Synthesis and Antimicrobial Activity of Albicidin Derivatives with Variations of the Central Cyanoalanine Building Block.

To investigate the pharmacophore regions of the antibiotic albicidin, derivatives with variations on the central amino acid were synthesized. Charged as well as uncharged residues were chosen to explore the influence of charge, chirality, and steric bulk. The bioactivity of the newly synthesized derivatives was determined by a microdilution technique to obtain minimum inhibitory concentrations (MIC) values. The compounds were also tested in a cell-free system to obtain information about their ability to inhibit their primary target, DNA gyrase. It was then shown that derivatives with uncharged side chains retain antibacterial activity, whereas incorporation of charged amino acid residues decreases the antibacterial activity dramatically, possibly due to restricted cell penetration of these derivatives. From the newly synthesized derivatives, the threonine derivative shows the most promising results in both tests. The information will help to develop the features of albicidin toward more drug-like structures.

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.

Streptocidins A-D, novel cyclic decapeptide antibiotics produced by Streptomyces sp. Tü 6071. II. Structure elucidation.

The structures of the new antibiotics streptocidins A approximately D were elucidated as cyclic decapeptides cyclo[L-Val1-L-Orn2-L-Leu3-D-Phe4-L-Pro5-L-Leu6-X7-L-Asn8-L-Gln9-X10] with X7=D-Trp (A, B, C) or D-Phe (D) and X10=L-Tyr (A), L-Trp (B, D), or D-Trp (C). The amino acid composition (including the configuration) of the substances was determined by chiral-phase GC-MS of the hydrolysates. The sequences were established by EDMAN degradation following linearisation of the cyclic peptides upon treatment with LiAlH4. NMR spectroscopic studies of streptocidins C and D confirmed the proposed sequences and provided conformational data which indicate a molecular topology of streptocidins C and D similar to those of tyrocidine A and gramicidin S.

Bioengineering of lanthipeptides in Escherichia coli: assessing the specificity of lichenicidin and haloduracin biosynthetic machinery.

The lichenicidin and haloduracin biosynthetic machinery specificity was investigated in vivo in Escherichia coli. Unlike previous reports using different hosts, it was found that the biosynthetic machineries of lichenicidin and haloduracin are highly specific to their dedicated peptide precursors. Likewise, the substitution of lichenicidin structural genes by chimeras of lichenicidin leader sequences and haloduracin core peptides did not yield mature haloduracin peptides. Despite these restrictions, it was found that the bifunctional enzyme HalT was able to process and export lichenicidin peptides. These findings corroborate the promiscuity of LanT enzymes reported for other lantibiotics, such as nukacin ISK-1 and lacticin 481.

Discovery of antibacterials and other bioactive compounds from microorganisms-evaluating methodologies for discovery and generation of non-ribosomal peptide antibiotics.

After decades of neglect in industrial research the comeback of natural products is due since improved screening approaches are at disposal, yielding a multitude of new compounds from natural sources. Besides traditional compound libraries peptides are characterized by an enormous structural complexity, thus increasing the chance of finding a hit in a screening. Emphasizing antibacterial compounds structural complexity is a prerequisite for their success. This review focuses on the screening approaches employed for the discovery of mostly antibacterial, non-ribosomal peptides derived from natural sources. Traditional screening methodologies as well as genetic approaches are discussed in this context. Utilizing genetic engineering methods e.g., precursor-directed biosynthesis, mutasynthesis, combinatorial biosynthesis, as well as chemoenzymatics to achieve greater structural diversity is thoroughly discussed and exemplified by recent discoveries.

Mutational biosynthesis--a tool for the generation of structural diversity in the biosynthesis of antibiotics.

Natural products represent an important source of drugs in a number of therapeutic fields, e.g. antiinfectives and cancer therapy. Natural products are considered as biologically validated lead structures, and evolution of compounds with novel or enhanced biological properties is expected from the generation of structural diversity in natural product libraries. However, natural products are often structurally complex, thus precluding reasonable synthetic access for further structure-activity relationship studies. As a consequence, natural product research involves semisynthetic or biotechnological approaches. Among the latter are mutasynthesis (also known as mutational biosynthesis) and precursor-directed biosynthesis, which are based on the cellular uptake and incorporation into complex antibiotics of relatively simple biosynthetic building blocks. This appealing idea, which has been applied almost exclusively to bacteria and fungi as producing organisms, elegantly circumvents labourious total chemical synthesis approaches and exploits the biosynthetic machinery of the microorganism. The recent revitalization of mutasynthesis is based on advancements in both chemical syntheses and molecular biology, which have provided a broader available substrate range combined with the generation of directed biosynthesis mutants. As an important tool in supporting combinatorial biosynthesis, mutasynthesis will further impact the future development of novel secondary metabolite structures.

The biosynthesis of glycopeptide antibiotics--a model for complex, non-ribosomally synthesized, peptidic secondary metabolites.

Glycopeptide antibiotics are a class of widely known natural compounds produced by Actinomycetes. Vancomycin, the first member of the glycopeptide family to be discovered, was described in 1955 and used as an antibiotic soon thereafter. During the past 50 years numerous contributions on the structure, mode of action, and therapeutic features of vancomycin have been published. Recently, there has been considerable progress in elucidating the biosynthesis of glycopeptide antibiotics by combining molecular biology and analytical chemistry methods. Here, we provide an overview of the current knowledge regarding biosynthetic glycopeptide assembly.

Impact of mass spectrometry on combinatorial chemistry.

In the past few years, the emergence of combinatorial chemistry has drawn increasing attention and a great deal of analytical research has been centered around this new methodology. These new methods capable of producing vast numbers of samples, which are in many cases highly complex, demand fast and reliable analytical techniques able to provide high quality information concerning sample compositions. Mass spectrometry (MS) is the method of choice to face these analytical challenges. In particular, the introduction of electrospray ionization (ESI and matrix assisted laser desorption/ionization (MALDI) have been the driving forces for many of the recent innovations, not only within the fields of the biosciences, but also in combinatorial chemistry. These ionization techniques are extremely versatile for the characterization of both single compound collections and compound mixture collections. The high-throughput capabilities, as well as many possible couplings with separation techniques (HPLC, CE) have been thus facilitated. However, mass spectrometry is not only limited to use as an instrument for synthesis control, but also plays an increasing role in the identification of active compounds from complex libraries. Recently, new initiatives for library analysis and screening have arisen from the application of the latest developments in mass spectrometry, Fourier transform ion cyclotron resonance (FTICR).

Hantzsch pyrrole synthesis on solid support.

An efficient method for solid-phase synthesis of pyrroles is described. Polystyrene Rink amide resin is acetoacetylated and converted into polymer bound enaminones upon treatment with primary amines. These then undergo a Hantzsch reaction with alpha-bromoketones to yield pyrroles. After cleavage with 20% trifluoroacetic acid in dichloromethane pyrrole-3-carboxamides are obtained in excellent purity.

Optical biosensors. Monitoring studies of glycopeptide antibiotic fermentation using white light interference.

This paper describes the design, characterization, and use of an optical biosensor suited for the process control of biotechnological processes. The detector principle is based on reflectometric interference spectroscopy (RIfS). RIfS enables a label-free, product-specific monitoring, with a future outline for on-line process control. The potential of the RIfS biosensor is exemplified by the qualitative and quantitative monitoring of the microbial production of vancomycin-type glycopeptide antibiotics.