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Chemistry & Biodiversity

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ISSN (Print) 1612-1872 - ISSN (Online) 1612-1880
Published by John Wiley and Sons

[1586 journals]

Spin‐Echo Electron Paramagnetic Resonance (EPR) Spectroscopy of a Pore‐Forming (Lipo)Peptaibol in Model and Bacterial Membranes
- Abstract: This review compiles the unusual structural and dynamic peculiarities of trichogin GA IV and its analogs in lipid bilayers. Different electron spin echo (ESE) spectroscopic techniques were employed to study a set of spin‐labeled analogs of trichogin GA IV in model and natural membranes. Pulsed electron‐electron double resonance (PELDOR) method enabled the elucidation of the peptide conformation, while the ESE envelope modulation (ESEEM) technique was applied to study the insertion of the site‐specifically spin‐labeled peptide into the core of the membrane. The latter technique was also used to examine the water accessibility for peptide‐attached spin labels at different levels of membrane depth. Finally, it will be shown that measurement of the ESE decays at different temperatures reveals molecular information on the mobility of the transmembrane lipopeptide aggregate. The experimental results are discussed in terms of the antibiotic and toxic activities of trichogin GA IV.
Contents: C&B 5/2013
Towards a Myriad of Peptaibiotics
The Comprehensive Peptaibiotics Database
- Abstract: Peptaibiotics are nonribosomally biosynthesized peptides, which – according to definition – contain the marker amino acid α‐aminoisobutyric acid (Aib) and possess antibiotic properties. Being known since 1958, a constantly increasing number of peptaibiotics have been described and investigated with a particular emphasis on hypocrealean fungi. Starting from the existing online ‘Peptaibol Database’, first published in 1997, an exhaustive literature survey of all known peptaibiotics was carried out and resulted in a list of 1043 peptaibiotics. The gathered information was compiled and used to create the new ‘The Comprehensive Peptaibiotics Database’, which is presented here. The database was devised as a software tool based on Microsoft (MS) Access. It is freely available from the internet at http://peptaibiotics‐database.boku.ac.at and can easily be installed and operated on any computer offering a Windows XP/7 environment. It provides useful information on characteristic properties of the peptaibiotics included such as peptide category, group name of the microheterogeneous mixture to which the peptide belongs, amino acid sequence, sequence length, producing fungus, peptide subfamily, molecular formula, and monoisotopic mass. All these characteristics can be used and combined for automated search within the database, which makes The Comprehensive Peptaibiotics Database a versatile tool for the retrieval of valuable information about peptaibiotics. Sequence data have been considered as to December 14, 2012.
Recent Results in Alamethicin Research
Unprecedented 17‐Residue Peptaibiotics Produced by Marine‐Derived Trichoderma atroviride
- Abstract: In the course of investigations on marine‐derived toxigenic fungi, five strains of Trichoderma atroviride were studied for their production of peptaibiotics. While these five strains were found to produce classical 19‐residue peptaibols, three of them exhibited unusual peptidic sodium‐adduct [M + 2 Na]2+ ion peaks at m/z between 824 and 854. The sequencing of these peptides led to two series of unprecedented 17‐residue peptaibiotics based on the model Ac‐XXX‐Ala‐Ala‐XXX‐XXX‐Gln‐Aib‐Aib‐Aib‐Ala/Ser‐Lxx‐Aib‐Pro‐XXX‐Aib‐Lxx‐[C129]. The C‐terminus of these new peptides was common to all of them, and its elemental formula C5H9N2O2 was established by HR‐MS. It could correspond to the cyclized form of Nδ‐hydroxyornithine which has already been observed at the C‐terminus of various peptidic siderophores. The comparison of the sequences of 17‐ and 19‐residue peptides showed similarities for positions 1–16. This observation seems to indicate a common biosynthesis pathway. Both new 17‐residue peptaibiotics and 19‐residue peptaibols exhibited weak in vitro cytotoxicities against KB cells.
Peptaibol Antiamoebin I: Spatial Structure, Backbone Dynamics, Interaction with Bicelles and Lipid‐Protein Nanodiscs, and Pore Formation in Context of Barrel‐Stave Model
- Abstract: Antiamoebin I (Aam‐I) is a membrane‐active peptaibol antibiotic isolated from fungal species belonging to the genera Cephalosporium, Emericellopsis, Gliocladium, and Stilbella. In comparison with other 16‐amino acid‐residue peptaibols, e.g., zervamicin IIB (Zrv‐IIB), Aam‐I possesses relatively weak biological and channel‐forming activities. In MeOH solution, Aam‐I demonstrates fast cooperative transitions between right‐handed and left‐handed helical conformation of the N‐terminal (1–8) region. We studied Aam‐I spatial structure and backbone dynamics in the membrane‐mimicking environment (DMPC/DHPC bicelles)1) by heteronuclear 1H,13C,15N‐NMR spectroscopy. Interaction with the bicelles stabilizes the Aam‐I right‐handed helical conformation retaining significant intramolecular mobility on the ms–μs time scale. Extensive ms–μs dynamics were also detected in the DPC and DHPC micelles and DOPG nanodiscs. In contrast, Zrv‐IIB in the DPC micelles demonstrates appreciably lesser mobility on the μs–ms time scale. Titration with Mn2+ and 16‐doxylstearate paramagnetic probes revealed Aam‐I binding to the bicelle surface with the N‐terminus slightly immersed into hydrocarbon region. Fluctuations of the Aam‐I helix between surface‐bound and transmembrane (TM) state were observed in the nanodisc membranes formed from the short‐chain (diC12 : 0) DLPC/DLPG lipids. All the obtained experimental data are in agreement with the barrel‐stave model of TM pore formation, similarly to the mechanism proposed for Zrv‐IIB and other peptaibols. The observed extensive intramolecular dynamics explains the relatively low activity of Aam‐I.
The Sequences of the Eleven‐Residue Peptaibiotics: Suzukacillins‐B
- Abstract: The filamentous fungus designated ‘Trichoderma viride’ strain 63 C‐1 simultaneously produces suzukacillins (SZs), two microheterogeneous groups of peptaibols, under submerged culture conditions. Both groups are readily distinguishable by TLC: the major group is designated SZ‐A, whereas the minor group with a higher Rf value is named SZ‐B. The peptide mixture was obtained from a MeOH extract of the mycelium. SZ‐B was separated from SZ‐A by Sephadex LH‐20 column chromatography. Although it provided one single spot on silica‐gel TLC plates, 15 individual peptides could be separated by C8 reversed‐phase (RP) HPLC, and their sequences were determined by HPLC/QqTOF‐ESI‐HRMS. Fourteen peptides exhibit the C‐terminal sequence Pro6‐Lxx‐Lxx‐Aib‐Pro‐Vxxol/Lxxol11, which is common for eleven‐residue peptaibols. The remaining peptide is tentatively assigned as a ten‐residue sequence, in which the C‐terminal 1,2‐amino alcohol is deleted, thus terminating in free proline. Nine of the peptides carry an Ac‐Aib residue at the N‐terminus, very frequently found in eleven‐residue peptaibols. Four peptides comprise the rare Ac‐Ala N‐terminus, and for two peptides, N‐terminal Ac‐D‐Iva residues were identified. One peptide contains a C‐terminal residue of yet undetermined structure. Comparison with previously reported eleven‐residue peptaibol sequences reveals that eight of the peptides represent new sequence analogs.
Sequences of Metanicins, 20‐Residue Peptaibols from the Ascomycetous Fungus CBS 597.80
- Abstract: Four linear 20‐residue peptaibols, named metanicins (MTCs) A–D, were isolated from submerged cultures of the ascomycetous fungus CBS 597.80. Structure elucidation was performed by a combination of fast‐atom‐bombardment mass spectrometry (FAB‐MS), electrospray ionization MS, Edman degradation of isolated fragments, and amino acid analysis by ion‐exchange and gas chromatography, and enantioselective HPLC. The sequences of MTC A(B) are (amino acid exchange in B and C in parentheses): Ac‐Aib‐Ala‐Aib‐Ala‐Aib‐Ala‐Gln‐Aib‐Val‐Aib‐Gly‐Leu‐Aib‐Pro‐Val‐Aib‐Aib(D‐Iva)‐Gln‐Gln‐Pheol and of MTC C(D) Ac‐Aib‐Ala‐Aib‐Ala‐Aib‐Ala‐Gln‐Aib‐Val‐Aib‐Gly‐Leu‐Aib‐Pro‐Val‐Aib‐Aib(D‐Iva)‐Gln‐Gln‐Pheol (Ac, acetyl; Aib, α‐aminoisobutyric acid; Iva, isovaline; Pheol, L‐phenylalaninol). The peptides are related, and some of the sequences are identical, to other 20‐residue peptaibols isolated from Trichoderma species. MTCs show moderate activities against Micrococcus luteus, Enterococcus faecalis, and Staphylococcus aureus, and very low activities against Bacillus subtilis. The producer has originally been identified and deposited as Metarhizium anisopliae var. anisopliae CBS 597.80. Although this identification has been withdrawn by Centralbureau voor Schimmelcultures (CBS) in the meantime, the accession number will be retained – independently from any taxonomic revisions.
Screening the Biosphere: The Fungicolous Fungus Trichoderma phellinicola, a Prolific Source of Hypophellins, New 17‐, 18‐, 19‐, and 20‐Residue Peptaibiotics
- Abstract: To investigate the significance of antibiotics for the producing organism(s) in the natural habitat, we screened a specimen of the fungicolous fungus Trichoderma phellinicola (syn. Hypocrea phellinicola) growing on its natural host Phellinus ferruginosus. Results revealed that a particular group of non‐ribosomal antibiotic polypeptides, peptaibiotics, which contain the non‐proteinogenic marker amino acid, α‐aminoisobutyric acid, was biosynthesized in the natural habitat by the fungicolous producer and, consequently, released into the host. By means of liquid chromatography coupled to electrospray high‐resolution time‐of‐flight mass spectrometry, we detected ten 20‐residue peptaibols in the specimen. Sequences of peptaibiotics found in vivo were independently confirmed by analyzing the peptaibiome of an agar plate culture of T. phellinicola CBS 119283 (ex‐type) grown under laboratory conditions. Notably, this strain could be identified as a potent producer of 39 new 17‐, 18‐, and 19‐residue peptaibiotics, which display the same building scheme as the 20‐residue peptaibols found in the specimen. Two of the 19‐residue peptaibols are tentatively assigned to carry tyrosinol, a novel C‐terminal residue, as deduced from high‐resolution tandem mass‐spectrometry data. For the new peptaibiotics produced by T. phellinicola, the name ‘hypophellin(s)’, based on the teleomorph name, is introduced.
Bis(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl) Ether as Coupling Reagent for Peptide Synthesis
- Abstract: Bis(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl) ether (4) was prepared by treatment of 2‐hydroxy‐4,6‐dimethoxy‐1,3,5‐triazine with 2‐chloro‐4,6‐dimethoxy‐1,3,5‐triazine in 61% yield. Ether 4, isoelectronic with pyrocarbonates, was found capable to activate carboxylic acids in the presence of 1,4‐diazabicyclo[2.2.2]octane (DABCO) to yield, under mild reaction conditions, superactive triazine esters. Versatility of this new coupling reagent was confirmed by condensation of lipophilic and sterically hindered carboxylic acids with amines in 71–98% yield, and by synthesis of peptides, including those containing Aib‐Aib sequence, in solution with high yield and high enantiomeric purity.
Attempts toward the Synthesis of the Peptaibol Antiamoebin by Using the ‘Azirine/Oxazolone Method’
- Abstract: The two segments, 1–9 and 10–16, of the peptaibol antibiotic antiamoebin I, i.e., the nonapeptide Ac‐Phe‐Aib‐Aib‐Aib‐D,L‐Iva‐Gly‐Leu‐Aib‐Aib‐OH (15) and the heptapeptide Z‐Hyp‐Gln‐D,L‐Iva‐Hyp‐Aib‐Pro‐Pheol (34), have been prepared as mixtures of the epimers containing D,L‐Iva. All α,α‐disubstituted α‐amino acids were introduced by the ‘azirine/oxazolone method’, in which amino or peptide acids are coupled with the corresponding 2H‐azirin‐3‐amines, followed by selective hydrolysis of the terminal amide bond. The amino acids Hyp and Gln were introduced as Z‐protected4) (2S,4R)‐4‐(tert‐butoxy)proline (19) and methyl N‐[bis(4‐methoxyphenyl)methyl]glutamine (26). Coupling of peptide segments was achieved via the ‘mixed anhydride’ method, the DCC/HOBt or TBTU/HOBt strategy. The crystal structure of the segment 6–9 was determined by X‐ray crystallography and displayed the presence of a β‐turn conformation.
Conformational Properties of Secondary Amino Acids: Replacement of Pipecolic Acid by N‐Methyl‐L‐alanine in Efrapeptin C
- Abstract: The efrapeptins, a family of naturally occurring peptides with inhibitory activities against ATPases, contain several α,α‐disubstituted α‐amino acids such as α‐aminoisobutyric acid (Aib) or isovaline (Iva) besides pipecolic acid (Pip), β‐Ala, Leu, Gly, and a C‐terminal heterocyclic residue. Secondary α‐amino acids such as proline are known to stabilize discrete conformations in peptides. A similar influence is ascribed to N‐alkyl α‐amino acids. We synthesized two efrapeptin C analogs with replacement of Pip by N‐methyl‐L‐alanine (MeAla) using a combination of solid‐ and solution‐phase techniques in a fragment‐condensation strategy to compare the conformational bias of both secondary amino acids. The solution conformation was investigated by vibrational circular dichroism (VCD) to probe whether the analogs adopt a 310‐helical conformation. The MeAla‐containing analogs [MeAla1,3]efrapeptin C and [MeAla1,3,11]efrapeptin C inhibit ATP hydrolysis by the A3B3 complex of A1A0‐ATP synthase from Methanosarcina mazei Gö1.
Spectroscopically Labeled Peptaibiotics. Synthesis and Properties of Selected Trichogin GA IV Analogs Bearing a Side‐Chain‐Monofluorinated Aromatic Amino Acid for 19F‐NMR Analysis
- Abstract: We prepared by solid‐phase methods, chromatographically purified, and characterized three analogs of the ten‐amino acid‐residue, membrane‐active, lipopeptaibiotic trichogin GA IV, each containing a single (4‐fluorophenyl)alanine in position 3, 7, or 10, where it replaces the hydrophobic residue Leu3, Leu7, or Ile10, respectively. We incorporated the fluorine probe based on the observation that the 19F‐NMR technique has been extensively utilized to analyze peptidemembrane interactions in biological systems. A detailed conformational investigation in solution, including a membrane‐mimetic environment, was performed on these compounds using FT‐IR absorption, CD, and 2D‐NMR, combined with molecular‐dynamics calculations. The experimentally observed, mixed 310/α‐helical structures unequivocally show that the principal conformational features of trichogin GA IV are preserved in all three analogs. Analogies and differences between the behavior of the natural lipopeptaibiotic and those of the peptides characterized by the side‐chain monofluorinated aromatic amino acid were found in membrane‐permeabilization experiments and antimicrobial assays. The results of a preliminary solution 19F‐NMR study support the view that the 19F label is an excellent reporter for changes in the helical environment of the peptide.
3D Structure, Dynamics, and Activity of Synthetic Analog of the Peptaibiotic Trichodecenin I
- Abstract: In this contribution, we report on the conformational preferences of synthetic analogs of the antimicrobial peptide trichodecenin I in solution. This 6‐amino acid residue long peptide is characterized by a single, strongly helicogenic Aib residue in the central part of the sequence and is rich in the conformationally mobile Gly residues. It has been reported that, in CHCl3 solution and in the crystal state, this peptaibiotic adopts a non‐helical, multiple β‐turn conformation, whereas a 310/α‐helical structure was obtained from an X‐ray diffraction study on a trichodecenin I analog (TDT4W6) containing the fluorescent Trp residue in position 6 (replacing Ile) and an equally helicogenic TOAC residue in position 4 (replacing Aib). In this work, we applied spectroscopic techniques and molecular‐dynamics calculations, in particular, on the fluorescent TDT4W6 trichodecenin I analog with the aim at investigating its 3D‐structural and dynamical features in solution. Our results revealed that TDT4W6 can be described by an ensemble of conformers quickly interconverting in the nanosecond time scale. The most populated cluster has a conformation similar to the NMR structure of native trichodecenin I in CHCl3. However, also helical‐like conformers are present, even if poorly populated and less stable under the analytical conditions.
Structural Characterization of the Short Peptaibols Trichobrachins by Molecular‐Dynamics Methods
- Abstract: A structural characterization was carried out by molecular‐dynamics methods for eight trichobrachin peptides, to identify the conformational features of these short peptaibols. For all peptides, the backbone and side‐chain conformations were investigated, different secondary structures, such as type‐I and ‐III β‐turns as well as β‐bend ribbon spirals, were determined in certain tetrapeptide units of the molecules, and the preferred rotamers of the side chains of amino acids were identified. Furthermore, the end‐to‐end and residueresidue distances were examined, as well as the fluctuations of backbone atoms were studied. Based on these results, the peptides were compared to one another. Our theoretical study indicated that trichobrachins could be characterized by typical structural properties, and both conformational similarities and dissimilarities were observed between these peptaibols. In summary, this structural investigation supplied a characterization of the various conformational features of eight trichobrachin peptides.