Purification and biochemical characterisation of some of the properties of recombinant human kynureninase

Recombinant human kynureninase (L-kynurenine hydrolase, EC 3.7.1.3) was purified to homogeneity (60-fold) from Spodoptera frugiperda (Sf9) cells infected with baculovirus containing the kynureninase gene. The purification protocol comprised ammonium sulfate precipitation and several chromatographic steps, including DEAF-Sepharose CL-6B, hydroxyapatite, strong anionic and cationic separations. The purity of the enzyme was determined by SDS/ PAGE, and the molecular mass verified by MALDI-TOF MS. The monomeric molecular mass of 52.4 kDa determined was &gt; 99.99% of the predicted molecular mass. A UV absorption spectrum of the holoenzyme resulted in a peak at 432 mn. The optimum pH was 8.25 and the enzyme displayed a strong dependence on the ionic strength of the buffer for optimum activity. This cloned enzyme was highly specific for 3-hydroxykynureiune (K-m = 3.0 muM +/- 0.10) and was inhibited by L-kynurenine (K-i = 20 muM), D-kynurenine (K-i = 12 muM) and a synthetic substrate analogue D,L-3,7-dihydroxydesaminokynurenine (K-i = 100 nM). The activity/concentration profile for kynureninase from this source was sigmoidal in all instances. There appeared to be partial inhibition by substrate, and excess pyridoxal 5'-phosphate was found to be inhibitory.</p

The Synthesis of 5-Fluorokynurenine and 6-Fluorokynurenic Acid as Metabolic Probes

(2S)-5-Fluorokynurenine 9 and 6-fluorokynurenic acid 14 were synthesised as fluorinated probes to enable the metabolism of kynurenine to be monitored in vivo by F-19 NMR spectroscopy. The ( 2S)-5-fluorokynurenine 9 was prepared using a Friedel-Crafts acylation of N-((t)butoxycarbonyl)-4-fluoroaniline with a chiral oxazolidine derivative 6, derived from 2S-aspartic acid. This represents a novel, and simple, method for the synthesis of kynurenine derivatives. The 6-fluorokynurenic acid 14 was synthesised using a Conrad-Limpach type synthesis. Preliminary biological studies are described.</p

Synthesis of multiply ¹³C-labeled furofuran lignans using ¹³C-labeled cinnamyl alcohols as building blocks

Plant lignans are currently being widely studied for their potential benefits for human health as their consumption has been correlated with lower risks for developing chronic diseases, such as breast cancer and coronary heart disease. However, studies of some classes of lignans, in particular the furofurans, are hampered by the lack of suitable standards to allow accurate analysis. Herein, we report the syntheses of two racemic C-13-labeled furofuran lignans [7,8,9-C-13(3)]medioresinol and [7,8, 9-C-13(3)]sesamin as internal standards for LC-MS analysis. The labeled furofuran lignans were constructed from triply labeled cinnamyl alcohols, using a radical cyclization method. (c) 2005 Elsevier Inc. All rights reserved.</p

Synthesis of multiply ¹³C-labeled furofuran lignans using ¹³C-labeled cinnamyl alcohols as building blocks

Plant lignans are currently being widely studied for their potential benefits for human health as their consumption has been correlated with lower risks for developing chronic diseases, such as breast cancer and coronary heart disease. However, studies of some classes of lignans, in particular the furofurans, are hampered by the lack of suitable standards to allow accurate analysis. Herein, we report the syntheses of two racemic C-13-labeled furofuran lignans [7,8,9-C-13(3)]medioresinol and [7,8, 9-C-13(3)]sesamin as internal standards for LC-MS analysis. The labeled furofuran lignans were constructed from triply labeled cinnamyl alcohols, using a radical cyclization method. (c) 2005 Elsevier Inc. All rights reserved.</p

Synthesis of [1-13C] and [1-15N] labelled D1-HomphenylalanineVia a Key Neber Rearrangement

A synthetic route involving a key Neber rearrangement is described for the preparation of both [1-C-13] and [1-N-15] DL-homophenylalanine (2-amino-4-phenylbutanoic acid), using suitably labelled sodium cyanide as the source of the isotopic label. These compounds have been prepared for use in studies on the biosynthesis of phenylethyl glucosinolate in Brassica napus. 3-Phenylpropanaldoxime, the initial biosynthetic product formed from homophenylalanine, was also prepared in N-15 labelled form.</p

Synthesis of [1-13C] and [1-15N] labelled D1-HomphenylalanineVia a Key Neber Rearrangement

A synthetic route involving a key Neber rearrangement is described for the preparation of both [1-C-13] and [1-N-15] DL-homophenylalanine (2-amino-4-phenylbutanoic acid), using suitably labelled sodium cyanide as the source of the isotopic label. These compounds have been prepared for use in studies on the biosynthesis of phenylethyl glucosinolate in Brassica napus. 3-Phenylpropanaldoxime, the initial biosynthetic product formed from homophenylalanine, was also prepared in N-15 labelled form.</p

Synthesis Of Deuterium Labelled Desulfoglucosinolates as Internal Standards for LC-MS Analysis

The syntheses of three deuterium labelled desulfoglucosinolates are described. These are the phenethyl, 1-methoxyindolyl and 4-methoxyindolyl derivatives. The compounds were prepared as internal standards for use in the quantitative LC-MS analysis of glucosinolates to improve the sensitivity of the analytical procedure. (C) 1999 Elsevier Science Ltd. All rights reserved.</p

A new short synthesis of coumestrol and its application for the synthesis of [6,6a,11a-¹³C₃]coumestrol

A convenient and simple two-step method for the synthesis of coumestrol has been established, which involves a base catalysed condensation of phenyl acetate with benzoyl chloride, followed by demethylation and subsequent tandem intramolecular cyclisation. This method was then employed for the efficient synthesis of multiply C-13-labelled coumestrol. (C) 2003 Elsevier Ltd. All rights reserved.</p