Making 1H-1H Couplings More Accessible and Accurate with Selective 2DJ NMR Experiments Aided by 13C Satellites.

1H-1H coupling constants are one of the primary sources of information for nuclear magnetic resonance (NMR) structural analysis. Several selective 2DJ experiments have been proposed that allow for their individual measurement at pure shift resolution. However, all of these experiments fail in the not uncommon case when coupled protons have very close chemical shifts. First, the coupling between protons with overlapping multiplets is inaccessible due to the inability of a frequency-selective pulse to invert just one of them. Second, the strong coupling condition affects the accuracy of coupling measurements involving third spins. These shortcomings impose a limit on the effectiveness of state-of-the-art experiments, such as G-SERF or PSYCHEDELIC. Here, we introduce two new and complementary selective 2DJ experiments that we coin SERFBIRD and SATASERF. These experiments overcome the aforementioned issues by utilizing the 13C satellite signals at natural isotope abundance, which resolves the chemical shift degeneracy. We demonstrate the utility of these experiments on the tetrasaccharide stachyose and the challenging case of norcamphor, for the latter achieving measurement of all JHH couplings, while only a few were accessible with PSYCHEDELIC. The new experiments are applicable to any organic compound and will prove valuable for configurational and conformational analyses.

Conformation and Affinity Modulations by Multiple Phosphorylation Occurring in the BIN1 SH3 Domain Binding Site of the Tau Protein Proline-Rich Region.

An increase in phosphorylation of the Tau protein is associated with Alzheimer's disease (AD) progression through unclear molecular mechanisms. In general, phosphorylation modifies the interaction of intrinsically disordered proteins, such as Tau, with other proteins; however, elucidating the structural basis of this regulation mechanism remains challenging. The bridging integrator-1 gene is an AD genetic determinant whose gene product, BIN1, directly interacts with Tau. The proline-rich motif recognized within a Tau(210-240) peptide by the SH3 domain of BIN1 (BIN1 SH3) is defined as 216PTPP219, and this interaction is modulated by phosphorylation. Phosphorylation of T217 within the Tau(210-240) peptide led to a 6-fold reduction in the affinity, while single phosphorylation at either T212, T231, or S235 had no effect on the interaction. Nonetheless, combined phosphorylation of T231 and S235 led to a 3-fold reduction in the affinity, although these phosphorylations are not within the BIN1 SH3-bound region of the Tau peptide. Using nuclear magnetic resonance (NMR) spectroscopy, these phosphorylations were shown to affect the local secondary structure and dynamics of the Tau(210-240) peptide. Models of the (un)phosphorylated peptides were obtained from molecular dynamics (MD) simulation validated by experimental data and showed compaction of the phosphorylated peptide due to increased salt bridge formation. This dynamic folding might indirectly impact the BIN1 SH3 binding by a decreased accessibility of the binding site. Regulation of the binding might thus not only be due to local electrostatic or steric effects from phosphorylation but also to the modification of the conformational properties of Tau.

Obtaining Pure 1H NMR Spectra of Individual Pyranose and Furanose Anomers of Reducing Deoxyfluorinated Sugars.

Due to tautomeric equilibria, NMR spectra of reducing sugars can be complex with many overlapping resonances. This hampers coupling constant determination, which is required for conformational analysis and configurational assignment of substituents. Given that mixtures of interconverting species are physically inseparable, easy-to-use techniques that enable facile full 1H NMR characterization of sugars are of interest. Here, we show that individual spectra of both pyranoside and furanoside forms of reducing fluorosugars can be obtained using 1D FESTA. We discuss the unique opportunities offered by FESTA over standard sel-TOCSY and show how it allows a more complete characterization. We illustrate the power of FESTA by presenting the first full NMR characterization of many fluorosugars, including of the important fluorosugar 2-deoxy-2-fluoroglucose. We discuss in detail all practical considerations for setting up FESTA experiments for fluorosugars, which can be extended to any mixture of fluorine-containing species interconverting slowly on the NMR frequency-time scale.

Relating Conformational Equilibria to Conformer-Specific Lipophilicities: New Opportunities in Drug Discovery.

Efficient drug discovery is based on a concerted effort in optimizing bioactivity and compound properties such as lipophilicity, and is guided by efficiency metrics that reflect both aspects. While conformation-activity relationships and ligand conformational control are known strategies to improve bioactivity, the use of conformer-specific lipophilicities (logp) is much less explored. Here we show how conformer-specific logp values can be obtained from knowledge of the macroscopic logP value, and of the equilibrium constants between the individual species in water and in octanol. This is illustrated with fluorinated amide rotamers, with integration of rotamer 19 F NMR signals as a facile, direct method to obtain logp values. The difference between logp and logP optimization is highlighted, giving rise to a novel avenue for lipophilicity control in drug discovery.

Expedited Nuclear Magnetic Resonance Assignment of Small- to Medium-Sized Molecules with Improved HSQC-CLIP-COSY Experiments.

Resonance assignment is a pivotal step for any nuclear magnetic resonance (NMR) analysis, such as structure elucidation or the investigation of protein-ligand interactions. Both 1H-13C heteronuclear single quantum correlation (HSQC) and 1H-1H correlation spectroscopy (COSY) two-dimensional (2D) experiments are invaluable for 1H NMR assignment, by extending the high signal dispersion of 13C chemical shifts onto 1H resonances and by providing a high amount of through-bond 1H-1H connectivity information, respectively. The recently introduced HSQC-CLIP(Clean In-Phase)-COSY method combines these two experiments, providing COSY correlations along the high-resolution 13C dimension with clean in-phase multiplets. However, two experiments need to be recorded to unambiguously identify COSY cross-peaks. Here, we propose novel variants of the HSQC-CLIP-COSY pulse sequence that edit cross-peak signs so that direct HSQC responses can be distinguished from COSY relay peaks, and/or the multiplicities of the 13C nuclei are reflected, allowing the assignment of all the peaks in a single experiment. The advanced HSQC-CLIP-COSY variants have the potential to accelerate and simplify the NMR structure-elucidation process of both synthetic and natural products and to become valuable tools for high-throughput computer-assisted structure determination.

Adamantane Containing Peptidoglycan Fragments Enhance RANTES and IL-6 Production in Lipopolysaccharide-Induced Macrophages.

We report the enhancement of the lipopolysaccharide-induced immune response by adamantane containing peptidoglycan fragments in vitro. The immune stimulation was detected by Il-6 (interleukine 6) and RANTES (regulated on activation, normal T cell expressed and secreted) chemokine expression using cell assays on immortalized mouse bone-marrow derived macrophages. The most active compound was a α-D-mannosyl derivative of an adamantylated tripeptide with L-chirality at the adamantyl group attachment, whereby the mannose moiety assumed to target mannose receptors expressed on macrophage cell surfaces. The immune co-stimulatory effect was also influenced by the configuration of the adamantyl center, revealing the importance of specific molecular recognition event taking place with its receptor. The immunostimulating activities of these compounds were further enhanced upon their incorporation into lipid bilayers, which is likely related to the presence of the adamantyl group that helps anchor the peptidoglycan fragment into lipid nanoparticles. We concluded that the proposed adamantane containing peptidoglycan fragments act as co-stimulatory agents and are also suitable for the preparation of lipid nanoparticle-based delivery of peptidoglycan fragments.

Probing Long-Range Anisotropic Interactions: a General and Sign-Sensitive Strategy to Measure 1 H-1 H Residual Dipolar Couplings as a Key Advance for Organic Structure Determination.

Residual dipolar couplings (RDCs) are amongst the most powerful NMR parameters for organic structure elucidation. In order to maximize their effectiveness in increasingly complex cases such as flexible compounds, a maximum of RDCs between nuclei sampling a large distribution of orientations is needed, including sign information. For this, the easily accessible one-bond 1 H-13 C RDCs alone often fall short. Long-range 1 H-1 H RDCs are both abundant and typically sample highly complementary orientations, but accessing them in a sign-sensitive way has been severely obstructed due to the overflow of 1 H-1 H couplings. Here, we present a generally applicable strategy that allows the measurement of a large number of 1 H-1 H RDCs, including their signs, which is based on a combination of an improved PSYCHEDELIC method and a new selective constant-time ß-COSY experiment. The potential of 1 H-1 H RDCs to better determine molecular alignment and to discriminate between enantiomers and diastereomers is demonstrated.

Fluorescent Pseudomonas and cyclic lipopeptide diversity in the rhizosphere of cocoyam (Xanthosoma sagittifolium).

Cocoyam (Xanthosoma sagittifolium (L.)), an important tuber crop in the tropics, is severely affected by the cocoyam root rot disease (CRRD) caused by Pythium myriotylum. The white cocoyam genotype is very susceptible while the red cocoyam has some field tolerance to CRRD. Fluorescent Pseudomonas isolates obtained from the rhizosphere of healthy red and white cocoyams from three different fields in Cameroon were taxonomically characterized. The cocoyam rhizosphere was enriched with P. fluorescens complex and P. putida isolates independent of the plant genotype. LC-MS and NMR analyses revealed that 50% of the Pseudomonas isolates produced cyclic lipopeptides (CLPs) including entolysin, lokisin, WLIP, putisolvin and xantholysin together with eight novel CLPs. In general, CLP types were linked to specific taxonomic groups within the fluorescent pseudomonads. Representative CLP-producing bacteria showed effective control against CRRD while purified CLPs caused hyphal branching or hyphal leakage in P. myriotylum. The structure of cocoyamide A, a CLP which is predominantly produced by P. koreensis group isolates within the P. fluorescens complex is described. Compared with the white cocoyam, the red cocoyam rhizosphere appeared to support a more diverse CLP spectrum. It remains to be investigated whether this contributes to the field tolerance displayed by the red cocoyam.

Synthesis and Conformational Properties of 3,4-Difluoro-l-prolines.

Fluorinated proline derivatives have found diverse applications in areas ranging from medicinal chemistry over structural biochemistry to organocatalysis. Depending on the stereochemistry of monofluorination at the proline 3- or 4-position, different effects on the conformational properties of proline (ring pucker, cis/ trans isomerization) are introduced. With fluorination at both 3- and 4-positions, matching or mismatching effects can occur depending on the relative stereochemistry. Here we report, in full, the syntheses and conformational properties of three out of the four possible 3,4-difluoro-l-proline diastereoisomers. The yet unreported conformational properties are described for (3 S,4 S)- and (3 R,4 R)-difluoro-l-proline, which are shown to bias ring pucker and cis/ trans ratios on the same order of magnitude as their respective monofluorinated progenitors, although with significantly faster amide cis/ trans isomerization rates. The reported analogues thus expand the scope of available fluorinated proline analogues as tools to tailor proline's distinct conformational and dynamical properties, allowing for the interrogation of its role in, for instance, protein stability or folding.

Conformation and Dynamics of the Cyclic Lipopeptide Viscosinamide at the Water-Lipid Interface.

Cyclic lipodepsipeptides or CLiPs from Pseudomonas are secondary metabolites that mediate a wide range of biological functions for their producers, and display antimicrobial and anticancer activities. Direct interaction of CLiPs with the cellular membranes is presumed to be essential in causing these. To understand the processes involved at the molecular level, knowledge of the conformation and dynamics of CLiPs at the water-lipid interface is required to guide the interpretation of biophysical investigations in model membrane systems. We used NMR and molecular dynamics to study the conformation, location and orientation of the Pseudomonas CLiP viscosinamide in a water/dodecylphosphocholine solution. In the process, we demonstrate the strong added value of combining uniform, isotope-enriched viscosinamide and protein NMR methods. In particular, the use of techniques to determine backbone dihedral angles and detect and identify long-lived hydrogen bonds, establishes that the solution conformation previously determined in acetonitrile is maintained in water/dodecylphosphocholine solution. Paramagnetic relaxation enhancements pinpoint viscosinamide near the water-lipid interface, with its orientation dictated by the amphipathic distribution of hydrophobic and hydrophilic residues. Finally, the experimental observations are supported by molecular dynamics simulations. Thus a firm structural basis is now available for interpreting biophysical and bioactivity data relating to this class of compounds.

Improved Isotopic Profiling by Pure Shift Heteronuclear 2D J-Resolved NMR Spectroscopy.

Quantitative information on the carbon isotope content of metabolites is essential for flux analysis. Whereas this information is in principle present in proton NMR spectra through both direct and long-range heteronuclear coupling constants, spectral overlap and homonuclear coupling constants both hinder its extraction. We demonstrate here how pure shift 2D J-resolved NMR spectroscopy can simultaneously remove the homonuclear couplings and separate the chemical shift information from the heteronuclear coupling patterns. We demonstrate the power of this method on cell lysates from different bacterial cultures and investigate in detail the branched chain amino acid biosynthesis.

Clean pure shift 2D J-resolved spectroscopy.

2D J-resolved spectroscopy is one of the oldest and conceptually most elegant ways to separate homonuclear scalar couplings from chemical shift information. In practice, the classical experiment suffers from a number of complications that limits accuracy and resolution, including phasetwist lineshapes, strong coupling artifacts, and the need for shearing the spectrum by 45° to obtain a (J,δ)-representation. Here, a novel pure shift 2DJ experiment based on the TSE-PSYCHE experiment is reported that deals with all these issues. Previous experiments proposed z-filtration to avoid excessive artifacts caused by chunked pure shift acquisition. It will be shown that these artifacts can also easily be avoided by means of the Pell-Keeler method. As opposed to its z-filtered counterparts, the new experiment provides pure shift 2DJ spectra that are free of artifacts from pulse imperfections and minimize responses related to strong coupling. In this way, multiplet analysis becomes possible at maximal resolution and a minimum of spectral complications.

Membrane Interactions of Natural Cyclic Lipodepsipeptides of the Viscosin Group.

Many Pseudomonas spp. produce cyclic lipodepsipeptides (CLPs), which, besides their role in biological functions such as motility, biofilm formation and interspecies interactions, are antimicrobial. It has been established that interaction with the cellular membrane is central to the mode of action of CLPs. In this work, we focus on the CLPs of the so-called viscosin group, aiming to assess the impact of the main structural variations observed within this group on both the antimicrobial activity and the interaction with model membranes. The antimicrobial activity of viscosin, viscosinamide A, WLIP and pseudodesmin A were all tested on a broad panel of mainly Gram-positive bacteria. Their capacity to permeabilize or fuse PG/PE/cardiolipin model membrane vesicles is assessed using fluorescent probes. We find that the Glu2/Gln2 structural variation within the viscosin group is the main factor that influences both the membrane permeabilization properties and the minimum inhibitory concentration of bacterial growth, while the configuration of the Leu5 residue has no apparent effect. The CLP-membrane interactions were further evaluated using CD and FT-IR spectroscopy on model membranes consisting of PG/PE/cardiolipin or POPC with or without cholesterol. In contrast to previous studies, we observe no conformational change upon membrane insertion. The CLPs interact both with the polar heads and aliphatic tails of model membrane systems, altering bilayer fluidity, while cholesterol reduces CLP insertion depth.

Aminophosphines: A Double Role in the Synthesis of Colloidal Indium Phosphide Quantum Dots.

Aminophosphines have recently emerged as economical, easy-to-implement precursors for making InP nanocrystals, which stand out as alternative Cd-free quantum dots for optoelectronic applications. Here, we present a complete investigation of the chemical reactions leading to InP formation starting from InCl3 and tris(dialkylamino)phosphines. Using nuclear magnetic resonance (NMR) spectroscopy and single crystal X-ray diffraction, we demonstrate that injection of the aminophosphine in the reaction mixture is followed by a transamination with oleylamine, the solvent of the reaction. In addition, mass spectrometry and NMR indicate that the formation of InP concurs with that of tetra(oleylamino)phosphonium chloride. The chemical yield of the InP formation agrees with this 4 P(+III) → P(-III) + 3 P(+V) disproportionation reaction occurring, since full conversion of the In precursor was only attained for a 4:1 P/In ratio. Hence it underlines the double role of the aminophosphine as both precursor and reducing agent. These new insights will guide further optimization of high quality InP quantum dots and might lead to the extension of synthetic protocols toward other pnictide nanocrystals.

Amino Acid-Based Stabilization of Oxide Nanocrystals in Polar Media: From Insight in Ligand Exchange to Solution ¹H NMR Probing of Short-Chained Adsorbates.

Ligand exchange is a crucial step between nanocrystal synthesis and nanocrystal application. Although colloidal stability and ligand exchange in nonpolar media are readily established, the exchange of native, hydrophobic ligands with polar ligands is less systematic. In this paper, we present a versatile ligand exchange strategy for the phase transfer of carboxylic acid capped HfO2 and ZrO2 nanocrystals to various polar solvents, based on small amino acids as the incoming ligand. To gain insight in the fundamental mechanism of the exchange, we study this system with a combination of FTIR, zeta potential measurements, and solution (1)H NMR techniques. The detection of surface-associated, small ligands with solution NMR proves challenging in this respect. Tightly bound amino acids are undetectable, but their existence can be proven through displacement with other ligands in titration experiments. Alternatively, we find that methyl moieties belonging to bound species can circumvent these limitations because of their more favorable relaxation properties as a result of internal mobility. As such, our results are not limited to amino acids but to any short-chained ligand and will therefore facilitate the rigorous investigation and understanding of various ligand exchange processes.

A General Method for Extracting Individual Coupling Constants from Crowded (1)H†NMR Spectra.

Couplings between protons, whether scalar or dipolar, provide a wealth of structural information. Unfortunately, the high number of (1)H-(1)H couplings gives rise to complex multiplets and severe overlap in crowded spectra, greatly complicating their measurement. Many different methods exist for disentangling couplings, but none approaches optimum resolution. Here, we present a general new 2D J-resolved method, PSYCHEDELIC, in which all homonuclear couplings are suppressed in F2, and only the couplings to chosen spins appear, as simple doublets, in F1. This approaches the theoretical limit for resolving (1)H-(1)H couplings, with close to natural linewidths and with only chemical shifts in F2. With the same high sensitivity and spectral purity as the parent PSYCHE pure shift experiment, PSYCHEDELIC offers a robust method for chemists seeking to exploit couplings for structural, conformational, or stereochemical analyses.

Ultrahigh-Resolution Diffusion-Ordered Spectroscopy.

Diffusion-ordered spectroscopy (DOSY) is an effective method for the analysis of intact mixtures, but the quality of results is critically limited by resolution in the NMR dimension. A new experiment integrating diffusion weighting into the PSYCHE method for pure shift NMR spectroscopy allows DOSY spectra to be measured with ultrahigh NMR resolution at improved sensitivity.

Impact of a stereocentre inversion in cyclic lipodepsipeptides from the viscosin group: a comparative study of the viscosinamide and pseudodesmin conformation and self-assembly.

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.

Rapid total synthesis of cyclic lipodepsipeptides as a premise to investigate their self-assembly and biological activity.

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.

Synthesis of an apionucleoside family and discovery of a prodrug with anti-HIV activity.

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 µM), 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.