The viscosin group covers a series of cyclic lipodepsipeptides (CLPs) produced by Pseudomonas bacteria, with a range of biological functions and antimicrobial activities. Their oligopeptide moieties are composed of both L- and D-amino acids. Remarkably, the Leu5 amino acid-centrally located in the nonapeptide sequence-is the sole residue found to possess either an L or D configuration, depending on the producing strain. The impact of this D/L switch on the solution conformation was investigated by NMR-restrained molecular modelling of the epimers pseudodesmin A and viscosinamide A. Although the backbone fold remained unaffected, the D/L switch adjusted the segregation between hydrophobic and hydrophilic residues, and thus the amphipathicity. It also influenced the self-assembly capacity in organic solvents. Additionally, several new minor variants of viscosinamide A from Pseudomonas fluorescens DR54 were identified, and an NMR assay is proposed to assess the presence of either an L- or D-Leu5.
We report the synthesis of a family of D- and L-furano-D-apionucleosides, their 3'-deoxy, as well as their 2',3'-dideoxy analogues with thymine and adenine nucleobases. Single carbon homologation of 1,2-O-isopropylidene-D-glycero-tetrafuranos-3-ulose (15) and optimized glycosylation conditions involving microwave irradiation were key to the successful synthesis of the target compounds. While all target nucleosides failed to show significant antiviral activity, we demonstrated that the triphosphate of 2',3'-deoxy-D-apio-D-furanoadenosine (1), in contrast to that of its D-apio-L-furanose epimer 2, was readily incorporated into a DNA template by HIV reverse transcriptase to act as a DNA chain terminator. This led us to convert adenine derivative 1 into two phosphoramidate prodrugs. ProTide 9b was found active against HIV-1 and HIV-2 (EC50 = 0.5-1.5 ?M), indicating that the lack of activity of the parent nucleoside, and possibly also other members of the D-apio-D-furanose nucleoside family must be sought in the inefficient cellular conversion to the monophosphate.
A rapid and efficient total synthesis is reported for the cyclic lipodepsipeptide pseudodesmin?A. This member of the Pseudomonas viscosin group is active against Gram-positive bacteria and features self-assembling properties. A conserved serine residue within the lactone macrocycle is exploited for initial immobilization on 2-chlorotrityl chloride resin through ether formation with the side-chain alcohol. Subsequent elongation proceeds through Fmoc solid-phase peptide synthesis, including automated incorporation of the enantioselectively synthesized (R)-3-hydroxydecanoic acid lipid tail. Following esterification to generate the incipient lactone bond, the macrocycle is formed by on-resin head-to-tail macrolactamization and cleaved from the resin to give the desired compound in good purity. The short and efficient synthesis route allows rapid generation of analogues by facile variation of both the peptide and lipid moieties with good control of epimerization while maximizing automation. Synthesis of the pseudodesmin?A enantiomer yields identical self-assembly and biological activity to that observed for the natural compound, showing that activity is not mediated by chiral interactions. A D-Asn8 analogue developed en route retains self-assembly, but loses activity. The synthesis strategy should be generally applicable for the rapid generation of analogues from various cyclic lipodepsipeptide groups, allowing an investigation of their self-assembling properties and structure-activity relationships.
The most favourable solvent suppression methods that have been applied to PGSTE experiments for the measurement of diffusion are WATERGATE and excitation sculpting. However, both methods come with significant J-modulation line-shape distortions on multiplets, a phenomenon that is known to be of particular concern for DOSY data processing. Here, two new PGSTE experiments are proposed that suppress both the solvent peak and J-modulation based on the perfect echo WATERGATE sequence. This allows narrow suppression bandwidths and thus measurement of diffusion on peaks close to the solvent peak. Both sequences perform admirably and the better option depends on the priority one puts on the quality of the solvent suppression or signal loss due to T2 weighting. Gradient-based solvent suppression in PGSTE experiments can often be compromised by the variable, diffusion-encoding gradient pulses. Special emphasis is put on how to maximise the robustness of the solvent suppression.
Two Pseudomonas strains, identified as closely related to Pseudomonas tolaasii, were isolated from milk of a farm with frequent false-positive Delvotest results for screening putative antibiotic residues in raw milk executed as part of the regulatory quality programme. Growth at 5 to 7°C of these isolates in milk resulted in high lipolysis and the production of bacterial inhibitors. The two main bacterial inhibitors have a molecular weight of 1168.7 and 1140.7 Da respectively, are heat-tolerant and inhibit Geobacillus stearothermophilus var. calidolactis, the test strain of most of the commercially available microbiological inhibitor tests for screening of antibiotic residues in milk. Furthermore, these bacterial inhibitors show antimicrobial activity against Staphylococcus aureus, Bacillus cereus and B. subtilis and also interfere negatively with yoghurt production. Following their isolation and purification with RP-HPLC, the inhibitors were identified by NMR analysis as cyclic lipodepsipeptides of the viscosin group. Our findings bring to light a new challenge for quality control in the dairy industry. By prolonging the refrigerated storage of raw milk, the keeping quality of milk is influenced by growth and metabolic activities of psychrotrophic bacteria such as pseudomonads. Besides an increased risk of possible spoilage of long shelf-life milk, the production at low temperature of natural bacterial inhibitors may also result in false-positive results for antibiotic residue screening tests based on microbial inhibitor assays thus leading to undue production loss.
We present a scalable synthesis of a versatile MTX reagent with an azide ligation handle that allows rapid ?-selective conjugation to yield MTX fusion compounds (MFCs) appropriate for MASPIT, a three-hybrid system that enables the identification of mammalian cytosolic proteins that interact with a small molecule of interest. We selected three structurally diverse pharmacologically active compounds (tamoxifen, reversine, and FK506) as model baits. After acetylene functionalization of these baits, MFCs were synthesized via a CuAAC reaction, demonstrating the general applicability of the MTX reagent. In analytical mode, MASPIT was able to give concentration-dependent reporter signals for the established target proteins. Furthermore, we demonstrate that the sensitivity obtained with the new MTX reagent was significantly stronger than that of a previously used non-regiomeric conjugate mixture. Finally, the FK506 MFC was explored in a cellular array screen for targets of FK506. Out of a pilot collection of nearly 2000 full-length human ORF preys, FKBP12, the established target of FK506, emerged as the prey protein that gave the highest increase in luciferase activity. This indicates that our newly developed synthetic strategy for the straightforward generation of MFCs is a promising asset to uncover new intracellular targets using MASPIT cellular array screening.
The rhizosphere isolate Pseudomonas putida BW11M1 produces a mixture of cyclic lipopeptide congeners, designated xantholysins. Properties of the major compound xantholysin A, shared with several other Pseudomonas lipopeptides, include antifungal activity and toxicity to Gram-positive bacteria, a supportive role in biofilm formation, and facilitation of surface colonization through swarming. Atypical is the lipopeptides capacity to inhibit some Gram-negative bacteria, including several xanthomonads. The lipotetradecadepsipeptides are assembled by XtlA, XtlB and XtlC, three co-linearly operating non-ribosomal peptide synthetases (NRPSs) displaying similarity in modular architecture with the entolysin-producing enzymes of the entomopathogenic Pseudomonas entomophila L48. A shifted serine-incorporating unit in the eight-module enzyme XtlB elongating the central peptide moiety not only generates an amino acid sequence differing at several equivalent positions from entolysin, but also directs xantholysins macrocyclization into an octacyclic structure, distinct from the pentacyclic closure in entolysin. Relaxed fatty acid specificity during lipoinitiation by XtlA (acylation with 3-hydroxydodec-5-enoate instead of 3-hydroxydecanoate) and for incorporation of the ultimate amino acid by XtlC (valine instead of isoleucine) account for the production of the minor structural variants xantholysin C and B, respectively. Remarkably, the genetic backbones of the xantholysin and entolysin NRPS systems also bear pronounced phylogenetic similarity to those of the P. putida strains PCL1445 and RW10S2, albeit generating the seemingly structurally unrelated cyclic lipopeptides putisolvin (undecapeptide containing a cyclotetrapeptide) and WLIP (nonapeptide containing a cycloheptapeptide), respectively. This similarity includes the linked genes encoding the cognate LuxR-family regulator and tripartite export system components in addition to individual modules of the NRPS enzymes, and probably reflects a common evolutionary origin. Phylogenetic scrutiny of the modules used for selective amino acid activation by these synthetases indicates that bacteria such as pseudomonads recruit and reshuffle individual biosynthetic units and blocks thereof to engineer reorganized or novel NRPS assembly lines for diversified synthesis of lipopeptides.
An efficient protocol for the construction of C-6-(hetero)aryl-substituted uridine phosphonate analogues utilizing an aerobic, ligand-free Suzuki-Miyaura cross-coupling reaction of a 6-iodo-precursor in aqueous media has been established. The method presents a modular approach toward the target compounds as demonstrated by the synthesis of a small library comprising 14 novel nucleoside phosphonates.
Proton nuclear magnetic resonance ((1)H-NMR)-based metabolomics enables the high-resolution and high-throughput assessment of a broad spectrum of metabolites in biofluids. Despite the straightforward character of the experimental methodology, the analysis of spectral profiles is rather complex, particularly due to the requirement of numerous data preprocessing steps. Here, we evaluate how several of the most common preprocessing procedures affect the subsequent univariate analyses of blood serum spectra, with a particular focus on how the standard methods perform compared to more advanced examples. Carr-Purcell-Meiboom-Gill 1D (1)H spectra were obtained for 240 serum samples from healthy subjects of the Asklepios study. We studied the impact of different preprocessing steps--integral (standard method) and probabilistic quotient normalization; no, equidistant (standard), and adaptive-intelligent binning; mean (standard) and maximum bin intensity data summation--on the resonance intensities of three different types of metabolites: triglycerides, glucose, and creatinine. The effects were evaluated by correlating the differently preprocessed NMR data with the independently measured metabolite concentrations. The analyses revealed that the standard methods performed inferiorly and that a combination of probabilistic quotient normalization after adaptive-intelligent binning and maximum intensity variable definition yielded the best overall results (triglycerides, R = 0.98; glucose, R = 0.76; creatinine, R = 0.70). Therefore, at least in the case of serum metabolomics, these or equivalent methods should be preferred above the standard preprocessing methods, particularly for univariate analyses. Additional optimization of the normalization procedure might further improve the analyses.
We present the use of 1-mm room-temperature probe technology to perform intermolecular interaction studies using chemical shift perturbation methods and saturation transfer difference (STD) spectroscopy using small sample volumes. The use of a small sample volume (5-10 µl) allows for an alternative titration protocol where individual samples are prepared for each titration point, rather than the usual protocol used for a 5-mm probe setup where the ligand is added consecutively to the solution containing the protein or host of interest. This allows for considerable economy in the consumption and cost of the protein and ligand amounts required for interaction studies. For titration experiments, the use of the 1-mm setup consumes less than 10% of the ligand amount required using a 5-mm setup. This is especially significant when complex ligands that are only available in limited quantities, typically because they are obtained from natural sources or through elaborate synthesis efforts, need to be investigated. While the use of smaller volumes does increase the measuring time, we demonstrate that the use of commercial small volume probes allows the study of interactions that would otherwise be impossible to address by NMR.
Pseudodesmin A is a cyclic lipodepsipeptide (CLP) of the viscosin group with a moderate in vitro biological activity. For several CLPs, including members of this group, this activity has been related to the ability to form ion pores in cellular membranes. As their size does not allow individual CLPs to span the membrane bilayer, individual monomers must somehow assemble into a larger structure. NMR spectroscopy has been used to demonstrate that in chloroform and other apolar organic solvents, pseudodesmin A monomers assemble into a supramolecular structure. These self-assembled structures can become sufficiently large to span the membrane bilayer as demonstrated with translational diffusion NMR spectroscopic measurements. With the aim to obtain more insight into the structural nature of this assembly, the solution conformation of pseudodesmin A was first determined by using ROESY (rOe) restraints measured in acetonitrile, in which no self-association occurs. The structure, which is found to be mostly similar to the previously described crystal structure, is shown to be retained within the supramolecular complex. Intermolecular rOe contacts obtained in chloroform together with chemical shift perturbation data provides structural insight into the organization of the self-associated complex. Based upon this analysis, a model for the organization of pseudodesmin A monomers in the supramolecular assembly is proposed, which is in agreement with the formation of bilayer spanning hydrophilic pores and provides the basis for a structure-function relationship for this type of CLPs. Finally, it is demonstrated that the differences previously reported between the crystal and solution conformation of the white line inducing principle (WLIP), a close analogue of pseudodesmin A, are the result of the use of dimethyl sulfoxide as solvent, whose strong hydrogen-bonding capacity induces conformational exchange.
The influence of the physicochemical properties of the vesicular bilayer on the sorption of poorly water soluble compounds was investigated with pulsed field gradient 1H nuclear magnetic resonance (PFG-NMR) for the case of phosphatidylcholine and dioctadecyldimethylammonium bromide (DODAB), using 4-ethylbenzyl alcohol as a model compound. Hereby, the effect of bilayer thickness at a constant physicochemical state was studied using a range of phosphatidylcholines of varying chain lengths, whereas DODAB was preferred to check the influence of the bilayer physicochemical state since this cationic lipid is characterized by three different states within the studied temperature range. When the phospholipid alkyl chain length was changed, no differences were observed in the sorption which was linked to the surface-mediated sorption. On the other hand, when the chemical composition was preserved but the temperature and thus the physical state of the bilayer were changed, the sorption in dioctadecyldimethylammonium bromide (DODAB) vesicles changed dramatically. From those experiments, a strong relationship between the ordering of the surfactant molecules and the sorption can be assumed.
Polysorbate surfactants (commercially available as Tween) are widely used in pharmaceutical, cosmetic and food products. They are generally considered as esters of ethoxylated sorbitan with fatty acids. Diffusion H1 NMR spectroscopy on a solution of polysorbate 20 in D2O revealed that only one diffusion coefficient was found for the fatty acyl part. Using the Stokes-Einstein equation, it became obvious that this diffusion behavior was caused by micelles. On the other hand, two significantly different diffusion coefficients were found for the methylene groups of ethylene oxide (EO). This indicates the presence of two distinct EO containing species in solution. Since the slowest diffusing EO species has the same diffusion coefficient as the fatty acyl part, it corresponds to the micellar (i.e. fatty acyl bound) ethoxylates. The diffusion coefficient of the fastest diffusing EO species was a factor of four larger than that of the slowly diffusing species and was attributed to water-soluble non-esterified ethoxylates. A solution of polysorbate 20 in the presence of NaOD was prepared to investigate if hydrolysis of the sorbitan ester could be the reason for the occurence of these hydrophilic ethoxylates. It was found that alkaline hydrolysis does lead to an increasing fraction of non-esterified ethoxylates, but is not the cause of its presence in untreated polysorbate samples since these species were also found in solutions of polyethylene glycol oleyl ether (commercially available as Brij), which are not susceptible to hydrolysis. Fractionation of the EO species present in polysorbate 20 into an amphiphilic and a hydrophilic fraction was only partly obtained by activated carbon adsorption. On the other hand, sequential extraction of aqueous polysorbate solutions by ethyl acetate and chloroform enabled a nearly complete fractionation. H1 NMR spectroscopy proved to be very useful since it allows in situ determination of the global composition of a surfactant sample, as well as quantification of both the amphiphilic and hydrophilic ethoxylate fractions via diffusion measurements.
The solubilization of the poorly water soluble anti-inflammatory drug flurbiprofen in non-ionic Tween 20 surfactant micellar solutions was studied by both (19)F and (1)H NMR spectroscopy in an acidic environment. These non-destructive techniques allowed us to investigate the effect of temperature cycling in situ. Using (19)F NMR, an increased solubilisation capacity was observed as the temperature increased. This effect became more pronounced above the cloud point, which was reduced by more than 30 degrees C in the presence of an excess of flurbiprofen. Upon clouding, peak splitting was observed in the (19)F spectrum, which indicates that two pools of solubilised flurbiprofen exist that are in slow exchange on the NMR frequency timescale. The clouding and solubilization processes were found to be reversible, albeit with slow kinetics. Based on chemical shift differences of both Tween 20 and flurbiprofen, as well as NOESY experiments, the flurbiprofen was found to be accumulated within the palisade layer of the Tween 20 micelles.
The secondary metabolite mediating the GacS-dependent growth-inhibitory effect exerted by the rice rhizosphere isolate Pseudomonas putida RW10S2 on phytopathogenic Xanthomonas species was identified as white-line-inducing principle (WLIP), a member of the viscosin group of cyclic lipononadepsipeptides. WLIP producers are commonly referred to by the taxonomically invalid name "Pseudomonas reactans," based on their capacity to reveal the presence of a nearby colony of Pseudomonas tolaasii by inducing the formation of a visible precipitate ("white line") in agar medium between both colonies. This phenomenon is attributed to the interaction of WLIP with a cyclic lipopeptide of a distinct structural group, the fungitoxic tolaasin, and has found application as a diagnostic tool to identify tolaasin-producing bacteria pathogenic to mushrooms. The genes encoding the WLIP nonribosomal peptide synthetases WlpA, WlpB, and WlpC were identified in two separate genomic clusters (wlpR-wlpA and wlpBC) with an operon organization similar to that of the viscosin, massetolide, and entolysin biosynthetic systems. Expression of wlpR is dependent on gacS, and the encoded regulator of the LuxR family (WlpR) activates transcription of the biosynthetic genes and the linked export genes, which is not controlled by the RW10S2 quorum-sensing system PmrR/PmrI. In addition to linking the known phenotypes of white line production and hemolytic activity of a WLIP producer with WLIP biosynthesis, additional properties of ecological relevance conferred by WLIP production were identified, namely, antagonism against Xanthomonas and involvement in swarming and biofilm formation.
We explored the influence of modifications of uridine 5-methylenephosphonate on biological activity at the human P2Y(2) receptor. Key steps in the synthesis of a series of 5-substituted uridine 5-methylenephosphonates were the reaction of a suitably protected uridine 5-aldehyde with [(diethoxyphosphinyl)methylidene]triphenylphosphorane, C-5 bromination and a Suzuki-Miyaura coupling. These analogues behaved as selective agonists at the P2Y(2) receptor, with three analogues exhibiting potencies in the submicromolar range. Although maximal activities observed with the phosphonate analogues were much less than observed with UTP, high concentrations of the phosphonates had no effect on the stimulatory effect of UTP. These results suggest that these phosphonates bind to an allosteric site of the P2Y(2) receptor.
A small series of 5-(hetero)aryl-modified nucleoside phosphonates was synthesized via an 8-step procedure including a Wittig reaction and Suzuki-Miyaura coupling. An unanticipated anomerization during phosphonate deprotection allowed us to isolate both anomers of the 5-substituted 2-deoxy-uridine phosphonates and assess their antiviral activity against a broad panel of viruses.
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