Influence of borate complexation on the electrophoretic behavior of 2-AA derivatized saccharides in capillary electrophoresis

A complete separation with baseline resolution of the 2-AA derivatized saccharides, including mono-, di-, and oligosaccharides, was achieved using 50 mM sodium phosphate-150 mM borate solution, pH 7.0 as running buffer by capillary electrophoresis. It was thought to be a result of the inclusion of 150 mM borate in the running electrolyte solution. The formation of borate complexes was observed by means of ¹¹B and ¹³C NMR spectroscopy and the electrophoretic mobilities of the various derivatives were calculated. It was found that steric factors play an important role in the stability of the formed borate complexes, which depends strongly on the configuration of the three vicinal hydroxyl groups at C-2, C-3, and C-4. 2-AA-Glc mainly forms stable 1,2-diester complexes with borate and 2-AA-Mal can form stable 1,2-monoesters. In turn, for 2-AA-Rib the formation of complexes is difficult to take place. The results implied that the configurational difference between the hydroxyl groups could cause the difference in formation of borate complexes leading to significant difference among saccharide molecules in their migration time on CE analysis.     

A novel,stereospecific synthesis of β-D-glucopyranosyl phosphate

In alkaline solution, periodate ions form complexes with three consecutive hydroxyl groups in an axial-equatorial-axial arrangement but not with three syn-axial hydroxyl groups. Borate ions form complexes with three syn-axial hydroxyl groups but not with the axeqax sequence. Accordingly, cis-inositol gives a 1,2,3-periodate and a 1,3,5-borate complex. Cations form complexes with either type of conformation, but complex formation provides little energy towards achieving it by ring inversion. Lanthanide ions have been used to establish the sites of complex formation.     

Reactions of biogenic amines with aqueous potassium dichromate. An application to the determination of dopamine.

The reaction of potassium dichromate with a series of phenols, aminophenols, catecholamines, indolealkylamines and metabolites of the latter two was studied. Reaction required the presence of aromatic ortho- or para-dihydroxy or -diamino groups. Potassium dichromate reacted not only with the vicinal hydroxyl groups of catecholamines but also with the 5-hydroxy group and the ring nitrogen in the indolealkylamine series. Reaction occurs immediately upon mixing the reagents; the colored products are insoluble in water and most common organic solvents. 3-O-methylated catecholamines and acids and 5-O-methylated indolealkylamines and acids did not react with dichromate. Physical and chemical data on the products of these reactions suggest lack of reaction with the side chain in the biogenic amines. A method using dichromate oxidation-products to determine dopamine concentrations in urine is presented.     

A comparative analysis of the interaction of borate ion with various polyols.

Although it is now generally accepted that borate ion, B(OH)₄^?, reacts with suitable polyols in aqueous solution to form two types of complexes with strongly acidic properties (1), several investigators have presented evidence to show the formation of two types of complexes is stoichiometrically unsatisfactory (2,3).Aside from its usefulness in structural studies, the reaction has important implications in numerous biochemical applications. Many compounds of biological importance contain hydroxyl groups in positions favorable for reaction with borate ion.As an extension of previous work (4) we report here the determination of solution stability constants of borate ion complexes with several biologically important polyols by means of a pH method and in two instances a calorimetric method.     

Isopropylidenation with 2,2-dimethoxypropane-N,N-dimethylformamide-p-toluenesulfonic acid selective substitution in certain aminocyclitols

The behavior of a variety of N-acetyl- and N-(benzyloxycarbonyl)-aminocyclitols with 2,2-dimethoxypropane-N,N-dimethylformamide-p-toluenesulfonic acid has been examined. Both cis- and trans-vicinal hydroxyl groups are readily bridged to give 1,3-dioxolanes. In one (sterically favorable) case, the reagent linked the nitrogen atom of an acetamido group with a vicinal hydroxyl group to give an N-acetyl-2,2- dimethyloxazolidine; this heterocyclic system is less labile to acid than are the 1,3-dioxolanes.     

The vicinal hydroxyl group is prerequisite for metal activation of Clostridium thermocellum acetylxylan esterase

Positional specificity of NodB-like domain of a multidomain xylanase U from Clostridium thermocellum (CtAxe) was investigated. Of three monoacetates of 4-nitrophenyl β-d-xylopyranoside the acetylxylan esterase domain showed a clear preference for the 2-acetate. Moreover, the enzyme was significantly activated by Co²⁺. Acetylated methyl β-d-xylopyranosides were deacetylated slightly better at position 3 than at position 2, suggesting that the enzyme binds the substrate with the small methyl aglycone also in the opposite orientation. Nevertheless, both positions 2 and 3 of methyl β-d-xylopyranoside were deacetylated much faster in the presence of the activating metal ion. In contrast, replacement of the hydroxyl group at either of these positions with fluorine or hydrogen, as well as acetylation of both positions, abolished the enzyme activity, regardless the absence or the presence of Co²⁺. Thus, the presence of the free vicinal hydroxyl group seems to be a prerequisite not only for an efficient deacetylation of position 2 or 3, but also for the activation of the enzyme with cobalt ion. The demonstrated involvement of the vicinal hydroxyl groups in the mechanism of deacetylation is in accord with 3-D structures of CtAxe as well as other CE4 metal-dependent deacetylases.     

Détermination des sites d'oxydation des dérivés du α-D-glucofuranose utilisés comme donneurs

The sites of oxidation, by catalytic transfer of H, of derivatives of 1,2-O-isopropylidene-α-D-glucofuranose suggest a regiospecific reaction. Compounds having vicinal hydroxyl groups at C-5 and C-6, or at C-3 and C-5, are oxidized at OH-5, whereas compounds having two hydroxyl groups at C-3 and C-6 or three hydroxyl groups give first aldehydes and then lactones.     

Phenolic O-methyltransferase from Lentinus lepideus (basidiomycete)

The fungus, Lentinus lepideus, produces crystalline methyl p-methoxycinnamate in stationary cultures. O-methylation and methyl ester formation of hydroxycinnamic acids were examined with enzyme preparations of the fungus. Using S-adenosylmethionine-¹⁴CH3, it was found that only the methyl esters of the hydroxycinnamic acids are substrates for O-methylation and not the free acids. Benzoic acids and their methyl esters are not substrates. The activity of the enzyme is p-specific and its specific activity decreases with increasing number of hydroxyl groups in the substrate. The same enzyme preparations catalyze the formation of the methyl ester of cinnamic acid from the free acid.     

Stereospecific complexation of uranyl ion with tartaric acids studied by NMR spectroscopy

A full pH range ¹H and ¹³C NMR study was performed on the complexation of UO₂²⁺ with (D,L)- and meso-tartaric acids, for variable concentrations and molar ratios, in comparison with (D)-tartaric acid. The main result is that, in spite of the already high number of complexes formed with the active ligand, an additional species occurs with the racemic mixture for which experimental evidence indicates a cyclic trimer structure. A smaller number of complexes is formed with meso-tartaric acid. Information on the conformation of bound ligand is also obtained.     

Relative reactivity of hydroxyl groups in inositol derivatives: role of metal ion chelation

O-Alkylation of myo-inositol derivatives containing more than one hydroxyl group via their alkali metal alkoxides (sodium or lithium) preferentially occurs at a hydroxyl group having a vicinal cis-oxygen atom. In general the observed selectivity is relatively higher for lithium alkoxides than for the corresponding sodium alkoxide. The observed regioselectivity is also dependent on other factors such as the solvent and reaction temperature. A perusal of the results presented in this article as well as those available in the literature suggests that chelation of metal ions by inositol derivatives plays a significant role in the observed regioselectivity. Steric factors associated with the axial or equatorial disposition of the reacting hydroxyl groups do not contribute much to the outcome of these O-alkylation reactions. These results could serve as guidelines in planning synthetic strategies involving other carbohydrates and their derivatives.     

Antifreeze glycoproteins from Antarctic fish. Inactivation by borate.

Antifreeze glycoprotein, which has previously been shown to be inactive in the presence of borate, migrates electrophoretically as the borate complex, presumably through formation of borate complexes with hydroxyl groups on the sugar side chains. Antifreeze glycoprotein (5 mg/ml) has been found to be completely active in the presence of 0.1 M borate at pH 7, but inactive at pH 9. A titration curve of pH versus the antifreeze activity of glycoprotein (5 mg/ml) in 0.1 M borate showed a progressive decrease in antifreeze activity as the pH was increased. Concomitant with decreases in activity were increases in binding of borate. At pH 9.0, nearly 2 mol of borate were complexed per glycotripeptide. Ultracentrifuge analyses showed similar molecular weights and laser quasi-elastic light scattering showed similar diffusions at pH 7.0 and 9.0 in borate and in the absence of borate. The binding of borate, rather than a change in conformation, is thus directly related to the loss of antifreeze activity. Alkaline borate also decreased hemagglutinating activity of Osage orange lectin and decreased the inhibition of the activity by the antifreeze glycoproteins.     

Localization of the carbohydrate residue of the S-layer glycoprotein from Clostridium thermohydrosulfuricum L111-69

The location of the covalently attached carbohydrate residue of the S-layer glycoprotein from Clostridium thermohydrosulfuricum L111-69 was determined by electron microscopical procedures after converting the hydroxyl groups of the carbohydrate chains into carboxyl groups by succinylation. The introduction of carboxyl groups was examined by labelling with polycationized ferritin (PCF; a net positively charged topographical marker for electron microscopy). Cyanogen bromide was used for activating vicinal hydroxyl groups of the carbohydrate chains which could then react with amino groups of amino carbonic acids or ferritin. The amount of covalently bound ferritin was determined by freeze-etching and UV-measurement. Both, succinylation experiments and covalent attachment of ferritin confirmed that at least a considerable portion of the carbohydrate residue must be located on the S-layer surface.     

Mode of action of acetylxylan esterase from Streptomyces lividans: a study with deoxy and deoxy-fluoro analogues of acetylated methyl beta-D-xylopyranoside

Streptomyces lividans acetylxylan esterase removes the 2- or 3-O-acetyl groups from methyl 2,4-di-O-acetyl- and 3,4-di-O-acetyl beta-D-xylopyranoside. When the free hydroxyl group was replaced with a hydrogen or fluorine, the rate of deacetylation was markedly reduced, but regioselectivity was not affected. The regioselectivity of deacetylation was found to be independent of the prevailing conformation of the substrates in solution as determined by 1H-NMR spectroscopy. These observations confirm the importance of the vicinal hydroxyl group and are consistent with our earlier hypothesis that the deacetylation of positions 2 and 3 may involve a common ortho-ester intermediate. Another possible role of the free vicinal hydroxyl group could be the activation of the acyl leaving group in the deacetylation mechanism. Involvement of the free hydroxyl group in the enzyme-substrate binding is not supported by the results of inhibition experiments in which methyl 2,4-di-O-acetyl beta-D-xylopyranoside was used as substrate and its analogues or methyl beta-D-xylopyranoside as inhibitors. The enzyme requires for its efficient action the trans arrangement of the free and acetylated hydroxyl groups at positions 2 and 3.     

X-ray studies on crystalline complexes involving amino acids and peptides: Part XVIII. Crystal structure of a new form of L-arginine D-glutamate and a comparative study of amino acid crystal structures containing molecules of the same and mixed chirality

The new form of L-arginine D-glutamate is monoclinic, P2₁, witha = 9.941(1),b = 4.668(2),c = 17.307(1) Å,β = 95.27(1)°, and Z = 2. In terms of composition, the new form differs from the old form in that the former is a monohydrate whereas the latter is a trihydrate. The structure has been solved by the direct methods and refined to R = 0.085 for 1012 observed reflections. The conformation of the arginine molecule is the same in both the forms whereas that of the glutamate ion is different. The change in the conformation of the glutamate ion is such that it facilitates extensive pseudosymmetry in the crystals. The molecules arrange themselves in double-layers stabilised by head-to-tail sequences involving main chains, in both the forms. However, considerable differences exist between the two forms in the interface, consisting of side chains and water molecules, between double-layers. A comparative study of the relationship between the crystal structures of L and DL amino acids on the one hand and that between the structures of LL and LD amino acid-amino acid complexes on the other, provides interesting insights into amino acid aggregation and the effect of chirality on it. The crystal structures of most hydrophobic amino acids are made up of double-layers and those of most hydrophilic amino acids contain single layers, irrespective of the chiralities of the amino acids involved. In most cases, the molecules tend to appropriately rearrange themselves to preserve the broad features of aggregation patterns when the chirality of half the molecules is reversed as in the structures of DL amino acids. The basic elements of aggregation in the LL and the LD complexes, are similar to those found in the crystals of L and DL amino acids. However, the differences between the LL and the LD complexes in the distribution of these elements are more pronounced than those between the distributions in the structures of L and DL amino acids.     

Conformational analysis of peracetylated hexononitriles

The conformations of six peracetylated hexononitriles in solution have been investigated by Fourier-transform, proton n.m.r. spectroscopy at 90 MHz, with iterative analysis and simulation of many of the spectra. The conformation of tetra-O-acetyl-L-arabinononitrile has been re-examined by the same methods. A shift reagent [Eu(fod)₃-d₃₀] and spectra at 220 MHz were used to improve spectral dispersion, where necessary. For practically all of the derivatives studied, the vicinal, proton-proton coupling-constants are consistent with a zigzag conformation in which the cyano group lies in the plane of the other carbon atoms of the chain, unless this conformation contains a parallel 1,3-interaction of substituents. Other conformers that are also consistent with the coupling constants observed are discussed, including rotamers about chain-terminal, carbon-carbon bonds.     

Trichoderma reesei CE16 acetyl esterase and its role in enzymatic degradation of acetylated hemicellulose

Background

Trichoderma reesei CE16 acetyl esterase (AcE) is a component of the plant cell wall degrading system of the fungus. The enzyme behaves as an exo-acting deacetylase removing acetyl groups from non-reducing end sugar residues.

Methods

In this work we demonstrate this exo-deacetylating activity on natural acetylated xylooligosaccharides using MALDI ToF MS.

Results

The combined action of GH10 xylanase and acetylxylan esterases (AcXEs) leads to formation of neutral and acidic xylooligosaccharides with a few resistant acetyl groups mainly at their non-reducing ends. We show here that these acetyl groups serve as targets for TrCE16 AcE. The most prominent target is the 3-O-acetyl group at the non-reducing terminal Xylp residues of linear neutral xylooligosaccharides or on aldouronic acids carrying MeGlcA at the non-reducing terminus. Deacetylation of the non-reducing end sugar may involve migration of acetyl groups to position 4, which also serves as substrate of the TrCE16 esterase.

Conclusion

Concerted action of CtGH10 xylanase, an AcXE and TrCE16 AcE resulted in close to complete deacetylation of neutral xylooligosaccharides, whereas substitution with MeGlcA prevents removal of acetyl groups from only a small fraction of the aldouronic acids. Experiments with diacetyl derivatives of methyl β-d-xylopyranoside confirmed that the best substrate of TrCE16 AcE is 3-O-acetylated Xylp residue followed by 4-O-acetylated Xylp residue with a free vicinal hydroxyl group.

General significance

This study shows that CE16 acetyl esterases are crucial enzymes to achieve complete deacetylation and, consequently, complete the saccharification of acetylated xylans by xylanases, which is an important task of current biotechnology.     

A study of the complexes of borate ions and some cyclitols using 11B-n.m.r. spectroscopy

Complexes between borate ion and cyclohexane-cis-1,2-diol, cyclohexane-cis,cis-1,3,5-triol, and myo- and epi-inositol have been investigated by ¹¹B-n.m.r. spectroscopy. Three different complexes of myo-inositol have been identified. Formation constants have been determined for the borate complexes of each cyclitol. Where the complex is formed from the less-stable chair conformer, MNDO calculations have been performed to determine the enthalpies of inversion. For myo-inositol, an iterative method of calculation gave a set of constants which provided a good match with experimental data and supported the proposed formulation of its borate complexes.     

Conformations of acyclic sugar derivatives : Part II. Determination of the conformations of alditol acetates in solution by the use of 250-MHz n.m.r. spectra

The p.m.r. spectra of all the fully acetylated pentitols and several fully acetylated hexitols have been analysed. Computation by iterative analysis and recourse to 250-MHz spectra were required in several cases. The vicinal coupling constants were used to determine the conformations of these compounds in solution. The planar zigzag conformation was found to be predominant only in those configurations (arabino, manno) which do not possess parallel 1,3-interactions between acetoxyl groups on alternate carbon atoms. The other compounds were found to be mixtures of two or more conformers, none of which has the planar zigzag conformation, except in the case of hexa-O-acetylallitol. The conformations of alditols in the crystalline state and of the alditol acetates in solution are compared.     

Free and bound hydroxyl and carboxyl groups in the cutin of Quercus suber leaves

The number of free and bound hydroxyl and carboxyl groups of the cutin of Quercus suber leaves was investigated by the lithium borohydride hydrogenolysis of mesyl-cutin compared with the lithium borohydride hydrogenolysis of untreated cutin. Fifty per cent of the vic-diol groups of the trihydroxy C₁₈ acid component and twenty five per cent of the secondary hydroxyl groups of the dihydroxy C₁₆ acid component are free. The rest of the secondary and all of the primary hydroxyl groups are esterified; all carboxyl groups are esterified.     

Synthesis, structural studies and solubility properties of zinc(II), nickel(II) and copper(II) complexes of bulky tris(triazolyl)borate ligands

Tris(triazolyl)borate (Ttz) ligands are sterically similar to tris(pyrazolyl)borate (Tp) but complexes of Ttz show improved solubility in water and alcohols due to their propensity for forming hydrogen bonds. Recently developed bulky tris(triazolyl)borate ligands can produce four and five coordinate transition metal complexes and serve as models for enzyme active sites in an aqueous environment. Herein we report the synthesis of such complexes, i.e. (Ttz^tBu,Me)ZnCl, (Ttz^tBu,Me)ZnBr, (Ttz^tBu,Me)NiCl, and (Ttz^tBu,Me)CuCl, which were analyzed by X-ray crystallographic and spectroscopic methods [Ttz^tBu,Me = tris(3-t-butyl-5-methyl-1,2,4-triazolyl)borate]. (Ttz^tBu,Me)ZnCl crystallizes as two different polymorphs with cubic and monoclinic symmetry. Both polymorphs of (Ttz^tBu,Me)ZnCl and (Ttz^tBu,Me)ZnBr have tetrahedral zinc atoms whereas the geometries at the metal in (Ttz^tBu,Me)NiCl and (Ttz^tBu,Me)CuCl are distorted tetrahedral. All complexes are methanol soluble and they also dissolve in methanol/water mixtures with up to 60% water.