A Colorimetric Assay of Lipoyl-N-?-Lysine Hydrolysis Activity Using 2,6-Dibromoquinone-4-Chlorimide

Lipoic acid is a coenzyme for pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, branched chain-ketoacid dehydrogenase, and the glycine cleavage system. Lipoic acid is covalently attached through an amide to the ?-amino group of specific lysine residues of these enzymes. Lipoamidase hydrolyzes the amide bond of lipoyl-N-?-lysine. Because of the difficulty in quantitating lipoic acid or lysine released by hydrolysis of lipoyl-N-?-lysine, a sensitive assay of lipoamidase activity was developed based on quantitation of lipoic acid liberated from lipoyl-?-lysine using 2,6-dibromoquinone-4-chlorimide (DBQC). This method involves acidification of the assay mixture with HCl and separation of lipoic acid from lipoyl-N-?-lysine by extraction into ethyl acetate where it can react with DBQC. This method is as sensitive as methods based on the reaction of lipoic acid with dinitrothiobenzoate and requires only a single extraction, but does not require reduction of the disulfide and the color reagent does not need to be prepared daily. Results obtained using this assay to quantitate lipoic acid released from lipoyl-N-p-aminobenzoate correlated excellently with results obtained using the Marshall–Bratton reaction to quantitatep-aminobenzoate. We have detected lipoyl-N-?-lysine hydrolysis activity that is distinct from that of biotinidase and bile salt-stimulated lipase in lymphoblasts from a patient with biotinidase deficiency. This assay can be used to measure lipoyl-N-?-lysine hydrolysis activity in tissues, especially those with little or no biotinidase activity.     

Synthesis of Triamino Acid Building Blocks with Different Lipophilicities

To obtain different amino acids with varying lipophilicity and that can carry up to three positive charges we have developed a number of new triamino acid building blocks. One set of building blocks was achieved by aminoethyl extension, via reductive amination, of the side chain of ortnithine, diaminopropanoic and diaminobutanoic acid. A second set of triamino acids with the aminoethyl extension having hydrocarbon side chains was synthesized from diaminobutanoic acid. The aldehydes needed for the extension by reductive amination were synthesized from the corresponding Fmoc-L-2-amino fatty acids in two steps. Reductive amination of these compounds with Boc-L-Dab-OH gave the C4-C8 alkyl-branched triamino acids. All triamino acids were subsequently Boc-protected at the formed secondary amine to make the monomers appropriate for the N-terminus position when performing Fmoc-based solid-phase peptide synthesis.     

The conformation of catalytically functional Schiff's bases: the pyruvate--lysine ketimine of 2-keto-3-deoxygluconate-6-phosphate aldolase of Pseudomonas putida

The reduction stereochemistry of the Schiff's base formed between pyruvate and the ?-amino of the catalytic lysine of 2-keto-3-deoxygluconate-6-P-phosphate aldolase of Pseudomonas putida was investigated. Reduction was stereoselective yielding 55.73% N⁶-[(1R)-and 44.27% N⁶-[(1S)-1-carboxyethyl]-S-lysine. Thus the reducing agent predominated slightly at the si face of the ketimine carbon. For comparison, the reduction stereochemistry of the pyruvate-lysine ketimine formed on d-amino acid oxidase during d-alanine turnover at pH 8.5 was also investigated. In this case reduction was random, consistent with nonactive site participation in that transimination reaction generating the ketimine, as postulated by other investigators of this enzyme.     

Analysis of adipimidate-crosslinked lysine

The chromatographic conditions for separation of N,N′-bislysyl(?-N)adipamidine and N-lysyl(?-N)adipamidinic acid, which were the products of acid hydrolysis of proteins treated with adipimidate esters, from other amino acids on an amino acid analyzer were established including their ninhydrin color values. Kinetics of decomposition of these lysine derivatives under the conditions of total acid hydrolysis of protein are also reported.     

Microwave-enhanced reductive amination via Schiff's base formation for block copolymer synthesis

Reductive amination via Schiff's base formation is a widely used reaction for laboratory and industrial applications ranging from protein immobilization to nanoparticle synthesis. One major limitation of this reaction is the slow kinetics and hence, it can take several days for the reaction to reach completion. Here we demonstrate that electromagnetic microwave can be used to accelerate the rate of reduction amination. To demonstrate proof of concept, we utilized the reductive amination between reducing end of dextran and primary amine from N-Boc-ethylenediamine as a model reaction. The reaction was conducted at room temperature to demonstrate that the enhancement was mainly due to electromagnetic effects of the microwave rather than thermal effects. We show that reductive amination reaction time was reduced from 72 h to 4 h using microwave irradiation. These results indicate non-thermal microwave effects to expedite reductive amination for synthesizing copolymers. The efficient conjugation of dextran using reductive amination provides an important tool for developing biocompatible copolymers using carbohydrates.     

2‐Picoline‐borane: A non‐toxic reducing agent for oligosaccharide labeling by reductive amination

Analysis of N‐glycans is often performed by LC coupled to fluorescence detection. The N‐glycans are usually labeled by reductive amination with a fluorophore containing a primary amine to allow fluorescence detection. Moreover, many of the commonly applied labels also allow improved mass spectrometric detection of oligosaccharides. For reductive amination, the amine group of the label reacts with the reducing‐end aldehyde group of the oligosaccharide to form a Schiff base, which is reduced to a secondary amine. Here, we propose the use of 2‐picoline‐borane as the reducing agent, as a non‐toxic alternative to the extensively used, but toxic sodium cyanoborohydride. Using dextran oligosaccharides and plasma N‐glycans, we demonstrate similar labeling efficacies for 2‐picoline‐borane and sodium cyanoborohydride. Therefore, 2‐picoline‐borane is a non‐toxic alternative to sodium cyanoborohydride for the labeling of oligosaccharides.     

Microorganisms hydrolyse amide bonds; knowledge enabling read-across of biodegradability of fatty acid amides

To get insight in the biodegradation and potential read-across of fatty acid amides, N-[3-(dimethylamino)propyl] cocoamide and N-(1-ethylpiperazine) tall oil amide were used as model compounds. Two bacteria, Pseudomonas aeruginosa PK1 and Pseudomonas putida PK2 were isolated with N-[3-(dimethylamino)propyl] cocoamide and its hydrolysis product N,N-dimethyl-1,3-propanediamine, respectively. In mixed culture, both strains accomplished complete mineralization of N-[3-(dimethylamino)propyl] cocoamide. Aeromonas hydrophila PK3 was enriched with N-(1-ethylpiperazine) tall oil amide and subsequently isolated using agar plates containing dodecanoate. N-(2-Aminoethyl)piperazine, the hydrolysis product of N-(1-ethylpiperazine) tall oil amide, was not degraded. The aerobic biodegradation pathway for primary and secondary fatty acid amides of P. aeruginosa and A. hydrophila involved initial hydrolysis of the amide bond producing ammonium, or amines, where the fatty acids formed were immediately metabolized. Complete mineralization of secondary fatty acid amides depended on the biodegradability of the released amine. Tertiary fatty acid amides were not transformed by P. aeruginosa or A. hydrophila. These strains were able to utilize all tested primary and secondary fatty acid amides independent of the amine structure and fatty acid. Read-across of previous reported ready biodegradability results of primary and secondary fatty acid amides is justified based on the broad substrate specificity and the initial hydrolytic attack of the two isolates PK1 and PK3.     

Concise preparation of <Emphasis Type="Italic">N</Emphasis><Superscript>α</Superscript>-Fmoc-<Emphasis Type="Italic">N</Emphasis><Superscript>ɛ</Superscript>-(Boc,methyl)-lysine and its application in the synthesis of site-specifically lysine monomethylated peptide

Summary. A concise preparation of N ^α-Fmoc-N ^ɛ-(Boc, methyl)-lysine and its application in the synthesis of site-specifically lysine monomethylated peptide is described. N ^α-Fmoc-N ^ɛ-(Boc, methyl)-lysine is obtained, via consecutive reductive benzylation and reductive methylation in a one-pot reaction, followed by debenzylation through catalytic hydrogenolysis and Boc protection in another one-pot reaction. A peptide containing monomethylated lysine is successfully synthesized by incorporating N ^α-Fmoc-N ^ɛ-(Boc, methyl)-lysine as a building block via solid-phase peptide synthesis.     

A fast, efficient and stereoselective synthesis of hydroxy-pyrrolidines

A five-step, protecting group free synthesis of 2,3-cis substituted hydroxy-pyrrolidines is presented. Key steps in the synthesis are the chemoselective formation of a primary amine via a Vasella reductive amination using ammonia as the nitrogen source, and the stereoselective formation of a cyclic carbamate from an alkenylamine. Improvement of the reductive amination, by way of the use of α-picoline borane as a more environmentally benign reducing agent, is also presented.     

Kinetics and mechanism of catalysis by proteolytic enzymes: The kinetics of hydrolysis of derivatives of l-lysine and S-(beta-aminoethyl)-l-cysteine(thialysine)by bovine trypsin

1. Several esters of the α-N-toluene-p-sulphonyl and N-benzoyl derivatives of l-lysine and S-(β-aminoethyl)-l-cysteine have been synthesized. 2. The kinetics of hydrolysis of the esters by bovine trypsin have been compared. Values of k₀ are similar for corresponding derivatives of the isosteric amino acids and deacylation of an acyl-enzyme appears to be rate-determining in each case. There are, however, some quantitative kinetic differences between the various series of substrates.     

Saccharopine and 2-aminoadipic acid in Reseda odorata

2(S),2′(S)-N⁶-(2′-Glutaryl)lysine (l-saccharopine) and 2(S)-2-aminoadipic acid have been isolated from Reseda odorata. When traditional isolation procedures are used l-pyrosaccharopine (5(S),5′(S)-N-(5′-amino-5′-carboxy-pentyl)-2-pyrrolidone-5-carboxylic acid) is formed from l-saccharopine by lactamisation. The degree of lactamisation during various isolation steps has been studied, The amino acids were identified by IR and PMR spectroscopy and the configurations established by enzymic and polarimetric analyses. The contents of saccharopine and 2-amino-adipic acid have been determined relative to the total nitrogen content at various stages in the growth cycle of R. odorata.     

Synthesis of N-(1-deoxyhexitol-1-yl)amino acids, reference compounds for the nonenzymic glycosylation of proteins

The N-(1-deoxy-D-mannitol-1-yl) and N-(1-deoxy-D-glucitol-1-yl) derivatives of L-valine, L-alanine, L-threonine, and L-leucine were prepared by reductive amination of D-mannose and D-glucose with the appropriate amino acids, in the presence of sodium cyanoborohydride. N epsilon-(1-Deoxy-D-mannitol-1-yl)- and N epsilon-(1-deoxy-D-glucitol-1-yl)-L-lysine were prepared by similar reactions of hexoses with N alpha-tert-butoxycarbonyl and N alpha-benzyloxycarbonyl-L-lysine, followed by removal of the protecting groups. The structures were confirmed by 1H-n.m.r. spectroscopy, which showed that each compound was completely free of its C-2 epimer. The synthetic compounds may be used as reference compounds for the identification of N-(1-deoxyhexitol-1-yl)amino acids formed when N-(1-deoxy-D-fructose-1-yl) groups of nonenzymically glycosylated proteins, of the hemoglobin A1c type, are reduced with sodium borohydride, and the protein is subjected to acid-catalyzed hydrolysis.     

Synthesis of iminoalditol and N-alkyl iminoalditol derivatives of ribopyranosides

Iminoalditol analogs of ribopyranosides were prepared by reduction of a vinylogous urethane intermediate formed from methyl 2-C-(5-O-methanesulfonyl-β-d-ribofuranosyl)acetate (1) by treatment with sodium azide in DMF at reflux. The N-alkylated analogs were synthesized either by N-alkylation of the corresponding parent iminoaldithol or, more efficiently, from the product of the reaction of 1 with various alkylamines. The latter process involves an S_N2 substitution at C-5 by the amine followed by an intramolecular hetero-Michael reaction under basic conditions. The ‘aglycon’ of the iminoalditol was also modified through amidation and esterification.     

1-Deoxynojirimycins with dansyl capped N-substituents as probes for Morbus Gaucher affected cell lines

Cyclization by double reductive amination of d-xylo-hexos-5-ulose with methyl 6-aminohexanoate gave (methoxycarbonyl)pentyl-1-deoxynojirimycin. Reaction of the terminal carboxylic acid with N-dansyl-1,6-diaminohexane provided the corresponding chain-extended fluorescent derivative. By reaction with bis(6-dansylaminohexyl)amine, the corresponding branched di-N-dansyl compound was obtained. Both compounds are strong inhibitors of d-glucosidases and could also be shown to distinctly improve, at sub-inhibitory concentrations, the activity of β-glucocerebrosidase in a Gaucher fibroblast (N370S) cell-line through chaperoning of the enzyme to the lysosome.     

Analysis of unsaturated fatty acids, and their hydroperoxy and hydroxy derivatives by high-performance liquid chromatography.

A simple procedure for the detection of endo-β-N-acetylglucosaminidase H activity is described. The method utilizes N-[¹⁴C]methylribonuclease B as substrate. This is prepared from ribonuclease B by reductive alkylation of free amine groups in the protein with [¹⁴C]formaldehyde. Because the carbohydrate moiety of ribonuclease B has α-mannosyl residues at nonreducing terminal positions, the radioactive molecule binds to Sepharose-concanavalin A. Endo-β-N-acetylglucosaminidase action releases this mannose-containing oligosaccharide by splitting the di-N-acetylchitobiosyl residue that links it with the peptide and thereby renders the radioactive portion of the molecule unreactive with Sepharose-concanavalin A. This forms the basis of a convenient assay for screening column fractions during the purification of the endoglycosidase. Although protease or α-mannosidase activity might also be detected by the procedure, no difficulties were presented by these enzymes when the assay was used for the preparation of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus.     

Enzymatic synthesis of L-6-hydroxynorleucine

L-6-Hydroxynorleucine, a key chiral intermediate used for synthesis of a vasopeptidase inhibitor, was prepared in 89% yield and > 99% optical purity by reductive amination of 2-keto-6-hydroxyhexanoic acid using glutamate dehydrogenase from beef liver. In an alternate process, racemic 6-hydroxynorleucine produced by hydrolysis of 5-(4-hydroxybutyl)hydantoin was treated with D-amino acid oxidase to prepare a mixture containing 2-keto-6-hydroxyhexanoic acid and L-6-hydroxynorleucine followed by the reductive amination procedure to convert the mixture entirely to L-6-hydroxynorleucine, with yields of 91 to 97% and optical purities of > 99%.     

Metabolism of L-beta-lysine in a Pseudomonas: conversion of 6-N-acetyl-L-beta-lysine to 3-keto-6-acetamidohexanoate and of 4-aminobutyrate to succinic semialdehyde by different transaminases.

Some kinetic properties of two new species of transaminase found in extracts of a β-lysine-utilizing Pseudomonas are reported. Transaminase A catalyzes transamination between 6-N-acetyl-l-β-lysine (3-amino-6-acetamidohexanoate) and α-ketoglutarate to form 3-keto-6-acetamidohexanoate and glutamate. Transaminase B catalyzes a reaction between 4-aminobutyrate and pyruvate to form succinic semialdehyde and alanine. The formation of both transaminases is induced by growth of the bacteria on l-β-lysine, although transaminase B is also produced in the absence of this substrate. Transaminase A requires pyridoxal phosphate for activity. The β-keto acid formed from acetyl-β-lysine by transaminase A has been purified and characterized by decarboxylation, conversion to a formazan, reduction to a stable β-hydroxy acid, and conversion of the latter to its methyl ester. Transaminase B, unlike previously reported transaminases utilizing 4-aminobutyrate, cannot use α-ketoglutarate as an amino group acceptor. This enzyme is not stimulated by addition of pyridoxal phosphate, but is inhibited by hydroxylamine or cyanide. Both transaminases appear to function in the main pathway of β-lysine degradation.     

Pseudo-halogen sugar derivatives: Synthesis and hofmann-rearrangement reactions; 6-O-[2-(N,N-dichlorocarbamoyl)-ethyl]-1,2:3,4-di-O-isopropylidene-α-d-galactopyranose

6-O-[2-(N,N-Dichlorocarbamoyl)ethyl]-1,2:3,4-di- O-isopropylidene-α-d-galactopyranose, a highly reactive pseudo-halogen, was conveniently prepared in 97% yield by addition of sodium hypochlorite to an aqueous acidic (pH <2) solution of 6-O-(2-carbamoylethyl)-1,2:3,4-di-O-isopropylidene-α-d-galactopyranose. Mild, reductive dechlorination or alkaline hydrolysis readily converted the nonpolar N,N-di-chloroamide sugar derivative into the corresponding water-soluble N-monochloroamide form. Hofmann rearrangement of the N-chloroamide group provides a synthetic route to novel binary sugar-derivatives having carbamoyl, ureylene, and allophanoyl linkages. Structural proof for the pseudo-halogens and their Hofmann-rearrangement products was obtained from i.r., ¹H-n.m.r., mass-spectral, and chemical data.     

Reactions of chitosan: 5. The reaction of chitosan with 2,4-dinitrofluorobenzene and determination of the extent of the reaction

The reaction between chitosan and 2,4-dinitrofluorobenzene has been studied and suitable conditions established for hydrolysis of the product prior to determination of the extent of reaction by u.v./visible spectroscopy. The chromophore system in $\text{N-(2,4-}\text{dinitrophenyl}\text{)-2-}\text{amino}\text{-2-}\text{deoxy-}\text{d}\text{-glucose}$, the final product from the acid hydrolysis of N-(2,4-dinitrophenyl)chitosan, is unstable to heating in solution in either water or aqueous acid. The temperature of hydrolysis should therefore not exceed 50°C and at this temperature the u.v./visible absorption spectrum of $\text{N-(2,4-}\text{dinitrophenyl}\text{)-2-}\text{amino}\text{-2-}\text{deoxy-}\text{d}\text{-glucose}$ is constant for up to 50 h. Complete reaction of the amine groups is not achieved under heterogeneous or homogeneous conditions, only approximately 50% of the available amine groups undergoing reaction under homogeneous conditions. This restricted reactivity results from the bulky N-(2,4-dinitrophenyl) residues shielding adjacent unreacted amine groups on the same chain, thereby preventing their reaction with 2,4-dinitrofluorobenzene. Such intramolecular steric hindrance would be expected to increase with increase in the free amine group content of the sample, due to the increase in the fraction of amine groups occurring in sequence length of two or more, and an inverse relationship between the total initial free amine group content and the percentage of these that react with 2,4-dinitrofluorobenzene has been found     

Identification and estimation of some uncommon diamino acids

Application of the amino acid analyzer is described for the separation, identification, and estimation of several uncommon dibasic amino acids that are eluted with a 0.35 m, pH 5.28, sodium citrate buffer between lysine and ammonia, namely, 2,5-diaminohexanoate, threo- and erythro-3,5-diaminohexanoate, 3,6-diaminohexanoate (β-lysine) and 2,4-diaminopentanoic acid. Conditions are also given for estimating d- and l-β-lysine as their l-glutamyl peptides, and for estimating the enantiomers of threo- and erythro-3,5-diaminohexanoate as the l-glutamyl derivative of the corresponding lactams.