Non‐Uniform Sampling and J‐UNIO Automation for Efficient Protein NMR Structure Determination

High‐resolution structure determination of small proteins in solution is one of the big assets of NMR spectroscopy in structural biology. Improvements in the efficiency of NMR structure determination by advances in NMR experiments and automation of data handling therefore attracts continued interest. Here, non‐uniform sampling (NUS) of 3D heteronuclear‐resolved [¹H,¹H]‐NOESY data yielded two‐ to three‐fold savings of instrument time for structure determinations of soluble proteins. With the 152‐residue protein NP_372339.1 from Staphylococcus aureus and the 71‐residue protein NP_346341.1 from Streptococcus pneumonia we show that high‐quality structures can be obtained with NUS NMR data, which are equally well amenable to robust automated analysis as the corresponding uniformly sampled data.     

Non-Stationary Complementary Non-Uniform Sampling (NOSCO NUS) for Fast Acquisition of Serial 2D NMR Titration Data

NMR spectroscopy offers unique benefits for ligand binding studies on isotopically labelled target proteins. These benefits include atomic resolution, direct distinction of binding sites and modes, a lowest detectable affinity limit, and function independent setup. Yet, retracing protein signal assignments from apo to holo states to derive exact dissociation constants and chemical shift perturbation amplitudes (for ligand docking and structure-based optimization) requires lengthy titration series of 2D heteronuclear correlation spectra at variable ligand concentration that may exceed the protein's lifetime and available spectrometer time. We present a novel method to overcome this critical limitation, based on non-stationary complementary non-uniform sampling (NOSCO NUS) combined with a robust particle swarm optimization algorithm. We illustrate its potential in two challenging studies with very distinct protein sizes and binding affinities, showing that NOSCO NUS can reduce measurement times by an order of magnitude to make such highly informative NMR titration studies more broadly feasible.     

Sensitivity enhancement for maximally resolved two‐dimensional NMR by nonuniform sampling

Resolving NMR signals which are separated in frequency on the order of their line widths requires obtaining the time domain free induction decay for a maximum time t_max = πT₂, where T₂ is the transverse relaxation time of the given signals. Unfortunately, samples acquired beyond ～1.26T₂ contribute more noise than signal to the data; and samples in the range of about (0.75–1.26)× T₂ have a negligible effect on the signal‐to‐noise ratio (SNR). Therefore, one must sacrifice SNR to reach evolution times of πT₂. One can preserve resolution in a shorter total experimental time by selecting a reduced set of samples from the Nyquist grid according to an exponential probability density which is on the order of the T₂ of the signals. This practice is widely termed nonuniform sampling (NUS). We derive analytic theory for the enhancement of the intrinsic SNR of NUS time domain data compared with uniformly sampled data when the total experimental times are equivalent. This theory is general for any t_max and exponential weighting and is further carefully validated with simulations. Enhancements of SNR in the time domain on the order of twofold are routinely available when t_max ～ πT₂ and are reflected in the subsequent maximum entropy reconstructed spectra. SNR enhancement by NUS is demonstrated to be helpful in enabling the acquisition of HMQC spectra of dilute bile salts in which high resolution in the indirect carbon dimension is required. Copyright © 2011 John Wiley & Sons, Ltd.     

Pure In‐Phase Heteronuclear Correlation NMR Experiments

A general NMR approach to provide pure in‐phase (PIP) multiplets in heteronuclear correlation experiments is described. The implementation of a zero‐quantum filter efficiently suppresses any unwanted anti‐phase contributions that usually distort the multiplet pattern of cross‐peaks and can hamper their analysis. The clean pattern obtained in PIP‐HSQMBC experiments is suitable for a direct extraction of coupling constants in resolved signals, for a peak‐fitting process from a reference signal, and for the application of the IPAP technique in non‐resolved multiplets.     

SPEED: single‐point evaluation of the evolution dimension

Two‐dimensional NMR spectroscopy can be speeded up by orders of magnitude by severely restricting the number of sampling operations in the evolution dimension–we demonstrate that just a single measurement may suffice. The frequencies evolving in the indirect dimension (t₁) are deduced from the amplitudes of the signals acquired in the direct dimension (t₂). Prior measurements of the one‐dimensional spectra are required. Results are presented for the two‐dimensional ¹³C‐HSQC spectrum of 2‐ethylindanone recorded at a single fixed setting of the evolution time, demonstrating a speed advantage of 120. The method can be extended to multidimensional spectra, with correspondingly greater gains in speed. Copyright © 2007 John Wiley & Sons, Ltd.     

Developing nonuniform sampling strategies to improve sensitivity and resolution in 1,1-ADEQUATE experiments

Nonuniform sampling (NUS) strategies are developed for acquiring highly resolved 1,1-ADEQUATE spectra, in both conventional and homodecoupled (HD) variants with improved sensitivity. Specifically, the quantile-directed and Poisson gap methods were critically compared for distributing the samples nonuniformly, and the quantile schedules were further optimized for weighting. Both maximum entropy and iterative soft thresholding spectral estimation algorithms were evaluated. All NUS approaches were robust when the degree of data reduction is moderate, on the order of a 50% reduction of sampling points. Further sampling reduction by NUS is facilitated by using weighted schedules designed by the quantile method, which also suppresses sampling noise well. Seed independence and the ability to specify the sample weighting in quantile scheduling are important in optimizing NUS for 1,1-ADEQUATE data acquisition. Using NUS yields an improvement in sensitivity, while also making longer evolution times accessible that would be difficult or impractical to attain by uniform sampling. Theoretical predictions for the sensitivity enhancements in these experiments are in the range of 5–20%; NUS is shown to disambiguate weak signals, reveal some ⁿJ_CC correlations obscured by noise, and improve signal strength relative to uniform sampling in the same experimental time. This work presents sample schedule development for applying NUS to challenging experiments. The schedules developed here are made available for general use and should facilitate the broader utilization of ADEQUATE experiments (including 1,1-, 1,n-, and HD- variants) for challenging structure elucidation problems.     

Charakterisierung der Protonen in polykristallinen Parawolframaten durch 1H‐MAS‐NMR‐Untersuchungen

Characterization of the Protons in Polycrystalline Paratungstates using ¹H MAS NMR Investigations ¹H MAS NMR experiments are used to characterize the non‐acid protons of the anions in polycrystalline paratungstates by means of the measured isotropic chemical shift values. The investigation of various hydrates of ammonium paratungstate allows a direct proof of protons in NH₄ ions and in water molecules while protons of the anions are not detectable. However, for both the potassium and the sodium paratungstates ¹H MAS NMR investigations detected the protons of water molecules and the non‐acid protons of the paratungstate anions. Additional ¹H broad‐line NMR experiments at 173 K support the interpretation of the results obtained by the ¹H MAS NMR investigations. For the NMR signal of the non‐acid protons of the paratungstate anion in the ¹H MAS NMR spectra of the potassium salt line‐splitting appears. This refers to the existence of two nonidentical positions of the protons in the crystal lattice and is in agreement with the results of the X‐ray structural analysis.     

Synthesis of new non‐symmetrical substituted 7,7′‐bisindolizines by the direct reaction of 4,4′‐bipyridinium‐ylides with dimethyl acetylenedicarboxylate

We report here on the synthesis of some novel non‐symmetrical substituted bisindolizines by 3+2 dipolar cycloaddition reaction. New compounds were prepared by the direct reaction of isolated non‐symmetrical substituted 4,4′‐bipyridinium bisylides with dimethyl acetylenedicarboxy late (DMAD). The obtained compounds can be used as precursors of fluorescent markers in fluorometric analysis.     

Decomposition of mixtures' spectra by multivariate curve resolution of rapidly acquired TOCSY experiments

A method is presented that allows for retrieving 1D spectra of the individual components of a mixture from a sparsely acquired 2D‐TOCSY spectrum. The decomposition of the 2D‐TOCSY data into pure 1D traces is achieved using a non‐negative matrix factorization algorithm, also known as multivariate curve resolution analysis. Here, we show that the algorithm can be applied to data processed in the direct dimension only. Thus, our method can be applied to non‐linearly sampled experiments or data acquired with few indirect points. An example is shown for the spectra of a mixture of six amino acids, acquired in 15 min. Copyright © 2010 John Wiley & Sons, Ltd.     

Fast and high-resolution stereochemical analysis by nonuniform sampling and covariance processing of anisotropic natural abundance 2D 2H NMR datasets

Natural abundance deuterium (NAD) 2D NMR spectroscopy using chiral or achiral liquid crystals is an efficient analytical tool for the stereochemical analysis of enantio- or diastereomers by the virtue of proton-to-deuterium substitution. In particular, it allows the measurement of enantiopurity of organic synthetic molecules or the determination of the natural isotopic (1)H/(2)H fractionation in biological molecules, such as fatty acid methyl esters (FAME). So far, the NAD 2D spectra of solutes were acquired by using uniform sampling (US) and processed by conventional 2D Fourier transform (FT), which could result in long measurement times for medium-sized analytes or low solute concentrations. Herein, we demonstrate that this conventional approach can be advantageously replaced by nonuniform sampling (NUS) processed by covariance (Cov) transform. This original spectral reconstruction provides a significant enhancement of spectral resolution, as well as a reduction of measurement times. The application of Cov to NUS data has required the introduction of a regularization procedure in the time domain for the indirect dimension. The analytical potential of combining Cov and NUS is demonstrated by measuring the enantiomeric excess of a scalemic mixture of 2-ethyloxirane and by determining the diastereomeric excess of methyl vernoleate, a natural FAME. These two organic compounds were aligned in a polypeptide (poly(γ-benzyl-L-glutamate)) mesophase. In the case of NAD 2D NMR spectroscopy, we show that Cov and NUS methods allow a decrease in measurement time by a factor of two compared with Cov applied to US data and a factor of four compared with FT applied to US data.     

Synthesis of meta‐Terphenyl‐2,2′′‐diols by Anodic C−C Cross‐Coupling Reactions

The anodic C?C cross‐coupling reaction is a versatile synthetic approach to symmetric and non‐symmetric biphenols and arylated phenols. We herein present a metal‐free electrosynthetic method that provides access to symmetric and non‐symmetric meta‐terphenyl‐2,2′′‐diols in good yields and high selectivity. Symmetric derivatives can be obtained by direct electrolysis in an undivided cell. The synthesis of non‐symmetric meta‐terphenyl‐2,2′′‐diols required two electrochemical steps. The reactions are easy to conduct and scalable. The method also features a broad substrate scope, and a large variety of functional groups are tolerated. The target molecules may serve as [OCO]^3? pincer ligands.     

1H and 13C spectral assignment of naphtho[2′,1′:5,6]‐naphtho[2′,1′:4,5]thieno[2,3‐c]quinoline using the IDR‐GHSQC‐TOCSY experiment

The assignment of the NMR spectra of the polynuclear heteroaromatic naphtho[2′,1′:5,6]naphtho‐[2′,1′:4,5]thieno[2,3‐c]quinoline is reported. The analysis was based on the homonuclear ROESY, heteronuclear direct GHSQC, IDR‐GHSQC‐TOCSY, and long‐range GHMBC experiments. The complete ¹H and ¹³C shift assignments are reported.     

Two Energetic Salts based on 5,5′‐Bitetrazole‐1,1′‐diolate: Syntheses,Characterization, and Properties

Two salts based on 1H,1′H‐5,5′‐bitetrazole‐1,1′‐diolate (BTO) anion with pyrazole ( 1 ) and imidazole ( 2 ) cations were synthesized with metathesis reactions. Structural characterization was accomplished for them by using the element analysis, Fourier transform infrared spectroscopy (FT‐IR), NMR and mass spectrum, and X‐ray single crystal diffraction. Thermal analysis for the title salts were determined by means of differential scanning calorimetry (DSC) and thermogravimetry‐derivative thermogravimetry (TG‐DTG) as well as the calculation of non‐isothermal kinetic parameters. Consequently, both salts shown acceptable thermal stabilities as the decomposition temperatures were over 200 °C. The enthalpies of formation were calculated for these salts using the measured combustion energies with a result of 70.6 kJ · mol^–1 for 1 and –47.8 kJ · mol^–1 for 2 , respectively. Impact and friction sensitivities were also tested and the results indicated that these salts both have low sensitivities (>40 J, 120 N). The title energetic salts possess acceptable performance, they can therefore be applied in the field of energetic materials.     

Regioselective synthesis of symmetrical pyridyl selenium compounds by bromine–magnesium exchange of bromopyridines using isopropyl magnesium chloride: X‐ray crystal structure of 2,2′,5,5′‐tetrabromo‐3,3′‐dipyridyldiselenide

2,2′‐Dipyridyl‐3,3′‐dipyridyl,5,5′‐dipyridyl‐diselenides have been synthesized by a convenient method employing non‐cryogenic conditions. Various bromopyridines (2‐Bromopyridine, 2,5‐dibromopyridines and 2,3,5‐Tribromopyridines) undergo selective monobromine–magnesium exchange to yield the corresponding pyridyl magnesium chlorides at room temperature upon treatment with ⁱPrMgCl. The resulting pyridyl magnesium chloride is quenched with elemental selenium, which upon further oxidation affords the above diselenides in good yields. The compounds prepared using this methodology have been characterized by elemental analysis, IR, NMR (¹H, ¹³C, ⁷⁷Se) and mass spectral analysis. The molecular structure of 2,2′,5,5′‐Tetrabromo‐3,3′‐dipyridyldiselenide has been established by single‐crystal X‐ray diffraction analysis. It exists as a dimeric form due to the non‐bonding interactions between the selenium of one pyridine moiety and the hydrogen of the other. Copyright © 2004 John Wiley & Sons, Ltd.     

Silver Ions in Non‐canonical DNA Base Pairs: Metal‐Mediated Mismatch Stabilization of 2′‐Deoxyadenosine and 7‐Deazapurine Derivatives with 2′‐Deoxycytidine and 2′‐Deoxyguanosine

Novel silver‐mediated dA?dC, dA*?dC, and dA*?dG base pairs were formed in a natural DNA double helix environment (dA* denotes 7‐deaza‐dA, 7‐deaza‐7‐iodo‐dA, and 7‐cyclopropyl‐7‐deaza‐dA). 7‐Deazapurine nucleosides enforce silver ion binding and direct metal‐mediated base pair formation to their Watson–Crick face. New phosphoramidites were prepared from 7‐deaza‐dA, 7‐deaza‐7‐iodo‐dA, and 7‐cyclopropyl‐7‐deaza‐dA, which contain labile isobutyryl protecting groups. Solid‐phase synthesis furnished oligonucleotides that contain mismatches in near central positions. Increased thermal stabilities (higher T_m values) were observed for oligonucleotide duplexes with non‐canonical dA*?dC and dA?dC pairs in the presence of silver ions. The stability of the silver‐mediated base pairs was pH dependent. Silver ion binding was not observed for the dA?dG mismatch but took place when mismatches were formed between 7‐deazaadenine and guanine. The specific binding of silver ions was confirmed by stoichiometric UV titration experiments, which proved that one silver ion is captured by one mismatch. The stability increase of canonical DNA mismatches might have an impact on cellular DNA repair.     

Self‐Assembly of Highly Stable Zirconium(IV) Coordination Cages with Aggregation Induced Emission Molecular Rotors for Live‐Cell Imaging

The self‐assembly of highly stable zirconium(IV)‐based coordination cages with aggregation induced emission (AIE) molecular rotors for in vitro bio‐imaging is reported. The two coordination cages, NUS‐100 and NUS‐101, are assembled from the highly stable trinuclear zirconium vertices and two flexible carboxyl‐decorated tetraphenylethylene (TPE) spacers. Extensive experimental and theoretical results show that the emissive intensity of the coordination cages can be controlled by restricting the dynamics of AIE‐active molecular rotors though multiple external stimuli. Because the two coordination cages have excellent chemical stability in aqueous solutions (pH stability: 2–10) and impressive AIE characteristics contributed by the molecular rotors, they can be employed as novel biological fluorescent probes for in vitro live‐cell imaging.     

Synthesis of Some N-Alkoxycarbonyl-N″-benzoyl-benzamidrazones（p-toluamidrazones） and 1,3,5-Trisubstituted 1,2,4-Triazole Derivatives from N-Benzoylimidates and their Antimicrobial and Anticancer Screening Studies

Some new N-alkoxycarbonyl-N″-benzoyl-benzamidrazones （p-toluamidrazones） 3a-3d, and 1,3,5-trisubstituted 1,2,4-triazole 4a-4h derivatives by starting from N-benzoylbenzimidates or N-benzoyl-p-toluimidates. The structures of compounds 3 and 4 were established on the basis of elemental analyses, IR, ^1H NMR, ^13C NMR and UV data. Antimicrobial experiments of the compounds performed by using agar-well diffusion and broth microdilution methods revealed that only compounds 3a-3d, 4a and 4b showed inhibitory effect only on Candida albicans ATCC 60193. However, compound 4b had also specific antibacterial activity against Staphylococcus aureus ATCC 25923. The other compounds showed neither antifungal nor antibacterial activities. Compounds 3a, 4a and 4b have been screened on three human tumor cell lines, breast cancer （MCF7）, non small cell lung cancer （NCI-H460）, and CNS cancer （SF-268） at the National Cancer Institute （NCI）, USA, which were found to exhibit low antiproliferative activity.     

Concurrent Asymmetric Reactions Combining Photocatalysis and Enzyme Catalysis: Direct Enantioselective Synthesis of 2,2‐Disubstituted Indol‐3‐ones from 2‐Arylindoles

The combination of photoredox and enzymatic catalysis for the direct asymmetric one‐pot synthesis of 2,2‐disubstituted indol‐3‐ones from 2‐arylindoles through concurrent oxidization and alkylation reactions is described. 2‐Arylindoles can be photocatalytically oxidized to 2‐arylindol‐3‐one with subsequent enantioselective alkylation with ketones catalyzed by wheat germ lipase (WGL). The chiral quaternary carbon center at C2 of the indoles was directly constructed. This mode of concurrent photobiocatalysis provides a mild and powerful strategy for one‐pot enantioselective synthesis of complex compounds. The experiments proved that other lipases containing structurally analogous catalytic triad in the active site also can catalyze the reaction in the same way. This reaction is the first example of combining the non‐natural catalytic activity of hydrolases with visible‐light catalysis for enantioselective organic synthesis and it does not require any cofactors.     

2,2′:3′,2′′‐Terthiophene‐Based all‐Thiophene Dendrons and Dendrimers: Synthesis,Structural Characterization,and Properties

The synthesis of generational dendritic oligothiophenes (DOTs) has been successfully achieved by a divergent/convergent approach that involves halogenation, boronation, and palladium‐catalyzed Suzuki coupling reactions. The key point in the presented synthetic approach is the use of trimethylsilyl (TMS) protecting groups, which allow for the core‐lithiation and subsequent boronation of the dendrons and for the peripheral ipso‐substitution with iodine monochloride or N‐bromosuccimide. In addition, the TMS protecting groups can be completely removed by using tetrabutylammonium fluoride, thus yielding only‐thiophene‐based dendrons and dendrimers. Due to their highly branched structure, all these synthesized DOTs are soluble in organic solvents. Chemical structures were confirmed by NMR spectroscopic, mass spectrometric, and elemental analysis. Concentration‐dependent ¹H NMR spectroscopic investigations revealed that the higher generation compounds tend to aggregate in solution. Such an aggregation behavior was further confirmed by measuring with MALDI‐TOF MS. Both MALDI‐TOF MS and gel‐permeation chromatography (GPC) analyses confirmed the monodispersity of the DOTs. Furthermore, GPC results revealed that these DOT molecules adopt a condensed globular molecular shape. Their optical and electronic properties were also investigated. The results indicated that these DOTs comprise various conjugated α‐oligothiophenes with different chain lengths, which results in the higher generation compounds showing broad and featureless UV/Vis absorption spectra and ill‐defined redox waves.     

Monomer–Dimer Equilibrium for the 5′–5′ Stacking of Propeller‐Type Parallel‐Stranded G‐Quadruplexes: NMR Structural Study

Guanine‐rich sequence motifs, which contain tracts of three consecutive guanines connected by single non‐guanine nucleotides, are abundant in the human genome and can form a robust G‐quadruplex structure with high stability. Herein, by using NMR spectroscopy, we investigate the equilibrium between monomeric and 5′–5′ stacked dimeric propeller‐type G‐quadruplexes that are formed by DNA sequences containing GGGT motifs. We show that the monomer–dimer equilibrium depends on a number of parameters, including the DNA concentration, DNA flanking sequences, the concentration and type of cations, and the temperature. We report on the high‐definition structure of a simple monomeric G‐quadruplex containing three single‐residue loops, which could serve as a reference for propeller‐type G‐quadruplex structures in solution.