Two-dimensional 1H Nuclear Magnetic Resonance Research Articles

Polyhydroxy Sterols Isolated From Ganoderma sinense Spores and Their Cytotoxic Activities

Background: The fruiting body of Ganoderma sinense Zhao, Xu et Zhang iscertified as an authoritative medical material in the Chinese Pharmacopoeia 2020 edition and has been used as a crude drug for a long time. G sinense spores are crucial part of the fruiting body of G sinense and exhibited antitumor and immune-enhancing activities according to our previous reports. However, there were few studies about the chemical composition of G sinense spores. This study was aimed at the structure determination and antitumor effect of the sterols isolated from G sinense spores. Methods: Modern chromatographic methods were applied to isolate compounds from the ruptured spores of G sinense. Their chemical structures were elucidated by analyses of one-dimensional and two-dimensional 1H nuclear magnetic resonance, and high-resolution electrospray ionization mass spectrometry data. The cytotoxicities of the isolated compounds against 3 tumor cell lines, human non-small cell lung cancer A549 and 95D cells, and human hepatocellular carcinoma HepG2 cells were measured by Cell Counting Kit-8. Results: A novel ergosterol derivative, (22 E,24 R)-3β,5β,6α,7α,14β-pentahydroxyergosta-8,22-dien-15-one (1), and 5 known sterols, (22 E,24 R)-3β,5α,9α,14β-tetrahydroxyergosta-7,22-dien-6-one (2), (22 E,24 R)-ergosta-7,9(11),22-triene-3β,5α,6β-triol (3), (22 E,24 R)-ergosta-7,9(11),22-triene-3β,5α,6β,14α-tetrol (4), β-daucosterine (5), and (22 E,24 R)-ergosta-7,22-diene-3β,5α,6β,9α-tetrol (6), were obtained from the ruptured spores of G sinense. Compound 1 exhibited cytotoxicity against A549 cells and HepG-2 cells with IC50 values of 65.12 ± 4.76 and 97.34 ± 6.36 μM, respectively. Compounds 1, 2, and 4 were obtained from G sinense for the first time. Conclusion: This paper is a continuation of an investigation of the chemical ingredients from G sinense spores and their antitumor effect. Only compound 1 showed weak cytotoxicities against A549 cells and HepG-2 cells.

Structural and Serological Characterization of the O Antigen of Proteus mirabilis Clinical Isolates Classified into a New Proteus Serogroup, O84.

Two closely related Proteus mirabilis smooth strains, Kr1 and Ks20, were isolated from wound and skin samples, respectively, of two infected patients in central Poland. Serological tests, using the rabbit Kr1-specific antiserum, revealed that both strains presented the same O serotype. Their O antigens are unique among the Proteus O serotypes, which had been described earlier, as they were not recognized in an enzyme-linked immunosorbent assay (ELISA) by a set of Proteus O1-O83 antisera. Additionally, the Kr1 antiserum did not react with O1-O83 lipopolysaccharides (LPSs). The O-specific polysaccharide (OPS, O antigen) of P. mirabilis Kr1 was obtained via the mild acid degradation of the LPSs, and its structure was established via a chemical analysis and one- and two-dimensional 1H and 13C nuclear magnetic resonance (NMR) spectroscopy applied to both initial and O-deacetylated polysaccharides, where most β-2-acetamido-2-deoxyglucose (N-acetylglucosamine) (GlcNAc) residues are non-stoichiometrically O-acetylated at positions 3, 4, and 6 or 3 and 6, and a minority of α-GlcNAc residues are 6-O-acetylated. Based on the serological features and chemical data, P. mirabilis Kr1 and Ks20 were proposed as candidates to a new successive O-serogroup in the genus Proteus, O84, which is another example of new Proteus O serotypes identified lately among serologically differentiated Proteus bacilli infecting patients in central Poland.

Characterization of an L-α,β-diaminopropionic acid polymer with comb-like structure isolated from a poly(ε-L-lysine)-producing Streptomyces sp.

Polymers of basic amino acids function as polycationic compounds under physiological conditions and exhibit intriguing biological properties, such as antimicrobial and antiviral activities, immunopotentiating ability, and DNA-binding activity. Poly(ε-L-lysine) (ε-PL) produced by some strains of Streptomyces spp. is a cationic homopolymer of L-lysine linking between ε-amino and α-carboxylic acid functional groups and has been used as a food preservative based on its biocompatibility and biodegradability. An ε-PL-producing strain of Streptomyces sp. USE-33 was found to secrete a novel polycationic substance into its culture broth along with ε-PL. High-performance liquid chromatography analyses and one- and two-dimensional 1H and 13C nuclear magnetic resonance (NMR) experiments, accompanied by NMR titration studies, revealed that the secreted substance was poly[β-(L-diaminopropionyl-L-diaminopropionic acid)], PAP, characterized by an isopeptide backbone linking between the β-amino and α-carboxylic acid groups of L-α,β-diaminopropionic acid (L-Dpr) with pendent L-Dpr residues. PAP had a molecular weight of 500 to 1400, and copolymers composed of the two amino acids L-Dpr and L-lysine were not detected in the producer strain USE-33. The strain coproduced high levels of the two poly(amino acid)s in the presence of glycerol, citrate, and ammonium sulfate at pH 4.0 in a two-stage cultivation procedure. PAP exhibited strong inhibitory activities against several yeasts and weaker activities against bacteria than ε-PL. PAP may share a number of biological functions with ε-PL, and the use of PAP along with ε-PL has potential as a specific and advanced material for technical applications in various fields.Key points• Novel cationic poly(amino acid) was found in an ε-PL-producing Streptomyces species.• The l-α,β-diaminopropionic acid polymer was characterized by a comb-like structure.• The novel poly(amino acid), PAP, exhibited antibacterial and antifungal activities.

Nuclear spin relaxation as a probe of zeolite acidity: a combined NMR and TPD investigation of pyridine in HZSM-5.

The relative surface affinities of pyridine within microporous HZSM-5 zeolites are explored using two-dimensional 1H nuclear magnetic resonance (NMR) relaxation time measurements. The dimensionless ratio of longitudinal-to-transverse nuclear spin relaxation times T1/T2 is shown to exhibit strong sensitivity to the silica/alumina ratio (SAR) of these zeolites, which is indicative of material acidity. This trend is interpreted in terms of increased pyridine surface affinity with decreasing SAR. Temperature programmed desorption (TPD) analysis corroborates this observation, revealing a distinct increase in the heat of desorption associated with adsorbed pyridine as a function of decreasing SAR. A direct correlation between NMR and TPD data suggests NMR relaxation time analysis can be a valuable tool for the non-invasive characterisation of adsorption phenomena in microporous solids.

Identification of compounds from terrestrial dissolved organic matter toxic to cyanobacteria

There is emerging evidence for the phytotoxicity of terrestrial dissolved organic matter (DOM), however its sources, transformations and ecological effects in aquatic ecosystems are poorly understood. DOM characterization by Nuclear Magnetic Resonance (NMR) spectroscopy has typically involved solid-state techniques, but poor resolution has often precluded identification of individual components. This study is the first to directly identify individual phytotoxic components using a novel combined approach of preparative HPLC fractionation of DOM (obtained from leaves of two common riparian trees, Casuarina cunninghamiana and Eucalyptus tereticornis). This was followed by chemical characterization of fractions, using one-dimensional (1D) and two-dimensional (2D) solution-state 1H NMR analyses. Additionally, the phytotoxic effect of the fractions was determined using cultures of the cyanobacteria Raphidiopsis (Cylindrospermopsis) raciborskii. The amino acid, proline, from Casuarina leachate was identified as phytotoxic, while for Eucalyptus leachate, it was gallic acid and polyphenols. These phytotoxicants remained in the leachates when they were incubated in sunlight or the dark conditions over 5 days. Our study identifies phytotoxic compounds with the potential to affect algal species composition, and potentially control nuisance R. raciborskii blooms.

Genetics of biosynthesis and structure of the K53 capsular polysaccharide of Acinetobacter baumannii D23 made up of a disaccharide K unit.

The KL53 capsular polysaccharide (CPS) gene cluster of Acinetobacter baumannii D23 was sequenced, and includes a single gtr gene encoding the glycosyltransferase Gtr2, and the itrA1 gene for ItrA1 that is known to initiate CPS biosynthesis with d-QuiNAc4NAc. The K53 CPS was isolated and studied by one- and two-dimensional 1H and 13C nuclear magnetic resonance (NMR) spectroscopy before and after O-deacetylation. The disaccharide K unit of the CPS was established as →3)-α-d-GalpNAcA4Ac-(1→3)-β-d-QuipNAc4NAc-(1→, where GalNAcA and QuiNAc4NAc indicate 2-acetamido-2-deoxygalacturonic acid and 2,4-diacetamido-2,4,6-trideoxyglucose, respectively. This established the linkage formed by Gtr2. The degree of 4-O-acetylation of d-GalNAcA by Atr18, encoded at the KL53 locus, is ~55 %.

Origin and Structural Characteristics of Tri-coordinated Extra-framework Aluminum Species in Dealuminated Zeolites.

Post-synthetic dealumination treatment is a common tactic adopted to improve the catalytic performance of industrialized zeolitic catalysts through enhancements in acidity and stability. However, among the possible extra-framework aluminum (EFAL) species in dealuminated zeolites such as Al3+, Al(OH)2+, Al(OH)2+, AlO+, AlOOH, and Al(OH)3, the presence of tri-coordinated EFAL-Al3+ species, which exhibit large quadrupolar effect due to the lack of hydrogen-bonding species, was normally undetectable by conventional one- and two-dimensional 1H and/or 27Al solid-state nuclear magnetic resonance (SSNMR) techniques. By combining density functional theory (DFT) calculations with experimental 31P SSNMR using trimethylphosphine (TMP) as the probe molecule, we report herein a comprehensive study to certify the origin, fine structure, and possible location of tri-coordinated EFAL-Al3+ species in dealuminated HY zeolite. The spatial proximities and synergies between the Brønsted and various Lewis acid sites were clearly identified, and the origin for the observed EFAL-Al3+ species with ultra-strong Lewis acidity was deduced to be at the expense of adjacent Brønsted acid sites. The excellent performance of such tri-coordinated EFAL species was furthermore confirmed by glucose isomerization reactions.

Two-dimensional 1H and 1H-detected NMR study of a heterogeneous biocatalyst using fast MAS at high magnetic fields

Nuclear magnetic resonance (NMR) is a powerful tool for investigating atomic-scale structure in heterogeneous or composite materials where long-range order is absent. In this work solid-state 1H and 1H-detected NMR experiments were performed with fast magic angle spinning (νR = 75 kHz) and at high magnetic fields (B0 = 20 T) and used to gain structural insight into a heterogeneous biocatalyst consisting of an enzyme, human carbonic anhydrase II (hCA II), covalently immobilized on epoxy-functionalized silica. Two-dimensional 1H-1H NOESY-type correlation experiments were able to provide information on 1H environments in silica, epoxy-silica and the immobilized enzyme. Two distinct signals originating from water protons were observed: water associated with the surface of the silica and the water associated with the immobilized enzyme. Additional two-dimensional 1H-1H double–single quantum (DQ-SQ) correlation experiments suggested that the immobilized enzyme is not in close contact with the silica surface. Most significantly, comparison of two-dimensional 1H-15N spectra of the immobilized enzyme and the solution-state enzyme confirmed that the structural integrity of the protein is well preserved upon covalent immobilization.

Accelerating two-dimensional nuclear magnetic resonance correlation spectroscopy via selective coherence transfer.

Nuclear magnetic resonance (NMR) spectroscopy serves as an important tool for both qualitative and quantitative analyses of various systems in chemistry, biology, and medicine. However, applications of one-dimensional 1H NMR are often restrained by the presence of severe overlap among different resonances. The advent of two-dimensional (2D) 1H NMR constitutes a promising alternative by extending the crowded resonances into a plane and thereby alleviating the spectral congestions. However, the enhanced ability in discriminating resonances is achieved at the cost of extended experimental duration due to necessity of various scans with progressive delays to construct the indirect dimension. Therefore, in this study, we propose a selective coherence transfer (SECOT) method to accelerate acquisitions of 2D correlation spectroscopy by converting chemical shifts into spatial positions within the effective sample length and then performing an echo planar spectroscopic imaging module to record the spatial and spectral information, which generates 2D correlation spectrum after 2D Fourier transformation. The feasibility and effectiveness of SECOT have been verified by a set of experiments under both homogeneous and inhomogeneous magnetic fields. Moreover, evaluations of SECOT for quantitative analyses are carried out on samples with a series of different concentrations. Based on these experimental results, the SECOT may open important perspectives for fast, accurate, and stable investigations of various chemical systems both qualitatively and quantitatively.

Characterization of natural organic matter in bentonite clays for potential use in deep geological repositories for used nuclear fuel

The Nuclear Waste Management Organization (NWMO) is developing a Deep Geological Repository (DGR) to contain and isolate used nuclear fuel in a suitable rock formation at a depth of approximately 500m. The design concept employs a multibarrier system, including the use of copper-coated used fuel containers, surrounded by a low-permeability, swelling clay buffer material within a low permeability, stable host rock environment. The natural organic matter (NOM) composition of the bentonite clays being considered for the buffer material is largely uncharacterized at the molecular-level. To gain a better understanding of the NOM in target clays from Wyoming and Saskatchewan, molecular-level methods (biomarker analysis, solid-state 13C NMR and solution-state 1H nuclear magnetic resonance (NMR)) were used to elucidate the structure and sources of NOM. Organic carbon content in three commercially available bentonites analyzed was low (0.11–0.41%). The aliphatic lipid distribution of the clay samples analyzed showed a predominance of higher concentration of lipids from vascular plants and low concentrations of lipids consistent with microbial origin. The lignin phenol vanillyl acid to aldehyde ratio (Ad/Al) for the National sample indicated an advanced state of lignin oxidation and NOM diagenesis. The 13C NMR spectra were dominated by signals in the aromatic and aliphatic regions. The ratio of alkyl/O-alkyl carbon ranged from 7.6 to 9.7, indicating that the NOM has undergone advanced diagenetic alteration. The absence lignin-derived phenols commonly observed in CuO oxidation extracts from contemporary soils and sediments as well as the lack of amino acids suggests that the material corresponding to the aromatic signal is not composed of lignin or proteins but may be derived from another source such as black carbon or some other non-extractable aromatic-rich NOM. The aliphatic signal appears to correspond to long-chain compounds with little side branching based on the results of the one-dimensional (1D) and two-dimensional (2D) solution-state 1H NMR analyses. Overall, the organic geochemical analyses suggest that the NOM is composed mainly of plant-derived waxes and highly aromatic carbon with low contributions from small molecules. The compounds identified by the molecular-level analysis of NOM in the clay samples are hypothesized to be recalcitrant but future studies should examine if these compounds may serve as a microbial substrate to further test the observations of this study. Furthermore, our study suggests that the NOM has undergone diagenesis and that marine NOM signatures are no longer recognizable or detectable. As such, future work may also examine the diagenesis of these deposits to further understand the NOM geochemistry and paleoenvironmental conditions in bentonite deposits.

Effects of time and temperature of firing on Fe-rich ceramics studied by Mössbauer spectroscopy and two-dimensional 1H-nuclear magnetic resonance relaxometry

The combined effects of firing temperature and soaking time on the microstructure of iron-rich porous ceramics have been studied by 57Fe-Mössbauer spectroscopy and 2D 1H nuclear magnetic resonance (NMR) relaxometry using a single-sided probe. Examining water-saturated ceramics using the relaxation correlation method, where longitudinal (T1) and transverse (T2) relaxation times are measured concurrently, provides information about firing-induced changes in both porosity (related to T1) and magnetic properties (related to T2). Comparing the information obtained from 1H-NMR analyses with that obtained from Mössbauer spectroscopy (which characterizes changes in iron-bearing species) shows that the T1-T2 NMR correlation technique is very sensitive to even subtle modifications in the magnetic behavior of Fe-bearing species. Moreover, the single-sided NMR approach allows us to perform millimeter-scale depth-resolved measurements, which can be used to non-invasively study the microstructural heterogeneities associated with non-uniform firing effects inside ceramics. This is in contrast to Mössbauer spectroscopy, which requires that the ceramic samples be ground.

Role of Propionates in Substrate Binding to Heme Oxygenase from Neisseria meningitidis: A Nuclear Magnetic Resonance Study

Heme oxygenase (HO) cleaves hemin into biliverdin, iron, and CO. For mammalian HOs, both native hemin propionates are required for substrate binding and activity. The HO from the pathogenic bacterium Neisseria meningitidis (NmHO) possesses a crystallographically undetected C-terminal fragment that by solution (1)H nuclear magnetic resonance (NMR) is found to fold and interact with the active site. One of the substrate propionates has been proposed to form a salt bridge to the C-terminus rather than to the conventional buried cationic side chain in other HOs. Moreover, the C-terminal dipeptide Arg208His209 cleaves spontaneously over ~24 h at a rate dependent on substituent size. Two-dimensional (1)H NMR of NmHO azide complexes with hemins with selectively deleted or rearranged propionates shows that all bind to NmHO with a structurally conserved active site as reflected in optical spectra and NMR nuclear Overhauser effect spectroscopy cross-peak and hyperfine shift patterns. In contrast to mammalian HOs, NmHO requires only a single propionate interacting with the buried terminus of Lys16 to exhibit full activity and tolerates the existence of a propionate at the exposed 8-position. The structure of the C-terminus is qualitatively retained upon deletion of the 7-propionate, but a dramatic change in the 7-propionate carboxylate (13)C chemical shift upon C-terminal cleavage confirms its role in the interaction with the C-terminus. The stronger hydrophobic contacts between pyrroles A and B with NmHO contribute more substantially to the substrate binding free energy than in mammalian HOs, "liberating" one propionate to stabilize the C-terminus. The functional implications of the C-terminus in product release are discussed.

Structural elucidation of an -1,2-mannosidase resistant oligosaccharide produced in Pichia pastoris

The N-glycosylation pathway in Pichia pastoris has been humanized by the deletion of genes responsible for fungal-type glycosylation (high mannose) as well as the introduction of heterologous genes capable of forming human-like N-glycosylation. This results in a yeast host that is capable of expressing therapeutic glycoproteins. A thorough investigation was performed to examine whether glycoproteins expressed in glycoengineered P. pastoris strains may contain residual fungal-type high-mannose structures. In a pool of N-linked glycans enzymatically released by protein N-glycosidase from a reporter glycoprotein expressed in a developmental glycoengineered P. pastoris strain, an oligosaccharide with a mass consistent with a Hexose(9)GlcNAc(2) oligosaccharide was identified. When this structure was analyzed by a normal-phase high-performance liquid chromatography (HPLC), its retention time was identical to a Man(9)GlcNAc(2) standard. However, this Hexose(9)GlcNAc(2) oligosaccharide was found to be resistant to α-1,2-mannosidase as well as endomannosidase, which preferentially catabolizes endoplasmic reticulum oligosaccharides containing terminal α-linked glucose. To further characterize this oligosaccharide, we purified the Hexose(9)GlcNAc(2) oligosaccharide by HPLC and analyzed the structure by high-field one-dimensional (1D) and two-dimensional (2D) (1)H NMR (nuclear magnetic resonance) spectroscopy followed by structural elucidation by homonuclear and heteronuclear 1D and 2D (1)H and (13)C NMR spectroscopy. The results of these experiments lead to the identification of an oligosaccharide α-Man-(1 → 2)-β-Man-(1 → 2)-β-Man-(1 → 2)-α-Man-(1 → 2) moiety as part of a tri-antennary structure. The difference in enzymatic reactivity can be attributed to multiple β-linkages on the α-1,3 arm of the Man(9)GlcNAc(2) oligosaccharide.

Poly(γ-l-diaminobutanoic acid), a novel poly(amino acid), coproduced with poly(ɛ-l-lysine) by two strains ofStreptomyces celluloflavus

Two poly(epsilon-L-lysine) (epsilon-PL) producer strains of Streptomyces celluloflavus secreted a novel polymeric substance into their culture broths along with epsilon-PL. Three types of HPLC analysis plus one- and two-dimensional 1H and 13C nuclear magnetic resonance experiments revealed that the secreted substance was poly(gamma-L-diaminobutanoic acid) (gamma-PAB), an L-alpha,gamma-diaminobutanoic acid (L-DAB) homopolymer linking between y-amino and alpha-carboxylic acid functional groups. The gamma-PABs from the two strains had an identical chemical structure, and the same number-average molecular weight of 2100-2200. No copolymers composed of the two amino acids L-DAB and L-lysine were found in either of the broths from the producers. Both strains coproduced high levels of the two poly(amino acid)s in the presence of SO4(2-) at pH 4.0 and 4.5 L min(-1) aeration in a 5-L jar fermentor. gamma-PAB exhibited strong inhibitory activities against various yeasts and weaker actions against bacteria than epsilon-PL. gamma-PAB may have various biological functions similar to epsilon-PL, and the use of gamma-PAB along with epsilon-PL would be advantageous for technical applications in various fields.

Three-Dimensional Structure of Conotoxin tx3a: An m-1 Branch Peptide of the M-Superfamily

The M-superfamily, one of eight major conotoxin superfamilies found in the venom of the cone snail, contains a Cys framework with disulfide-linked loops labeled 1, 2, and 3 (-CC (1) C (2) C (3) CC-). M-Superfamily conotoxins can be divided into the m-1, -2, -3, and -4 branches, based upon the number of residues located in the third Cys loop between the fourth and fifth Cys residues. Here we provide a three-dimensional solution structure for the m-1 conotoxin tx3a found in the venom of Conus textile. The 15-amino acid peptide, CCSWDVCDHPSCTCC, has disulfide bonds between Cys (1) and Cys (14), Cys (2) and Cys (12), and Cys (7) and Cys (15) typical of the C1-C5, C2-C4, and C3-C6 connectivity pattern seen in m-1 branch peptides. The tertiary structure of tx3a was determined by two-dimensional (1)H NMR in combination with the combined assignment and dynamics algorithm for nuclear magnetic resonance (NMR) applications CYANA program. Input for structure calculations consisted of 62 inter- and intraproton, five phi angle, and four hydrogen bond constraints. The root-mean-square deviation values for the 20 final structures are 0.32 +/- 0.07 and 0.84 +/- 0.11 A for the backbone and heavy atoms, respectively. Surprisingly, the structure of tx3a has a "triple-turn" motif seen in the m-2 branch conotoxin mr3a, which is absent in mr3e, the only other member of the m-1 branch of the M-superfamily whose structure is known. Interestingly, injection of tx3a into mice elicits an excitatory response similar to that of the m-2 branch peptide mr3a, even though the conotoxins have different disulfide connectivity patterns.

Microstructure of N,N′-bis(cetyldimethyl)-α,ω-propanediammonium dibromide micelle and its dynamics in solution studied by 1H NMR

The characteristics of N,N′-bis(cetyldimethyl)-α,ω-alkane (propane and butane) diammonium dibromide (16-3-16 and 16-4-16) aqueous solutions were studied by one- and two-dimensional 1H nuclear magnetic resonance (NMR). The measurements of self-diffusion coefficient and inter-proton distance at 318 K suggest that 16-3-16 spherical micelles are formed in the dilute solution at a concentration of 0.26 mmol/l and the polar head groups of the surfactant are in a saw-toothed form staying at the surface of the micelle to overcome the strong electrostatic repulsion force. Relaxation measurements obviously show that the spacer chain is rigid in the surface layer of the hydrophobic micellar core, and the side alkyl chains of 16-3-16 are packed more tightly than those of 16-4-16 in the micellar core. The line-shape analysis of the methyl protons at the end of the side alkyl chain of 16-3-16 and 16-4-16 suggests that two possible momentary morphologies of their side alkyl chains are situated in the micelle, respectively.

The Structure of a Novel Insect Peptide Explains Its Ca2+ Channel Blocking and Antifungal Activities,

Diapause-specific peptide (DSP), derived from the leaf beetle, inhibits Ca2+ channels and has antifungal activity. DSP acts on chromaffin cells of the adrenal medulla in a fashion similar to that of omega-conotoxin GVIA, a well-known neurotoxic peptide, and blocks N-type voltage-dependent Ca2+ channels. However, the amino acid sequence of DSP has little homology with any other known Ca2+ channel blockers or antifungal peptides. In this paper, we analyzed the solution structure of DSP by using two-dimensional 1H nuclear magnetic resonance and determined the pairing of half-cystine residues forming disulfide bonds. The arrangement of the three disulfide bridges in DSP was distinct from that of other antifungal peptides and conotoxins. The overall structure of DSP is compact due in part to the three disulfide bridges and, interestingly, is very similar to those of the insect- and plant-derived antifungal peptides. On the other hand, the disulfide arrangement and the three-dimensional structure of DSP and GVIA are not similar. Nevertheless, some surface residues of DSP superimpose on the key functional residues of GVIA. This homologous distribution of hydrophobic and charged side chains may result in the functional similarity between DSP and GVIA. Thus, we propose here that the three-dimensional structure of DSP can explain its dual function as a Ca2+ channel blocker and antifungal peptide.

Novel carbazole phenoxy-based methacrylates to produce high-refractive index polymers

A novel, high-refractive index homopolymer was produced by incorporating carbazole and phenol into the methacrylate monomer structure. The reaction of phenol with 9-(2,3-epoxypropyl)-carbazole, followed by the reaction of the carbazole phenoxy-based intermediate with methacryloyl chloride or methacrylic anhydride, and recrystallization from methanol, produced a good yield of highly pure carbazole phenoxy functionalized methacrylate monomer. Subsequent free radical polymerization or UV photopolymerization of the functionalized methacrylate monomer, in addition to copolymerizations with methyl methacrylate, provided for high-refractive index materials well suited for lightweight optical applications. Unlike N-vinyl carbazole, the novel carbazole phenoxy-based methacrylate readily copolymerized with methyl methacrylate. Statistical copolymers of carbazole based methacrylates with methyl methacrylate were produced by free radical solution polymerization in DMAC or by photopolymerization in DMF. The carbazole phenoxy-based methacrylate monomer was characterized for molecular weight using gel permeation chromatography (GPC), for melting point and glass transition temperature using differential scanning calorimetry (DSC), for decomposition using thermal gravimetric analysis (TGA), and for chemical composition by one- and two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy and by elemental analysis. The AIBN initiated carbazole phenoxy-based methacrylate polymerization was followed using in situ FTIR, which showed the reaction to be complete within 40 min in DMAC at 90 °C. Refractive indices of the carbazole based methacrylate homopolymers and copolymers ranged from 1.52 to 1.63. PhotoDSC was used to determine the heat of polymerization (Δ H p) for the carbazole phenoxy-based methacrylate (Δ H p=−39.4 kJ/mol). The carbazole phenoxy-based methacrylate homopolymer had a surprisingly high onset of decomposition temperature ( T onset=316 °C). 13C NMR spectroscopy experiments and molecular modeling were used to explore the configuration of the polymerized carbazole phenoxy-based methacrylate. The lack of head-to-head linkages due to steric considerations reasonably explains the high thermal stability observed for the carbazole phenoxy-based methacrylate polymer.

Isolation, gene expression and solution structure of a novel moricin analogue, antibacterial peptide from a lepidopteran insect, Spodoptera litura

An antibacterial peptide was isolated from a lepidopteran insect, Spodoptera litura. The molecular mass of this peptide was determined to be 4489.55 by matrix assisted laser desorption/ionization-time of flight mass (MALDI-TOF MS) spectrometry. The peptide consists of 42 amino acids and the sequence has 69–98% identity to those of moricin-related peptides, antibacterial peptides from lepidopetran insects. Thus, the peptide was designated S. litura (Sl) moricin. Sl moricin showed a broad antibacterial spectrum against Gram-positive and negative bacteria. Sl moricin gene was inducible by bacterial injection and expressed tissue—specifically in the fat body and hemocytes. Furthermore, the solution structure of Sl moricin was determined by two-dimensional (2D) 1H-nuclear magnetic resonance (NMR) spectroscopy and hybrid distance geometry-simulated annealing calculation. The tertiary structure revealed a long α-helix containing eight turns along nearly the full length of the peptide like that of moricin, confirming that Sl moricin is a new moricin-like antibacterial peptide. These results suggest that moricin is present not only in B. mori but also in other lepidopteran insects forming a gene family.

Spectroscopic Characterization of Ozonated Sunflower Oil

Ozonation reactions are very important in vegetable oil chemistry since their ozonation products are involved in antimicrobial effect in therapeutical uses for several microbiological etiology diseases. Information on the spectroscopic characterization of the products generated by ozonolysis of sunflower oil is limited. In the present study ozonized sunflower oil with 650 mmol-equiv/kg of peroxide index is chemically characterized. Ozonation of sunflower oil produced ozonides, aldehydes and hydroperoxides which were identified by 1H, 13C and two-dimensional 1H Nuclear Magnetic Resonance (NMR). The virgin sunflower oil and ozonized sunflower oil show very similar 1H NMR spectra except for the resonances at δ = 9.74 and δ = 9.63 ppm that correspond to both triplet from aldehydic protons, δ = 5.6 ppm (olefinic signal from hydroperoxides), and δ = 5.15 ppm (multiplet from ozonides methylic protons). Other resonance assignments are based on the connectivities provided by the proton scalar coupling constants. These are the following: δ = 3.15 ppm (doublet from methylenic group in α position respect to olefinic proton), δ = 2.45 ppm (multiplet from methylenic group allylic to ozonides methynic protons) and δ = 1.62 ppm (multiplet methylenic protons in β position respect to ozonides methynic protons). From the 13C NMR and 1H-13C two- dimensional spectrum of the ozonized sunflower oil, the presence of ozonides was confirmed by the signals δ = 103.43 and δ = 103.49 ppm, respectively. The others new signals found in δ = 42.5 and δ = 42.76 ppm confirm the presence of methylenic carbons from hydroperoxides and ozonides. These results indicate that NMR Spectroscopy can provide valuable information about the amount of reaction compounds of ozonized vegetable oil. From the chemical structural elucidation of ozonated sunflower oils, relevant biochemical and chemical information can be achieved.