Investigations on unconventional hydrogen bonds in RNA binding proteins: The role of CH⋯OC interactions

We have investigated the roles played by CH⋯OC interactions in RNA binding proteins. There was an average of 78 CH⋯OC interactions per protein and also there was an average of one significant CH⋯OC interaction for every 6 residues in the 59 RNA binding proteins studied. Main chain–Main chain (MM) CH⋯OC interactions are the predominant type of interactions in RNA binding proteins. The donor atom contribution to CH⋯OC interactions was mainly from aliphatic residues. The acceptor atom contribution for MM CH⋯OC interactions was mainly from Val, Phe, Leu, Ile, Arg and Ala. The secondary structure preference analysis of CH⋯OC interacting residues showed that, Arg, Gln, Glu and Tyr preferred to be in helix, while Ala, Asp, Cys, Gly, Ile, Leu, Lys, Met, Phe, Trp and Val preferred to be in strand conformation. Most of the CH⋯OC interacting polar amino acid residues were solvent exposed while, majority of the CH⋯OC interacting non polar residues were excluded from the solvent. Long and medium-range CH⋯OC interactions are the predominant type of interactions in RNA binding proteins. More than 50% of CH⋯OC interacting residues had a higher conservation score. Significant percentage of CH⋯OC interacting residues had one or more stabilization centers. Sixty-six percent of the theoretically predicted stabilizing residues were also involved in CH⋯OC interactions and hence these residues may also contribute additional stability to RNA binding proteins.     

Intramolecular C(sp3)H amination of arylsulfonyl azides with engineered and artificial myoglobin-based catalysts

The direct conversion of aliphatic CH bonds into CN bonds provides an attractive approach to the introduction of nitrogen-containing functionalities in organic molecules. Following the recent discovery that cytochrome P450 enzymes can catalyze the cyclization of arylsulfonyl azide compounds via an intramolecular C(sp³)H amination reaction, we have explored here the CH amination reactivity of other hemoproteins. Various heme-containing proteins, and in particular myoglobin and horseradish peroxidase, were found to be capable of catalyzing this transformation. Based on this finding, a series of engineered and artificial myoglobin variants containing active site mutations and non-native Mn- and Co-protoporphyrin IX cofactors, respectively, were prepared to investigate the effect of these structural changes on the catalytic activity and selectivity of these catalysts. Our studies showed that metallo-substituted myoglobins constitute viable CH amination catalysts, revealing a distinctive reactivity trend as compared to synthetic metalloporphyrin counterparts. On the other hand, amino acid substitutions at the level of the heme pocket were found to be beneficial toward improving the stereo- and enantioselectivity of these Mb-catalyzed reactions. Mechanistic studies involving kinetic isotope effect experiments indicate that CH bond cleavage is implicated in the rate-limiting step of myoglobin-catalyzed amination of arylsulfonyl azides. Altogether, these studies indicate that myoglobin constitutes a promising scaffold for the design and development of CH amination catalysts.     

Precursor De13.1 from Conus delessertii defines the novel G gene superfamily

Peptide de13a was previously purified from the venom of the worm-hunting cone snail Conus delessertii from the Yucatán Channel, México. This peptide has eight cysteine (Cys) residues in the unique arrangement CCCCCCCC, which defines the cysteine framework XIII (“” represents one or more non-Cys residues). Remarkably, δ-hydroxy-lysine residues have been found only in conotoxin de13a, which also contains an unusually high proportion of hydroxylated amino acid residues. Here, we report the cDNA cloning of the complete precursor De13.1 of a related peptide, de13b, which has the same Cys framework and inter-Cys spacings as peptide de13a, and shares high protein/nucleic acid sequence identity (87%/90%) with de13a, suggesting that both peptides belong to the same conotoxin gene superfamily. Analysis of the signal peptide of precursor De13.1 reveals that this precursor belongs to a novel conotoxin gene superfamily that we chose to name gene superfamily G. Thus far superfamily G only includes two peptides, each of which contains the same, distinctive Cys framework and a high proportion of amino acid residues with hydroxylated side chains.     

Unlocking nature’s CH bonds

In an idealistic setting, it can be imagined that if every CH bond on an organic molecule could be selectively functionalized, the fields of chemical synthesis and drug discovery would be forever revolutionized. With the purpose of investigating the practicality of this idealistic scenario, our group has endeavored to unlock the potential of nature’s CH bonds by developing palladium-catalyzed, site selective CH insertions that can be incorporated into both known and new catalytic cycles. To this end, we have developed a number of catalytic transformations that not only provide rapid diversification of simple starting materials and natural products through CH functionalization, but streamline the synthesis of a variety of natural products with biological activity and expand upon methods to access highly valuable enantiopure materials.     

Water-soluble N-carboxymethylchitosan derivatives: Preparation,characteristics and its application

In this work, only N-substituted chitosan derivatives (water-soluble N-carboxymethylchitosan derivatives: N-CMC) with different degrees of substitution were obtained by reaction of a fully deacetylated chitosan (derived from deacetylation of chitosan using decrystallized method) with monochloroacetic acid at pH 8 and temperature of 90 °C. The structure of N-carboxymethylchitosan and chitosan was characterized by IR, ¹H, ¹³C and ¹H–¹³C NMR-HSQC spectra. In the IR spectrum of the N-carboxymethylchitosan, the appearance of peak at 1742 cm^?1 was assigned for CO group of NHCH₂COOH of substituted chitosan. In the ¹H NMR spectra, the peaks at about 3.81÷4.06 ppm, assigned for CH₂ groups of NHCH₂ and N(CH₂)₂, were the major feature, while in the ¹H–¹³C NMR-HSQC spectra, signals of CH₂ confirmed the presence of these two different substituted CH₂ groups. The degree of substitution (DS) of N-monosubstitution (DS_N-mono) decreased from 0.47 to 0.03 meanwhile that of N,N-disubstitution (DS_N,N-di) increased from 0.52 to 0.96 since the mass ratio of chitosan/monochloroacetic acid changing from 1/1 to 1/4. The N-carboxymethylchitosan derivatives have been used for adsorption Cu(II) ion from aqueous solution. The results shown that the optimum conditions for adsorption Cu(II) ion in nitrate solution were pH 6.5, temperature of 30 °C, for 60–90 min and the substituted chitosan derivative having DS_N-mono of 0.16 and DS_N,N-di of 0.81 had maximum adsorption capacity of 192 mg Cu(II) per gram of N-CMC.     

Impacts of sulfur regulation in vivo on arsenic accumulation and tolerance of hyperaccumulator Pteris vittata

The impacts of the regulation of sulfur (S) metabolism in vivo on arsenic (As) and S species and on As accumulation by Pteris vittata L. were investigated using a synchrotron-based X-ray-absorption fine structure method. The S assimilation inhibitor l-buthionine-sulfoximine (BSO) markedly inhibited As reduction, doubling arsenate (As(V)) content in P. vittata rhizoids. The resulting As transport blockage in rhizoids, decreased As movement to aboveground tissues by 47%. The significant impact of BSO demonstrated the vital role of sulfhydryl groups (SH) as reductants in As(V) reduction and confirmed the importance of As(V) reduction in As accumulation in this fern. The S metabolism accelerant O-acetyl-l-serine resulted in the appearance of large amounts of As–SH in rhizoids and had no obvious impact on As accumulation, but with As stress conditions, effectively increased plant biomass, possibly through chelation of excess As with SH. Thus, SH appeared able to act as both a reductant and a chelator of As in P. vittata, and the ratio of SH to As may have been a factor that determined the specific role of SH in P. vittata under these conditions.     

Synthesis and characterization of cis-dioxomolybdenum(VI) complexes with sterically bulky tripodal tetradentate ligands

A new series of sterically bulky tripodal tetradentate ligands have been synthesized. The ligands were found to react readily with MoO ₂(acac) ₂ in absolute methanol to yield the corresponding cis- dioxoMo(VI) complexes. These complexes were characterized by IR, ¹H and ¹³C NMR, UV-Vis spectroscopy, elemental analysis and cyclic voltammetry (CV). Cyclic voltammetry was performed in DMF and CH ₂Cl ₂. In CH ₂Cl ₂ the ease of reduction was found to decrease in the order MoO ₂[LNO ₂ (SCH ₃)]> MoO ₂[LNO ₂(OCH ₃)]> MoO ₂ [LNO ₂ (N(CH ₃) ₂)]. The CV results are consistent with that expected for a S,O,N substitution pattern.     

Influences of conformations of peptides on stereoinversions and/or isomerizations of aspartic acid residues

Recently, non-enzymatic stereoinversions of aspartic acid (Asp) residues in proteins and peptides have been reported. Here, we performed replica exchange molecular dynamics (REMD) simulations of model peptides (exon 6, 26A-1, and 26A-2) extracted from elastin to investigate their structural features, thereby revealing the factor that influences stereoinversions. For REMD trajectories, we calculated distances between carboxyl carbon in Asp and amide nitrogen in the (n + 1) residue (CN distances). Because bond formation between carbon and nitrogen is indispensable to the formation of a succinimide intermediate the distance between them seems to play an important role in stereoinversion. Moreover, we calculated polar surface areas (PSAs) for the trajectories, finding that CN distances and PSA were different for each peptide, with the longest CN distance and smallest PSA observed for exon 6 peptide, where stereoinversion of Asp is the slowest. Although the average CN distance was shorter for exon 26A-1 peptide than for exon 26A-2 peptide, the number of conformations with CN distances <3.0 Å was greater for exon 26A-2 peptide than for exon 26A-1 peptide. Furthermore, PSA for amide nitrogen of the (n + 1) residue was larger for exon 26A-2 peptide than for exon 26A-1 peptide. These results indicated that the flexibility of Asp and (n + 1) residues and hydrophilicity of peptides, especially in the (n + 1) residue, play important roles in the stereoinversion of Asp. This article is part of a Special Issue entitled: D-Amino acids: biology in the mirror, edited by Dr. Loredano Pollegioni, Dr. Jean-Pierre Mothet and Dr. Molla Gianluca.     

Synthesis and characterization of two isomers of Os3(CO)10(C5H4N-2-C(H)NR) and the conversion to ortho-metallated HOs3(C5H3N-2-C(H)NR)(CO)9. Part III. X-ray Structure of HOs3(C5H3N-2-C(H)Ni-Pr)(CO)9

Os₃(CO)₁₀(MeCN)₂ reacts at room temperature in MeCN or toluene with R-Pyca2 to yield two isomers of Os₃(CO)₁₀(R-Pyca) that differ in the bonding of the R-Pyca ligand to the Os₃(CO)₁₀ unit. In all cases Os₃(CO)₁₀(R-Pyca(4e)) (isomer A; 4a: R = c-Pr, 4b: R = i-Pr, 4c: R = neo-Pent, 4d: R = t-Bu), containing a chelating 4e donating R-Pyca ligand and three OsS bonds, could be isolated. In the case of R = c-Pr and R = i-Pr Os₃(CO)₁₀(R-Pyca(6e)) (isomer B; 5a: R = c-Pr, 5b: R = i-Pr), in which only two OsS bonds are present and the R-Pyca ligand is bonded as a 6e donating ligand bridging two non-bonded Os atoms, could be isolated as a minor product.At 70 °C Os₃(CO)₁₀(R-Pyca(4e)) (4b and 4d) loses one carbonyl and the pyridine moiety of the R-Pyca ligand is ortho-metallated to form HOs₃(C₅H₃N-2-C(H)NR)(CO)₉ (6b: R = i-Pr and 6d: R = t-Bu). Under the same conditions Os₃(CO)₁₀(i-Pr-Pyca(6e)) (5b) reacts to Os₂(CO)₆(6e)) (7b) containing a bridging 6e donating ligands. The latter two reactions were followed with FT-IR spectroscopy in a high temperature IR cell.The structure of the complexes in solution have been studied by ¹H and ¹C NMR and IR spectroscopy. The stoichiometries of 4a and 5a were determined by FAB-mass spectrometry while an exact mass determination was carried out for 4a.The crystal structure of 6b has been determined. Crystal of 6b are monoclinic, space group P2₁/n, with a = 7.808(2),b = 17.613(3),c = 16.400(8)Å, β = 94.09(3)° and Z = 4. The structure was refined to R = 0.039. The molecule contains a triangular array of osmium atoms [Os(1)Os(2) = 2.898(2)Å, Os(1)Os(3) = 2.886(2)Åand Os(2)O(3) = 2.911(2)Å] and nine terminally bonded carbonyl ligands. The C₅H₃N-2-C(H)N-i-Pr ligand is chelate bonded to Os(2) with the pyridine and imine nitrogens atoms axially and equatorially coordinated respectively [Os(2)N(1) = 2.00(2)Åand Os(2)N(2) = 2.11(2)Å]. The i-Pr-Pyca ligand is ortho-metallated at C(1) and forms a four membered ring containing Os(2), Os(3), C(1) and N(1), the Os(3)C(1) distance being 2.12(2)Å. The hydride, which could not be located unequivocally from a difference Fourier map is proposed to bridge the Os(2)(3) bond on the basis of stereochemical considerations.     

Four- versus five-coordination in platinum(II) complexes of α-diimines and the crystal structure of [PtClMe(i-PrNCHCHNi-Pr)]

The complex [PtCIMe(i-PrNCHCHNi-Pr)] and its unstable five-coordinate ethylene adduct have been prepared and characterized by ¹H NMR. The crystal and molecular structure of the former has been determined. The complex crystallizes in the orthorhombic space group Pca2 ₁, with a = 12.138(6), b = 9.601(6), c = 10.586(6)Å, Z = 4. Refinement converged to a final R index of 0.059. The geometrical parameters of the structure are compared with those of a related complex and discussed in relation to the stability of the five-coordinate olefin adducts.     

Rational derivation of CETP self-binding helical peptides by π-π stacking and halogen bonding: Therapeutic implication for atherosclerosis

The human cholesteryl ester transfer protein (CETP) transfers cholesteryl ester from high-density lipoprotein (HDL) to other lipoproteins and has been established as an attractive target for reducing the risk of atherosclerosis. Here, an amphipathic α-helix peptide, namely SBH-peptide (⁴⁶⁵EHLLVDFLQSLS⁴⁷⁶), was derived from the C-terminal tail of CETP. The peptide exhibits self-binding capability towards the CETP. Crystal structure analysis, molecular dynamics (MD) simulations and ab initio electron correlation characterizations of CETP–SBH-peptide complex system revealed that the Phe471 residue plays a key role in SBH-peptide binding, which can form a π-π stacking with the Phe197 residue of CETP. In addition, substitution of the hydrogen atom H4 of Phe471 with halogen atoms, in particular the bromine atom Br4, can constitute a geometrically satisfactory halogen bonding with the oxygen atom O of CETP Ile193 residue. Fluorescence polarization assays substantiated that (i) mutation of the aromatic Phe471 to small Ala residue would impair the SBH-peptide affinity with K_d change from 10.5 to 26.4 μM, indicating that the π-π stacking should exist in Phe471⋯Phe197 adduct, and (ii) substitution with Br4 can considerably improve SBH-peptide affinity by ∼3-fold, but the SBH-peptide binding does not change essentially upon substitution with Br3 (a negative control that is theoretically unable to form the halogen bonding), indicating that the rationally designed halogen bonding should form between the Phe471(Br4) residue of SBH-peptide and the Ile193 residue of CETP protein.     

Vancomycin resistance: Modeling backbone variants with d-Ala-d-Ala and d-Ala-d-Lac peptides

To seek vancomycin analogs with broader antibacterial activity, effects of backbone modifications for the agylcon 2 on binding with d-Ala-d-Ala- and d-Ala-d-Lac-containing peptides were investigated by Monte Carlo/free energy perturbation (MC/FEP) calculations. The experimental trend in binding affinities for 2 with three tripeptides was well reproduced. Possible modifications of the peptide bond between residues 4 and 5 were then considered, specifically for conversion of the OCNH linkage to CH₂NH₂⁺ (6), FCCH (7), HCCH (8), and HNCO (9). The MC/FEP results did not yield binding improvements for 7, 8, and 9, though the fluorovinyl replacement is relatively benign. The previously reported analog 6 remains as the only variant that exhibits improved affinity for the d-Ala-d-Lac sequence and acceptable affinity for the d-Ala-d-Ala sequence.     

Characterizations of divalent lanthanoid iodides in tetrahydrofuran by UV-Vis,fluorescence and ESR spectroscopy

Spectrochemical properties of LnI ₂ (Ln = Sm, Eu, Yb) prepared with lanthanoid metal and diiodoethane in THF were characterized by means of UV—Vis, fluorescence and ESR spectroscopy. A comparative investigation has also been made concerning LnI ₃ (Ln= LaLu) and the Grignard-type compounds RLnI (R = Et, Ph; Ln = Eu, Yb). LnI ₂ (Ln = Sm, Eu, Yb) was also prepared from reaction of lanthanoid metal and iodine in THF.     

Radical scavenging and anti-inflammatory activities of (hetero)arylethenesulfonyl fluorides: Synthesis and structure-activity relationship (SAR) and QSAR studies

A series of (hetero)arylethenesulfonyl fluorides (1–58) were synthesized and screened for their in vitro antioxidant (DPPH, ABTS and DMPD methods) and anti-inflammatory activities. The results revealed that compounds 4, 15, 16, 24, 25, 26, 38, 39, 40, and 54 exhibited excellent antioxidant activity using all the three performed antioxidant methods, which were superior to the standard antioxidants ascorbic acid and gallic acid. Compounds 6–9, 11, 18, 19, 21, 22, 30, 39, 40, 44, 45, 48–50, 54, 55 and 57 displayed promising anti-inflammatory activity, which were better than the reference drug indomethacin. Preliminary structure–activity relationship (SAR) revealed that compounds containing electron donating (OH and OCH₃) groups on the phenyl ring possessed excellent antioxidant properties while compounds containing electron-withdrawing (Cl, NO₂, F and Br) groups on the phenyl ring were found to be most potent anti-inflammatory agents. The presence of SO₂F group played a crucial role in increases both antioxidant and anti-inflammatory activities.     

Interaction of a quercetin derivative - lensoside Aβ with liposomal membranes

Lensoside Aβ, representing the flavonol glycosides, is a compound isolated from the aerial parts of edible lentil (Lens culinaris) cultivar Tina. This substance arouses interest because so far there is very little data about secondary metabolites isolated from the leaves and stems of this plant. Additionally, bioactive potential of flavonoids is directly coupled with the membranes as a primary target of their physiological and pharmacological activity. The aim of this study was to investigate the effect of lensoside Aβ on lipid membranes. Interaction of examined compound with liposomes formed with dipalmitoylphosphatidylcholine (DPPC) was investigated with application of FTIR spectroscopy and ¹H NMR technique. Molecular localization and orientation of lensoside Aβ in a single lipid bilayer system represented by giant unilamellar vesicles, was also investigated with application of confocal fluorescence lifetime imaging microscopy (FLIM).FTIR analysis revealed that the tested compound incorporates into DPPC membranes via hydrogen bonding to lipid polar head groups in the PO₂ group region and the COPOC segment. Furthermore ¹H NMR analysis showed ordering effect in both the hydrophobic alkyl chains region and the polar heads of phospholipids. FLIM investigation has revealed roughly parallel orientation of its molecules in the membranes. This suggests that one of the possible physiological functions of this flavonol could be screening a cell against short-wavelength radiation.     

Enhancements of enantio and diastereoselectivities in reduction of (Z)-3-halo-4-phenyl-3-buten-2-one mediated by microorganisms in ionic liquid/water biphasic system

Reductions of (Z)-C₆H₅CHCXC(O)CH₃ (X = Cl, Br) mediated by Saccharomyces cerevisiae, Candida albicans, Rhodotorula glutinis, Geotrichum candidum and Micrococcus luteus gave the corresponding halohydrins through consecutive reduction reactions of CC and CO bonds. In general, the reactions performed in the biphasic system water/[(bmim)PF₆] gave better diastereoselectivity and enantioselectivity than in pure water.     

Influence of microbial associations on selenium localization and speciation in roots of Astragalus and Stanleya hyperaccumulators

Selenium (Se) hyperaccumulator plants can accumulate and tolerate Se up to 1% of their dry weight. Since little is known about below-ground processes of Se uptake and metabolism in hyperaccumulators, X-ray absorption spectromicroscopy was used to characterize the chemical composition and spatial distribution of Se in roots of Astragalus and Stanleya hyperaccumulators. Selenium was present throughout the roots, with the highest levels in the cortex. The main form of Se (48–95%) in both species collected from naturally seleniferous soil was an organic CSeC compound, likely methyl-selenocysteine. In addition, surprisingly high fractions (up to 35%) of elemental Se (Se⁰) were found, a form so far not reported in plants but commonly produced by Se-tolerant bacteria and fungi. Four fungi collected from hyperaccumulator roots were characterized with respect to their Se tolerance and ability to produce Se⁰, and then used to inoculate hyperaccumulators in a controlled greenhouse study. The roots of the greenhouse-grown Astragalus and Stanleya contained mainly CSeC; in most plants no Se⁰ was detected, with the exception of Astragalus nodules and roots of Astragalus inoculated with Alternaria astragali, an Se⁰-producing fungus. Apparently, Se⁰-producing endosymbionts including nitrogen-fixing bacteria and endophytic fungi or bacteria in the root can affect Se speciation in hyperaccumulator roots. Microbes that affect plant Se speciation may be applicable in phytoremediation and biofortification, especially if they are promiscuous and affect Se tolerance in crop species.     

Investigation of transition metal-catalyzed nitrene transfer reactions in water

Transition metal-catalyzed nitrene transfer is a powerful method for incorporating new CN bonds into relatively unfunctionalized scaffolds. In this communication, we report the first examples of site- and chemoselective CH bond amination reactions in aqueous media. The unexpected ability to employ water as the solvent in these reactions is advantageous in that it eliminates toxic solvent use and enables reactions to be run at increased concentrations with lower oxidant loadings. Using water as the reaction medium has potential to expand the scope of nitrene transfer to encompass a variety of biomolecules and highly polar substrates, as well as enable pH control over the site-selectivity of CH bond amination.