Toward the assembly of heparin and heparan sulfate oligosaccharide libraries: efficient synthesis of uronic acid and disaccharide building blocks

The monosaccharide moieties found in heparin (HP) and heparan sulfate (HS), glucosamine and two kinds of uronic acids, glucuronic and iduronic acids, were efficiently synthesized by use of glucosamine hydrochloride and glucurono-6,3-lactone as starting compounds. In the synthesis of the disaccharide building block, the key issues of preparation of uronic acids (glucuronic acid and iduronic acid moieties) were achieved in 12 steps and 15 steps, respectively, without cumbersome C-6 oxidation. The resulting monosaccharide moieties were utilized to the syntheses of HP/HS disaccharide building blocks possessing glucosamine-glucuronic acid (GlcN-GlcA) or iduronic acid (GlcN-IdoA) sequences. The disaccharide building blocks were also suitable for further modification such as glycosylation, selective deprotection, and sulfation.     

Direct and specific recognition of glycosaminoglycans by antibodies after their separation by agarose gel electrophoresis and blotting on cetylpyridinium chloride-treated nitrocellulose membranes

A method for the immunodetection of several natural complex polysaccharides (glycosaminoglycans) after their separation by conventional agarose gel electrophoresis, blotting and immobilizing on nitrocellulose membranes derivatized with the cationic detergent cetylpyridinium chloride (CPC), and direct and specific immunodetection by antibodies is described. This new approach is based on the principles that were used to develop the Western blot, and is applied to the separation of the glycosaminoglycans purified from normal human urine. After migration in agarose gel electrophoresis, chondroitin sulfate samples of different origin were blotted and transferred onto nitrocellulose membranes treated with CPC. Immunodetection was performed using the anti-chondroitin-6-sulfate antibody that specifically recognizes intact chondroitin-6-sulfate. By calculating the ratio between the antibody staining (epitope) and alcian blue staining (mass), the epitope density expressed as a percentage, i.e., the number of repetitive epitopes per mass, was obtained. These values were in agreement with the quantitation of 6-sulfated groups of chondroitin sulfate performed by the evaluation of unsatured disaccharide-6-sulfate (DeltaDi6S) produced after treatment with chondroitinase ABC and separated by high-performance liquid chromatography (HPLC). Furthermore, immunodetection of heparan sulfate was performed using the anti-heparan sulfate antibody.     

Advances in the separation,sensitive detection,and characterization of heparin and heparan sulfate

Elucidation of the relationship between the structure and biological function of the glycosaminoglycans (GAGs) heparin and heparan sulfate (HS) presents an important analytical challenge mainly due to the difficulty in determining their fine structure. Heparin and HS are responsible for mediation of a wide range of biological actions through specific binding to a variety of proteins including those involved in blood coagulation, cell proliferation, differentiation and adhesion, and host–pathogen interactions. Therefore, there is a growing interest in characterizing the microstructure of heparin and HS and in elucidating the molecular level details of their interaction with peptides and proteins. This review discusses recent developments in the analytical methods used for sensitive separation, detection, and structural characterization of heparin and HS. A brief discussion of the analysis of contaminants in pharmaceutical heparin is also presented.     

PhBCl2 promoted reductive opening of 2,4-O-p-methoxybenzylidene: new regioselective differentiation of position 2 and 4 of α-l-iduronyl moieties in disaccharide building blocks

We describe a new protocol for the challenging differentiation of the position 2^′ and 4^′ of l-iduronyl moieties located at the nonreducing end of various disaccharide building blocks. This methodology is based on the introduction of a 2^′,4^′-O-p-methoxybenzylidene group, followed by a totally regioselective reductive opening of this acetal by the PhBCl₂/Et₃SiH reagent system. l-Iduronyl moieties protected by a 4^′-O-p-methoxybenzyl group were thus obtained regioselectively and efficiently.     

Late-Stage Functionalisation of Peptides on the Solid Phase by an Iodination-Substitution Approach

The functionalisation of peptides at a late synthesis stage holds great potential, for example, for the synthesis of peptide pharmaceuticals, fluorescent biosensors or peptidomimetics. Here we describe an on-resin iodination-substitution reaction sequence on homoserine that is also suitable for peptide modification in a combinatorial format. The reaction sequence is accessible to a wide range of sulfur nucleophiles with various functional groups including boronic acids, hydroxy groups or aromatic amines. In this way, methionine-like thioethers or thioesters and thiosulfonates are accessible. Next to sulfur nucleophiles, selenols, pyridines and carboxylic acids were successfully used as nucleophiles, whereas phenols did not react. The late-stage iodination-substitution approach is not only applicable to short peptides but also to the more complex 34-amino-acid WW domains. We applied this strategy to introduce 7-mercapto-4-methylcoumarin into a switchable Zn^II responsive WW domain to design an iFRET-based Zn^II sensor.     

Comparative Study of the Reactivity of Iodinating Agents in Solution and Solid Phase

The results of reactions of a series of aromatic substrates with iodine, iodine(I) chloride, and N-iodosuccinimide in solution and solid phase were compared for the first time. In all cases, the general relations holding in the iodination process were similar. Iodine(I) chloride was found to chlorinate anthracene. A high efficiency of solid-phase iodination of β-diketones was demonstrated using dibenzoylmethane as an example.__________Translated from Zhurnal Organicheskoi Khimii, Vol. 41, No. 6, 2005, pp. 876–880.Original Russian Text Copyright © 2005 by Krasnokutskaya, Lesina, Gorlushko, Filimonov.     

超声提取/固相萃取测定固体介质中合成麝香及有机磷酸酯阻燃剂/增塑剂

基于超声提取与固相萃取技术,建立了沉积物和污泥中合成麝香与有机磷酸酯增塑剂/阻燃剂联合前处理方法,并采用气相色谱-质谱对目标化合物进行定性定量分析。考察了不同溶剂对目标化合物萃取效率的影响,确定以丙酮与乙腈-水(1∶3)的组合作为超声萃取溶剂。研究了基质效应和老化效应对目标化合物回收率的影响。结果表明,不同加标浓度下,4种常见合成麝香和9种常用有机磷酸酯的回收率为(27%±12%)~(117%±9%)。     

氨基酸和多肽在碱性硅胶载体上的固相衍生研究

为进行复杂体系中痕量生理活性物质 (如氨基酸和多肽等 )的高灵敏度分析 ,往往需要对其进行柱前或柱后的荧光衍生 -高效液相色谱或毛细管电泳分析 [1] .在一个存在着竞争反应的体系中 ,为保证样品有足够的反应产率 ,往往需使衍生试剂过量很多 ,这就使得衍生后的样品中必然含有高浓度的衍生试剂及其水解后形成的副产物 ,从而大大地干扰了分离与分析 .为解决这一问题 ,通常可采取溶剂萃取[2～ 5] 或加入 1 -金刚烷胺 [6 ,7] 或羟胺 [8,9] 等方法除去过量试剂 .但这些额外的处理使衍生方法更加烦琐 ,有时还导致收率的降低 .也有使用固相化的衍…     

离子交换固相比色法测定氰化物浸液中的微量金

本文采用Ｄ２９０大孔强碱性阴离子交换树脂首先预富集王水介质中的微量金，然后在ｐＨ３．２条件下用硫代米酮（ＴＭＫ）显色，从而建立了固相比色法测定微量金的方法，应用于金矿石氰化物浸液中微量金的测定，结果令人满意。     

A Simple Approach Toward Solution of the Problems of Signal Instability and Interferences Observed with Spectrophotofluorometers

Abstract

Unexpected fluorescence signal changes observed by analysts are frequently due to lamp instability and interferences from other equipment near the fluorometer; The addition of a constant voltage transformer between the line and the power supply is an inexpensive and simple procedure to provide long term stability of the lamp, which leads to more reliable use of the fluorometer. Interference problems were significantly reduced by adding a filter system to the instrument.

The problem of long term stability and interferences from nearby electronic instrumentation are frequently observed by individuals using spectrophotofluorometers. Unexpected fluorescence intensity differences for samples which are believed to contain similar quantities of an analyte are an outward manifestation of these problems, which may lead to the incorrect report of the concentration of the compound of interest. We have observed such difficulties in a study of the effect of solvent composition on the luminescence of tyrosine and tryptophan (which will be reported separately) and would like to report our difficulties and the remedies to the problem, We feel that this situation is quite widespread, based on informal discussions with other individuals.

The observed fluorescence signal due to a given solution was found to vary quite drastically (up to ± 20%) as a cuvet containing an identical sample was placed in the sample chamber at various times during a day. In some cases, such drastic changes could be observed with measurements taken 15 minutes apart. The changes were not due to solute decomposition, solvent interaction with the solute, photochemical decomposition or thermal decomposition of the solute, as these changes were not reproducible, and appeared to be independent of the solute and the solvent composition.

A block of MgO was placed in the sample compartment and the fluorometer was set up for front surface emission measurement, so that the refleactance could be monitored over a period of time. The reflectance intensity therefore mirrored the lamp intensity, and any change in lamp intensity would appear as a change in the reflectance observed as a function of time. It was observed that random changes in intensity did take place, with such changes occurring over a very short time period. The reflectance intensity changes were quite random with respect to time, and individual shifts were often as much as ± 10%.

When we turned the Xe lamp off and placed a flashlight bulb powered by a 6V lantern battery in the sample chamber, the light measured by the photomultiplier did not exhibit any abrupt changes over a period of 16 hours. This confirmed that the changes in signal intensity observed with the various samples and the reflectance experiments were due to changes in the intensity of the xenon lamp. It was determined that variations in the observed reflectance could be correllated with variations in line voltage.

Our instrument is used in a large laboratory with a number of other instruments within 5 meters. We observe that noise from “external sources” was bothersome, especially on higher amplification steps of the photomultiplier amplifier. In general, the variation in signal was ± 3% full scale at the highest amplification.

We report below our solutions to the problems described above as we feel that our solutions are of interest to those using spectrophotofluorometers.     

Studies on the Effects of Salt and Surfactant in Wet Phase Separation of Polysulfone

This study investigates the effect of additives in the nonsolvent water in terms of cloud point during the phase inversion of Polysulfone (PS) in dimethyl formamide (DMF). The exponential pattern is observed with PS concentration (0.2 to 0.6% (w/w)). It needs a low amount of water to get the cloud point at low temperature. The cloud point varied with the nature of water matrix and depended on the amount of salt, as well as the PS amount. The presence of salts (sodium chloride and sodium sulfate) lowers the cloud point of the solution. The network distribution of the particles at the cloud point is disturbed in the presence of salt. The requirement is more for Sodium lauryl sulfate (SLS) added water to reach the cloud point in the low range of PS solution up to 0.3% PS (w/w). The morphological and distribution pattern of PS particles are very different compared to PS particles produced from pure water. XRD study of PS particles produced from the mixed water system reflects relatively more amorphous character with respect to PS particles from pure water. The presence of both surfactant and salts in water systems also influences the cloud point in synergistic manner.     

Halogen‐Bonded Supramolecular Capsules in the Solid State,in Solution,and in the Gas Phase

Supramolecular capsules were assembled by neutral halogen bonding (XB) and studied in the solid state, in solution, and in the gas phase. The geometry of the highly organized capsules is shown by an X‐ray crystal structure which features the assembly of two XB hemispheres, geometrically rigidified by H‐bonding to eight MeOH molecules and encapsulation of two benzene guests. To enhance capsular association strength, tuning the XB donor is more efficient than tuning the XB acceptor, due to desolvation penalties in protic solvents, as shown for a tetraquinuclidine XB acceptor hemisphere. With a tetra(iodoethynyl) XB donor and a tetralutidine XB acceptor, the association in deuterated benzene/acetone/methanol 70:30:1 at 283 K reaches K _a=(2.11±0.39)×10⁵ m ⁻¹ (ΔG =−6.9±0.1 kcal mol⁻¹). The stability of the XB capsules in the gas phase was confirmed by electrospray ionization mass spectrometry (ESI‐MS). A new guest binding site was uncovered within the elongated iodoethynyl capsule.     

Multiple Multidentate Halogen Bonding in Solution,in the Solid State,and in the (Calculated) Gas Phase

The binding properties of neutral halogen‐bond donors (XB donors) bearing two multidentate Lewis acidic motifs toward halides were investigated. Employing polyfluorinated and polyiodinated terphenyl and quaterphenyl derivatives as anion receptors, we obtained X‐ray crystallographic data of the adducts of three structurally related XB donors with tetraalkylammonium chloride, bromide, and iodide. The stability of these XB complexes in solution was determined by isothermal titration calorimetry (ITC), and the results were compared to X‐ray analyses as well as to calculated binding patterns in the gas phase. Density functional theory (DFT) calculations on the gas‐phase complexes indicated that the experimentally observed distortion of the XB donors during multiple multidentate binding can be reproduced in 1:1 complexes with halides, whereas adducts with two halides show a symmetric binding pattern in the gas phase that is markedly different from the solid state structures. Overall, this study demonstrates the limitations in the transferability of binding data between solid state, solution, and gas phase in the study of complex multidentate XB donors.     

Comparison of the Reactivity of the Three Distonic Isomers of the Pyridine Radical Cation Toward Tetrahydrofuran in Solution and in the Gas Phase

The reactivity of the three distonic isomers of the pyridine radical cation toward tetrahydrofuran is compared in solution and in the gas phase. In solution, the distonic ions were generated by UV photolysis at 300 nm from iodo-precursors in acidic 50:50 tetrahydrofuran/water solutions. In the gas phase, the ions were generated by collisionally activated dissociation (CAD) of protonated iodo-precursors in an FT-ICR mass spectrometer, as described in the literature. The same major reaction, hydrogen atom abstraction, was observed in solution and in the gas phase. Attempts to cleave the iodine atom from the 2-iodopyridinium cation in the gas phase and in solution yielded the 2-pyridyl cation in addition to the desired 2-dehydropyridinium cation. In the gas phase, this ion was ejected prior to the examination of the desired ion’s chemical properties. This was not possible in solution. This study suggests that solvation effects are not significant for radical reactions of charged radicals. On the other hand, the even-electron ion studied, the 2-pyridyl cation, shows substantial solvation effects. For example, in solution, the 2-pyridyl cation forms a stable adduct with tetrahydrofuran, whereas in the gas phase, only addition/elimination reactions were observed.      

硫酸锌与组氨酸固液反应的热动力学研究

用微热量计对硫酸锌与组氨酸在水中的固液反应进行了热动力学研究。通过实验和计算得出了该反应的热动力学参数（活化焓,活化熵及活化自由能）,速率常数和动力学参数（活化能,指前因子及反应级数）,并对温度改变对该反应的影响及配合物的合成条件进行了讨论。     

Conformational Transformation Exhibited by the Peptide Extracted from Crystalline Region of Bombyx mori Silk Fibroin in Solid and Solution States

The conformational transformation of a 30-residue peptide H（Ala-Gly-Ser-Gly-AIa-Gly）5OH, i.e., （AGSGAG）5, extracted from highly crystalline region of Bombyx mori （B. mori） silk fibroin was described by using the high resolution solid state 13^C NMR, and CD spectroscopies. Based on the conformation-dependent 13^C NMR chemical shifts of the Ala, Gly and Ser residues and the line-shape analysis of the conformation sensitive Ala Cβ resonance, the peptide revealed a strong preference for silk Ⅱ structural form, i,e,, an antiparallel fl-sheet structure （φ= - 140±20°and ψ= 135±20°） in solid state. On the contrary, the CD spectra of this peptide in the two non-native hexafluorinated fibre spinning solvents, hexafluoroisopropanol （HFIP） and hexafluoroacetone （HFA）, exhibited the existence of an unusual tightly-folded conformation resembling 310-helix （φ=- 60±20° and ψ=-30±20°）, as judged from the R ratio of [θ]222/[θ]203 in HFIP solution, whereas a dynamically averaged unordered structure in HFA, Taken together, the information inclined to hypothesis that the primary structure of the highly crystalline regions of B. mori silk fibroin may be easily accessible to the large conformational changes, which in turn may be critical for facilitating the structural transformation from unprocessed silk fibroin （silk I form） to processed silk fiber （silk Ⅱform）.     

A New Microscale Method for the Conversion of Phosphorus Oxyacids to Their Fluorinated Analogues,Using Cyanuric Fluoride in Solution and on Solid Support

Cyanuric fluoride, in solution or loaded onto a Wang resin, is successfully used as a fluorinating agent for phosphorus oxy acids. The reaction is very efficient with high yields and easy workup procedures, thereby in general generating products in quantitative yields. The cyanuric fluoride is proven suitable for micromolar scale synthesis of analytical standards, particularly in its resin-bound form.     

Synthesis,Thermal Isomerization in Solution and in Solid Phase of the Complexes [Pt(RCN)2Cl2]

The interaction of the clathrate Pt₆Cl₁₂ · 0.1 EtCl · 5.7H₂O with RCN nitriles results in cis-[Pt(PhCH₂CN)₂Cl₂] and in [Pt(RCN)₂Cl₂] (R = CH₂CO₂Et, Ph) complexes as a mixture of cis- and trans-isomers which separated and characterized. Cis-[Pt(MeCN)₂Cl₂] has been synthesized using a well known technique of K₂[PtCl₄] interaction with acetonitrile in water. Heating cis-[Pt(RCN)₂Cl₂] (R = Me, CH₂Ph, CH₂CO₂Et) in the solid phase leads to cis-trans isomerization. In case of cis-[Pt(PhCN)₂Cl₂] thermal conversion results in trans-[Pt(PhCN)₂Cl₂] but the process of geometrical isomerizations accompained by a considerable decomposition of starting material and/or final products. Boiling of cis-[Pt(PhCH₂CN₂)Cl₂] in mixture of EtNO₂? PhCH₂CN or cis-[Pt(EtCO₂CH₂CN)₂Cl₂] in MeNO₂ or EtNO₂ solutions results in complete cis-trans conversion. Similarily heating of cis-[Pt(RCN)₂Cl₂] (R = Me, Ph) in solution produces an equilibrium mixture of cis- and transisomers.