streptomyces viridochromogenes Tü57 and production of new antibiotics

BACKGROUND Streptomyces viridochromogenes Tü57 is the producer of avilamycin A. The antibiotic consists of a heptasaccharide side chain and a polyketide-derived dichloroisoeverninic acid as aglycone. Molecular cloning and characterization of the genes governing the avilamycin A biosynthesis is of major interest as this information might set the direction for the development of new antimicrobial agents. RESULTS A 60-kb section of the S. viridochromogenes Tü57 chromosome containing genes involved in avilamycin biosynthesis was sequenced. Analysis of the DNA sequence revealed 54 open reading frames. Based on the putative function of the gene products a model for avilamycin biosynthesis is proposed. Inactivation of aviG4 and aviH, encoding a methyltransferase and a halogenase, respectively, prevented the mutant strains from producing the complete dichloroisoeverninic acid moiety resulting in the accumulation of new antibiotics named gavibamycins. CONCLUSIONS The avilamycin A biosynthetic gene cluster represents an interesting system to study the formation and attachment of unusual deoxysugars. Several enzymes putatively responsible for specific steps of this pathway could be assigned. Two genes encoding enzymes involved in post-PKS tailoring reactions were deleted allowing the production of new analogues of avilamycin A.

Elucidation of the function of two glycosyltransferase genes (lanGT1 and lanGT4) involved in landomycin biosynthesis and generation of new oligosaccharide antibiotics

BACKGROUND The genetic engineering of antibiotic-producing Streptomyces strains is an approach that became a successful methodology in developing new natural polyketide derivatives. Glycosyltransferases are important biosynthetic enzymes that link sugar moieties to aglycones, which often derive from polyketides. Biological activity is frequently generated along with this process. Here we report the use of glycosyltransferase genes isolated from the landomycin biosynthetic gene cluster to create hybrid landomycin/urdamycin oligosaccharide antibiotics. RESULTS Production of several novel urdamycin derivatives by a mutant of Streptomyces fradiae Tü2717 has been achieved in a combinatorial biosynthetic approach using glycosyltransferase genes from the landomycin producer Streptomyces cyanogenus S136. For the generation of gene cassettes useful for combinatorial biosynthesis experiments new vectors named pMUNI, pMUNII and pMUNIII were constructed. These vectors facilitate the construction of gene combinations taking advantage of the compatible MunI and EcoRI restriction sites. CONCLUSIONS The high-yielding production of novel oligosaccharide antibiotics using glycosyltransferase gene cassettes generated in a very convenient way proves that glycosyltransferases can be flexible towards the alcohol substrate. In addition, our results indicate that LanGT1 from S. cyanogenus S136 is a D-olivosyltransferase, whereas LanGT4 is a L-rhodinosyltransferase.

Mass, NMR and IR spectroscopic characterization of pentedrone and pentylone and identification of their isocathinone by-products

This study presents and discusses the mass spectrometric, nuclear magnetic resonance spectroscopic and infrared spectroscopic data of the designer drugs pentedrone (2-methylamino-1-phenylpentan-1-one) and its methylenedioxy analog pentylone (2-methylamino-1-(3,4-methylenedioxyphenyl)pentan-1-one). The structure elucidation of the aliphatic parts was carried out by product ion spectroscopy of the immonium ion with m/z=86 formed after electron ionization, and by one- and two-dimensional (1)H- and (13)C-NMR spectroscopy on the hydrochloride salts to verify the structure of the alkyl side chain and to determine the methylenedioxy position in the aromatic ring of pentylone. Furthermore, two typical cathinone synthesis by-products were detected besides the main compounds. Their mass spectra are discussed and for one of them (1-methylamino-1-phenylpentan-2-one (isopentedrone)) a NMR assignment was possible in the existing mixture.

Spectroscopic characterization of 3,4-methylenedioxypyrrolidinobutyrophenone: A new designer drug with α-pyrrolidinophenone structure

This study presents and discusses the infrared spectroscopic, the nuclear magnetic resonance spectroscopic and mass spectrometric data of the designer drug 3,4 methylenedioxypyrrolidinobutyrophenone (MDPBP), a homolog of 3,4 methylenedioxypyrovalerone (MDPV). MDPBP was first seized in Germany in the year 2009. The structure elucidation of the aliphatic part of MDPBP was carried out by product ion spectrometry of the immonium ion with m/z=112 formed after electron ionization, and by one- and two-dimensional (1)H- and (13)C NMR spectroscopy.

Structure elucidation of a new designer benzylpiperazine: 4-Bromo-2,5-dimethoxybenzylpiperazine

A new designer benzylpiperazine was seized in Germany for the first time. Interpreting the results of gas chromatography-mass spectroscopy (GC-MS), product ion spectroscopy (GC-MS/MS), and nuclear magnetic resonance (NMR) spectroscopy the compound was 4-bromo-2,5-dimethoxybenzylpiperazine. The structure of the new benzylpiperazine was finally proved by two-dimensional NMR correlations and by GC-MS after synthesis of two of the possible isomers from commercially available starting materials. Additionally mass spectroscopic data after liquid chromatography-mass spectroscopy (LC-MS/MS) using electrospray ionization (ESI) as well as ultraviolet (UV) spectral data of the new compound are presented. A small quantity of the new benzylpiperazine was seized in very high purity along with other also very pure designer drugs in Hamburg, Germany.

CHARACTERIZATION OF BIGUANIDES BY 15N NMR SPECTROSCOPY

The monoprotonated biguanides phenformin hydrochloride, buformin hydrochloride and metformin hydrochloride were investigated by 15N NMR spectroscopy. The structure of all hydrochlorides is best described by a 1,3‐diazabutadienic skeleton with three amino groups and its corresponding mesomeric forms. There is no evidence for the presence of other tautomeric forms in solution. Copyright

A new isocoumarin from Hypericum annulatum

A new isocoumarin, annulatomarin (1), together with the known physcion and β-sitosterol were isolated from the aerial parts of Hypericum annulatum. The structure of the new compound was established as 6,8-dihydroxy-7-methoxy-3-phenyl-3,4-dihydro-1H-isochromen-1-one on the basis of detailed spectroscopic analysis. Annulatomarin exhibited a modest growth-inhibitory activity in vitro against human chronic myeloid leukaemia LAMA-84 cells with an IC50 = 111 µM.

Metabolism of benzamidoxime (N-hydroxyamidine) in human hepatocytes and role of UDP-glucuronosyltransferases

N-Hydroxyamidines (amidoximes) can act as pro-drugs of amidines (e.g. ximelagatran, a novel direct thrombin inhibitor). This known pro-drug principle is based on the N-reduction of an oral bioavailable amidoxime to its active form. Previous study of the metabolism of the model substrate benzamidoxime by pig hepatocytes demonstrated the formation of benzamidoxime-O-glucuronide in addition to the well-established N-reduction. The objective of the present work was to investigate the glucuronidation of benzamidoxime by using cultivated cryopreserved human hepatocytes. Furthermore, the involvement of human UDP-glucuronosyltransferases (UGTs) was examined by incubating benzamidoxime in the presence of eight human hepatic recombinant UGT enzymes. Metabolites were analysed by liquid chromatography/mass spectrometry using electrospray ionization and compared with authentic synthetic compounds. For the first time, the O-glucuronidation of benzamidoxime was demonstrated in cultures of human hepatocytes. UGT1A9 is the most efficient enzyme conjugating benzamidoxime, whereas the conversion activities of UGT1A1 and UGT1A3 were 60-fold lower. Human hepatocytes form two non-mutagenic compounds: benzamidine, as the predominating metabolite, and benzamidoxime-O-glucuronide to a lesser extent. N-oxidation of benzamidine was not detected.

Bromoanaindolone, a novel antimicrobial exometabolite from the cyanobacterium Anabaena constricta

A new brominated indole alkaloid, designated as bromoanaindolone, was isolated from culture media of the cyanobacterium Anabaena constricta and was identified as 6-bromo-3-hydroxy-3-methyl-indol-2-one with a slight excess of the (3R) enantiomer. The molecular structure was elucidated on the basis of IR, MS and NMR data. This extracellular metabolite of A. constricta possessed antimicrobial (anticyanobacterial and antibacterial) activity in different test systems, such as suspension and porous matrix tests.

METABOLISM OF N-HYDROXYGUANIDINES (N-HYDROXYDEBRISOQUINE) IN HUMAN AND PORCINE HEPATOCYTES: REDUCTION AND FORMATION OF GLUCURONIDES

The biotransformation of N-hydroxydebrisoquine, a model substrate for N-hydroxyguanidines, was studied in vitro with cultured and characterized porcine and human hepatocytes. The objective of the present work was to compare the N-oxidative and N-reductive metabolism of this compound using a monolayer culture system with previously described microsomal studies and to investigate the phase 2 metabolism, in particular, the glucuronidation of this class of compounds. At the same time, the suitability of pig hepatocytes as a model system for the human metabolism could be investigated. Two glucuronides of the parent compound N-hydroxydebrisoquine were analyzed. For the first time, one of these phase 2 metabolites could be identified as an O-glucuronide of an N-hydroxyguanidine by comparing it to a synthesized authentic compound. The involvement of certain human UDP-glucuronosyltransferases (UGTs) was evaluated by incubating the substrate with eight human hepatic recombinant UGT enzymes. Metabolites were determined by a newly developed LC-MS (liquid chromatography/mass spectrometry) analysis using electrospray ionization (ESI). The known microsomal reduction of the N-hydroxylated compound was also demonstrated with hepatocytes. The N-hydroxylation of the corresponding reduced compound (debrisoquine), which was previously described with microsomes, could not be detected in hepatocytes. There was no qualitative difference in the formation of the described derivatives by human and porcine hepatocytes. All phase 2 metabolites identified in hepatocyte culture were also formed by glucuronosyltransferases. In culture, the N-reduction of the N-hydroxylated substrate is the dominating reaction, indicating a predominance of N-reduction in vivo.