Nuclear magnetic resonance spectroscopy of 3 beta,7 beta-dihydroxy-5-cholen-24-oic acid multi-conjugates: unusual bile acid metabolites in human urine

Complete ¹H and ¹³C resonance assignments were made for a new type of 3β,7β-dihydroxy-5-cholen-24-oic acid doubly conjugated with sulfuric acid at C-3 and N-acetylglucosamine at C-7 and its glycine- and taurine-amidated triple-conjugates by the combined use of several homonuclear and heteronuclear shift-correlated 2D NMR techniques. The effects of sulfation at C-3, N-acetylglucosaminidation at C-7, and aminoacyl amidation at C-24 on the ¹H and ¹³C chemical shifts and signal multiplicity were clarified. The shielding data serving to characterize each of the bile acid multi-conjugates are also discussed.     

Degradation of Hyaluronic Acid, Poly- and Mono-Saccharides, and Model Compounds by Hypochlorite: Evidence for Radical Intermediates and Fragmentation

Degradation of hyaluronic acid by oxidants such as HO^· and HOCl/ClO⁻ is believed to be important in the progression of rheumatoid arthritis. While reaction of hyaluronic acid with HO^· has been investigated extensively, reaction with HOCl/ClO⁻ is less well defined. Thus, little is known about the site(s) of HOCl/ClO⁻ attack, the intermediates formed, or the mechanism(s) of polymer degradation. In this study reaction of HOCl/ClO⁻ with amides, sugars, polysaccharides, and hyaluronic acid has been monitored by UV-visible (220–340 nm) and EPR spectroscopy. UV-visible experiments have shown that HOCl/ClO⁻ reacts preferentially with N-acetyl groups. This reaction is believed to give rise to transient chloramide (R—NCl—C(O)—R′) species, which decompose rapidly to give radicals via either homolysis (to produce N· and Cl·) or heterolysis (one-electron reduction, to give N· and Cl⁻) of the N—Cl bond. The nature of the radicals formed has been investigated by EPR spin trapping. Reaction of HOCl/ClO⁻ with hyaluronic acid, chondroitin sulphates A and C, N-acetyl sugars, and amides gave novel, carbon-centered, spin adducts, the formation of which is consistent with selective initial attack at the N-acetyl group. Thus, reaction with hyaluronic acid and chondroitin sulphate A, appears to be localized at the N-acetylglucosamine sugar rings. These carbon-centered radicals are suggested to arise from rapid rearrangement of initial nitrogen-centered radicals, formed from the N-acetyl chloramide, by reactions analogous to those observed with alkoxyl radicals. The detection of increasing yields of low-molecular-weight radical adducts from hyaluronic acid and chondroitin sulphate A with increasing HOCl/ClO⁻ concentrations suggests that formation of the initial nitrogen-centered species on the N-acetylglucosamine rings, and the carbon-centered radicals derived from them, brings about polymer fragmentation.     

Reaction of peroxynitrite with hyaluronan and related saccharides

The effects of peroxynitrite on hyaluronan has been studied by using an integrated spectroscopical approach, namely electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), and mass spectrometry (MS). The reaction has been performed with the polymer, the tetrasaccharide oligomer as well as with the monosaccharides N-acetylglucosamine and glucuronic acid. The outcome of the presence of molecular oxygen and carbon dioxide has been also evaluated. Although ₁H-NMR and ESI-MS experiments did not revealed peroxynitrite-mediated modification of hyaluronan as well as of related saccharides, from spin-trapping EPR experiments it was concluded that peroxynitrite induce the formation of C-centered carbon radicals, most probably by the way of its hydroxyl radical-like reactivity. These EPR data support the oxidative pathway involved in the degradation of hyaluronan, a probable event in the development and progression of rheumatoid arthritis.     

Identification of a wheat germ agglutinin-sensitive ATPase in yeast nuclei

We have found that wheat germ agglutinin (WGA), a lectin that specifically binds to N-acetylglucosamine residues inhibits the in vitro transport of plasmid DNA, pJDB219, into yeast nuclei. Histochemical staining of the isolated nuclei with biotinylated WGA and streptavidin-biotinylated peroxidase complex revealed the presence of WGA-binding materials around the nuclear pore under an electron microscope. Using WGA-agarose column chromatography of yeast nuclear extracts, a novel Mg²⁺-dependent ATPase was isolated. Its activity was highly sensitive to WGA and stimulated by Nonidet P-40 or phosphatidylserine. We suggest that the WGA-sensitive ATPase plays a role in yeast nuclear transport of DNA.     

Characteristic properties of N-Carboxybutyl chitosan

N-Carboxybutyl chitosans obtained from levulinic acid (4-oxo-pentanoic acid) and five crustacean chitosans (heteropolymers of N-acetylglucosamine and glucosamine) have been instrumentally characterized and found to have degree of N-carboxyalkylation 0·27. They dissolve in water and in water-ethanol mixtures without the need of any acid and give more viscous solutions than the corresponding chitosans. Their compatibility with other polymers and with salts has been surveyed, and the soluble chelates of Cr(III) and Pb(II) have been studied. The bacteriostatic activity of the N-carboxybutyl chitosans, together with other favorable properties, such as the viscosifying action, the enhanced film-forming ability, the moisturizing effect and the stabilization of emulsions, make these novel modified chitosans most suitable as functional cosmetic ingredients.     

H NMR study of the interaction of N,N′,N″-triacetyl chitotriose with Ac-AMP2, a sugar binding antimicrobial protein isolated from Amaranthus caudatus

The interaction between Ac-AMP2, a lectin-like small protein with antimicrobial and antifungal activity isolated from Amaranthus caudatus, and N,N′,N″-triacetyl chitotriose was studied using ¹H NMR spectroscopy. Changes in chemical shift and line width upon increasing concentration of N,N′,N″-triacetyl chitotriose to Ac-AMP2 solutions at pH 6.9 and 2.4 were used to determine the interaction site and the association constant K_a. The most pronounced shifts occur mainly in the C-terminal half of the sequence. They involve the aromatic residues Phe¹⁸, Tyr²⁰ and Tyr²⁷ together with their surrounding residues, as well as the N-terminal Val-Gly-Glu segment. Several NOEs between Ac-AMP2 and the N,N′,N″-triacetyl chitotriose resonances are reported.     

Synthesis of 2-(2-adamantyl)piperidines and structure anti-influenza virus A activity relationship study using a combination of NMR spectroscopy and molecular modeling

The 2-(2-adamantyl)piperidines 13 and 15a-c were synthesized and evaluated for anti-influenza virus A and B activity. The parent N-H compound 13 was 3–4 times more active than amantadine and rimantadine against H₂N₂ influenza A. N-alkylation of 13 resulted in derivatives 15a-c that were devoid of biological activity. This dramatic reduction in biological activity may be attributed to the different conformational properties between N-H and N-alkyl piperidines, as deduced from the combination of computational chemistry and NMR spectroscopy.     

Screening for plant lectins by latex agglutination tests

The latex agglutination test has been applied as a detection system for lectins, the method being especially useful in locations where the dependence on blood for hemagglutination tests could be minimised. The binding of various glycoproteins and sugars individually to the latex particles facilitated the agglutination with lectins having varying sugar specificities. The glycoproteins used were ovalbumin, horseradish peroxidase, porcine mucin and fetuin, while N-acetylglucosamine, N-acetylgalactosamine comprised the sugars used for binding to latex. The sensitivity of the latex agglutination tests was comparable with that of hemagglutination tests. Sugar binding specificity of the lectins could also be determined by inhibition of the agglutination in the presence of corresponding free sugars. The method proved to be useful in screening crude seed extracts for the presence of lectins.     

Stereognostic coordination chemistry 4 the design and synthesis of a selective uranyl ion complexant

A new approach to ligand design for the sequestration of metal-oxo cations has been called stereognostic coordination chemistry, in that the ligand incorporates a traditional Lewis base coordination to the metal center and a hydrogen bond donor to interact with the oxo group. This paper reports the synthesis of ligands that are more rigid and sterically predisposed to bind the targeted UO₂²⁺ cation. These are the tripod ligands tris-N,N′,N′′-[2-(2-carboxy-phenoxy)ethyl]-1,4,7-triazacyclononane bis-hydrochloride (ETAC · 2HCl) and tris-N,N′,N′′-[2-(2-carboxy-4-decyl-phenoxy)ethyl]-1,4,7-triazacyclononane tris-hydrochloride (DETAC · 3HCl), which chelate uranyl with a tris-carboxylate coordination sphere and provide a hydrogen bond donor through a protonated amine on the triazacyclononane macrocycle to interact with one uranyl oxo atom. Structural models predict that upon uranyl binding the hydrogen bond donor must point directly towards the oxo atom, enforcing a stereognostic interaction. Both ETAC and DETAC chelate the uranyl ion; DETAC is a powerful extractant and will quantitatively extract uranyl into an organic phase at pH 1.9 and above. The extraction coefficient is estimated to be 10¹⁴ in neutral aqueous conditions. Vibrational spectra of ¹⁸O labeled UO₂²⁺ have been used to probe the stereognostic coordination to uranyl utilizing hydrogen bonding.     

Molecular dynamics of N,N′-di-p-fluorophenyltriazenido complex of platinum (II)

The triazenide complex of Pt(II) trans-(o-Tol)Pt(PEt₃)₂N₃Ar₂(1) (Ar = p-FC₆H₄) was synthesized by reaction of (o-Tol)Pt(PEt₃)₂BF₄ with Ar₂N₃Na. The ¹H, ¹⁹F and ³¹P NMR spectra of this complex in toluene-d₈ were studied at different temperatures. Two kinds of dynamic processes were observed. The first one is the intramolecular N,N′ migration of the (o-Tol)Pt(PEt₃)₂ group, detected by ¹⁹F NMR. The second process, revealed by ¹H, ¹⁹P NMR, is the rotation around the partially double N(2)–N(3) bond. Thermodynamic parameters for these processes were calculated from dynamic NMR spectra.     

Preparation of sugarcane bagasse hemicellulosic succinates using NBS as a catalyst

The chemical modification of native sugarcane bagasse hemicelluloses with succinic anhydride using N-bromosuccinimide as a catalyst and N,N-dimethylacetamide/lithium chloride system as solvent was studied. The parameters optimised included succinic anhydride concentration by the molar ratio of succinic anhydride/anhydroxylose units in native hemicelluloses from 1:1 to 9:1, reaction time 0.5–6 h, NBS concentration 0.5–3.0%, and reaction temperature 25–85 °C required in the process. Results were also compared with other catalysts such as pyridine, DMAP, H₂SO₄, and other two tertiary amine catalysts, N-methyl pyrrolidine, and N-methyl pyrrolidinone. The degree of substitution of succinylated hemicelluloses ranged between 0.19 and 1.39, depending on the experimental conditions. FT-IR and ¹H and ¹³C NMR spectroscopic characterization of the esterified polymers indicated a monoester substitution. The thermal stability of the succinylated hemicelluloses decreased upon chemical modification.     

Synthesis and NMR structural analysis of O-succinyl derivative of low-molecular-weight κ-carrageenan

Low-molecular-weight (LMW) κ-carrageenan was achieved through mild hydrochloric acid hydrolysis of κ-carrageenan. The acylation of LMW κ-carrageenan was performed by use of tetrabutylammonium (TBA) salt of the anionic polysaccharide fragments, succinic anhydride, 4-dimethylaminopyridine and tributylamine under homogeneous conditions in N,N-dimethylformamide at 80 °C. Investigation of FT-IR spectrum of the succinylated LMW κ-carrageenan showed that a monoester derivative with succinyl group was formed when LMW κ-carrageenan reacted with succinic anhydride. The ¹H and ¹³C NMR spectroscopy has been used to characterize the fine structure of O-succinyl derivative of the LMW κ-carrageenan. The ¹³C and ¹H NMR chemical shifts of disaccharide unit of O-succinyl LMW κ-carrageenan have been fully assigned using 2D NMR spectroscopic techniques.     

Preparation, spectral characterization, cytotoxic and thermal studies of platinum (IV) thiohydrazone complexes

Platinum (IV) complexes of type [Pt(L)₂Cl₂] [where, L=benzaldehyde-N,N-diphenyl-N-thiohydrazone (L¹), salicylaldehyde-N,N-diphenyl-N-thiohydrazone (L²), acetaphenone-N,N-diphenyl-N-thiohydrazone (L³) and cyclohexanone-N,N-diphenyl-N-thiohydrazone (L⁴)] have been synthesized. The thiohydrazones can exist as thione–thiol tautomer and coordinates as a bidentate N–S ligand. The ligands found to act in monobasic bidentate fashion. Analytical data reveals that metal to ligand stoichiometry is 1:2. The complexes have been characterized by elemental analysis, IR, mass, electronic, ¹H NMR spectroscopic studies, and TG/DTA study. Various kinetic and thermodynamic parameters like order of reaction (n), activation energy (E_a), apparent activation entropy (S^#) and heat of reaction (ΔH) have also been carried out for one complex. Cytotoxic study has also been carried out for one complex.     

Synthesis of graft copolymer (k-carrageenan-g-N,N-dimethylacrylamide) and studies of metal ion uptake, swelling capacity and flocculation properties

Graft copolymer of k-carrageenan and N,N-dimethylacrylamide has been synthesized by free radical polymerization using peroxymonosulphate/glycolic acid redox pair in an inert atmosphere. The grafting parameters i.e. grafting ratio, add on and efficiency decrease with increase in concentration of k-carrageenan from 0.6 to 1.4 g dm⁻³ and hydrogen ion from 3 × 10⁻³ to 7 × 10⁻³ mol dm⁻³, but these grafting parameters increase with increase in concentration of N,N-dimethylacrylamide from 16 × 10⁻² to 32 × 10⁻² mol dm⁻³, and peroxymonosulphate from 0.8 × 10⁻² to 2.4 × 10⁻² mol dm⁻³. The metal ion sorption, swelling behaviour and flocculation properties have been studied. The intrinsic viscosity of pure and grafted samples has been measured by using Ubbelohde capillary viscometer. Flocculation capability of k-carrageenan and k-carrageenan-g-N,N-dimethylacrylamide for both coking and non-coking coals has been studied for the treatment of coal mine waste water. The graft copolymer has been characterized by Infrared (IR) spectroscopy and thermogravimetric analysis.     

Hydrogen bonding in nucleosides and nucleotides

An analysis of the hydrogen bonding in 76 nucleoside and 11 nucleotide crystal structures shows that the hydrogen bond lengths fall into well-defined categories according to the nature of the donor or acceptor groups. The shortest bonds are those involving P---OH or O=P groups. For donor groups, the sequence in bond lengths is

P—OH<C—OH< N−H<O_w(H)—H<N(H)—H<C−H

There are ten examples of two centre

H—H…O

bonds, which are comparable in length with P---OH …O bonds. The acceptor seqeunce is

O=P<OH₂<OH₂<O=CO(H)C<N N(H₂)C<Cl^…<O<S=C

The number of three-centre bonds, about 24%, is comparable to that observed in the carbohydrates and the amino acids. Most hydrogen bonds are involved in short finite chains. Only in the nucleotides are cyclic hydrogen bonding schemes observed.     

Synthesis and conformational analysis of Aib-containing peptide modelling for N-glycosylation site in N-glycoprotein

A tetrapetide containing an Aib residue, Boc-Asn-Aib-Thr-Aib-OMe, was synthesized as a peptide model for the N-glycosylation site in N-glycoproteins. Backbone conformation of the peptide and possible intramolecular interaction between the Asn and Thr side chains were elucidated by means of n.m.r. spectroscopy. Temperature dependence of NH proton chemical shift and NOE experiments showed that Boc-Asn-Aib-Thr-Aib-OMe has a tendency to form a β-turn structure with a hydrogen bond involving Thr and Aib⁴ NH groups. Incorporation of Aib residues in the peptide model promotes folding of the peptide backbone. With folded backbone conformation, carboxyamide protons of the Asn residue are not involved in hydrogen bond network, while the OH group of the Thr residue is a candidate for a hydrogen bond in DMSO-d₆ solution.     

1-Alkyl-3-amino-5-aryl-1H-[1,2,4]triazoles: novel synthesis via cyclization of N-acyl-S-methylisothioureas with alkylhydrazines and their potent corticotropin-releasing factor-1 (CRF(1)) receptor antagonist activities.

Cyclizations of alkylhydrazines with N-acyl-S-methylisothioureas, readily synthesized from acyl chlorides, sodium thioisocyanate, dialkylamines then methyl iodide in a one-pot reaction, gave 1-alkyl-3-dialkylamino-5-phenyltriazoles 7 as major products. The regioisomers were assigned through the use of NOE NMR experiments. While bearing a N-bis(cyclopropyl)methyl-N-propylamino group, this series of compounds shows very good binding affinity on the human CRF₁ receptor. Among them, 1-methyl-3-[N-bis(cyclopropyl)methyl-N-propylamino]-5-(2,4-dichlorophenyl)-1H-[1,2,4]triazole 7a had the best binding affinity for the CRF₁ receptor (K_i=9 nM).     

Conformational characterization of square planar nickel(II) tetraaza macrocyclic complexes by proton NMR. Crystal structure of [Ni(13aneN4)]ZnCl4

Conformations in solution of several diamagnetic nickel(II) complexes of macrocyclic tetraaza ligands are elucidated using proton NMR. There are six possible configurational isomers of planar [Ni(13aneN₄)]²⁺ (13aneN₄ = 1,4,7,10-tetraazacyclotridecane due to the orientation of the N---H protons above or below the plane of the macrocyle. Using NMR it is shown that in aqueous solution the [Ni(13aneN₄)]²⁺ complex has the R,S,R,S or trans-II configuration. A single-crystal X-ray study demonstrates the same configuration of the nitrogen atoms in the complex [Ni(13aneN₄)]ZnCl₄. In the case of the 14-membered ring macrocyle cyclam (cyclam = 1,4,8,11-tetraazacyclotetradecane), previous NMR studies revealed the presence, in aqueous solution, of the previously unobserved trans-I or R,S,R,S isomer, whose spectrum is examined in greater detail here. Solution structures of nickel(II) complexes of bicyclam (1,5,8,12-tetraazabicyclo[10.2.2]hexadecane) and dachden (N, N′-bis(2-aminoethyl)-1,4-diazacycloheptane) are also reported.     

The structure of the O-antigenic chain of the lipopolysaccharide of Rhizobium trifolii 4s

The structure of the O-antigen chain of the lipopolysaccharide (LPS) of Rhizobium trifolii 4s has been determined by a combination of chemical and spectroscopic methods. The glycosyl components were found to be -rhamnose, N-acetyl- -glucosamine, and N-acetyl- -mannosamine in 3:1:1 molar proportion, as determined by gas chromatography and gas chromatography-mass spectrometry of alditol acetate and persilylated (R)-2-hydroxybutyl glycoside derivatives. The linkage positions and configurations of the glycosyl residues were obtained by 1D and 2D NMR spectroscopy. The polymer has a pentasaccharide repeating-unit containing rhammose and N-acetylglucosamine in the main chain and N-acetylmannosamine as the sole-side chain component. This latter residue is linked to a main-chain rhamnose residue. This result was suggested by NMR spectroscopy and confirmed by periodate oxidation. The sequence was deduced by 1D and 2D NMR NOE experiments and by partial hydrolysis studies. The repeating unit of the polysaccharide is shown. This constitutes the first complete structure of an O-antigenic chain of the lipopolysaccharide of any strain of Rhizobium trifolii.

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Modified chitosan carrying phosphonic and alkyl groups

The introduction of an alkyl chain onto a water soluble modified chitosan (N-methylene phosphonic chitosan) offer the presence of hydrophobic and hydrophilic branched for controlling solubility properties. A simple methodology for the preparation of a new chitosan derivative surfactant, N-lauryl-N-methylene phosphonic chitosan, has been developed. Its chemical identity was determined by FTIR and confirmed by ¹H and ¹³C NMR. The degree of lauryl substitution was estimated to be 0.33. As a result of the amphiphilic properties, like surface activity typical for surfactants, this derivative opens new perspectives in pharmaceutical and cosmetic field.