Heteronuclear 2D NMR Experiments Research Articles

Serendipitous N,S-difunctionalization of triazoles with trifluoromethyl-β-diketones: access to regioisomeric 1-trifluoroacetyl-3-aryl-5-(2-oxo-2-arylethylthio)-1,2,4-triazoles as DNA-groove binders.

In the present research work, a serendipitous regioselective synthesis of DNA targeting agents, 1-trifluoroacetyl-3-aryl-5-(2-oxo-2-arylethylthio)-1,2,4-triazoles, has been achieved through the one-pot cascade reaction of 3-mercapto[1,2,4]triazoles with trifluoromethyl-β-diktetones in presence of NBS instead of the cyclized thiazolo[3,2-b][1,2,4]triazole. The present protocol offered a unique approach for functionalizing both N-acylation and S-alkylation in a concerted fashion. The structures of the regioisomeric products were thoroughly characterized by heteronuclear 2D NMR experiments. Facile scalability and excellent atom economy through easily available starting reactants are the notable features of the present sustainable protocol. Targeting tumor cell DNA with minor groove-binding small molecules has proven highly effective in the recent past, drawing significant attention for combating tumor-related afflictions. In this context, the synthesized analogs were primarily screened for their ability to bind with the DNA duplex d(CGCGAATTCGCG)2 using molecular modeling tools. Additionally, the most promising compound 14m was deployed as a probe for DNA sensing and interaction mechanisms with calf thymus (ct)DNA through various spectral techniques at a physiologic temperature of 37 °C. It has been found that the compound demonstrated a strong binding affinity (Kb = 1 × 105 M-1) with double-helical DNA, particularly within the minor groove, resulting in the formation of a stable complex through static quenching (Kq = 5.86 ± 0.11 × 1012 M-1 s-1). The fluorescent displacement assay confirmed that the quencher binds to the minor groove of ctDNA, further supported by circular dichroism and viscosity studies.

An expeditious on-water regioselective synthesis of novel arylidene-hydrazinyl-thiazoles as DNA targeting agents

A series of twenty novel (E)-arylidene-hydrazinyl-thiazole derivatives has been synthesized employing α-bromo-β-diketones, thiosemicarbazide, and aromatic/heteroaromatic aldehydes with a simple and facile one-pot multicomponent reaction passageway. This organic transformation proceeds efficiently in aqueous media and demonstrated a large functional group tolerance. The structures and stereochemistry of the regioisomeric product were rigorously characterized using heteronuclear 2D NMR experiments. The binding potential of the synthesized analogs with B-DNA dodecamer d(CGCGAATTCGCG)2 was primarily screened using molecular modeling tools and further, mechanistic investigations (either groove or intercalation) were performed using various spectroscopic techniques such as UV–Visible, Fluorescence, and Circular dichroism. The absorption spectra showed a hyperchromic shift in the absorption maxima of ctDNA with successive addition of thiazole derivatives, implying groove binding mode of interactions, further supported by displacement assay and circular dichroism analysis. Furthermore, steady-state fluorescence analysis revealed the static mode of quenching and moderate bindings between the ligand and DNA biomolecule. The competitive studies showed that the derivatives having a pyridinyl (heteroaromatic) group in their structure, bind with the nucleic acid of calf-thymus (ctDNA) more effectively in the minor groove region as compared with the aromatic substitutions.

High-Resolution Nuclear Magnetic Resonance Spectroscopy with Picomole Sensitivity by Hyperpolarization on a Chip.

We show that high-resolution NMR can reach picomole sensitivity for micromolar concentrations of analyte by combining parahydrogen-induced hyperpolarization (PHIP) with a high-sensitivity transmission line microdetector. The para-enriched hydrogen gas is introduced into solution by diffusion through a membrane integrated into a microfluidic chip. NMR microdetectors, operating with sample volumes of a few μL or less, benefit from a favorable scaling of mass sensitivity. However, the small volumes make it very difficult to detect species present at less than millimolar concentrations in microfluidic NMR systems. In view of overcoming this limitation, we implement PHIP on a microfluidic device with a 2.5 μL detection volume. Integrating the hydrogenation reaction into the chip minimizes polarization losses to spin-lattice relaxation, allowing the detection of picomoles of substance. This corresponds to a concentration limit of detection of better than , unprecedented at this sample volume. The stability and sensitivity of the system allow quantitative characterization of the signal dependence on flow rates and other reaction parameters and permit homo- (1H-1H) and heteronuclear (1H-13C) 2D NMR experiments at natural 13C abundance.

Characterization and quantification of microstructures of a fluorinated terpolymer by both homonuclear and heteronuclear two-dimensional NMR spectroscopy.

Fluoropolymers are usually insoluble in organic solvents. Insolubility of fluoropolymers limits basic characterization such as microstructural investigations. In the family of fluoropolymers, terpolymer of tetrafluorethylene (TFE), hexafluoropropylene (HFP), and vinylidene fluoride (VDF), named THV is one of the newest members. There are nine grades of THV available. Among the nine grades, THV-221 G is an ideal model polymer for basic characterization purposes. THV-221 G is soluble in solvents such as acetone and ethyl acetate. In the current report, both homonuclear and heteronuclear 2D NMR experiments were employed in solution on THV-221 G. The homonuclear gradient correlation spectroscopy NMR measurement revealed that THV has two adjacent TFE units in addition to TFE-HFP sequence orders. The fraction of the microstructures is quantified by the analysis of 1D solution (19)F NMR spectrum. Further, the gradient heteronuclear single quantum coherence experiment helped with the clarification of chemical environments of the units TFE, HFP, and VDF. The 1D solution (13)C NMR spectrum was helpful in clarifying sequence assignments of VDF. It is concluded that THV is a random polymer with a limited fraction of TFE-TFE and TFE-HFP sequence orders in addition to head-to-tail polymerization of VDF unit.

Smenamides A and B, Chlorinated Peptide/Polyketide Hybrids Containing a Dolapyrrolidinone Unit from the Caribbean Sponge Smenospongia aurea. Evaluation of Their Role as Leads in Antitumor Drug Research

An in-depth study of the secondary metabolites contained in the Caribbean sponge Smenospongia aurea led to the isolation of smenamide A (1) and B (2), hybrid peptide/polyketide compounds containing a dolapyrrolidinone unit. Their structures were elucidated using high-resolution ESI-MS/MS and homo- and heteronuclear 2D NMR experiments. Structures of smenamides suggested that they are products of the cyanobacterial metabolism, and 16S rRNA metagenomic analysis detected Synechococcus spongiarum as the only cyanobacterium present in S. aurea. Smenamides showed potent cytotoxic activity at nanomolar levels on lung cancer Calu-1 cells, which for compound 1 is exerted through a clear pro-apoptotic mechanism. This makes smenamides promising leads for antitumor drug design.

Iterative One‐Pot α‐Glycosylation Strategy: Application to Oligosaccharide Synthesis

AbstractBased on the combined use of dimethylformamide (DMF) modulation and neighboring group participation, three iterative one‐pot α‐glycosylation methods, i.e., one‐pot (α,α)‐, one‐pot (β,α)‐, and one‐pot (α,β)‐glycosylations, were developed. These methods are applicable to a range of thioglycosyl donors, confer stereocontrol in α‐/β‐glycosidic bond formation, and thus provide for rapid access to oligosaccharides with various permutations of anomeric configurations. The utility of these one‐pot glycosylation methods is demonstrated in the synthesis of eight non‐natural and natural oligosaccharide targets, including the core 1 serine conjugate, core 8 serine conjugate, the D‐Gal‐α(1→3)‐D‐Glc‐α(1→3)‐L‐Rha trisaccharide unit of the cell wall component in Streptococcus pneumoniae, and the D‐Glc‐α(1→2)‐D‐Glc‐α(1→3)‐D‐Glc trisaccharide terminus of the N‐linked glycan precursor. Confirmation of the anomeric configurations of these oligosaccharides is evidenced by 1H, 13C, 13C‐non‐proton decoupling, and heteronuclear correlation 2D NMR experiments. Global deprotection of selected oligosaccharide targets is illustrated.

Structure and absolute configuration of new acidic metabolites from Stachys ehrenbergii

Two novel metabolites have been isolated from the aerial parts of Stachys ehrenberiigii. Their structures and stereochemistry were elucidated using a combination of 13C and 1H homo and heteronuclear 2D NMR experiments and mass analysis. The development of an enantioselective synthesis of 3-(2′-acetoxy-4-phenylbut-3′-enoylamino)propionic acid allowed to confirm the structure and assign the ( R) absolute configuration at C-2′ of the natural product.

13C Direct‐Detection Biomolecular NMR Spectroscopy in Living Cells

A direct response: Exclusively heteronuclear 13C direct-detection 2D NMR experiments are performed on 13C,15N-enriched proteins in E. coli cells. Unfolded proteins or protein fragments provide well-resolved carbonyl detection (see picture), whereas folded proteins or structured motifs do not give any detectable spectrum.

Investigation of the Experimental Limits of Small-Sample Heteronuclear 2D NMR

Practical experimental performance limits for an ensemble of heteronuclear 2D NMR experiments using a state-of-the-art 600 MHz 1.7 mm Bruker TCI Micro CryoProbe are reported. In the specific case of multiplicity-edited GHSQC, it was possible to acquire data on a 540 ng sample of strychnine (1; ∼1.6 nmol), prepared by serial dilution, which was used as a model compound. The experiments discussed also included GCOSY, (13)C reference spectra, (1)H-(13)C GHMBC, IDR-GHSQC-TOCSY, 1,1-ADEQUATE, and (1)H-(15)N GHMBC.

RETRACTED: Identification of 3-O-acetylglycerol, a novel structural element in bacterial polysaccharides

We have discovered a novel bacterial polysaccharide structural element, 3-O-acetylglycerol, in the Streptococcus pneumoniae ST11A polysaccharide: This moiety was elucidated through a combination of homonuclear and heteronuclear 1D and 2D NMR experiments using (1)H, (13)C, and (31)P in various combinations. The 3-O-acetylglycerol moiety is substoichiometrically O-acetylated in ST11A; yet, key connectivities that unequivocally show O-acetylation at the glycerol are provided by the long-range correlations from the acetate methyl groups to the glycerol in the (1)H-(13)C HMBC spectrum. Additionally, we clarify the (1)H-(31)P assignments previously presented.

High-precision heteronuclear 2D NMR experiments using 10-ppm spectral window to resolve carbon overlap

The acquisition of a complementary heteronuclear 2D NMR experiment with 10-ppm carbon window allows chemists to improve by a factor 20-25 the spectral resolution and determine carbon chemical shifts with five figures from 2D spectra.

Acremines H–N, novel prenylated polyketide metabolites produced by a strain of Acremonium byssoides

Five novel metabolites, acremines H–N, have been isolated from malt extract–peptone–glucose agar cultures of a strain of Acremonium byssoides. Their structures and stereochemistry were elucidated using a combination of 13C and 1H homo and heteronuclear 2D NMR experiments. Acremines H–N inhibited the germination of sporangia of Plasmopara viticola.

Application of NMR Spectroscopy and Mass Spectrometry to the Structural Elucidation of Modified Flavan‐3‐ols and Their Coupling Reaction Products*

NMR and MS techniques were used for the unambiguous structural elucidation of synthesized modified monomeric and dimeric flavan‐3‐ols presenting different substituents on the A‐ring C8 position. The full characterization of the synthesized compounds was achieved by concerted use of NMR and ESI‐MS techniques. Assignments of proton and carbon atoms was achieved through analysis of the 1D 1H and 13C NMR spectra combined with homo‐ and heteronuclear 2D NMR experiments. In each case, HMBC correlation between proton H2 and carbon C8a was observed allowing assignment of this carbon, which represents the key for attribution of the A‐ring carbon atoms. The synthesis and structural characterization of activated monomeric and dimeric flavanols were also achieved and used as precursors for preparation of natural and modified dimeric procyanidin derivatives. The preparation of various dimeric species involving modified flavanols was explored through different coupling reactions. The structures of the compounds formed were characterized on the basis of their MS and NMR spectral analysis. Dimeric species were characterized through proton–proton and proton–carbon correlations, which distinguished between the different flavanol moieties and established their sequences.

Synthesis and Spectroscopic Structural Elucidation of New Quinoxaline Derivatives

A quinoxaline‐2,3‐dione derivative was synthesized, and its chemical structure was determined through spectral analysis. Alkylation of this compound under phase transfer catalysis (PTC) conditions yielded monoalkylated and diakylated adducts. The monolalkylation process was shown to be regioselective occurring on the quinoxalic nitrogen atom rather than on its pyrazolic analogue. The full characterization of the synthesized compounds was studied by concerted use of NMR and MS techniques. Assignments of proton and carbon atoms were achieved through analysis of the 1D 1H and 13C NMR spectra combined with homo‐ and hetero-nuclear 2D NMR experiments. Determination of the alkylation site was achieved through long‐range proton–carbon coupling correlations spectroscopy.

Computer optimized spectral aliasing in the indirect dimension of 1H– 13C heteronuclear 2D NMR experiments. A new algorithm and examples of applications to small molecules

A new algorithm for optimizing spectral width in the indirect dimension of heteronuclear 2D experiments is introduced. It takes a list of carbon chemical shifts as input and calculates the optimal spectral width and number of time increments to use in the carbon dimension of 2D experiments such as HSQC, HMBC, etc. When using optimized conditions where signals are better distributed along the carbon dimension, the number of time increments needed to resolve all of the signals is reduced by one to two orders of magnitude. This decreases the experimental time by the same factors and makes the acquisition of spectra such as HSQC–TOCSY, HSQC–NOESY, etc. more practical. The new algorithm allows users to limit the maximal t 1 evolution time when relaxation is a concern, and can take lists of carbons that do not need to be resolved. For any carbon, insights regarding the position of signals in the proton dimension increase the efficiency of the optimization by allowing the overlap of pairs of carbons with incompatible proton dispersions. The application of a second optimization using a fully-resolved spectrum as a source of proton dispersion for the carbons allows the number of time increments to be reduced further. Application to cyclosporine A shows that the time taken to acquire fully resolved HSQC spectra can be 126 times less than would be required in a full-width spectrum with the same resolution. The most interesting applications concern experiments where series of HSQC-based experiments have to be acquired, for example in relaxation time measurements. It is shown that the acquisition of quickly acquired series of selective-TOCSY–HSQC can facilitate assignment in carbohydrates. Computer-optimized spectral aliasing (COSA) generally requires no modification of the pulse sequence and can therefore be easily applied by non specialists.

Acremines A–F, novel secondary metabolites produced by a strain of an endophytic Acremonium, isolated from sporangiophores of Plasmopara viticola in grapevine leaves

Six novel metabolites, acremines A–F have been isolated from agar cultures of a strain of Acremonium sp. Their structures and stereochemistry were elucidated using a combination of 13C and 1H homo and heteronuclear 2D NMR experiments and X-ray analysis. Acremines A–D inhibited the germination of sporangia of Plasmopara viticola.

A new 3,4-seco-lupane derivative from Lasianthus gardneri.

A new seco-ring A lupane triterpene derivative (1), along with lupenone, lupeol, beta-sitosterol, ursolic acid, and stigmasterol 3-O-beta-d-glucoside, were isolated from a methanol extract of mature stems of Lasianthus gardneri, a shrub from the family Rubiaceae growing in Sri Lanka. The structure and stereochemistry of the new compound were determined using a combination of (13)C and (1)H homo- and heteronuclear 2D NMR experiments and from mass spectral data. The structure of 1 was confirmed by partial synthesis from lupeol.

Ligand-induced changes in dynamics in the RT loop of the C-terminal SH3 domain of Sem-5 indicate cooperative conformational coupling.

We report the effects of peptide binding on the (15)N relaxation rates and chemical shifts of the C-SH3 of Sem-5. (15)N spin-lattice relaxation time (T(1)), spin-spin relaxation time (T(2)), and ((1)H)-(15)N NOE were obtained from heteronuclear 2D NMR experiments. These parameters were then analyzed using the Lipari-Szabo model free formalism to obtain parameters that describe the internal motions of the protein. High-order parameters (S(2) > 0.8) are found in elements of regular secondary structure, whereas some residues in the loop regions show relatively low-order parameters, notably the RT loop. Peptide binding is characterized by a significant decrease in the (15)N relaxation in the RT loop. Concomitant with the change in dynamics is a cooperative change in chemical shifts. The agreement between the binding constants calculated from chemical shift differences and that obtained from ITC indicates that the binding of Sem-5 C-SH3 to its putative peptide ligand is coupled to a cooperative conformational change in which a portion of the binding site undergoes a significant reduction in conformational heterogeneity.

Assignment of pH‐dependent NMR spectra of the scorpiand macrocycle: an application of titration profiles and spectral linewidths

AbstractRoutine homo‐ and heteronuclear 2D NMR experiments (COSY, HMQC, TOCSY and ROESY) were performed for the complex macrocyclic pentaamine ligand 1‐(2′‐aminoethyl)‐1,4,8,11‐tetraazacyclotetradecane (1, scorpiand), derived from 1,4,8,11‐tetraazacyclotetradecane (cyclam). However, some its 13C and 1H resonances were distinguished only based on the similarity between profiles of relevant pH–chemical shift plots and/or linewidth considerations. Following this empirical method relying on the assumption that titration curves of nuclei in comparable chemical environments have comparable shapes, it was possible to assign all chemical shifts (13C, pH 0.25–13.4; 1H, pH 10–11.5) for some different protonated species Hn1n+. A multiple experimental approach used for recognizing their NMR signals is offered as a general assignment procedure in the case of polyazamacrocycles. Copyright © 2002 John Wiley & Sons, Ltd.

ISOLATION AND STRUCTURAL ANALYSIS OF NEW FRUCTANS PRODUCED BY CHICORY

This report describes a new series of oligosaccharides, which is formed in chicory roots under forcing conditions and during in vitro experiments using purified chicory 1-FFT (fructan:fructan 1-fructosyl transferase). It was demonstrated that the three smallest members of this new series (disaccharide, trisaccharide and tetrasaccharide) contain exclusively β-D-fructosyl residues after hydrolysis. The present data demonstrate that the smallest compound is levanbiose and that the other oligomers of this new series of fructans do not belong to the linear 2→6 linked levan-oligosaccharides nor to the linear 2→1 linked inulo-oligosaccharides. A combination of several chromatographic techniques yielded a fraction that contained only the compound with degree of polymerisation (DP) 2 (levanbiose, β-D-fructofuranosyl-(2→6)-D-fructofuranose), and a mixture of DP 3 of the new series and 1-kestose. Using homonuclear and heteronuclear 2D NMR experiments the complete 1H and 13C NMR assignments of levanbiose and DP 3 were obtained. From High Performance Anion Exchange Chromatography (HPAEC) and NMR experiments of DP 3 of the new series it was concluded that the molecule contains a β-D-fructofuranosyl residue 2→1 linked to the non-reducing moiety of levanbiose, and thus has to be named β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→6)-D-fructofuranose. The simple and regular pattern of the HPAEC retention times of the new oligosaccharides suggests that it is a homologous series of oligomers build by 2→1 elongation with β-D-fructofuranosyl residues at the non-reducing residue of levanbiose.