Preparation of omega trideuterated sulfidopeptide leukotrienes: Analysis by FAB/MS

Facile preparation of leukotrienes C4, D4 and E4 containing carbon‐13 and nitrogen‐15 for quantification of cysteinyl leukotrienes by mass spectrometry

“…The reaction of labeled glutathione with commercially available leukotriene A 4 methyl ester provides labeled leukotriene C 4 methyl ester, which is converted to labeled leukotriene C 4 by hydrolysis under alkaline conditions (Figure 1). Using the same strategy, leukotriene D 4 synthesis requires labeled cysteinyl-glycine, but this was not the preferred procedure because of three reasons: (1) we found that labeled leukotriene C 4 could be completely converted to labeled leukotriene D 4 by incubation with commercially available transpeptidase in the presence of a mixture of amino acids as glutamyl acceptors ( Figure 1). It is more efficient and faster to carry out a single solid-phase peptide synthesis of glutathione rather than having to cleave two portions of resin and carry out two peptide purifications to also obtain labeled cysteinyl-glycine; (2) yields for the solid-phase synthesis of cysteinyl-glycine were very low (55%), the major product being the dipeptide-derived diketopiperazine.…”

“…Using commercially available reagents, heavy isotopelabeled cysteinyl leukotrienes (leukotriene C 4 , D 4 and E 4 ) are readily prepared in amounts sufficient for thousands of liquid chromatography/electrospray ionization mass spectrometry quantitative analyses of leukotrienes in biological samples. The use of chemically identically but isotopically differentiated cysteinyl leukotrienes as internal standards provides the most accurate way to quantify these eicosanoids.…”

“…The cysteinyl leukotrienes (leukotrienes C 4 , D 4 and E 4 ) are well-known eicosanoids (derived from arachidonic acid) that bind to G protein-coupled receptors and mediate a number of proinflammatory events, especially in the airways. 1 The quantification of cysteinyl leukotrienes is often carried out in studies of inflammation in experimental animals and in humans.…”

“…Although heavy atom-substituted prostaglandins and leukotriene B 4 are commercially available, there are no commercial sources of labeled cysteinyl leukotrienes. The latter are typically quantified by using a single internal standard, deuterated leukotriene B 4 , and determining, by separate analysis, the relative ionization efficiencies of the cysteinyl leukotriene relative to that for leukotriene B 4 . However, the addition of heavy atom-substituted internal standard for each molecular species at the earliest step possible to the biological sample is superior since it accounts for any loss of analyte due to absorption to container walls, enzymatic or non-enzymatic degradation of analyte and other factors.…”

“…The synthesis of cysteinyl leukotrienes containing deuterium in the fatty acid portion has been reported (for example, see Prakash et al 4 ), but this requires a fairly involved synthesis of deuterated leukotriene A 4 methyl ester, which can then be reacted with cysteine, cysteinyl-glycine or glutathione to form deuterated leukotriene E 4 , D 4 and C 4 , respectively. In this report, we describe the facile synthesis of cysteinyl leukotrienes containing carbon-13 and nitrogen-15 in the cysteine residue.…”