Modification of hyaluronic acid with aromatic amino acids

Hyaluronic acid was modified with aromatic amino acids (5-aminosalicylic, 4-aminosalicylic, anthranilic, and p-aminobenzoic) in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The modified glycans contained 9–43% of arylamide groups and 10–33% of isoureidocarbonyl groups depending on the nature of the amino acid. Reduction with sodium borohydride allowed the conversion of isoureidocarbonyl groups into hydroxymethyl groups.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 90–95.Original Russian Text Copyright © 2005 by Ponedelkina, Odinokov, Vakhrusheva, Golikova, Khalilov, Dzhemilev.     

Chemical modification of heparin

Heparin was modified at carboxyl groups by reaction with several pharmacologically important amino-containing compounds in aqueous medium in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. In dependence on the nature of the amine and the ratio of reagents, conjugates containing 36-100% amide and 0-25% isoureidocarbonyl groups were synthesized. Isoureidoarylamide groups are present, along with amide moieties, in the products of heparin modification by hydroxyl-containing aromatic amines. The conjugate of heparin with p-aminobenzoic acid contained oligomeric arylamide.     

Chemical modification of heparin

Heparin was modified at carboxyl groups by reaction with several pharmacologically important amino-containing compounds in aqueous medium in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. In dependence on the nature of the amine and the ratio of reagents, conjugates containing 36–100% amide and 0–25% isoureidocarbonyl groups were synthesized. Isoureidoarylamide groups are present, along with amide moieties, in the products of heparin modification by hydroxyl-containing aromatic amines. The conjugate of heparin with p-aminobenzoic acid contained oligomeric arylamide.     

Hydroxyl radical adduct of 5-aminosalicylic acid: a potential marker of ozone-induced oxidative stress.

The use of 5-aminosalicylic acid in assessment of reactive oxygen species formation was investigated by in vitro Fenton and ozonation reactions, and by in vivo ozone-exposure experiments. Enzymatic hydroxylation was evaluated by a microsomal assay. Fischer 344 male rats (250 g) injected with 5-aminosalicylic acid (100 mg x kg(-1) i.p.; 30 min) were exposed to ozone (0, 1, 2 ppm; nose only, 2 h); bronchoalveolar lavage, lung homogenates, and plasma were recovered. Oxidation products of 5-aminosalicylic acid were as follows: salicylic acid, by deamination; 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid, from radical or enzymatic hydroxylation; 5-amino-2-hydroxy-N,N'-bis(3-carboxy-4-hydroxyphenyl)-1,4-benzoquinonediimine, a condensation product of oxidized 5-aminosalicylic acid; and 5-amino-2,3,4,6-tetrahydroxybenzoic acid, attributed to hydroxyl radical attack without deamination, identified by HPLC electrochemical (HPLC-EC) detector system analysis and by GC-MS analysis of trimethylsilyl derivatives. 5-Aminotetrahydroxybenzoic acid was not formed enzymatically. 5-Aminotetrahydroxybenzoic acid, but not 5-aminosalicylic acid, was significantly elevated in bronchoalveolar lavage (+86%) and lung homogenates (+56%) in response to 2 ppm ozone (p < 0.05); no significant changes were detected in plasma. The data indicate that hydroxylation of 5-aminosalicylic acid is a potential specific probe for in vivo oxidative stress.     

Conjugation of chondroitin sulfates with amines

Conjugation of chondroitin sulfates with pharmacologically important amines in a water medium in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide was studied. Conjugates with amide and isoureidocarbonyl groups were synthesized. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 5; see also http://www.maik.ru     

On the antioxidant properties of therapeutic drugs: quenching of singlet molecular oxygen by aminosalicylic acids

The ability of the widely employed therapeutic drugs 4-aminosalicylic acid and 5-aminosalicylic acid to act as singlet molecular oxygen (O(2)((1)delta(g))) scavengers was investigated at pH 7 and pH 12. The isomer 3-aminosalicylic acid was also included in the study for comparative purposes. All three compounds quench photochemically generated O(2)((1)delta(g)) with rate constants in the range of 10(7)-10(8) x M(-1)s(-1), depending on the experimental conditions. No chemical reaction (oxidation of the aminosalicylic acids) was detected at the neutral pH, whereas at pH 12 both chemical and physical interactions with O(2)((1)delta(g)) operated. The physical process implies the de-activation of the oxidant species without destruction of the aminosalicylic acid. The quotients between the overall and reactive rate constants for O(2)((1)delta(g)) quenching at pH 12 (k(r)/k(t) ratios), which account for the actual effectiveness of photodegradation, were relatively low (0.22, 0.04, and 0.06 for 3-, 4- and 5-aminosalicylic acids, respectively). This indicates that the drugs, particularly the 4- and 5-amino derivatives, de-activate the excited oxygen species, at both pH values studied, mainly in a physical fashion, preventing its photodegradation and providing an antioxidative protection for possible photo-oxidizable biological targets in the surroundings.     

Chemical modification studies on a lectin from winged-bean [Psophocarpus tetragonolobus (L.) DC] tubers.

The effect of chemical modification on a D(+)-galactose-specific lectin isolated from winged-bean tubers was investigated to identify the type of amino acid involved in its haemagglutinating activity. Various anhydrides of dicarboxylic acids, such as acetic anhydride, succinic anhydride, maleic anhydride and citraconic anhydride, modified 57-68% of the amino groups of the winged-bean tuber lectin. Treatment with N-acetylimidazole modified only 45% of the total amino groups. Reductive methylation of free amino groups modified 57% of the amino groups. Modification of the amino groups of the lectin by acetic anhydride and succinic anhydride did not lead to any significant change in the haemagglutinating activity (greater than or equal to 75% active). However, citraconylation and maleylation of the lectin led to a significant decrease in the haemagglutinating activity (less than or equal to 20% active). Acetylation and succinylation (3-carboxypropionylation) of the lectin led to a decrease in the pI value of the native lectin from approx. 9.5 to approx. 4.5. Treatment of the lectin with N-bromosuccinimide led to the modification of two and four tryptophan residues per molecule in the absence and in the presence of 8 M-urea respectively. The immunological identity of all the modified lectin preparations showed no gross structural changes except the lectin modified with N-bromosuccinimide in the presence of urea at pH 4.0.     

Chemical modification of lysine epsilon-NH2-groups in horseradish peroxidase. Its effect on enzyme stability. Temperature dependence of thermo-inactivation constants for native and modified peroxidase]

Thermostability of horseradish peroxidase modified by acetic, propionic, butyric, valeric and succinic anhydrides and trinitrobenzolsulfonic acid (TNBS) is studied within the temperature range of 56-80 degrees C. Acylation of 4 amino groups and arylation of 3 amino groups with TNBS are found to stabilize the enzyme, while modification of 6 groups decreases the enzyme stability. Chemical modification of peroxidase does not change its pH-dependence with respect to enzyme thermostability. Thermodynamic activation parameters of irreversible thermoinactivation are determined for native and modified peroxidase. Native peroxidase has deltaH not equal to = 30+/-1 kcal/mole and deltaS not equal to = 14 e. e.; modified by acid anhydrides peroxidase has deltaH not equal to within 64-87 kcal/mole and deltaS not equal to within 110-178 e. e. depending on the nature of a modifying agent. The effect of the structure of a radical introduced into the enzyme molecule, and of a number of modified epsilon-amino groups on thermoinactivation deltaH not equal to and deltaS not equal to values is discussed.     

A specific property of bacteria in the genus Klebsiella--a color reaction with 5-aminosalicylic acid in the nutrient medium

For the first time bacteria of the genus Klebsiella have been found to possess a specific property, characteristic of this genus only, i.e. the capacity of giving color reaction with 5-aminosalicylic acid. This reaction can be observed in all Klebsiella species under study: K. pneumoniae (94.4 +/- 2.3%), K. ozaenae (93.3 +/- 4.5%), K. rhinoscleromatis (100%), K. oxytoca (88.0 +/- 4.9%), K. mobilis (92-5 +/- 4.3% of the strains). In all other bacteria under study (40 species, 22 genera and 7 families) the reaction is negative. The test for the color reaction with 5-aminosalicylic acid confirms the belonging of K. mobilis (Enterobacter aerogenes) to the genus Klebsiella, thus making it possible to simplify and accelerate the identification of Klebsiella.     

Ester synthesis at extraordinarily low temperature of -3 degrees C by modified lipase in benzene

The lipoprotein lipase from Pseudomonas fluorescens was modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine. The modified lipase in which 55% of the amino groups in the enzyme molecule were coupled with polyethylene glycol was found to be soluble in benzene and catalyzed the reactions of ester synthesis, ester exchange, aminolysis and ester hydrolysis in benzene. The modified lipase had an extraordinary temperature-dependency: enzymic activity for methyl laurate synthesis from methyl alcohol and lauric acid increased with decreasing temperature and attained the maximum at the extremely low temperature of -3 degrees C. The optimum temperature for hydrolysis of methyl laurate was as low as -4 degrees C.     

Modification of E. coli L-asparaginase with polyethylene glycol: disappearance of binding ability to anti-asparaginase serum.

L-Asparaginase from Escherichia coli A-1-3 was modified with activated polyethylene glycols (2-0-methoxypolyethylene glycol-4,6-dichloro-s-triazine) with molecular weights of 750, 1900 and 5000. The modification of asparaginase to 73 amino groups out of the total 92 amino groups in the molecule with polyethylene glycol of 5000 daltons gave rise to a complete loss of the binding ability towards anti-asparaginase serum from rabbit. This modified asparaginase retained the enzymic activity (7%) and had a resistivity against trypsin. Asparaginases modified with polyethylene glycols of 750 and 1900 daltons did not show a substantial change of the immunogenic properties.     

Effect of hydrophobicity of acyl groups on the activity and stability of acylated thermolysin

1. Normal carboxylic acids of different hydrophobicities and similar chain lengths were prepared and used for the modification of amino groups of thermolysin (EC 3.4.24.4). They were 4,7,10,13-tetraoxatetradecanoic acid, 4,7,10-trioxatetradecanoic acid, 4,7-dioxatetradecanoic acid and 4-oxatetradecanoic acid. 2. The modified enzymes were isolated by gel filtration. They had 6--7 acyl groups per molecule. Acylation of amino groups with 4-oxatetradecanoic acid and tetradecanoic acid made the enzyme insoluble. 3. The most hydrophilic enzyme derivative had similar enzyme activity and higher heat resistance than the native enzyme. The most hydrophobic derivative showed lower Km (50%) and V (40%) values for proteinase activity and lower heat resistance than the former derivative. The trioxa-derivative had intermediate characteristics. The results are discussed with respect to effects on stability and activity of the enzyme.     

Neocarzinostatin: effect of modification of side chain amino and carboxyl groups on chemical and biological properties.

The antitumor protein neocarzinostatin (NCS), isolated from Streptomyces carzinostaticus, is a single chain polypeptide with 109 amino acid residues. Complete acylation of the amino groups (alanine-1 and lysine-20) was observed when NCS was allowed to react with 3-(4-hydroxyphenyl)-propionic acid N-hydroxysuccinimide ester at pH 8.5. Since the ensuing bis[(alanine-1, lysine-20)-3-(4-hydroxyphenyl)]-propionamide NCS was fully active in antibacterial potency and in the inhibition of growth of leukemic (CCRF-CEM) cells in vitro, it appears that the two amino groups in the protein are not essential for biological activity. Radiolabeled NCS was prepared by using a tritiated or 125I-labeled acylating agent. Since the CD spectra of native and bis(alanine-1, lysine-20)-amino modified NCS were indistinguishable, there is presumably no change in the native conformation of the protein due to acylation. Reaction of NCS with ammonium chloride in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at pH 4.75 converted all the 10 carboxyl groups into carboxamides and produced a protein derivative of basic character. This modification caused a change in the native conformation of the protein accompanied by a loss in biological inhibitory activities.     

Colon-specific, mutual azo prodrug of 5-aminosalicylic acid with L-tryptophan: synthesis, kinetic studies and evaluation of its mitigating effect in trinitrobenzenesulfonic acid-induced colitis in rats

Mutual azo prodrug of 5-aminosalicylic acid with l-tryptophan was synthesized by coupling l-tryptophan with salicylic acid, for targeted drug delivery to the inflamed gut tissue in inflammatory bowel disease. The structure of synthesized prodrug was confirmed by elemental analysis, IR and NMR spectroscopy. In vitro kinetic studies in HCl buffer (pH 1.2) showed negligible release of 5-aminosalicylic acid, whereas in phosphate buffer (pH 7.4) 18% release was observed over a period of 7 h. In rat fecal matter, 87.9% of 5-aminosalicylic acid was released with a half-life of 143.6 min, following first order kinetics. The azo conjugate was evaluated for its ulcerogenic potential by Rainsford's cold stress method. The ameliorating effect of the azo conjugate and therapeutic efficacy of the carrier system was evaluated in trinitrobenzenesulfonic acid-induced experimental colitis model. The synthesized prodrug was found to be equally effective in mitigating the colitis in rats as that of sulfasalazine without the ulcerogenicity of 5-aminosalicylic acid.     

Identification of cysteine as the reactive group in pyruvate kinase alkylated by 5-chloro-4-oxopentanoic acid.

4-Hydroxypentanoic acid alanine thioether was synthesized and characterized by n.m.r. spectroscopy. This derivative corresponded to the modified amino acid obtained by allowing 5-chloro-4-oxo[3,5-3H]pentanoic acid to react with rabbit muscle pyruvate kinase. Performic acid oxidation of 4-oxo[3,5-3H]pentanoic acid alanine thioether in pyruvate kinase gave [3H]succinate (67%) and [3H]carboxymethylcysteine (33%) as expected. Evidence is presented to show that NaBH4 reduction followed by periodate oxidation and analysis of radioactive formaldehyde production may provide a convenient method for distinguishing between thiol and amino alkylation by halogenomethyl ketone compounds. Peptide 'mapping' confirms that the modification by 5-chloro-4-oxopentanoic acid occurs primarily at one region of pyruvate kinase.     

Synthesis, kinetic studies and pharmacological evaluation of mutual azo prodrug of 5-aminosalicylic acid with D-phenylalanine for colon specific drug delivery in inflammatory bowel disease

Mutual azo prodrug of 5-aminosalicylic acid with d-phenylalanine was synthesized by coupling D-phenylalanine with salicylic acid, for targeted drug delivery to the inflamed gut tissue in inflammatory bowel disease. The structure of synthesized prodrug was confirmed by elemental analysis, IR and NMR spectroscopy. In vitro kinetic studies in HCl buffer (pH 1.2) showed negligible release of 5-aminosalicylic acid, whereas in phosphate buffer (pH 7.4) only 15% release was observed over a period of 7h. In rat fecal matter the release of 5-aminosalicylic acid was almost complete (85%), with a half-life of 160.1 min, following first order kinetics. The azo conjugate was evaluated for its ulcerogenic potential by Rainsford's cold stress method. Therapeutic efficacy of the carrier system and the mitigating effect of the azo conjugate were evaluated in trinitrobenzenesulfonic acid-induced experimental colitis model. The synthesized prodrug was found to be equally effective in mitigating the colitis in rats as that of sulfasalazine without the ulcerogenicity of 5-aminosalicylic acid.     

Spectroscopic characterization of amino acid and amino acid ester-Schiff-base complexes of oxovanadium and their catalysis in sulfide oxidation

α-Amino acid Schiff-base complexes of oxovanadium(IV), whose ligands have amino acid side chains with coordinating functional groups, retained coordination geometries in which the amino acid side chains were probably coordinated in the axial position with a phenolate oxygen, a carboxylate oxygen, an imine nitrogen, and a solvent being bound in the equatorial plane. As for amino acid ester Schiff-base complexes, the amino acid side chains were coordinated in the equatorial plane in the place of the carboxyl group in the case of the amino acid Schiff-base complexes. The amino acid Schiff-base complexes of oxovanadium(V) were present as dimers in dichloromethane. Peroxo complexes prepared from the Schiff-base complexes of oxovanadium(V) converted methyl phenyl sulfide to the corresponding sulfoxide in 80-90% yield in CDCl₃ and in 30-70% yield in CD₃OD in 30 min. They converted the sulfide in a stereoselective manner yielding the sulfoxide in small enantiomeric excess (5-20%).     

A single-step purification of rat pancreatic and salivary amylase by affinity chromatography

Optimal conditions for the conjugation of carboxyl groups on low molecular weight molecules to reactive amino groups on rabbit immunoglobulin G (IgG) using a modified carbodiimide reaction have been investigated. Reaction of [¹⁴C]hippuric acid with N-ethyl-N′-(dimethylaminopropyl) carbodiimide at pH 5 followed by adjustment to pH 8 and coupling with rabbit IgG resulted in the formation of hippuric acid-IgG conjugates with less than 10% intra- and intermolecular IgG crosslinking. More than 93% of the bonds linking hippuric acid to IgG were stable to hydroxylamine hydrolysis, indicating the peptide properties of these bonds. This two-step process permitted a defined number of hippuric acid moieties to be loaded onto a single IgG molecule and should provide a useful method for the conjugation of molecules containing carboxyl groups to amino groups on a variety of polypeptides.     

Identification and characterization of an NADPH-cytochrome P450 reductase derived peptide involved in binding to cytochrome P450

The amino acids of cytochrome P450 reductase involved in the interaction with cytochrome P450 were identified with a differential labeling technique. The water-soluble carbodiimide EDC (1-ethyl-3-[3- (dimethylamino)propyl]-carbodiimide) was used with the nucleophile methylamine to modify carboxyl residues. When the modification was performed in the presence of cytochrome P450, there was no inhibition in the ability of the modified reductase to bind to cytochrome P450. However, subsequent modification of the reductase in the absence of cytochrome P450 caused a fourfold increase in the Km and an 80% decrease in kcat/Km (relative to the reductase modified in the first step), for the interaction with cytochrome P450. These effects are attributed to the modification of approximately 3.2 mol of carboxyl residues per mole of reductase. Tryptic peptides generated from the modified reductase were purified by reverse phase high-performance liquid chromatography and characterized. Amino acid sequencing and analysis suggest that the peptide which contains approximately 40% of the labeled carboxyl residues corresponds to amino acid residues 109-130 of rat liver NADPH-cytochrome P450 reductase. One or more of the seven carboxyl containing amino acids within this peptide is presumably involved in the interaction with cytochrome P450.     

Concentration and composition of free amino acids and osmolalities of porcine oviductal and uterine fluid and their effects on development of porcine IVF embryos

The concentration of free amino acids and the osmolalities in porcine oviductal (OF) and uterine fluids (UFs) on day 3 (D3) and day 5 (D5) were measured by HPLC and Vapor Pressure Osmometer, respectively. Based on these measurements we designed new media based on PZM3 by modifying the amino acid composition and osmolality. The effectiveness of the modified PZM3 on the development of porcine IVF embryos was then investigated. A total of 24 free amino acids were measured, including 20 protein and 4 nonprotein amino acids (beta-alanine, taurine, ornithine, and citrulline). There was no significant difference in the total concentration of amino acids among D3OF (13.06 +/- 3.63 mmol/L), D3UF (10.54 +/- 5.16 mmol/L), or D5UF (10.23 +/- 6.69 mmol/L). But the total concentration of amino acids in D5OF (5.89 +/- 1.47 mmol/L) was significantly lower than the three fluids above. Some individual amino acids varied significantly depending on where they were collected and from which day. The blastocyst rates of porcine IVF embryos were not improved when embryos were cultured in PZM3 with amino acids at D3OF (PZM3-D3OF, 20.3 +/- 7.9%) or D5UF (PZM3-D5UF, 14.3 +/- 10.7%) concentrations or in PZM3-D3OF for the first 48 (20.5 +/- 15.1), 72 (25.6 +/- 10.4), and 96 (18.7 +/- 10.0) hr and then transferred into PZM3-D5UF compared with PZM3 with Sigma amino acid solution (PZM3-SAA) (30.8 +/- 9.1%). However, when IVF embryos were cultured in PZM3-D5UF, the average nuclear number per blastocyst (57.6 +/- 8.3) was increased compared to PZM3-SAA (40.5 +/- 3.5). The osmolalities in D3OF, D3UF, D5OF, and D5UF were 318 +/- 8, 320 +/- 32, 321, and 293 +/- 8 mOsM, respectively. When the IVF embryos were cultured in PZM3-SAA and PZM3-D3OF at a variety of osmolalities (150-360 mOsM), higher blastocyst rates were obtained at 270-300 mOsM in the PZM3-SAA group (24.6-33.9%) and 270-290 mOsM in PZM3-D3OF group (22.4-24.2%). The blastocyst rate gradually decreased when the osmolality was increased or decreased in both groups. When the embryos were cultured in PZM3-SAA at 330 mOsM for the first 72 hr and then transferred to 250 mOsM (33.3 +/- 3.4%), the blastocyst rate was higher than original PZM3 (21.2 +/- 2.2%) (288 mOsM).