Two-dimensional NMR Spectroscopy Research Articles

Fine structures of different dextrans assessed by isolation and characterization of endo-dextranase liberated isomalto-oligosaccharides

Chromatographic analysis of endo-dextranase liberated branched oligosaccharides proved to be a valuable approach to differentiate dextrans from fermented food products or isolated lactic acid bacteria. Because these hydrolysis products also yield valuable information on the dextran fine structures, several branched isomalto-oligosaccharides were liberated from different dextrans and chromatographically purified. Mass spectrometry and two-dimensional NMR spectroscopy were used for structural characterization of the oligomers. Isomalto-oligosaccharides from L. reuteri TMW 1.106 dextrans were exclusively O4-branched. Furthermore, they contained only monomeric side chains and at least one unsubstituted backbone unit between ramified residues. In contrast, O3-branched oligosaccharides with monomeric as well as elongated side chains were isolated. The varying abundance of the O3-branched oligosaccharides suggests that side chain length is a major factor for structural differences between dextrans. Overall, we demonstrated that chromatographic analysis of endo-dextranase liberated isomalto-oligosaccharides provides valuable information on the structural properties and supplements conventional methods such as methylation analysis.

Synthesis, characterization, cytotoxic activity, and 19F NMR spectroscopic investigations of (OC-6-33)-diacetato(ethane-1,2-diamine)bis(3,3,3-trifluoropropanoato)platinum(IV) and its platinum(II) counterpart

Tetracarboxylatoplatinum(IV) complexes are interesting representatives of potential anticancer active platinum(IV) drugs. Revealing higher kinetic inertness in comparison to their cytotoxic platinum(II) counterparts, they offer an opportunity to reduce toxic and deactivating side reactions. Platinum(IV) complexes are generally considered as prodrugs, which have to be reduced in order to exert their anticancer activity. Two model complexes, a platinum(II) and platinum(IV) complex featuring two equatorial 3,3,3-trifluoropropanoato (tfpa) and in the case of the latter two axial acetato ligands, were synthesized and characterized in detail by multinuclear (1H, 13C, 15N, 19F, 195Pt) one- and two-dimensional NMR spectroscopy, high-resolution electrospray ionization mass spectrometry, elemental analysis and X-ray diffraction analysis. Cytotoxicity was evaluated in three human cancer cell lines (A549, SW480 and CH1/PA-1) by the MTT colorimetric assay, exhibiting IC50 values down to the low micromolar range. The fluorinated ligands in equatorial positions allowed detailed and highly sensitive 19F NMR spectroscopic investigations. Besides reaction studies in the presence of small molecules, e.g. ascorbic acid and 5′-GMP, the reduction of the platinum(IV) complex was additionally investigated in two cancer cell extracts deriving from the cell lines SW480 and CH1/PA-1 in order to estimate the intracellular reduction behavior. A significant increase in the rate of reduction in cell extracts (depending on the chosen cell line) compared to the reduction with ascorbic acid could be demonstrated.

Ultrafast Single-Scan 2D NMR Spectroscopic Detection of a PHIP-Hyperpolarized Protease Inhibitor.

Two-dimensional NMR spectroscopy is one of the most important spectroscopic tools for the investigation of biological macromolecules. However, due to the low sensitivity of NMR spectroscopy, it takes usually from several minutes to many hours to record such spectra. Here, the possibility of detecting a bioactive derivative of the sunflower trypsin inhibitor-1 (SFTI-1), a tetradecapeptide, by combining parahydrogen-induced polarization (PHIP) and ultrafast 2D NMR spectroscopy is shown. The PHIP activity of the inhibitor was achieved by labeling with O-propargyl-l-tyrosine. In 1D PHIP experiments a signal enhancement of a factor of approximately 1200 compared to standard NMR was found. This enhancement permits measurement of 2D NMR correlation spectra of low-concentrated SFTI-1 in less than 10 seconds, employing ultrafast single-scan 2D NMR detection. As experimental examples PHIP-assisted ultrafast single-scan TOCSY spectra of SFTI-1 are shown.

NMR model structure of the antimicrobial peptide maximin 3.

Maximin 3 is a 27-residue-long cationic antimicrobial peptide found in the skin secretion and brain of the Chinese red-belly toad Bombina maxima. The peptide is of biological interest as it possesses anti-HIV activity, not found in the other maximin peptides, in addition to antimicrobial, antitumor and spermicidal activities. The three-dimensional structure of maximin 3 was obtained in a 50/50% water/2,2,2-trifluoroethanol-d3 mixture using two-dimensional NMR spectroscopy. Maximin 3 was found to adopt an α-helical structure from residue G1 to A22, and a coil structure with a helical propensity in the C-terminal tail. The peptide is amphipathic, showing a clear separation between polar and hydrophobic residues. Interactions with sodium dodecyl sulfate micelles, a widely used bacterial membrane-mimicking environment, were modeled using molecular dynamics simulations. The peptide maintained an α-helical conformation, occasionally displaying a flexibility around residues G9 and G16, which is likely responsible for the peptide's low haemolytic activity. It is found to preferentially adopt a position parallel to the micellar surface, establishing a number of hydrophobic and electrostatic interactions with it.

A Phthalocyanine-ortho-Carborane Conjugate for Boron Neutron Capture Therapy: Synthesis, Physicochemical Properties, and in vitro Tests.

Boronated molecular systems can be applied to boron neutron capture therapy (BNCT). Among these systems, carborane-containing phthalocyanines (Pcs) are the most promising BNCT agents. Herein we report the new zinc (II) complex of the hexacationic Pc 6, which has been obtained as iodide salt through quaternization of the neutral precursor with methyl iodide. Compound 6 was synthesized over a sequence of four steps. The complex, and its precursors as well, were characterized by a combination of spectroscopic techniques, and their structures assessed by 1 H, 13 C, 11 B, and two-dimensional NMR spectroscopy experiments. Together with a marked tendency to aggregate, 6 showed appreciable solubility in water. Singlet oxygen quantum yield (ΦΔ ) of 0.38, and fluorescence quantum yield (ΦF ) of 0.13 were obtained for 6 in a DMF solution. The complex proved to be very effective in enriching UMR-106 cells with 10 B, showing very good performance even in case of very low concentrations exposure, i. e. 1 ppm, that moreover resulted in a mild cytotoxic effect. Such a feature can be related to the polycationic nature of the complex, and hence to the well-known propensity of positively charged species to enter the cellular membrane or to adhere to its external surface.

Lysine-based dendrimer with double arginine residues.

Due to their well-defined structure, multivalency, biocompatibility, and low toxicity, lysine dendrimers can be used as safe and efficient nanocarriers for drug and gene delivery. One useful strategy for improving the gene delivery properties of dendrimers is modification with arginine amino acid (Arg) residues. Incorporation of Arg residues could be favorable for the enhancement in transfection efficiency of lysine based dendrimers. In this work, we have synthesized a new second-generation poly-l-lysine dendrimer with repeating units containing two linear Arg residues between neighboring lysine branching points (Lys-2Arg dendrimer) and studied its physicochemical properties. We confirmed the structure of Lys-2Arg dendrimer using various one- and two-dimensional 1H and 13C NMR spectroscopy methods. Comparison of T1H relaxation data for Lys-2Arg and Lys-2Lys dendrimers showed that the replacement of double Lys residues with double Arg residues resulted in a sharp decrease in the mobility of methylene groups in side segments and in the main chain of ε-Lys inner segments. We suggest that this unexpected effect is caused by a guanidine–guanidine pairing effect in water, which leads to entanglements between dendrimer branches.

Expression and Characterization of <i>Manduca sexta</i> Stress Responsive Peptide-1; An Inducer of Antimicrobial Peptide Synthesis

In response to stress conditions such as wounding or infections in insects, several short peptides are processed to act as cytokines that induce AMP gene expression. To study their structure-activity relationship, immune inducibility, tissue specificity, stress responsiveness, and development relatedness, we chemically synthesized Manduca sexta stress response peptide-1, a 25-residue peptide with one disulfide bond (SRP1: FGVRVGTCPSGYVRRGTFCFPDDDY). Upon injection of the SRP1 into naïve larvae, several antimicrobial peptide genes were expressed at higher levels. The mRNA levels of SRP1 increased significantly in hemocytes and fat body after larvae were challenged with a mixture of bacteria and β-1,3-glucan. The expression patterns of SRP1 and its target genes are somewhat different from SRP2's, suggesting overlapping yet distinct functions. We elucidated the 3D structure of SRP1 in solution by two-dimensional 1H-1H NMR spectroscopy. The tertiary structure of SRP1 consists of two short β-strands at Y12-R15 and F18-F20, one type-II β-turn at R15-F18 in its well-defined core and is stabilized by a covalent disulfide bond between C8 and C19. The conformational ensemble of SRP1 from extensive atomistic simulation in explicit solvent (with 3.0 μs total effective sampling) shows high consistency with experimental intramolecular NOEs of the core region. The SRP1 core adopts a fold similar to the carboxyl-terminal subdomain of epidermal growth factor (EGF), suggesting that SRP1 may interact with EGF receptor-like molecules to trigger its biological function.

Two new iridoid glycosides from Callicarpa nudiflora

Two new iridoid glycosides, callicoside E (1) and callicoside F (2), were isolated from the leaves of Callicarpa nudiflora. Their structures were established by one- and two-dimensional NMR spectroscopy and mass spectrometry. In an in vitro bioassay, compounds 1 and 2 showed an pronounced hepatoprotective activity against d-galactosamine-induced toxicity in WB-F344 rat hepatic epithelial stem-like cells.

SYNTHESIS OF NEW DERIVATIVES OF 3-AZABICYCLO[3.3.1]NONANES BASIS ON N-(2-HYDROXY-3,5-DINITROPHENYL)ACETAMIDE σ-ADDUCT

A number of new derivatives of N-(3-R-1,5-dinitro-8-oxo-3-azabicyclo[3.3.1]non-6-en-7-yl)acetamides have been synthesized by Mannich condensation of hydride σ-adduct of the N-(2-hydroxy-3,5-dinitrophenyl)acetamide with formaldehyde and primary amines. The synthesis was carried out with two stages. In the first stage, under the action of sodium tetrahydride borate on a solution of N-(2-hydroxy-3,5-dinitrophenyl)acetamide, the C = C bonds of the aromatic ring were reduced to form a 3-charge hydride adduct. The resulting diaduct was isolated from the solution and, while cooling with ice, was introduced into Mannich-condensation with formaldehyde and a solution of the primary amine or amino acid. When the reaction mixture was acidified with dilute orthophosphoric acid to pH 4–5, precipitates of the target products precipitated. After recrystallization from ethanol, the yield of the target products, depending on the substituent at the nitrogen atom, ranged from 55 to 90%. This method is distinguished by relative simplicity, availability of reagents and allows under mild conditions to transfer from the aromatic system activated by nitro groups to 3-azabicyclo[3.3.1]nonane derivatives, containing promising from the point of view of further functionalization nitro, carbonyl and amino groups. The structure of the compounds obtained was proved by IR, 1H-, 13C-, two-dimensional correlation NMR spectroscopy, as well as elemental analysis data. In the IR spectra of the obtained substances, the characteristic absorption bands of amide I (1629-1633 cm-1) and amide II (1560-1570 cm-1), as well as antisymmetric (1549-1556 cm-1) and symmetric (1370-1377 cm-1) oscillations of nitro groups were observed. In the NMR spectra in the weakest field, the broadened signal of the proton NH is observed (δ 9.51-9.57 ppm), followed by the singlet signal of the proton at the double bond (δ 8.15-8.16 ppm). The protons of the methylene groups of the bicyclic system are diastereotopic. Therefore, their signals are mutually split into broadened doublets located in the region of 2.66–3.46 ppm.

Design, Synthesis and Antileishmanial Activity of Naphthotriazolyl-4- Oxoquinolines.

Leishmaniasis is a neglected public health problem caused by several protozoanspecies of the genus Leishmania. The therapeutic arsenal for treating leishmaniasis is quite limited, raising concerns about the occurrence of resistant strains. Furthermore, most of these drugs were developed more than 70 years ago and suffer from poor efficacy and safety and are not well adapted to the needs of patients. Therefore, research on novel natural or synthetic compounds with antiparasitic activity is urgently needed. In this paper, we evaluated the effect and the mechanism of action of naphthotriazolyl-4-oxoquinolines on promastigotes and intracellular amastigotes of Leishmania amazonensis. The naphthotriazolyl-4-oxoquinoline derivatives were obtained in good to moderate yields via the [3+2] cycloaddition reaction between 1,4-naphtoquinone and azido-4- oxoquinoline derivatives. HMPA at 100°C was established as the best solvent and temperature condition for this reaction. The structures of the compounds were confirmed by spectral analyses (infrared spectroscopy, one- and two-dimensional ¹H and ¹³C NMR spectroscopy, and high-resolution mass spectrometry). The compounds exhibited promising activities with IC50 values ranging from 0.7 to 2.0 µM against intracellular amastigotes of Leishmania amazonensis. The most selective compound was the Npentyl- substituted derivative, which showed a Selectivity Index (SI) of 8.6, making it less toxic than pentamidine (SI 4.5). Our results demonstrated that all compounds, except the N-propyl-substituted derivative, induce ROS production by parasites early in the culture. As a proof of concept, we demonstrated that the most selective compound was able to interfere with sterol biosynthesis in L. amazonensis. The naphthotriazolyl-4-oxoquinoline derivatives were obtained in good to moderate yields. These conjugates have potent in vitro antileishmanial activity involving at least two different mechanisms of action, making them promising lead compounds for the development of new therapeutic alternatives for leishmaniasis.

Structure and gene cluster of the O antigen of Escherichia coli F17, a candidate for a new O-serogroup

Escherichia coli F17 isolated from horse feces was studied in respect to the O antigen (O polysaccharide) structure and genetics. The lipopolysaccharide was isolated by phenol-water extraction of bacterial cells and cleaved by mild acid hydrolysis to yield the O polysaccharide, which was studied by sugar analysis and selective solvolysis with CF3CO2H along with one- and two-dimensional 1H and 13C NMR spectroscopy. The O polysaccharide was found to have a branched pentasaccharide repeat (O-unit) containing one residue each of d-galactose, d-mannose, l-rhamnose, d-glucuronic acid, and N-acetyl-d-glucosamine; about 2/3 units bear a side-chain glucose residue. To our knowledge, the F17 O-polysaccharide structure established is unique among known bacterial polysaccharide structures. The O-antigen gene cluster of E. coli F17 between the conserved genes galF and gnd was sequenced and found to be 99% identical to that of E. coli 102,755 assigned to a novel OgN8 genotype (A. Iguchi, S. Iyoda, K. Seto, H. Nishii, M. Ohnishi, H. Mekata, Y. Ogura, T. Hayashi, Front. Microbiol. 7 (2016) 765). Genes in the cluster were annotated taking into account the F17 O-polysaccharide structure. The data obtained confirm that E. coli F17 and E. coli strains belonging to the OgN8 genotype can be considered as a candidate to a new E. coli O-serogroup. The O antigen of this novel type was demonstrated to make for an effective shield protecting the intimate outer membrane surface of bacteria from direct interaction with bacteriophages.

Isolation and characterization of an exopolysaccharide‐producing Leuconostoc citreum strain from artisanal cheese

High molar mass exopolysaccharides (EPS) produced from sucrose by lactic acid bacteria (LAB) are of great interest as natural additives to use in foods, medical and pharmaceutical industry. This study aimed to identify the EPS produced by Leuconostoc citreum L3C1E7 isolated from Pico cheese and characterize the strain for technological and probiotic potential. Purified EPS was isolated from the culture of L. citreum L3C1E7 by ethanol precipitation, with a yield of 520mgml-1 . The EPS-producing strain had a mucoid phenotype and average molecular weight of 5·88×106 Da. The structural characterization of the purified EPS was determined by 1 H, 13 C and two-dimensional NMR spectroscopy. EPS was composed of alternating α-(1→6)-linked and α-(1→3)-linked D-glucopyranyl units, suggesting the existence of an alternan. The strain was slow acidifying, produced diacetyl and displayed high esterase/lipase and aminopeptidase activities, which promote the desirable flavours in dairy products. Moreover, L. citreum showed moderate resistance to the adverse conditions of the gastrointestinal (GI) tract and high adhesion to GI cells. This work provides a better understanding of EPS produced by L. citreum and the potential application of EPS-producing strain in food and/or as a probiotic culture. SIGNIFICANCE AND IMPACT OF THE STUDY: Some LAB strains are known to use extracellular glycoside-hydrolase enzymes for synthesizing a diversity of exopolysaccharides (EPS) with potential application as natural additives to foods. Previous studies have identified an EPS-producing Leuconostoc citreum strain with immunomodulatory properties. This work provides a better understanding of EPS produced by this strain and the potential application of the strain in food fermentation and/or as a probiotic culture.

Proline provides site-specific flexibility for in vivo collagen

Fibrillar collagens have mechanical and biological roles, providing tissues with both tensile strength and cell binding sites which allow molecular interactions with cell-surface receptors such as integrins. A key question is: how do collagens allow tissue flexibility whilst maintaining well-defined ligand binding sites? Here we show that proline residues in collagen glycine-proline-hydroxyproline (Gly-Pro-Hyp) triplets provide local conformational flexibility, which in turn confers well-defined, low energy molecular compression-extension and bending, by employing two-dimensional 13C-13C correlation NMR spectroscopy on 13C-labelled intact ex vivo bone and in vitro osteoblast extracellular matrix. We also find that the positions of Gly-Pro-Hyp triplets are highly conserved between animal species, and are spatially clustered in the currently-accepted model of molecular ordering in collagen type I fibrils. We propose that the Gly-Pro-Hyp triplets in fibrillar collagens provide fibril “expansion joints” to maintain molecular ordering within the fibril, thereby preserving the structural integrity of ligand binding sites.

Escherichia coli O106, a new member of a group of enteric bacteria sharing an O-polysaccharide backbone structure

O-Polysaccharides (O-antigens) of a number of genetically related Escherichia coli O-serogroups (O17, O44, O73, O77, and O106) and Salmonella enterica O:6,14 possess an identical main chain composed of d-GlcNAc and d-Man residues and differ from each other by the absence or presence of glucose side chains at various positions. Using two-dimensional NMR spectroscopy, we established the structure of the O-polysaccharide of E. coli O106 having two glucose side chains in a hexasaccharide repeating unit.

Synthesis, Structural, Spectral, and Electrochemical Studies of Selenabenziporphyrin and Its Pd(II) Complex.

A new nonaromatic selenabenziporphyrin was synthesized by (3 + 1) condensation of m-benzitripyrrane and 2,5-bis[( p-tolyl)hydroxymethyl] selenophene under mild trifluoroacetic acid-catalyzed reaction conditions. The selenabenziporphyrin was characterized by high-resolution mass spectrometry, one- and two-dimensional NMR spectroscopy, and X-ray crystallography. The crystal structure revealed that the macrocycle was planar with moderately tilted m-phenylene ring and that the phenylene ring completely blocks the macrocyclic π-delocalization. The selenabenziporphyrin exhibits one broad absorption band at 645 nm along with one sharp band at 415 nm, and electrochemical studies revealed that the macrocycle was electron-deficient. The selenabenziporphyrin readily forms organometallic Pd(II) complex when treated with PdCl2 in CH3CN/CHCl3 at reflux followed by recrystallization. The X-ray structure revealed that the Pd(II) ion was coordinated with two pyrrole "N"s, selenophene "Se", and m-phenylene ring "C" in square-planar fashion, and the complex retained its nonaromatic nature. The Pd(II) complex exhibits ill-defined absorption bands, and it was more electron-deficient than free-base selenabenziporphyrin macrocycle. Time-dependent density functional theory studies supported the experimental observations.

An Electric Trap: Electron‐Rich Carbonyl Axis Ends Slow Threading/Dethreading Exchange Dynamics of Pillar[5]arene Ring along Axis

AbstractViologen axes with negative carbonyl ends such as carboxylic acid and carbonylethyl groups were synthesized. The threading/dethreading exchange dynamics of the pillar[5]arene ring and these axes were studied by variable‐temperature 1H NMR and two‐dimensional exchange spectroscopy NMR spectroscopies. The exchange rate increased on introduction of electron‐rich carbonyl ends into the axis: the exchange rate constants of the pillar[5]arene ring along the carboxylic acid‐ended and carbonylethyl‐ended viologen axes were ca. 2000 and 360 times smaller, respectively, than that along the alkyl‐ended viologen axis.

Chemical characterization and serological properties of a unique O-polysaccharide of the Proteus mirabilis Sm 99 clinical strain – Identification of a new, O81, serotype

The current serological classification scheme of the medically important bacteria from the genus Proteus consists of 80 O serogroups, the last four of which (O77–O80) were created from clinical strains from Łódź, Poland. There are more serologically unique strains isolated from patient that do not fit into the existing scheme, such as Proteus mirabilis strain Sm 99 isolated from urine of a 74-year-old woman in Łódź. Serological investigation involving ELISA and Western blotting failed to classify the Proteus mirabilis strain Sm 99 into any of the 80 Proteus O serogroups. Sugar analysis along with two-dimensional NMR spectroscopy showed that the O-polysaccharide is composed of branched pentasaccharide repeating units containing one residue each of d-Glc, d-GlcNAc, d-GalNAc, d-glucuronic acid, and 4-[(R)-3-hydroxybutanoylamino]-4,6-dideoxy-d-glucose. The chemical and serological data show that the O antigen of P. mirabilis Sm 99 is unique among the known Proteus O antigens. Based on this finding, it is proposed to extend the current serological classification scheme of Proteus by adding a new serogroup, O81, which at present consists of P. mirabilis strain Sm 99 only.

Structure of the K82 Capsular Polysaccharide from Acinetobacter baumannii LUH5534 Containing a d-Galactose 4,6-Pyruvic Acid Acetal.

Type K82 capsular polysaccharide (CPS) was isolated from Acinetobacter baumannii LUH5534. The structure of a linear tetrasaccharide repeating unit of the CPS was established by sugar analysis along with one- and two-dimensional 1H and 13C NMR spectroscopy. Proteins encoded by the KL82 capsule gene cluster in the genome of LUH5534 were assigned to roles in the synthesis of the K82 CPS. In particular, functions were assigned to two new glycosyltransferases (Gtr152 and Gtr153) and a novel pyruvyltransferase, Ptr5, responsible for the synthesis of d-galactose 4,6-(R)-pyruvic acid acetal.

Network Structure and Connectivity in SnO-P2O5 Glasses: Results from 2D 31P and 119Sn NMR Spectroscopy.

The compositional evolution of the network structure and connectivity in binary SnO-P2O5 glasses with 35 ≤ mol % SnO ≤ 55 is studied using two-dimensional 119Sn and 31P NMR spectroscopy. The phosphate Q n species concentrations, as obtained from the analyses of the 31P NMR spectral line shapes suggest that the network modification can be described in terms of a binary Q-speciation model. On the other hand, the 119Sn NMR spectra suggest that the Sn-O coordination environment in these glasses is composition dependent. Sn is present in both 3-fold (trigonal pyramid) and 4-fold (trigonal bipyramid) coordinations with oxygen in these glasses. The relative fraction of the trigonal pyramidal environment increases with increasing SnO content. The unusually low glass transition temperature Tg of these glasses (241-266 °C) is argued to be related to the low coordination number of Sn, leading to a relatively sparsely connected structural network.

Structure characterization of the mannofucogalactan isolated from fruit bodies of Quinine conk Fomitopsis officinalis

The mannofucogalactan as a major component of water extract was obtained from fruit bodies of Fomitopsis officinalis by extraction with boiling water followed by deproteination, decoloration, and purification using anion-exchange chromatography and size exclusion chromatography. Its structure was characterized using the data of monosaccharide composition, methylation analysis, one- and two-dimensional NMR spectroscopy. The studied polysaccharide was a branched mannofucogalactan with a backbone composed of partially 3-O-methylated 1,6-O-linked α-D-galactopyranosyl residues. Almost every second residue in the backbone was substituted at O-2 by 3-O-α-D-mannopyranosyl-α-L-fucopyranosyl and β-D-galactopyranosyl residues. The non-reducing terminal α-L-fucopyranosyl units, which were identified by GC–MS analyses, appeared to be the part of mannofucogalactan side chains also.