Amino acid end-group in commercial heparin. III. Determination of the number of amino groups of amino acid and hexosamine residues per heparin molecule

The reactions of heparin with 2,4,6-trinitrobenzenesulfonic acid (TNBS) were studied spectrometrically. Seven different commercial heparins were used in this study. The amino groups react with TNBS to form equimolar amounts of trinitrophenylated (TNP) amino groups and bisulfite ions. The TNP-amino groups further react with bisulfite ions to form the monosubstituted anionic sigma complex. The absorption spectrum with two maxima at approximately 350 nm and approximately 420 nm, characteristic of either the TNP-amino groups or the complex, was analyzed for the reaction of TNBS with heparin. It was shown that the reactivities of TNBS with amino groups from α-amino acid and hexosamine residues are greatly different. By combining the results of the reaction kinetics and the reaction of heparin with Sanger's reagent, the number of the α-amino groups and the free amino groups in hexosamine residues were determined. These data have been performed with a range of heparins from different commercial sources, of different activities and physical characteristics. No correlation was found between the free amino contents of these heparins and biological potency. © 1993 John Wiley & Sons, Inc.     

Chemical modification by 2,4,6-trinitrobenzenesulfonic acid (TNBS) of an essential amino group in 3-ketovalidoxylamine A C-N lyase

3-Ketovalidoxylamine A C-N lyase of Flavobacterium saccharophilum is a monomeric protein with a molecular weight of 36000, and contains 32 amino groups and no cysteine or cystine residues. The enzyme was inactivated by 2,4,6-trinitrobenzenesulfonic acid (TNBS) following pseudo-first order kinetics. Substrate of the lyase, p-nitrophenyl-3-ketovalidamine, protected the enzyme against the inactivation, suggesting that the modification occurred at or near the active site. Although several amino groups were modified by TNBS, a plot of log (reciprocal of the half-time of inactivation) versus log (concentration of TNBS) suggested that one amino group has an essential role in catalysis.     

Chromatographic analysis of the trinitrophenyl derivatives of insulin

The insulin molecule was derivatised by reaction with trinitrobenzenesulphonic acid (TNBS), which is known to react predominantly with free primary amino groups. The products of the reaction were analysed by reversed-phase chromatography and by further derivatisation with dansyl chloride. Under the conditions of these experiments, TNBS was found to react preferentially with glycine at position A1. This finding is discussed in terms of the tertiary structure and immunogenicity of this derivative.     

The reaction of glutamic acid and trinitrobenzenesulfonic acid--kinetic study and analytical application

Some kinetic aspects of the reaction of glutamic acid (GLU) and Trinitrobenzenesulfonic acid (TNBS) were studied spectrophotometrically (420 nm), under pseudo-first order reaction conditions. The effect of GLU, TNBS, SO(2-)(3) and H(+) had been investigated. Working curves for the initial rates IR versus [GLU] are linear in the range 7.5-30.0 mM. The regression equation is IR = (8 +/- 8)E - 6 + (39 +/- 0.5).[GLU] and the correlation coefficient r = 0.9998. The limit of quantitation is 1.5 microM GLU and the relative standard deviation of the method 1.2%. The kinetics of the interfering TNBS hydrolysis reaction in alkaline range of pH, as well as the effect of sulfite concentration on the main reaction, are also presented. The analytical application of the reaction for the kinetic spectrophotometric assay of GLU and other amino acids, as well as TNBS, is presented and the relevant advantages and disadvantages of the method are discussed.     

活性导向的化学探针在氨基酸反应活性表征中的应用进展

原创药物的研制得益于蛋白质新靶标的发现,而新靶标的发现依赖于高可信度、高通量的药物-蛋白质相互作用分析方法。蛋白质作为生命功能的执行者,其表达量、空间定位与结构差异直接影响药效的发挥。目前,超过85%的蛋白质尚被认为是无法成药的,主要原因是缺少药物分子靶向的空腔以及相应的反应活性位点。因此,基于蛋白质组学层次实现对氨基酸反应活性位点的表征成为原创共价靶向药物设计的关键,也是克服难以成药靶标蛋白问题的关键。近年来,质谱技术的飞速发展极大地推动了基于蛋白质组学技术的药物-靶蛋白相互作用研究。其中基于活性的蛋白质组分析(ABPP)策略是利用活性位点导向的化学探针分子在复杂样品中实现功能状态酶和药物靶标等蛋白质的检测。基于化学探针的开发和质谱定量技术的发展,ABPP技术在氨基酸反应活性表征研究中展现出重要的应用潜力,将助力于药物新靶标的发现和药物先导化合物的开发。ABPP策略主要基于蛋白质的活性特征进行富集,活性探针作为ABPP策略的核心,近年来取得了飞速进展。该文回顾了ABPP策略的发展历程,重点介绍基于广谱活性探针的ABPP技术在多种氨基酸反应活性筛选领域的研究进展,并对其在药物靶点发现中...     

On the relative acidity and basicity of the amino groups of the nucleic acid bases

Quantum chemical calculations are reported on the deprotonation and protonation of the amino groups of the nucleic acid bases adenine, guanine and cytosine, in an attempt to compare the relative reactivities of these groups. In the light of renewed interest in the amino groups as reactive sites for certain carcinogenic and carcinostatic agents, we discuss the possible significance of our results for the interpretation of these molecular interactions.     

Formation,structure, and elasticity of loosely crosslinked epoxy-amine networks. I. Statistics of formation

Using the theory of branching processes based on cascade substitution, relations are derived for structural parameters characterizing network formation from diepoxide, monoepoxide, and diamine for various initial compositions. The different reactivities of the hydrogen atoms of the primary and secondary amino groups (the latter being formed in the reaction) and possibly different reactivities of epoxy groups in diepoxide and monoepoxide are taken into account. Relations are derived for the critical conversion at the gel point and for changes as a function of conversion in the average molecular weight in the pregel stage, changes in the sol and gel contents, effective functionality of the crosslinks, and concentration of elastically active network chains. The derivation includes the trapping factor that appears in the theory of permanent interchain interactions (trapped entanglements) of the Langley type. An analysis of the approximation usually employed in the statistical theory based on network formation from dyads of structural units shows that this approach is quite satisfactory for small differences in the reactivities of epoxy groups in diepoxide and monoepoxide.     

N Reactivity vs. O reactivity in aqueous chlorination

Nitrogenated compounds react with hypochlorous acid yielding N-chloro compounds. In principle, α-amino acids chlorination may take place on the oxygen of the carboxylic group or on the nitrogen of the amino group. In this framework, we discuss the different reactivities of nitrogen and oxygen towards chlorine, and come to the conclusion that the nitrogen is the preferred reaction site in α-amino acids chlorination. © 1994 John Wiley & Sons, Inc.     

Amino acid end-group in commercial heparin. I. Determination of the number of amino and hydroxyl end groups in heparin

Three commercial heparins of different molecular weights and anticoagulant activities were dinitrophenylated (DNP) with 2,4-dinitrofluorobenzene under aqueous conditions. The absorption spectra observed for DNP heparins in a 1% NaHCO₃ solution consisted of the two absorption spectra characteristic of DNP—amino (N—DNP) and DNP—hydroxyl (O—DNP) groups. The number of N—DNP, O—DNP, and (N + O)—DNP groups were determined as well as the number of N—DNP groups per heparin chain; different values (i.e., 0.16, 0.17, and 0.55) for the latter were obtained with the three heparins. Further calculations showed that two of the heparins had approximately two (N + O)—DNP groups per chain (i.e., 2.1 and 1.8) whilst the third sample, considered to be a “cruder” heparin, had a value of 1.4.     

Methodology for determining degree of hydrolysis of proteins in Hydrolysates: a review

Degree of hydrolysis (DH) is defined as the proportion of cleaved peptide bonds in a protein hydrolysate. Several methods exist for determining DH; the most commonly used of these include the pH-stat, trinitrobenzenesulfonic acid (TNBS), o-phthaldialdehyde (OPA), trichloroacetic acid soluble nitrogen (SN-TCA), and formol titration methods. The pH-stat method is based on the number of protons released during hydrolysis; the TNBS, OPA, and formol titration methods are based on the measurement of amino groups generated from hydrolysis. The SN-TCA method measures the amount of TCA-soluble nitrogen, rather than DH. The pH-stat is the simplest and most commonly used method, but does not determine peptide bonds directly. In addition, the accuracy of the method depends on the type of hydrolytic enzymes used, the size of the hydrolyzed peptides, and the reaction temperature. Generally, the TNBS and OPA methods compare well and do directly determine DH. However, the assumption that the response factor for all derivatized N-terminal amino acids is similar may lead to inaccuracies. In conclusion, there is no consensus as to the best method for determining the DH of protein hydrolysates; consequently, there is a need for a standardized approach if interstudy comparisons are to be made.     

Zwitterion formation in gas-phase cyclodextrin complexes

Protonated complexes of amino acids and underivatized beta-cyclodextrin, produced by electrospray ionization and trapped in the Fourier transform mass spectrometer, undergo formation of ternary complexes when reacted with alkyl amine. Based on the reactivities of the protonated amino acid complexes with alkylamines, the reactivities of the corresponding amino acid esters, and partially derivatized beta-cyclodextrin hosts, we conclude that the ternary complexes are salt-bridge zwitterionic species composed of amino acid zwitterions and protonated alkylamine all interacting with the hydroxyl groups on the narrow rim of the cyclodextrin. Molecular modeling calculations and experimental results suggest that the interactions of the amino acids with the rims contribute greatly to the formation of the zwitterionic species.     

Lewis Acid mediated reactions of zirconacyclopentadienes with aldehydes: one-pot synthetic route to indene and cyclopentadiene derivatives from aldehydes and benzyne or alkynes

One-pot procedures for the preparation of highly substituted indenes, tetrahydroindenes, and cyclopentadienes have been developed by using a combination of zirconocene-mediated Cbond;C-bond-forming reactions with Lewis acid mediated activation of carbonyl groups. The carbonyl groups of aldehydes were deoxygenated in the reaction and behaved formally as a one-carbon unit. A variety of Lewis acids were checked and showed different reactivities in this reaction.     

Mass fragmentographic quantitation of glutamic acid and gamma-aminobutyric acid in cerebellar nuclei and sympathetic ganglia of rats.

A method for the simultaneous quantitation of glutamic acid and gamma-aminobutyric acid (GABA) in tissue by mass fragmentography has been developed. The amino and carboxylic groups of the two amino acids were in a convenient one-step reaction derivatized with pentafluoropropionic anhydride and hexafluoroisopropanol. Deuterium-labeled glutamic acid and GABA and a homologue of GABA have been used as internal standards. The usefulness of the technique has been demonstrated by measurements in parts of rat cerebellum and in the superior cervical ganglion.     

Chain extension of amino acid skeletons: preparation of ketomethylene isosteres

Ketomethylene isosteric replacements for peptide bonds were generated through a zinc carbenoid-mediated chain extension reaction in which a variety of amino acid-derived β-keto esters are converted to γ-keto esters in a single step. The reaction tolerates a variety of protecting groups and amino acid side chains with no epimerization of the amino acid stereocenter.     

Phospholipase D modified with a polyethylene glycol derivative

Phospholipase D from Streptomyces sp. AA586, PLDP, was modified with methoxypolyethylene glycol succinimidylsuccinate (ss-PEG), an active derivative of polyethylene glycol. By titration with trinitrobenzene sulfonate (TNBS), approximately 70% of the free amino groups in the enzyme protein were shown to be modified by treatment with ss-PEG. By this modification, the molecular weight of the enzyme was increased, judging from the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration with Toyopearl HW-55F and TNBS titration. Due to loss of cationic charges, the enzyme protein became eluted faster in high performance liquid chromatography with CM-Toyopearl. By modification with ss-PEG, the enzyme became fairly thermostable, while pH-stability and optimal pH were not influenced. The value of Km for phosphatidylcholine of the hydrolytic reaction increased 2-fold, whereas that of the transphosphatidyl reaction was not significantly altered.     

Quantum semiempirical studies on the formation of aromatic polyamides, 1. Effect of structural parameters of diamines on the polyamidation reaction

A semiempirical quantum-mechanical study of structural and electronic parameters of aromatic diamines was performed using the method AM1. The diamines have been considered as condensation monomers, and the reactivities of both amino groups against acyl chlorides have been computed. The study shows that the amidation reaction is controlled mainly by the global charge on the amino group and, in a much smaller extent, by the frontier orbitals. This behaviour is consistent with the large energy gap between the HOMO 1

1 HOMO: highest occupied molecular orbital.

of the diamine and the LUMO 2

2 LUMO: lowest unoccupied molecular orbital.

of the acyl chloride. It has been found also that the electronic nature of the substituents, affecting the charge of the amino groups, greatly affects the carbons directly joined to these groups. This permits to correlate the reactivities with the calculated charges on these carbons:     

To probe the origin of activation effect of carboxylic acid and (+)-NLE in tridentated titanium catalyst systems

The activation effect of carboxylic acid and positive nonlinear effect ((+)-NLE) in tridentated titanium catalyst systems for hetero Diels-Alder reaction and aldol-type reactions has been first elucidated on the basis of X-ray crystal structural analysis of homochiral (pseudo-octahedral coordination geometry with C2 symmetry) and heterochiral (pseudo-octahedral coordination geometry with C1 symmetry) titanium complexes and the observation of their different reactivities with carboxylic acid additive.     

Protease-catalyzed monoacylation of 2-O-alpha-D-Glucopyranosyl-L-ascorbic acid in pyridine

2-O-alpha-D-Glucopyranosyl-6-O-octanoyl-L-ascorbic acid was enzymatically synthesized from 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) and vinyl octanoate with a protease from Bacillus subtilis in pyridine. Furthermore, with various linear saturated fatty acid vinylesters as acyl donors, AA-2G was also converted to their corresponding 6-O-acyl AA-2G in the same manner. The reactivities of transacylation decreased with increasing length of the acyl groups. Thus, short chain acyl groups were transferred to AA-2G by this protease more efficiently than were long chain acyl groups. This enzymatic method is recommended for the synthesis of 6-Acyl-AA-2G with short or medium length chain acyl groups.     

Room-temperature polycondensation of β-amino acid derivatives VI. Synthesis of various N-(hydroxyethyl) nylons

Various N-(hydroxyethyl)amino acid esters having a methyl substituent or phenyl group between amine and ester groups have been synthesized and their polycondensation behavior was investigated. These substituted amino acid esters gave amorphous polyamides which were soluble in alcohol. A model reaction between N-(hydroxyethyl)-amine and carboxylic acid ester was carried out in order to elucidate the role of hydroxyethyl group on the polycondensation. It was found that the amidation reaction took place rapidly at room temperature when the alkyl group of the carboxylic acid was small. N-(Hydroxyethyl) polyamides were obtained from N,N′-(bishydroxyethyl)-dicamines and dicarboxylic acid esters. The reaction mechanism of the room-temperature polycondensation reaction is discussed.     

α-Amino acids as acid components in the Passerini reaction: influence of N-protection on the yield and stereoselectivity

The Passerini reaction offers an easy access to depsipeptides, when both acid and isocyanide are derived from α-amino acids. However, racemisation of isocyanides derived from α-amino acid esters severely limits their use in the Passerini reaction. In order to overcome this limitation, a study on the influence of the α-amino acid N-protecting group on the yield and diastereoisomeric ratio of the product of the Passerini reaction was performed. Six different protecting groups were tested. Their influence turns out to be crucial and is not constant when the amino acid is changed. After optimisation, the Passerini reaction products with cyclohexanone as the carbonyl component were obtained with 99% yield and >98% de.