GNPS-Guided Discovery of Madurastatin Siderophores from the Termite-Associated Actinomadura sp. RB99**

In this study, we analyzed if Actinomadura sp. RB99 produces siderophores that that could be responsible for the antimicrobial activity observed in co-cultivation studies. Dereplication of high-resolution tandem mass spectrometry (HRMS/MS) and global natural product social molecular networking platform (GNPS) analysis of fungus-bacterium co-cultures resulted in the identification of five madurastatin derivatives (A1, A2, E1, F, and G1), of which were four new derivatives. Chemical structures were unambiguously confirmed by HR-ESI-MS, 1D and 2D NMR experiments, as well as MS/MS data and their absolute structures were elucidated based on Marfey's analysis, DP4+ probability calculation and total synthesis. Structure analysis revealed that madurastatin E1 ( 2 ) contained a rare 4-imidazolidinone cyclic moiety and madurastatin A1 ( 5 ) was characterized as a Ga³⁺-complex. The function of madurastatins as siderophores was evaluated using the fungal pathogen Cryptococcus neoformans as model organism. Based on homology models, we identified the putative NRPS-based gene cluster region of the siderophores in Actinomadura sp. RB99.     

Rapid and efficient screening of adsorbent for oligopeptide using molecular docking and isothermal titration calorimetry

A rapid and efficient method based on molecular docking and isothermal titration calorimetry (ITC) was developed to identify effective adsorbents for the target peptide Ser‐Glu‐Ala‐Asp‐His (SEADH). Preliminary screening of five candidate adsorbents using molecular docking revealed that three suitable structures (A1, A2, and A3) either with or without coordination of Zn²⁺ should be effective. The three selected structures were then prepared and further screened by evaluating their affinities for the peptide SEADH using ITC. The screening results revealed that the adsorbent A2 coordinated with Zn²⁺ was the best adsorbent, and subsequent static adsorption experiments confirmed the reliability of the screening method. Further ITC analysis, combined with molecular docking, was performed to provide the possible adsorption mechanism.     

Capillary electrophoresis-mass spectrometry of citrus endophytic bacteria siderophores

CE-ESI-MS with a liquid sheath interface and IT mass analyzer was used for analysis of siderophores from different strains of Methylobacterium spp. citrus endophyte extracts. Three Methylobacterium strains were investigated according to positive bioassay tests. Bacteria cultures were grown under Fe(III) absence (siderophore producing cultures) and under Fe(III) presence (control cultures). Siderophores were extracted from culture supernatant with polystyrene resins. BGE and sheath-liquid composition were optimized, respectively, in order to assure both, best peak resolution and ESI-MS sensitivity. The best analysis conditions were obtained with 100 mmol/L ammonium bicarbonate at pH 8 as BGE and methanol:H(2)O 25:75 + 0.05% formic acid as sheath liquid. CZE-ESI-MS analysis revealed two possible siderophores, according to bacterium species, presenting M(r) of 1004.3 and 798.3 Da.     

几种化学计量学方法在放线菌及其次生代谢产物抗肿瘤活性筛选中的应用

当今肿瘤的诊断与治疗对人类健康非常重要. 在放线菌次生代谢产物内寻找抗肿瘤抗生素的过程中, 如何从种类多、数量大的土壤放线菌株中确定目标菌株, 筛选是关键. 将化学计量学方法用于大批量的放线菌及其次生代谢产物的筛选数据分析, 首先通过主成分分析获得具有抗肿瘤活性菌株对6种肿瘤细胞抑制作用的一些关系与特异性, 其次在进行菌株次生代谢产物的分子模型筛选数据分析时, 利用比较分析发现有较好肿瘤抑制作用者, 基本与细胞筛选结果吻合. 二者结合建立了一种简便适当的分析模式, 能较快地从大批量放线菌株的筛选中找到具有抗肿瘤活性的有研究价值的目标放线菌株.     

Electrospray ionisation-mass spectrometry of hydroxamate siderophores

Electrospray ionisation-mass spectrometry (ESI-MS) was applied to the detection of the iron complexes of the hydroxamate type siderophores ferrioxamine (FO), ferrichrome (FC) and iron(III) rhodotoluate (FR). Mass spectra of the three siderophores produced by ESI-MS were dominated by the protonated (M + 1)+ parent ions, except for FR at pH 4.3, which was present as the positively charged 1:1 complex. On collision with He ions, fragmentation proceeded largely via cleavage of C-N bonds. Flow injection analysis of the siderophores with detection by ESI-MS produced detection limits of 1.9 fmol for FO, 31.1 fmol for FC and 524 fmol for FR.     

Characterization of pyoverdins and their hydrolytic degradation products by fast atom bombardment and tandem mass spectrometry

All types of pyoverdins (siderophores produced by Pseudomonas strains) have the following ‘chromophore’ substructure in common: (1S)-5-amino-2,3-dihydro-8,9-dihydroxy-lH-pyrimido[l,2-α]quinoline-1-carboxyhic acid. Its hydrolysis product, (1S)-2,3-dihydro-5,8,9-trihydroxy-lH-pyrimido[l,2-α]quinoline-1-carboxylic acid, has been isolated and analysed for the first time by means of fast atom bombardment (FAB) and tandem mass spectrometry. This allows the pyoverdin chromophore to be identified. It was established that the fragmentation of pyoverdin [M + H]⁺ ions under FAB conditions is initiated by a retro-Diels-Alder decomposition of the tetrahydropyrimidine ring of the chromophore moiety, contributing to the structure elucidation of pyoverdins.     

Mixing and Matching Siderophore Clusters: Structure and Biosynthesis of Serratiochelins from Serratia sp. V4

Interrogation of the evolutionary history underlying the remarkable structures and biological activities of natural products has been complicated by not knowing the functions they have evolved to fulfill. Siderophores-soluble, low molecular weight compounds-have an easily understood and measured function: acquiring iron from the environment. Bacteria engage in a fierce competition to acquire iron, which rewards the production of siderophores that bind iron tightly and cannot be used or pirated by competitors. The structures and biosyntheses of "odd" siderophores can reveal the evolutionary strategy that led to their creation. We report a new Serratia strain that produces serratiochelin and an analog of serratiochelin. A genetic approach located the serratiochelin gene cluster, and targeted mutations in several genes implicated in serratiochelin biosynthesis were generated. Bioinformatic analyses and mutagenesis results demonstrate that genes from two well-known siderophore clusters, the Escherichia coli enterobactin cluster and the Vibrio cholera vibriobactin cluster, were shuffled to produce a new siderophore biosynthetic pathway. These results highlight how modular siderophore gene clusters can be mixed and matched during evolution to generate structural diversity in siderophores.     

Iron Coordination Properties of Gramibactin as Model for the New Class of Diazeniumdiolate Based Siderophores

Gramibactin (GBT) is an archetype for the new class of diazeniumdiolate siderophores, produced by Paraburkholderia graminis, a cereal-associated rhizosphere bacterium, for which a detailed solution thermodynamic study exploring the iron coordination properties is reported. The acid-base behavior of gramibactin as well as its complexing ability toward Fe³⁺ was studied over a wide range of pH values (2≤pH≤11). For the latter the ligand-competition method employing EDTA was used. Only two species are formed: [Fe(GBT)]⁻ (pH 2 to 9) and [Fe(GBT)(OH)₂]³⁻ (pH≥9). The formation of [Fe(GBT)]⁻ and its occurrence in real systems was confirmed by LC-HRESIMS analysis of the bacteria culture broth extract. The sequestering ability of gramibactin was also evaluated in terms of the parameters pFe and pL_0.5. Gramibactin exhibits a higher sequestering ability toward Fe³⁺ than EDTA and of the same order of magnitude as hydroxamate-type microbial siderophores, but smaller than most of the catecholate-type siderophores and much higher than the most known phytosiderophores.     

Complexation of hydroxamate-based siderophores with cobalt(II/III): growth inhibitory effect of cobalt(III)-desferricoprogen complex on fungi

Solution equilibrium results for Co(II) and Co(III) complexes of two natural hydroxamate-based siderophores, the exocyclic desferricrocin (DFR) and the endocyclic triacetylfusarinine (TAF) are presented. The three hydroxamate chelating functions of TAF were found to complete the octahedral coordination sphere of a Co(II) ion in stepwise processes, but following the coordination of two hydroxamates of DFR practically in one step, the third function, most probably because of sterical reasons, remained uncoordinated. A comparison with corresponding results for the previously studied acyclic desferrioxamine B (DFB) and desferricoprogen (DFC) provided some information about the effects of the molecular framework of siderophores on their cobalt-binding ability. The oxidation of the central metal ion under basic conditions and investigation of the cobalt(III) complexes by cyclic voltammetry were also made. Compared to Fe(III), by several orders of magnitude, higher stability complexes were formed with Co(III). The possibility of any effect of the Co(III)-siderophore complex on microbial Fe(III) uptake was tested by investigation of the antifungal effect of Co(III)-DFC in comparison with that of CoCl₂ on two fungi cultures, Penicillium brevicompactum and Aspergillus fumigatus.     

Complexation of Pu(IV) with the natural siderophore desferrioxamine B and the redox properties of Pu(IV)(siderophore) complexes

The bioavailability and mobility of Pu species can be profoundly affected by siderophores and other oxygen-rich organic ligands. Pu(IV)(siderophore) complexes are generally soluble and may constitute with other soluble organo-Pu(IV) complexes the main fraction of soluble Pu(IV) in the environment. In order to understand the impact of siderophores on the behavior of Pu species, it is important to characterize the formation and redox behavior of Pu(siderophore) complexes. In this work, desferrioxamine B (DFO-B) was investigated for its capacity to bind Pu(IV) as a model siderophore and the properties of the complexes formed were characterized by optical spectroscopy measurements. In a 1:1 Pu(IV)/DFO-B ratio, the complexes Pu(IV)(H2DFO-B)4+, Pu(IV)(H1DFO-B)3+, Pu(IV)(DFO-B)2+, and Pu(IV)(DFO-B)(OH)+ form with corresponding thermodynamic stability constants log beta1,1,2 = 35.48, log beta1,1,1 = 34.87, log beta1,1,0 = 33.98, and log beta1,1,-1 = 27.33, respectively. In the presence of excess DFO-B, the complex Pu(IV)H2(DFO-B)22+ forms with the formation constant log beta2,1,2 = 62.30. The redox potential of the complex Pu(IV)H2(DFO-B)22+ was determined by cyclic voltammetry to be E1/2 = -0.509 V, and the redox potential of the complex Pu(IV)(DFO-B)2+ was estimated to be E1/2 = -0.269 V. The redox properties of Pu(IV)(DFO-B)2+ complexes indicate that Pu(III)(siderophore) complexes are more than 20 orders of magnitude less stable than their Pu(IV) analogues. This indicates that under reducing conditions, stable Pu(siderophore) complexes are unlikely to persist.     

Development of a spectrophotometric determination of siderophores using flow-injection analysis

A method has been developed for the spectrophotometric determination of siderophores using flow-injection analysis (FIA) based on the reaction of siderophores with the ternary complex Eriochrome Cyanine R-Fe(III)-cetyltrimethylammonium bromide. 2,3-Dihydroxybenzoic acid, 2,3-dihydroxynaphthalene, and tolypocine were used as the model iron-binding ligands. The calibration curve for one of the siderophores (tolypocine) is linear in the concentration range 2.6 x 10(-6)-1.5 x 10(-4)M. The determination limit (10sigma) for tolypocine was 2.6 x 10(-6)M. The applicability of the method was demonstrated on the determination of the complexation ability of siderophores produced by some entomopathogenic fungi. Samples can be analysed at a rate of 30 samples per hour.     

3-Thiolated 2-azetidinones: synthesis and in vitro antibacterial and antifungal activities

A series of 3-thiolated β-lactams were synthesized by [2+2] ketene-imine cycloaddition reaction from S-substituted mercaptoacetic acids and Schiff bases. Then, some of the 3-methylthio β-lactams were converted to 3-(methylsulfinyl) β-lactams and 3-(methylsulfonyl) β-lactams using m-CPBA under different reaction conditions. All the compounds were characterized by spectral data and elemental analyses and were evaluated for their in vitro antibacterial and antifungal activities against pathogenic strains including Staphylococcus aureus (Methicillin resistant strain). The preliminary screening results indicated that some of these compounds demonstrated moderate to very good antibacterial and antifungal activities.     

Complexes of Fe(III) and Ga(III) Derived from the Cyclic 6- and 7-Membered Hydroxamic Acids Found in Mixed Siderophores

Six- and seven-membered cyclic hydroxamic acids are found as terminal binding units in different families of siderophores, including exochelins and mycobactins. The simplest models of these preorganized chelating ligands were known, but their coordination chemistry with Fe³⁺, the target metal ion of siderophores, had never been reported. Four complexes were synthesized and studied: two Fe³⁺ complexes, one with the six-membered ring hydroxamate PIPO⁻ and one with the seven-membered ring hydroxamate AZEPO⁻, and the two corresponding Ga³⁺ complexes. X-ray diffraction studies showed that the interligand repulsion energies were better minimized in the case of the AZEPO⁻ complexes whatever the metal cation considered, and that the Fe−O bond distances were shorter in [Fe(AZEPO)₃] by comparison with [Fe(PIPO)₃].     

Rapid identification of siderophores by combined thin-layer chromatography/matrix-assisted laser desorption/ionization mass spectrometry

The investigation of a combined thin-layer chromatography/matrix-assisted laser desorption/ionization mass spectrometry (TLC/MALDI-MS) method for the analysis of siderophores from microbial samples is described. The investigated siderophores were enterobactin, ferrioxamine B, ferrichrome, ferrirhodin, rhodotorulic acid and coprogen. Solid-phase extraction was employed to recover the siderophores from the microbial samples. After visualization of the spots via spraying with ferric chloride or chrome azurol sulfonate assay solution, the MALDI matrix was applied to the gel surface. Several TLC/MALDI experimental parameters were optimized, such as type and concentration of MALDI matrix, as well as the type and composition of solvent to facilitate analyte transport from the inside of the TLC gel to the surface. The impact of these parameters on sensitivity, precision and ion formation of the various siderophores was studied. The detection limits for the investigated siderophores were in the range 1-4 pmol. These values were about 4-24 times higher than the detection limits obtained directly from stainless steel MALDI targets. The differences were most likely due to incomplete transport of the 'trapped' analyte molecules from the deeper layers of the TLC gel to the surface and into the matrix layer. In addition, chromatographic band broadening spread the analyte further in TLC as compared with the steel plates, resulting in less analyte per surface area. The identification of the siderophores was aided by concurrently applying a Ga(III) nitrate solution to the TLC plate during the visualization step. The resulting formation of Ga(III) complexes lead to distinctive (69)Ga/(71)Ga isotope patterns in the mass spectra. The versatility of the TLC/MALDI-MS assay was demonstrated by using it to analyze siderophores in a Pseudomonas aeruginosa sample. An iron-binding compound was identified in the sample, namely pyochelin (2-(2-o-hydroxyphenyl-2-thiazolin-4-yl)-3-methylthiazolidine-4-carboxylic acid).     

Combinatorial biomimetics. optimization of a composition of copper(II) poly-L-histidine complex as an electrocatalyst for O2 reduction by scanning electrochemical microscopy

A simple approach to prepare and characterize biomaterial-based electrocatalysts for oxygen reduction was carried out. Poly-l-histidine was used as a matrix and ligand to complex Cu2+ to mimic the active sites of laccases. A modified glassy carbon (GC) electrode with Cu2+-poly-l-histidine complex decreases the oxygen reduction overpotential as compared with the bare GC electrode. An array of Cu2+-poly-l-histidine spots with different compositions was deposited on a GC substrate, and their catalytic activity for oxygen reduction was evaluated by a scanning electrochemical microscopy-based screening technique. The electrocatalytic activities of complexes for oxygen reduction strongly depended on the mole ratio of Cu2+ to poly-l-histidine and the applied potential of the substrate.     

Convergent synthesis of complex diketopiperazines derived from pipecolic acid scaffolds and parallel screening against GPCR targets

A convergent approach to highly functionalized diketopiperazines (DKPs) using enantioenriched pipecolic acids is described. Scandium triflate-catalyzed [4 + 2] aza-annulation was employed to produce stereochemically well-defined building blocks. A resin "catch and release" strategy was devised to convert annulation products to pipecolic acid monomers. Complex diketopiperazines were efficiently assembled utilizing one-pot cyclodimerization of pipecolic acids. Massively parallel screening of the complex DKPs against a panel of molecular targets identified novel ligands for a number of G-protein-coupled receptors (GPCRs).     

Studies on the genetic variation of the green unicellular alga Haematococcus pluvialis (Chlorophyceae) obtained from different geographical locations using ISSR and RAPD molecular marker

Haematococcus pluvialis (Flotow) is a unicellular green alga, which is considered to be the best astaxanthin-producing organism. Molecular markers are suitable tools for the purpose of finding out genetic variations in organisms; however there have been no studies conducted on ISSR or RAPD molecular markers for this organism. The DNA of 10 different strains of H. pluvialis (four strains from Iran, two strains from Finland, one strain from Switzerland and three strains from the USA) was extracted. A genetic similarity study was carried out using 14 ISSR and 12 RAPD primers. Moreover, the molecular weights of the bands produced ranged from 0.14 to 3.4 Kb. The PCA and dendrogram clustered the H. pluvialis strains into various groups according to their geographical origin. The lowest genetic similarity was between the Iran2 and USA2 strains (0.08) and the highest genetic similarity was between Finland1 and Finland2 (0.64). The maximum numbers of bands produced by the ISSR and RAPD primers were 35 and 6 bands, respectively. The results showed that ISSR and RAPD markers are useful for genetic diversity studies of Haematococcus as they showed geographical discrimination.     

Rapid qualitative and quantitative analyses of flavanone aglycones in Fructus aurantii by HPLC ion-trap MS

IT-MS operated in the positive mode was applied for the rapid characterization/quantification of the flavanones in extracts from Fructus aurantii. APCI-MS and CID MS/MS provide unequivocal molecular weight (MW) data of these compounds and useful information about their structures (diagnostic fragment ions). Main fragment pathways include neutral losses of H2O, C2H2O, and B-ring as well as a retro-Diels-Alder (RDA) fragment giving rise to [1,3A + H], [1,3B+H]+, and [1,4B-H2 + H]+ ions, which form the characteristic MS/MS "fingerprint" of flavanone aglycones. When screening extracts of F. aurantii for flavanone aglycones, eight target compounds were characterized using this fingerprint. Meanwhile, ESI-MS in full-scan mode was developed and validated for the quantification of the main flavanone aglycones in F. aurantii. This method is simple, accurate, fast and requires only 16 min per sample for direct detection and quantification of naringenin and hesperetin. All the results and these characteristic fragments showed that the IT-MS is a powerful tool for the structural characterization and quantitative determination offlavanone aglycones.     

Tren-based analogues of bacillibactin: structure and stability

Synthetic analogues were designed to highlight the effect of the glycine moiety of bacillibactin on the overall stability of the ferric complex as compared to synthetic analogues of enterobactin. Insertion of a variety of amino acids to catecholamide analogues based on a Tren (tris(2-aminoethyl)amine) backbone increased the overall acidity of the ligands, causing an enhancement of the stability of the resulting ferric complex as compared to TRENCAM. Solution thermodynamic behavior of these siderophores and their synthetic analogues was investigated through potentiometric and spectrophotometric titrations. X-ray crystallography, circular dichroism, and molecular modeling were used to determine the chirality and geometry of the ferric complexes of bacillibactin and its analogues. In contrast to the Tren scaffold, addition of a glycine to the catechol chelating arms causes an inversion of the trilactone backbone, resulting in opposite chiralities of the two siderophores and a destabilization of the ferric complex of bacillibactin compared to ferric enterobactin.