Brian J. Fitzsimmons

Leukotriene A5 is a substrate and an inhibitor of rat and human neutrophil LTA4 hydrolase

The epoxide 5(S) trans-5,6 oxido, 7,9 trans-11,14,17 cis eicosatetraenoic acid (leukotriene A5) was chemically synthesized and demonstrated to be both a substrate and an inhibitor of partially purified rat and human LTA4 hydrolase. Both rat and human LTA4 hydrolase utilized leukotriene A5 less effectively as a substrate than leukotriene A4. Incubation of leukotriene A5 (10 microM) or leukotriene A4 (10 microM) with rat neutrophils demonstrated formation of 123 pmol LTB5/min/10(7) cells and 408 pmol LTB4/min/10(7) cells respectively. Purified rat neutrophil LTA4 hydrolase incubated with 100 microM leukotriene A5 produced 22 nmol LTB5/min/mg protein and when incubated with 100 microM leukotriene A4 produced 50 nmol LTB4/min/mg protein. Human neutrophil LTA4 hydrolase incubated with 100 microM leukotriene A5 produced 24 nmol LTB5/min/mg protein and when incubated with 100 microM leukotriene A4 produced 52 nmol LTB4/min/mg protein. Leukotriene A5 was an inhibitor of the formation of leukotriene B4 from leukotriene A4 by both the rat and human neutrophil LTA4 hydrolase. Excess leukotriene A5 prevented covalent coupling of [3H] leukotriene A4 to LTA4 hydrolase suggesting inhibition may involve covalent coupling of leukotriene A5 to the LTA4 hydrolase.

Activation of leukocyte movement and displacement of [3H]leukotriene B4 from leukocyte membrane preparations by (12R)-and (12S)-hydroxyeicosatetraenoic acid

Both (12R)- and (12S)-hydroxyeicosatetraenoic acid were demonstrated to produce aggregation of rat leukocytes and enhance human leukocyte chemokinesis. (12R)-Hydroxyeicosatetraenoic acid was 10-20-fold more potent than (12S)-hydroxyeicosatetraenoic acid but at least 500-fold less potent than leukotriene B4 in these assays. These relative potencies are correlated with the potencies of (12R)- and (12S)-hydroxyeicosatetraenoic acid for competition of [3H]leukotriene B4 binding to rat and human leukocyte membrane preparations.

[4+2] Cycloaddition reaction of bis (trichloroethyl) azodicarboxylate and glycals: preparation of a C1-C1 2-amino disaccharide

Abstract Bis (trichloroethyl) azodicarboxylate is an efficient diene in the [4+2] cycloaddition reaction with glycals. It offers several advantages over the previously described dibenzyl azodicarboxylate (DBAD) which was used to prepare 2-amino glycosides.

Total synthesis of lipoxin B: assignment of stereochemistry

Abstract Four isomers of lipoxin B were synthesized using carbohydrate precursors. An authentic sample of lipoxin B (from human leukocytes) was found to be a mixture of all-trans isomers 1 a and 1c .

Synthesis of lipoxins: Total synthesis of conjugated trihydroxy eicosatetraenoic acids

Abstract The first synthesis of a conjugated trihydroxy eicosatetraenoic acid, a possible structure for Lipoxin A is described. A biomimetic approach was utilized.

Metabolism and excretion of peptide leukotrienes in the anesthetized rat

The metabolism and excretion of the peptide leukotrienes C4, D4, E4 and N-acetylleukotriene E4 have been studied in the anesthetized rat. The intravenous administration of [3H]leukotriene C4 (2.6 X 10(-11) mol/kg) showed a rapid clearance of radioactivity from the blood and a time-related biliary excretion, recovering 69 +/- 1.6% (n = 6) over 60 min. Less than 1% of total radioactivity was recovered in the urine over the same time period. Similarly, the intravenous administration of [3H]leukotriene D4 (2.5 X 10(-11) mol/kg), [3H]leukotriene E4 (2.5 X 10(-11) mol/kg) and N-acetyl[3H]leukotriene E4 (2.1 X 10(-11) mol/kg) showed a 62 +/- 7.5% (n = 4), 52 +/- 1.5% (n = 4) and 37 +/- 4.6% (n = 5) biliary recovery of radioactivity, respectively, after 60 min. Examination of bile identified leukotriene D4 and N-acetylleukotriene E4 as the main products, although substantial radioactivity, which probably represents unidentified polar products, was present at the solvent fronts of the reverse-phase HPLC. Time course studies indicated a relatively rapid conversion of leukotriene C4 to leukotriene D4, while leukotriene D4 metabolism appeared to be much slower. Leukotriene E4 was a minor product, suggesting that the N-acetylation process is rapid. Incubation of [3H]leukotriene C4 in rat plasma and whole blood in vitro resulted in a slow conversion of leukotriene C4 to leukotriene D4 and leukotriene E4 only. These data suggest that the majority of the leukotriene metabolism and excretion in vivo in the anesthetized rat occurs predominantly in the hepatic system. We conclude that this model is suitable for the measurement of in vivo production of peptide leukotrienes.

Evidence of in-vivo W-oxidation of peptide leukotrienes in the rat: biliary excretion of 20-CO2H N-acetyl LTE4

In a previous study in our laboratory it was observed that after [3H] LTC4 administration (luCi/kg i.v.) to the anesthetized rat, significant amounts of injected radioactivity (approximately 25%) were associated with previously unidentified biliary polar metabolite(s). In the present study we describe the isolation and characterization of the predominant polar metabolite. Rats were injected with synthetic LTC4 (20 microgram/kg i.v.) and bile collected over 30 min. After extraction and purification (2 step RP-HPLC procedure), the retention time of the metabolite was compared (plus coinjections) and found to be identical with synthetic 20-CO2H N-Ac LTE4 in two RP-HPLC systems. Also, the UV spectrum of the biologically derived metabolite was compared and found identical to the synthetic material, giving a characteristic conjugated triene absorption in the UV with a max of 281 nm and shoulders at 270 and 290 nm. Further, the trimethyl ester derivative of the metabolite showed identical chromatographic behaviors in 2 reverse and 2 normal phase HPLC systems compared with synthetic 20-CO2H N-Ac LTE4 trimethyl ester. We conclude omega-oxidation of peptide leukotrienes occurs in the rat and that 20-CO2H N-Ac LTE4 is an in vivo product of LTC4 metabolism.

Leukotriene B3, leukotriene B4 and leukotriene B5; Binding to leukotriene B4 receptors on rat and human leukocyte membranes

Specific high-affinity binding sites for [3H]-leukotriene B4 have been identified on membrane preparations from rat and human leukocytes. The rat and human leukocyte membrane preparations show linearity of binding with increasing protein concentration, saturable binding and rapid dissociation of binding by excess unlabelled leukotriene B4. Dissociation constants of 0.5 to 2.5 nM and maximum binding of 5000 fmoles/mg protein were obtained for [3H] leukotriene B4 binding to these preparations. Displacement of [3H]-leukotriene B4 by leukotriene B4 was compared with displacement by leukotriene B3 and leukotriene B5 which differ from leukotriene B4 only by the absence of a double bond at carbon 14 or the presence of an additional double bond at carbon 17, respectively. Leukotriene B3 was shown to be equipotent to leukotriene B4 in ability to displace [3H]-leukotriene B4 from both rat and human leukocyte membranes while leukotriene B5 was 20-50 fold less potent. The relative potencies for the displacement of [3H]-leukotriene B4 by leukotrienes B3, B4 and B5 on rat and human leukocyte membranes were shown to correlate well with their potencies for the induction of the aggregation of rat leukocytes and the chemokinesis of human leukocytes.

Biochemical Studies on Mammalian Lipoxygenasesa,b

The arachidonate cascade provides biochemists with a number of materials of enzymological interest. Mammalian tissues have several lipoxygenases, each with a positional and stereochemical specificity of arachidonate oxygenation. Some lipoxygenases are involved in the synthesis of bioactive oxyeicosanoids, while others are still of unknown physiological significance. Fatty acid cyclooxygenase is an atypical lipoxygenase incorporating two molecules of oxygen into arachidonic acid and producing a prostaglandin peroxide.

Analogs of leukotriene B4: Effects of modification of the hydroxyl groups on leukocyte aggregation and binding to leukocyte leukotriene B4 receptors

The syntheses and agonist and binding activities of 5(S)-hydroxy- 6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (12-deoxy LTB4), 5(S), 12(S)-dihydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (12-epi LTB4), 12(R)-hydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5-deoxy LTB4), 5(R), 12(S)-dihydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5-epi LTB4), 6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5, 12-deoxy LTB4) are described. These leukotriene B4 analogs were all able to aggregate rat leukocytes and compete with [3H]-leukotriene B4 for binding to rat and human leukocyte leukotriene B4 receptors with varying efficacy. The analog in which the 12-hydroxyl group was removed was severely reduced both in agonist action (aggregation) and binding. The epimeric 12-hydroxyl analog demonstrated better agonist and binding properties than the analog without a hydroxyl at this position. In contrast, in the case of the 5-hydroxyl the epimeric hydroxyl analog had greatly reduced agonist and binding activities while the 5-deoxy analog demonstrated potency only several fold less than leukotriene B4 itself. The dideoxy leukotriene B4 analog was more than a thousand fold less active than leukotriene B4 as an agonist and in binding to the leukotriene B4 receptor. These results show that binding to the leukocyte leukotriene B4 receptor requires a hydroxyl group at the 12 position in either stereochemical orientation but that the presence of a hydroxyl at the 5 position is less important. However, the epimeric C5 leukotriene B4 analog clearly interacts unfavourably with the binding site of the leukotriene B4 receptor.