Amino acid end-group in commercial heparin. II. Reaction kinetics of the amino end groups with 2,4,6-trinitrobenzenesulfonic acid

Three different commercial heparins were trinitophenylated with 2,4,6-trinitrobenzenesulfonic acid (TNBS) under aqueous conditions. The reaction kinetics of amino groups in heparin with TNBS showed that the reactivities of amino groups were significantly different for free amino groups on heparin, compared to reactivities in peptides and amino acid residues attached to heparin molecules. With TNBS, unreactive amino groups were always present during the reaction.     

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.     

Synthesis of novel amino squaric acids via addition of dianion enolates derived from N-Boc amino acid esters

Novel α-amino squaric acid analogs were synthesized by initial addition reaction of a dianion enolate generated from N-Boc amino acid tert-butyl ester to squaric acid diisopropyl ester, and subsequent decarboxylation of the resulting carboxylic acid moiety.     

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.     

Design of radical-resistant amino acid residues: a combined theoretical and experimental investigation

Ab initio calculations have been used to design radical-resistant amino acid residues. Optimized structures of free and protected amino acids and their corresponding alpha-carbon-centered radicals were determined with B3-LYP/6-31G(d). Single-point RMP2/6-31G(d) calculations on these structures were then used to obtain radical stabilization energies, to examine the effect of steric repulsion between the side chains and amide carbonyl groups on the stability of alpha-carbon-centered peptide radicals. Relative to glycine, the destabilization for alanine and valine residues was found to be approximately 9 and 18 kJ mol(-1), respectively, which correlates with the reactivity of analogous amino acid residues in peptides toward hydrogen atom abstraction in conventional free radical reactions. To design amino acid residues that would resist radical reactions, strategies by which the steric effects could be magnified were considered. This resulted in the identification of tert-leucine and 3,3,3-trifluoroalanine as suitable molecules. With these amino acid residues, the destabilization of the alpha-carbon-centered radicals relative to that of glycine is increased substantially to approximately 36 and 41 kJ mol(-1), respectively. The theoretical predictions have been supported by experimental observations: a tert-leucine derivative was shown to be very slow to react with N-bromosuccinimide, while the corresponding trifluoroalanine derivative was found to be inert.     

Selective reaction of amino acid sulfoximines with nitrous acid; facile stereospecific conversion to amino acid sulfoxides

N-Unsubstituted sulfoximines of amino acids can he readily converted to the corresponding amino acid sulfoxides in high yields by treatment with stoichiometric quantities of nitrous acid. Under the conditions employed, reaction with L-methionine-S (or R)-sulfoximine is specific for the sulfoximine nitrogen atom, and no reaction occurs at the α-amino group. Conversion to the sulfoxide proceeds with complete retention of configuration at the sulfur atom.     

Mechanistic study of asymmetric Michael addition of malonates to enones catalyzed by a primary amino acid lithium salt

A mechanistic study was carried out for the asymmetric Michael addition reaction of malonates to enones catalyzed by a primary amino acid lithium salt to elucidate the origin of the asymmetric induction. A primary β-amino acid salt catalyst, O-TBDPS β-homoserine lithium salt, exhibited much higher enantioselectivity than that achieved with the corresponding catalysts derived from α- and γ-amino acids for this reaction. Detailed studies of the transition states with DFT calculations revealed that the lithium cation and carboxylate group of the β-amino acid salt catalyst have important roles in achieving high enantioselectivity in the Michael addition reaction of malonates to enones.     

Synthesis of novel fullerene α-amino acid conjugates

Aspartic acid and glutamic acid with protected α-amino and α-carboxyl groups had been used to react with the activated hydroxyl group of N-substituted 3,4-fullero pyrrolidine. The products were deprotected, affording two monofuUerene α-amino acids, monofullerene aspartic acid （mFas） and monofullerene glutamic acid （mFgu）. Then a bifullerene glutamic acid conjugate （bFguC） was synthesized by reaction of mFgu containing protected amino group with N-substituted 3,4-fullero pyrrolidine.     

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.     

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.     

A novel synthesis of cyclic α-amino aldehydes, amino alcohols, and α-amino acid methyl esters from cyclic ketones through sulfinylaziridines

Treatment of 1-chlorovinyl p-tolyl sulfoxides, which were synthesized from cyclic ketones and chloromethyl p-tolyl sulfoxide in three steps in good yields, with N-lithio arylamines gave sulfinylaziridines in high yields. On treatment with N-lithio aniline or N-lithio p-chloroaniline, the sulfinylaziridines gave α-amino aldehydes in high yields. The α-amino aldehydes were converted to amino alcohols and α-amino acid methyl esters in moderate to good yields. This procedure offers an efficient method for synthesis of cyclic α-quaternary α-amino aldehydes, amino alcohols, and α-amino acid derivatives from cyclic ketones.     

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.     

Analysis of the complex formation of heparin with protamine by light scattering and analytical ultracentrifugation: implications for blood coagulation management

Heparin, a linear glycosaminoglycan, is used in different forms in anticoagulation treatment. Protamine, a highly positive charged peptide containing about 32 amino acids, acts as an antagonist for heparin to restore normal blood coagulation. The complex formation of protamine with heparin was analyzed by a combination of analytical ultracentrifugation and light scattering. Titration of heparin with protamine in blood plasma preparations results in a drastic increase of turbidity, indicating the formation of nanoscale particles. A similar increase of turbidity was observed in physiological saline solution with or without human serum albumin (HSA). Particle size analysis by analytical ultracentrifugation revealed a particle radius of approximately 30 nm for unfractionated heparin and of approximately 60 nm for low molecular weight heparin upon complexation with excess protamine, in agreement with atomic force microscopy data. In the absence of HSA, larger and more heterogeneous particles were observed. The particles obtained were found to be stable for hours. The particle formation kinetics was analyzed by light scattering at different scattering angles and was found to be complete within several minutes. The time course of particle formation suggests a condensation reaction, with sigmoidal traces for low heparin concentrations and quasi-first-order reaction for high heparin concentrations. Under all conditions, the final scattering intensity reached after several minutes was found to be proportional to the amount of heparin in the blood plasma or buffer solution, provided that excess protamine was available and no multiple scattering occurred. On the basis of a direct relation between particle concentration and the heparin concentration present before protaminization, a light scattering assay was developed which permits the quantitative analysis of the heparin concentration in blood plasma and which could complement or even replace the activated clotting time test, which is currently the most commonly used method for blood coagulation management.     

Diastereoselective syntheses of 2-amino propargyl alcohols. Chiral building blocks for enantiopure amino γ-lactones and 5-hydroxy-piperidinone derivatives

α-Dibenzylamino aldehydes, derived from the corresponding natural α-amino acids, react with metal acetylides to yield anti-amino propargyl alcohols in good yield and diastereomeric excess. syn Amino alcohols are prepared from the anti diastereoisomers and all of them are elaborated in few steps to enantiopure amino lactones and hydroxy-piperidin-2-ones.     

Detection of a stable nitroxide during chemical depolymerization of heparin

The use of low-molecular-weight heparins (LMWHs) has become common, since compared to unfractionated heparin (UFH), they have a much longer plasma half-life and lower incidence of side effects. LMWHs are derived from the depolymerization of UFH, obtained either chemically, physically or enzymatically. We employed electron spin resonance (ESR) spectroscopy to study the depolymerization of UFH by copper in the presence of hydrogen peroxide. A stable nitroxide radical was detected. This could be generated by the hydroxyl radical attack either to the N-SO⁻₃ group or to free amino groups present in the UFH preparation.     

The decarboxylative Strecker reaction

α-Amino acids react with aldehydes in the presence of a cyanide source to form α-amino nitriles in what can be considered a decarboxylative variant of the classical Strecker reaction. This unprecedented transformation does not require the use of a metal catalyst and provides facile access to valuable α-amino nitriles that are inaccessible by traditional Strecker chemistry.     

Preparation and Synthetic Applications of Sterically Hindered Secondary Amines

The preparation of sterically hindered secondary amino esters from the reaction of N-protected α-amino aldehydes and 3-, or 2-oxo esters with α-amino esters by reductive amination is described. The resulting amino esters were converted to the β-lactam or acylated to form N-acyl secondary amides.     

Studies on some radical transfer reactions by entrapping the radicals as polymer end groups. II. Reactions of ȮH radicals with amino acids

The reaction of ?H radicals with a number of aliphatic amino acids has been studied by entrapping the resultant radicals as end groups of poly(methyl methacrylate) that have been detected and estimated by the sensitive dye partition technique. The rate constants of the reaction (in mol^?1 L S^?1) of 7 amino acids at 25°C and at pH 1.00 have been determined as 8.33 × 10⁸ for glycine, 2.56 × 10⁹ for β-alanine, 2.01 × 10⁹ for β-alanine, 3.99 × 10⁹ for 4-amino butyric acid, 7.56 × 10⁹ for (1+) valine, 1.42 × 10¹⁰ for (1?) leucine, and 5.98 × 10¹⁰ for 6-amino caproic acid. Glycine, α-alanine, β-alanine, and 4-amino butyric acid produced radicals that underwent deamination and incorporated only carboxyl-bearing end groups in the polymer. The other amino acids, leucine, valine, and 6-amino caproic acid, produced at least two types of radicals, radicals that underwent deamination and those that remained intact, and incorporated in the polymer both carboxyl- and amine-bearing end groups but in different amounts. The latter type of radicals were about 29% from 6-amino caproic acid, 23% from leucine, and 18% from valine. The change of pH from 0.80 to 2.72 did not produce any significant change in the end group profile of the polymer obtained, indicating no appreciable change in the rate of the reaction of ?H radicals with the simplest amino acid glycine in the pH range studied.     

Semi-synthetic Heparins with 2-Deoxy-2-sulfamino-α-l-iduronic Acid Residues: Chemical Reactivity and Biological Activity1

Abstract

Ammonolysis of the epoxide rings of 2,3-anhydro-α-L-guluronic acid residues, generated in alkaline medium from 2-O-sulfated α-L-iduronic acid residues of heparin, quantitatively afforded 2-amino-2-deoxy-α-L-iduronic acid residues. N-sulfation of these residues by TMA·SO₃ complex led to a formal replacement of the original 2-O-sulfate groups of heparin with N-sulfates, without configurational changes. These modified uronic acid residues (no longer amenable to alkaline epoxidation) can be easily N-desulfated. The presence of negative or positive charges at position 2 of the newly generated 2-amino-2-deoxy-α-L-iduronic acid residues influences the in vivo antithrombotic activity and haemorrhagic effects in different ways. A free amino group mainly decreases the haemorrhagic properties of heparin, while a negatively charged N-sulfate group decreases the coagulation parameters.     

Cyclohexane ring as a tool to select the presentation of the carbohydrate moiety in glycosyl amino acids

The design of mimic molecules that resemble natural products can be a useful tool to help understand the key aspects in molecular recognition processes that are difficult to access by using natural derivatives. We present the synthesis and the conformational analysis of different glucosylated diamide amino acids that simulate glycopeptides with β-O-linked glucose and contain the nonnatural β-hydroxycyclohexane-α-amino acid. The study, using NMR experiments, X-ray spectroscopy, and molecular dynamics simulations, reveals that the cyclohexane ring allows some naturally occurring ways of presentation of the carbohydrate to be fixed, or to stabilize some novel conformations. In addition, different chair conformations for the cyclohexane-α-amino acid moiety can be set, in particular, those with high population of conformers in which the bulky groups are located at axial positions. Moreover, to increase the scope of these cyclohexane derivatives, two dipeptides incorporating the glycomimics have been synthesized and further glycosylated to obtain the corresponding α-O-glycopeptides. These features can have important implications for the design of new drugs and for understanding the complex molecular processes that take place between glycopeptides and their biological targets.