Investigation of Two Zr-p-NO2Bn-DOTA Isomers via NMR and Quantum Chemical Studies.

A combination of NMR studies and quantum chemical calculations were employed to investigate the structure and energetics of Zr4+ chelates of pNO2Bn-DOTA. We have demonstrated that two discrete regioisomeric chelates are generated during the complex formation. The nitrobenzyl substituent can adopt either an equatorial corner or side position on the macrocyclic ring. These regioisomers are incapable of interconversion and were isolated by HPLC. The corner isomer is more stable than the side, and the SAP conformer of both regioisomers is energetically more favorable than the corresponding TSAP conformer.

Lentzeacins A-E, New Bacterial-Derived 2,5- and 2,6-Disubstituted Pyrazines from a BGC-Rich Soil Bacterium Lentzea sp. GA3-008.

Pyrazines (1,4-diazirines) are an important group of natural products that have tremendous monetary value in the food and fragrance industries and can exhibit a wide range of biological effects including antineoplastic, antidiabetic and antibiotic activities. As part of a project investigating the secondary metabolites present in understudied and chemically rich Actinomycetes, we isolated a series of six pyrazines from a soil-derived Lentzea sp. GA3-008, four of which are new. Here we describe the structures of lentzeacins A-E (1, 3, 5 and 6) along with two known analogues (2 and 4) and the porphyrin zincphyrin. The structures were determined by NMR spectroscopy and HR-ESI-MS. The suite of compounds present in Lentzea sp. includes 2,5-disubstituted pyrazines (compounds 2, 4, and 6) together with the new 2,6-disubstituted isomers (compounds 1, 3 and 5), a chemical class that is uncommon. We used long-read Nanopore sequencing to assemble a draft genome sequence of Lentzea sp. which revealed the presence of 40 biosynthetic gene clusters. Analysis of classical di-modular and single module non-ribosomal peptide synthase genes, and cyclic dipeptide synthases narrows down the possibilities for the biosynthesis of the pyrazines present in this strain.

X-ray Crystallography and Unexpected Chiroptical Properties Reassign the Configuration of Haliclonadiamine.

Haliclonadiamine and papuamine are bis-indane marine natural products isolated from the marine sponge Haliclona sp. Their relative structures were previously reported to differ by inversion at only one of their eight shared stereocenters. Here X-ray crystallography shows the opposite to be true: papuamine has a 1R,3S,8R,9S,14S,15R,20S,22R configuration, while haliclonadiamine has a 1S,3R,8S,9R,14R,15S,20R,22R configuration. Paradoxically the ECD of each structure displays a negative Cotton effect. X-ray crystallography reveals the two structures adopt similar conformations of their 13-membered macrocyclic core that comprises a configurationally relevant diene. B97x-D/Def2-TZVPP-(MeOH)-calculated ECD supports the diene configuration with the macrocycle dominating the ECD Cotton effect for haliclonadiamine and papuamine. Additional crystallographic and chiroptical analyses of three sponge samples from geographically distant locations indicate this pair of natural products always exists as a configurationally related couple. The co-discovery of a biosynthetic precursor, halichondriamine C, present in these same Haliclona samples must be considered when discussing any biosynthetic pathway. Taken together, this work justifies a reassignment of haliclonadiamine's structure and opens the question of how this complex stereochemical relationship between haliclonadiamine and palauamine arises biosynthetically.

Regioisomerization of Antimalarial Drug WR99210 Explains the Inactivity of a Commercial Stock.

WR99210, a former antimalarial drug candidate now widely used for the selection of Plasmodium transfectants, selectively targets the parasite's dihydrofolate reductase thymidine synthase bifunctional enzyme (DHFR-TS) but not human DHFR, which is not fused with TS. Accordingly, WR99210 and plasmids expressing the human dhfr gene have become valued tools for the genetic modification of parasites in the laboratory. Concerns over the ineffectiveness of WR99210 from some sources encouraged us to investigate the biological and chemical differences of supplies from two different companies (compounds 1 and 2). Compound 1 proved effective at low nanomolar concentrations against Plasmodium falciparum parasites, whereas compound 2 was ineffective, even at micromolar concentrations. Intact and fragmented mass spectra indicated identical molecular formulae of the unprotonated (free base) structures of compounds 1 and 2; however, the compounds displayed differences by thin-layer chromatography, reverse-phase high-performance liquid chromatography, and UV-visible spectroscopy, indicating important isomeric differences. Structural evaluations by 1H, 13C, and 15N nuclear magnetic resonance spectroscopy confirmed compound 1 as WR99210 and compound 2 as a dihydrotriazine regioisomer. Induced fit computational docking models showed that compound 1 binds tightly and specifically in the P. falciparum DHFR active site, whereas compound 2 fits poorly to the active site in loose and varied orientations. Stocks and concentrates of WR99210 should be monitored for the presence of regioisomer 2, particularly when they are not supplied as the hydrochloride salt or are exposed to basic conditions that may promote rearrangement. Absorption spectroscopy can serve for assays of the unrearranged and rearranged triazines.

Isolation, Purification, Characterization and Direct Conjugation of the Lipid†A-Free Lipopolysaccharide of Vibrio cholerae O139.

The lipopolysaccharide (LPS) of Vibrio cholerae O139, strain CIRS245, was isolated conventionally, and the lipid A was removed by mild acid hydrolysis (0.1 m NaOAc buffer containing 1 % SDS, pH 4.2, 95 °C, 8 h). The crude product was a complex mixture consisting mainly of constituent fragments of the O-specific polysaccharide-core (OSPc). The OSPc was only a minor component in the mixture. Two-stage purification of the crude OSPc by HPLC gave pure OSPc fragment of the LPS, as shown by NMR spectroscopy, analytical HPLC and ESI-MS. This material is the purest OSPc fragment of the LPS from Vibrio cholerae O139 reported to date. The purified OSPc was readily converted to the corresponding methyl squarate derivative and the latter was conjugated to BSA. The conjugate, when examined by ELISA, showed immunoreactivity with sera from patients in Bangladesh recovering from cholera caused by V. cholerae O139, but not O1.

Demystifying P2Y1 Receptor Ligand Recognition through Docking and Molecular Dynamics Analyses.

We performed a molecular modeling analysis of 100 nucleotide-like bisphosphates and 46 non-nucleotide arylurea derivatives previously reported as P2Y1R binders using the recently solved hP2Y1R structures. We initially docked the compounds at the X-ray structures and identified the binding modes of representative compounds highlighting key patterns in the structure-activity relationship (SAR). We subsequently subjected receptor complexes with selected key agonists (2MeSADP and MRS2268) and antagonists (MRS2500 and BPTU) to membrane molecular dynamics (MD) simulations (at least 200 ns run in triplicate, simulation time 0.6-1.6 µs per ligand system) while considering alternative protonation states of nucleotides. Comparing the temporal evolution of the ligand-protein interaction patterns with available site-directed mutagenesis (SDM) data and P2Y1R apo state simulation provided further SAR insights and suggested reasonable explanations for loss/gain of binding affinity as well as the most relevant charged species for nucleotide ligands. The MD analysis also predicted local conformational changes required for the receptor inactive state to accommodate nucleotide agonists.

Tulongicin, an Antibacterial Tri-Indole Alkaloid from a Deep-Water Topsentia sp. Sponge.

Antibacterial-guided fractionation of an extract of a deep-water Topsentia sp. marine sponge led to the isolation of two new indole alkaloids, tulongicin A (1) and dihydrospongotine C (2), along with two known analogues, spongotine C (3) and dibromodeoxytopsentin (4). Their planar structures were determined by NMR spectroscopy. Their absolute configurations were determined through a combination of experimental and computational analyses. Tulongicin (1) is the first natural product to contain a di(6-Br-1H-indol-3-yl)methyl group linked to an imidazole core. The coexistence of tri-indole 1 and bis-indole alcohol 2 suggests a possible route to 1. All of the compounds showed strong antimicrobial activity against Staphylococcus aureus.

Design and synthesis of small molecule-sulfotyrosine mimetics that inhibit HIV-1 entry.

In the absence of a cure or vaccine for HIV/AIDS, small molecule inhibitors remain an attractive choice for antiviral therapeutics. Recent structural and functional studies of the HIV-1 surface envelope glycoprotein gp120 have revealed sites of vulnerability that can be targeted by small molecule and peptide inhibitors, thereby inhibiting HIV-1 infection. Here we describe a series of small molecule entry inhibitors that were designed to mimic the sulfated N-terminal peptide of the HIV-1 coreceptor CCR5. From a panel of hydrazonothiazolyl pyrazolinones, we demonstrate that compounds containing naphthyl di- and tri-sulfonic acids inhibit HIV-1 infection in single round infectivity assays with the disulfonic acids being the most potent. Molecular docking supports the observed structure activity relationship, and SPR confirmed binding to gp120. In infectivity assays treatment with a representative naphthyl disulfonate and a disulfated CCR5 N-terminus peptide results in competitive inhibition, with combination indices >2. In total this work shows that gp120 and HIV-1 infection can be inhibited by small molecules that mimic the function of, and are competitive with the natural sulfated CCR5 N-terminus.

Polybrominated Diphenyl Ethers: Structure Determination and Trends in Antibacterial Activity.

Antibacterial-guided fractionation of the Dictyoceratid sponges Lamellodysidea sp. and two samples of Dysidea granulosa yielded 14 polybrominated, diphenyl ethers including one new methoxy-containing compound (8). Their structures were elucidated by interpretation of spectroscopic data of the natural product and their methoxy derivatives. Most of the compounds showed strong antimicrobial activity with low- to sub-microgram mL(-1) minimum inhibitory concentrations against drug-susceptible and drug-resistant strains of Staphylococcus aureus and Enterococcus faecium, and two compounds inhibited Escherichia coli in a structure-dependent manner.

The isotridecanyl side chain of plusbacin-A3 is essential for the transglycosylase inhibition of peptidoglycan biosynthesis.

Plusbacin-A3 (pb-A3) is a cyclic lipodepsipeptide that exhibits antibacterial activity against multidrug-resistant Gram-positive pathogens. Plusbacin-A3 is thought not to enter the cell cytoplasm, and its lipophilic isotridecanyl side chain is presumed to insert into the membrane bilayer, thereby facilitating either lipid II binding or some form of membrane disruption. Analogues of pb-A3, [(2)H]pb-A3 and deslipo-pb-A3, were synthesized to test membrane insertion as a key to the mode of action. [(2)H]pb-A3 has an isotopically (2)H-labeled isopropyl subunit of the lipid side chain, and deslipo-pb-A3 is missing the isotridecanyl side chain. Both analogues have the pb-A3 core structure. The loss of antimicrobial activity in deslipo-pb-A3 showed that the isotridecanyl side chain is crucial for the mode of action of the drug. However, rotational-echo double-resonance nuclear magnetic resonance characterization of [(2)H]pb-A3 bound to [1-(13)C]glycine-labeled whole cells of Staphylococcus aureus showed that the isotridecanyl side chain does not insert into the lipid membrane but instead is found in the staphylococcal cell wall, positioned near the pentaglycyl cross-bridge of the cell-wall peptidoglycan. Addition of [(2)H]pb-A3 during the growth of S. aureus resulted in the accumulation of Park's nucleotide, consistent with the inhibition of the transglycosylation step of peptidoglycan biosynthesis.

Structure-Activity Studies of 1H-Imidazo[4,5-c]quinolin-4-amine Derivatives as A3 Adenosine Receptor Positive Allosteric Modulators.

We previously reported 1H-imidazo[4,5-c]quinolin-4-amines as A3 adenosine receptor (A3AR) positive allosteric modulators (PAMs). A3AR agonists, but not PAMs, are in clinical trials for inflammatory diseases and liver conditions. We synthesized new analogues to distinguish 2-cyclopropyl antagonist 17 (orthosteric interaction demonstrated by binding and predicted computationally) from PAMs (derivatives with large 2-alkyl/cycloalkyl/bicycloalkyl groups). We predicted PAM binding at a hydrophobic site on the A3AR cytosolic interface. Although having low Caco-2 permeability and high plasma protein binding, hydrophobic 2-cyclohept-4-enyl-N-3,4-dichlorophenyl, MRS7788 18, and 2-heptan-4-yl-N-4-iodophenyl, MRS8054 39, derivatives were orally bioavailable in rat. 2-Heptan-4-yl-N-3,4-dichlorophenyl 14 and 2-cyclononyl-N-3,4-dichlorophenyl 20 derivatives and 39 greatly enhanced Cl-IB-MECA-stimulated [35S]GTPγS binding Emax, with only 12b trending toward decreasing the agonist EC50. A feasible route for radio-iodination at the p-position of a 4-phenylamino substituent suggests a potential radioligand for allosteric site binding. Herein, we advanced an allosteric approach to developing A3AR-activating drugs that are potentially event- and site-specific in action.

Intramyocardial triglyceride quantification by magnetic resonance spectroscopy: In vivo and ex vivo correlation in human subjects.

Accumulation of triglycerides (TG) in heart tissue has been associated with changes in left ventricular function. Proton magnetic resonance spectroscopy is currently the only noninvasive in vivo method to measure myocardial triglycerides content. The primary aim of this study was to determine if these in vivo measurements are specific to myocardial triglycerides in human subjects. Thus, in vivo proton magnetic resonance spectroscopy measurements were conducted on orthotopic heart transplant patients (n = 8) immediately before they underwent routine biopsies and ex vivo measurements were made on the endomyocardial biopsy samples. The correlation coefficient between the two measurements was 0.97, with P < 0.005, demonstrating for the first time the specificity of the in vivo measurement in human heart. From accompanying reliability experiments, the standardized typical error for the in vivo proton magnetic resonance spectroscopy method was estimated to be 7.0%, with a 95% confidence interval from 5.5 to 9.4%. These results suggest that proton magnetic resonance spectroscopy provides a specific and reliable measurement of myocardial triglycerides content and is suitable for routine studies.

Vertirhodins A-F, C-Linked Pyrrolidine-Iminosugar-Containing Pyranonaphthoquinones from Streptomyces sp. B15-008.

Six novel pyranonaphthoquinones, vertirhodins A-F (1-6), were discovered from a soil-derived Streptomyces sp. B15-008. Their chemical structures and absolute configurations were determined using nuclear magnetic resonance and comparison of experimental and theoretical electronic circular dichroism spectra. The vertirhodins feature an unusual C-8 N-methyl-2-pyrrolidinemethanol moiety, a 5,14-epoxide rarely seen in streptomyces-derived natural products, and a C-13 hydroxyl group that forms the semiquinone. A plausible ver biosynthetic gene cluster was identified through whole genome sequencing and provides insights into these features.

Plant cell-wall cross-links by REDOR NMR spectroscopy.

We present a new method that integrates selective biosynthetic labeling and solid-state NMR detection to identify in situ important protein cross-links in plant cell walls. We have labeled soybean cells by growth in media containing l-[ring-d(4)]tyrosine and l-[ring-4-(13)C]tyrosine, compared whole-cell and cell-wall (13)C CPMAS spectra, and examined intact cell walls using (13)C{(2)H} rotational echo double-resonance (REDOR) solid-state NMR. The proximity of (13)C and (2)H labels shows that 25% of the tyrosines in soybean cell walls are part of isodityrosine cross-links between protein chains. We also used (15)N{(13)C} REDOR of intact cell walls labeled by l-[ε-(15)N,6-(13)C]lysine and depleted in natural-abundance (15)N to establish that the side chains of lysine are not significantly involved in covalent cross-links to proteins or sugars.

Chain packing in polycarbonate glasses.

Chain packing in homogeneous blends of carbonate (13)C-labeled bisphenol A polycarbonate with either (i) CF(3)-labeled bisphenol A polycarbonate or (ii) ring-F-labeled bisphenol A polycarbonate has been characterized using (13)C{(19)F} rotational-echo double-resonance (REDOR) nuclear magnetic resonance. In both blends, the (13)C observed spin was at high concentration, and the (19)F dephasing or probe spin was at low concentration. In this situation, an analysis in terms of a distribution of isolated heteronuclear pairs of spins is valid. Nearest-neighbor separation of (13)C and (19)F labels was determined by accurately mapping the initial dipolar evolution using a shifted-pulse version of REDOR. Based on the results of this experiment, the average distance from a ring-fluorine to the nearest (13)C=O is more than 1.2 A greater than the corresponding CF(3)-(13)C=O distance. Next-nearest and more-distant-neighbor separations of labels were measured in a 416-rotor-cycle constant-time version of REDOR for both blends. Statistically significant local order was established for the nearest-neighbor labels in the methyl-labeled blend. These interchain packing results are in qualitative agreement with predictions based on coarse-grained simulations of a specially adapted model for bisphenol A polycarbonate. The model itself has been previously used to determine static and dynamic properties of polycarbonate with results in good agreement with those from rheological and neutron scattering experiments.

(15)N{(31)P} REDOR NMR studies of the binding of phosphonate reaction intermediate analogues to Saccharomyces cerevisiae lumazine synthase.

Lumazine synthase catalyzes the reaction of 5-amino-6-D-ribitylamino-2,4(1H,3H)-pyrimidinedione(1) with (S)-3,4-dihydroxybutanone 4-phosphate (2) to afford 6,7-dimethyl-8-D-ribityllumazine(3), the immediate biosynthetic precursor of riboflavin. The overall reaction implies a series of intermediates that are incompletely understood. The 15N{31P} REDOR NMR spectra of three metabolically stable phosphonate reaction intermediate analogues complexed to Saccharomyces cereVisiae lumazine synthase have been obtained at 7 and 12 T. Distances from the phosphorus atoms of the ligands to the side chain nitrogens of Lys92, His97, Arg136, and His148 have been determined. These distances were used in combination with the X-ray crystal coordinates of one of the intermediate analogues complexed with the enzyme in a series of distance-restrained molecular dynamics simulations. The resulting models indicate mobility of the Lys92 side chain, which could facilitate the exchange of inorganic phosphate eliminated from the substrate in one reaction, with the organic phosphate-containing substrate necessary for the next reaction.

A versatile custom cryostat for dynamic nuclear polarization supports multiple cryogenic magic angle spinning transmission line probes.

Dynamic nuclear polarization (DNP) with cryogenic magic angle spinning (MAS) provides significant improvements in NMR sensitivity, yet presents unique technical challenges. Here we describe a custom cryostat and suite of NMR probes capable of manipulating nuclear spins with multi-resonant radiofrequency circuits, cryogenic spinning below 6 K, sample exchange, and microwave coupling for DNP. The corrugated waveguide and six transfer lines needed for DNP and cryogenic spinning functionality are coupled to the probe from the top of the magnet. Transfer lines are vacuum-jacketed and provide bearing and drive gas, variable temperature fluid, two exhaust pathways, and a sample ejection port. The cryostat thermally isolates the magnet bore, thereby protecting the magnet and increasing cryogen efficiency. This novel design supports cryogenic MAS-DNP performance over an array of probes without altering DNP functionality. We present three MAS probes (two supporting 3.2 mm rotors and one supporting 9.5 mm rotors) interfacing with the single cryostat. Mechanical details, transmission line radio frequency design, and performance of the cryostat and three probes are described.

Limits of a localized magnetic resonance spectroscopy assay for ex vivo myocardial triacylglycerol.

Localized magnetic resonance spectroscopy (LMRS) promises a powerful non-invasive means to determine myocardial triacylglycerol (TAG) in a clinical setting. Here, the linearity, specificity, robustness, precision, and accuracy of an ex vivo mouse-heart LMRS TAG assay are assessed by quantifying the spatial, spectral, and relaxation-induced uncertainties. The protocol, which is based on localization by adiabatic selective refocusing (LASER) using frequency offset corrected inversion (FOCI) pulses, alternating gradient polarity, and simple post-processing, is shown to have good characteristics. The presented protocol has a benchmark, phantom-based, accuracy of 3%, and when applied to ex vivo mouse hearts the accuracy is 6%, making the LMRS assay comparable to the typical destructive bioanalytical assay.

Dual Mode of Action for Plusbacin A3 in Staphylococcus aureus.

We have used C{F}, N{F}, and N{P} rotational-echo double resonance NMR to determine the location and conformation of 19F and 15N double-labeled plusbacin A3 and of double-labeled deslipo-plusbacin A3, each bound to the cell walls of whole cells of Staphyloccocus aureus grown in media containing [1-13C]glycine. The 31P is primarily in wall teichoic acid. Approximately 25% of plusbacin headgroups (the cyclic depsipeptide backbone) are in a closed conformation (N-F separation of 6 Å), while 75% are in a more open conformation (N-F separation of 12 Å). The closed headgroups have no contact with wall teichoic acid, whereas the open headgroups have a strong contact. This places the closed headgroups in hydrophobic regions of the cell wall and the open headgroups in hydrophilic regions. None of the plusbacin tails have contact with the 31P of either wall teichoic acid or the cell membrane and thus are in hydrophobic regions of the cell wall. In addition, both heads and tails of plusbacin A3 have contact with the glycyl 13C incorporated in cell-wall peptidoglycan pentaglycyl bridges and with 13C-labeled purines near the membrane surface. We interpret these results in terms of a dual mode of action for plusbacin A3: first, disruption of the peptidoglycan layer nearest to the membrane surface by closed-conformation plusbacin A3 leading to an inhibition of chain extension by transglycosylation; second, thinning and disruption of the membrane (possibly including disruption of ATP-binding cassette transporters embedded in the membrane) by open-conformation plusbacin A3, thereby leading to release of ATP to the hydrophilic regions of the cell wall and subsequent binding by plusbacin A3.