Facile preparation of leukotrienes C4, D4 and E 4 containing carbon-13 and nitrogen-15 for quantification of cysteinyl leukotrienes by mass spectrometry

Article abstract of DOI:10.1002/jlcr.1414

With the recent ability to use combined liquid chromatography/electrospray tandem mass spectrometry to analyze for several eicosanoids in biological samples in a single and rapid experiment, heavy isotope-labeled eicosanoids are needed as internal standards in order to quantify eicosanoid analytes. The present study describes a practical preparation of cysteinyl leukotrienes (leukotriene C₄, D₄ and E₄) with three ¹³C atoms and one ¹⁵N atom in the cysteinyl residue. The method involves solid-phase peptide synthesis to make glutathione with heavy isotopes in the cysteinyl residue and reaction of this tripeptide with commercially available leukotriene A₄ methyl ester to give labeled leukotriene C₄ methyl ester, which is hydrolyzed to labeled leukotriene C₄. Labeled leukotriene E₄ is prepared in the same way with the use of labeled cysteine. Labeled leukotriene D₄ is prepared by treatment of labeled leukotriene C₄ with commercially available γ-glutamyl transpeptidase. Copyright

Full text of DOI:10.1002/jlcr.1414

Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 729–733.
Published online 17 July 2007 in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1414
JLCR
Note
Facile preparation of leukotrienes C4, D4 and E4 containing
carbon-13 and nitrogen-15 for quantification of cysteinyl
leukotrienes by mass spectrometry
FARIDEH GHOMASHCHI1, JAMES G. BOLLINGER1 and MICHAEL H. GELB1,2,
*
1 Department of Chemistry, Campus Box 351700, University of Washington, Seattle, WA 98195, USA
2 Department of Biochemistry, University of Washington, Seattle, WA 98195, USA
Received 24 April 2006; Revised 1 June 2007; Accepted 5 June 2007
Abstract: With the recent ability to use combined liquid chromatography/electrospray tandem mass spectrometry to
analyze for several eicosanoids in biological samples in a single and rapid experiment, heavy isotope-labeled
eicosanoids are needed as internal standards in order to quantify eicosanoid analytes. The present study describes a
practical preparation of cysteinyl leukotrienes (leukotriene C4, D4 and E4) with three 13C atoms and one 15N atom in
the cysteinyl residue. The method involves solid-phase peptide synthesis to make glutathione with heavy isotopes in
the cysteinyl residue and reaction of this tripeptide with commercially available leukotriene A4 methyl ester to give
labeled leukotriene C4 methyl ester, which is hydrolyzed to labeled leukotriene C4. Labeled leukotriene E4 is
prepared in the same way with the use of labeled cysteine. Labeled leukotriene D4 is prepared by treatment of labeled
leukotriene C4 with commercially available g-glutamyl transpeptidase. Copyright # 2007 John Wiley & Sons, Ltd.
Keywords: cysteinyl leukotrienes; mass spectrometry; eicosanoids; inflammation; lipid mediators
Introduction
accurate quantification can only be carried out by
inclusion of a chemically identical, but isotopically
distinguished, internal standard in the sample.
Although heavy atom-substituted prostaglandins and
leukotriene B4 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 B4, and
determining, by separate analysis, the relative ioniza-
tion efficiencies of the cysteinyl leukotriene relative to
that for leukotriene B4. 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. For example, cysteinyl leukotrienes are readily
degraded by hydrolases present on the surface of
inflammatory cells and are known to stick to metal
surfaces, such as the injection needle of the autoinjec-
tor at the front end of the liquid chromatograph.
The cysteinyl leukotrienes (leukotrienes C4, D4 and E4)
are well-known eicosanoids (derived from arachidonic
acid) that bind to G protein-coupled receptors and
mediate a number of proinflammatory events, espe-
cially in the airways.1 The quantification of cysteinyl
leukotrienes is often carried out in studies of inflam-
mation in experimental animals and in humans. This is
typically done by some form of immunoassay, such as
radioimmunoassay using commercially available kits.
However, recent developments using combined liquid
chromatography/electrospray
ionization
tandem
mass spectrometry allow a large set of eicosanoids
(leukotrienes, prostaglandins and other lipid media-
tors) to be quantified in a single multiplex analysis.2,3
Since different molecular species ionize in the
mass spectrometer source with different efficiencies,
*Correspondence to: Michael H. Gelb, Department of Chemistry,
Campus Box 351700, University of Washington, Seattle, WA 98195,
USA. E-mail: gelb@chem.washington.edu
Contract/grant sponsor: National Institutes of Health; contract/grant
numbers: HL36235, HL50040
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
Copyright # 2007 John Wiley & Sons, Ltd.

730 F. GHOMASHCHI ET AL.
fairly involved synthesis of deuterated leukotriene A4
methyl ester, which can then be reacted with cysteine,
cysteinyl-glycine or glutathione to form deuterated
leukotriene E4, D4 and C4, respectively. In this report,
we describe the facile synthesis of cysteinyl leuko-
trienes containing carbon-13 and nitrogen-15 in the
cysteine residue. These are readily made by preparing
the labeled cysteine-containing materials, which can be
reacted with commercially available leukotriene A4
methyl ester to form the desired labeled cysteinyl
leukotrienes.
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 diketopiper-
azine. Diketopiperazine formation occurs on occasion
during solid-phase synthesis of dipeptides, probably as
a result of intramolecular attack of the dipeptide amino
terminus onto the carbonyl carbon of the ester invol-
ving the C-terminal amino acid and the resin-bound
leaving group. This cyclization can occur either during
removal of the FMOC group from the N-terminal amino
acid or during the subsequent step of side-chain
deprotection combined with cleavage of the peptide
from the resin under acidic conditions; (3) we obtained
very low yields of leukotriene D4 methyl ester (51–2%)
for the reaction of leukotriene A4 methyl ester with
cysteinyl-glycine. The major products were leukotriene
B4 methyl ester (resulting from hydrolysis of leuko-
triene A4 methyl ester) and the disulfide derived from
cysteinyl-glycine. It seems that disulfide formation
from this dipeptide occurs more readily than disulfide
formation from glutathione or from cysteine during the
synthesis of leukotriene C4 methyl ester and leuko-
triene E4 methyl ester, respectively.
Results and discussion
The preparation of isotopically substituted leukotriene
C4 with heavy isotope in the cysteinyl portion requires
that labeled glutathione be prepared. This was carried
out using standard FMOC-based solid-phase peptide
synthesis. Labeled and protected cysteine ([1-13C,
2-13C, 3-13C, 15N]N-FMOC-Cys(S-trityl from Cambridge
Isotopes, Inc.)) was used because it is common to all of
the cysteinyl leukotrienes. During this process, the
cysteine residue is protected with a trityl group on its
sulfhydryl. The cleavage of the peptide from the resin
and side-chain deprotection is best carried out in the
presence of scavengers, such as thioanisole and 1,2-
dithioethane, to prevent capture of the trityl cation by
the cysteine sulfhydryl and other peptide nucleophiles.
It is important that 1,2-dithioethane be completely
removed since it can react with leukotriene A4 methyl
ester during the subsequent step. This was accom-
plished by trapping the glutathione on a strong cation
exchange column (Dowex 50 in the Hþ form) and
washing with water to elute the 1,2-dithioethane in the
void volume. The glutathione is then eluted by washing
with aqueous NH4OH. The glutathione at this point still
contains protecting group fragments and is purified by
high-performance liquid chromatography (HPLC) on a
reverse-phase (C18) column. Glutathione elutes in the
void volume, and the more hydrophobic protecting
group fragments are retained on the column. HPLC
purification is preferred over the use of a solid-phase
extraction cartridge since some of the impurities elute
just after the void volume peak. The reaction of labeled
glutathione with commercially available leukotriene A4
methyl ester provides labeled leukotriene C4 methyl
ester, which is converted to labeled leukotriene C4 by
hydrolysis under alkaline conditions (Figure 1).
Leukotriene E4 was prepared via reaction of leuko-
triene A4 methyl ester with commercially available
labeled cysteine followed by alkaline hydrolysis of the
methyl ester. The labeled cysteine could also be
obtained by deprotection of the same protected cysteine
that was used in the solid-phase synthesis of the
labeled glutathione. However, the use of commercially
available cysteine is more practical as it avoids the
steps needed to remove protecting group fragments
from the cysteine that form during deprotection of N-
FMOC-cysteine(S-trityl).
All three cysteinyl leukotrienes were purified by
HPLC on a C18 reverse-phase column. This is an
important step so that leukotriene B4 is removed. Since
leukotriene B4 is not labeled with stable isotopes, it
must be removed from the labeled cysteine leuko-
trienes to avoid contamination of biological samples (in
cases where sample-derived leukotriene B4 is being
quantified). Leukotriene B4 methyl ester forms in
small amounts by hydrolysis of leukotriene A4 methyl
ester during the reaction of the latter with the thiol
component.
Using the same strategy, leukotriene D4 synthesis
requires labeled cysteinyl-glycine, but this was not the
preferred procedure because of three reasons: (1) we
found that labeled leukotriene C4 could be completely
converted to labeled leukotriene D4 by incubation with
commercially available transpeptidase in the presence
All three labeled cysteinyl leukotrienes gave the
expected peak for the (M-Hþ)ꢀ ion in negative-
ion electrospray mass spectrometry (see Experimental).
For all three cysteinyl leukotrienes, the peak at (M-Hþ)ꢀ
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 729–733
DOI: 10.1002.jlcr

HEAVY ATOM-LABELED CYSTEINYL LEUKOTRIENES 731
Figure 1 Synthesis of labeled cysteinyl leukotriene methyl esters. The black dots indicate the location of the 13C and 15N isotopes.
Saponification leads to the labeled cysteinyl leukotrienes (not shown).
plus 1 was 17% as intense as the (M-Hþ)ꢀ, and no
peaks were observed at (M-Hþ)ꢀ plus 2 indicating no
cross-contamination of the labeled cysteine leuko-
trienes into the mass channel for the non-labeled
species. Thus, these labeled cysteinyl leukotrienes are
appropriate for use as internal standards to quantify
the amount of non-labeled cysteinyl leukotrienes in
biological samples (see Henderrson et al.,3 for an
example). Major fragment ions for all three labeled
cysteinyl leukotrienes include loss of H2O and the lipid
fragment due to cleavage of the Cb–S bond of the
cysteinyl residue.
Piperidine (20% by vol.) in DMF (10 ml) was added to
the resin, and the vial was swirled briefly. After 1 min,
the liquid was removed, and the resin was treated with
a second portion of piperidine (20%) in DMF for 30 min
with mixing on a rotating wheel. The resin was washed
4 times with 10 ml portions of DMF and then dried in a
vacuum desiccator.
Anhydrous HOBt (Novabiochem Inc., 0.27 g, 2 mmol)
and TBTU (Novabiochem Inc., 0.64 g, 2 mmol) were
dissolved in 5 ml DMF by stirring. The solution was
stored in a capped vial for up to 2 weeks at room
temperature. Gly-Wang resin (72 mg, 56 mmol Gly) was
placed in vial. The vial was charged with 50 mg of
[1-13C, 2-13C, 3-13C, 15N]N-FMOC-Cys(S-trityl) (Cam-
bridge Isotopes, Inc.), followed by 0.21 ml of HOBt/
TBTU/DMF solution, followed by 29 ml of diisopropy-
lethylamine (Aldrich) and finally followed by 0.4 ml
DMF. The capped vial was mixed on a rotating wheel
overnight (ꢁ12 h) at room temperature. The resin was
washed 6 times with 4 ml portions of DMF. The resin
was treated with 20% of piperidine in DMF as above.
The resin was washed 6 times with 4 ml portions of
DMF and then 3 times with 4 ml portions of ether. The
resin was dried in a vacuum desiccator.
Experimental
Synthesis of [1-13C, 2-13C, 3-13C, 15N]Cys-Glutathione
All steps with peptide synthesis resin were carried out
at room temperature in a 5-ml glass vial with a Teflon
septum-lined screw cap. To wash the resin, the vial was
centrifuged at ꢁ800 rpm for ꢁ30 s in
a clinical
centrifuge, and most of the supernatant was removed
with a Pasteur pipet, being careful not to take resin
beads. DMF is anhydrous grade (Aldrich). FMOC-Gly-
Wang Resin (Novabiochem Inc., 0.78 mmol/g) (0.5 g)
was washed 4 times with 10 ml portions of DMF.
A vial containing dried Cys-Gly-Wang resin (37 mg,
29 mmol peptide) (see above) was charged with
Copyright # 2007 John Wiley & Sons, Ltd.
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DOI: 10.1002.jlcr

732 F. GHOMASHCHI ET AL.
N-FMOC-Glu, with its a-COOH group protected as its
t-butyl ester (Bachem, Inc.) (49 mg, 116 mmol), 0.29 ml
of HOBt/TBTU/DMF solution (116 mmol) (see above)
and 40 ml of diisopropylethylamine (232 mmol). The
capped vial was mixed overnight at room temperature
on a rotating wheel. The resin was washed with DMF,
treated with piperidine/DMF and washed with DMF
and ether as above. The resin was dried briefly in a
vacuum desiccator and then treated with 1 ml of
aliquots of triethylamine with spotting of a small
solution aliquot onto moistened pH paper. Into a
second vial, 50 mg of leukotriene A4 methyl ester
(solution in hexane/1% triethylamine, Cayman Che-
micals) was added. The solvent was removed with a
stream of N2. To this vial, the solution of glutathione
was immediately added. The capped vial was kept at
room temperature for 4 h in the dark. Then 0.5 ml of
0.1 M K2CO3 in water was added, and the capped vial
was kept at room temperature for 3 h in the dark. The
mixture was brought to pH ꢁ5 (pH paper) with small
aliquots of 96% formic acid. The solution was applied to
a C18 solid-phase extraction cartridge (50 mg solid
phase, Waters Inc., which was pre-conditioned by
washing with 1 ml of methanol/0.1% formic acid and
then with two portions of water/0.1% formic acid, 1 ml
each). After application of the reaction mixture and
centrifugation, the cartridge was washed with two
portions of water/0.1% formic acid (1 ml each) (the
solution was first delivered to the empty reaction vial
and then transferred to the cartridge), and the product
was eluted into a clean receiving tube using two 1 ml
portions of methanol/0.1% formic acid (again rinsing
the empty reaction vial prior to transfer to the
cartridge). The eluant was concentrated to dryness in
a Speed-Vac. The residue was dissolved in 100 ml of
methanol and submitted to purification by HPLC
(two injections of 50 ml each). The column (Zorbax
SB-C18, 2.1 ꢂ 100 mm, 3.5 m, Agilent Inc.) was
equilibrated with 20% solvent B (acetonitrile with
0.1% formic acid) and 80% solvent A (water, Milli-Q,
Millipore, Inc.). The solvent program was 0–10 min,
20% B, 10–15 min 38% B, 15–25 min 65% B, 25–
35 min 100% B. The absorbance was monitored at
280 nm. The product eluted at ꢁ22 min. The product
solution was concentrated to dryness in a Speed-Vac,
and the residue was dissolved in ethanol (prior to use,
the ethanol was subjected to several cycles of vacuum
degassing, N2 purging). The solution was stored in
aliquots in flame-sealed ampules or Teflon-septum
capped glass vials under N2 at ꢀ808C. The yield is
15–20%. The structure was confirmed based on HPLC
retention time identical to leukotriene C4 authentic
standard (Cayman Chemicals). Negative-ion electro-
spray ionization mass spectrometry gave the expected
value for (M-Hþ)ꢀ¼ 628:5 and a collisional-induced
dissociation spectrum identical to that of the authentic
standard.
trifluoroacetic
acid/thioanisole/1,2-ethanedithiol/
water (85/5/5/5 by vol.) for 1 h at room temperature.
The solution was filtered through a glass wool-plugged
Pasteur pipet. Most of the liquid was removed with a
stream of N2 at room temperature (in a fume hood,
stench). To the residue was added 1 ml water (not all
solid dissolves), and the mixture was applied to a
column of AG 50W-X8 strong cation-exchange resin
(BioRad, 1.5 ꢂ 1.5 cm bed volume). The resin was
previously washed with ꢁ10 ml of 1 N NaOH, then with
water until the eluant was at neutral pH (pH paper),
then with ꢁ10 ml of 1 N HCl, and finally with water
until the eluant was at neutral pH. After loading the
crude peptide, the column was washed with five
portions of water (6 ml each). The column was washed
with five portions of 6 N aqueous NH4OH (6 ml each),
collecting each wash into a separate glass tube. The six
fractions were brought to dryness in a Speed-Vac
concentrator (Savant Instruments, Inc.). Negative-ion
electrospray ionization mass spectrometry analysis on
a Bruker Esquire ion trap instrument showed that the
glutathione peptide was present in fractions 1 and 2
(mainly as the reduced form, i.e. non-disulfide). The
residue in fractions 1 and 2 was dissolved in 0.7 ml of
water, and the solution was submitted to HPLC on a
C18 column (Vydac 218TP1010, 1 ꢂ 25 cm) that was
previously equilibrated with water containing 0.05%
trifluoroacetic acid. The eluant was monitored with a
UV detector set to 214 nm. The desired peptide eluted
in the void volume, with the first impurity UV peak
appearing just after the void volume peak. The void
volume fraction was brought to dryness in a Speed-Vac.
[1-13C, 2-13C, 3-13C, 15N]Cys-Leukotriene C4
The labeled glutathione was dissolved in 1 ml of water.
The concentration of free SH was determined by
standard Ellman assay with 5,50-dithio-bis(2-nitroben-
zoic acid) using a 2 ml aliquot of the glutathione solution
and standard cysteine solution for calibration. An
aliquot of the stock solution of labeled glutathione
(0.5 mmol) was transferred to a small glass vial and the
solution was brought to dryness in a Speed-Vac. To the
vial, 20 ml of methanol/water (6/1, v/v) was added. The
pH was adjusted to 9–10 by the addition of 0.5 ml
The concentration of labeled leukotriene C4 was
measured by spiking a known volume of stock solution
with a known amount of non-labeled leukotriene C4
(Caychem Chemicals, Inc.) and measuring the relative
parent ion signals by negative-ion electrospray mass
spectrometry.
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 729–733
DOI: 10.1002.jlcr

HEAVY ATOM-LABELED CYSTEINYL LEUKOTRIENES 733
[1-13C, 2-13C, 3-13C, 15N]Cys-Leukotriene D4
[1-13C, 2-13C, 3-13C, 15N]Cys-Leukotriene E4
The conversion of leukotriene C4 to leukotriene D4 was
carried out enzymatically, essentially as described in
Anderson et al.5 The enzyme is g-glutamyl transpepti-
dase (Sigma Cat. G-9270, 11 units/mg powder). The
enzyme stock is prepared by dissolving 0.8 mg of
powder in 1 ml of purified water (Milli-Q). The reaction
buffer contains 40 mM sodium phosphate, pH 7.4,
0.5 mM l-alanine, 0.12 mM l-arginine, 0.25 mM l-
asparagine, 0.02 mM l-aspartic acid, 0.04 mM l-cy-
stine, 0.04 mM l-glutamate, 0.4 mM l-glutamine,
0.08 mM l-histidine, 0.06 mM l-isoleucine, 0.15 mM
l-leucine, 0.2 mM l-lysine, 0.04 mM l-methionine,
0.13 mM l-phenylalanine, 0.19 mM l-serine, 0.2 mM
l-threonine, 0.05 mM l-tryptophan, 0.08 mM l-tyro-
sine, 0.2 mM l-valine, and 0.4 mM l-glycine. Gluta-
mine is added immediately before the addition of
enzyme. The reaction mixtures contain 5 mg of [1-13C,
2-13C, 3-13C, 15N]Cys-Leukotriene C4, 214 ml of reac-
tion buffer and 11 ml of enzyme stock solution. The
mixture is incubated at 378C for 30 min and then
stored at ꢀ208C prior to purification. The sample is
centrifuged at ꢁ12 000 ꢂ g for 1 min to remove
any solid (none observed) prior to injection of half
This was prepared, stored and quantified as for
leukotriene C4 using labeled cysteine ([1-13C, 2-13C,
3-13C, 15N]Cys, Cambridge Isotopes, Inc.). The yield is
40–50%. The structure was confirmed based on HPLC
retention time identical to leukotriene E4 authentic
standard (Cayman Chemicals). Negative-ion electrospray
ionization mass spectrometry gave the expected value for
(M-Hþ)ꢀ¼ 442:2 and a collisional-induced dissociation
spectrum identical to that of the authentic standard.
Conclusions
Using commercially available reagents, heavy isotope-
labeled cysteinyl leukotrienes (leukotriene C4, D4 and
E4) are readily prepared in amounts sufficient for
thousands of liquid chromatography/electrospray io-
nization mass spectrometry quantitative analyses of
leukotrienes in biological samples. The use of chemically
identically but isotopically differentiated cysteinyl leuko-
trienes as internal standards provides the most accurate
way to quantify these eicosanoids. The use of internal
standards corrects for any loss of analytes due to sample
processing or adsorption to surfaces of the liquid
chromatography/mass spectrometry instrument.
of the mixture onto
a reverse-phase C18 HPLC
column (Zorbax SB-C18, 2.1 ꢂ 100 mm, 3.5 mm,
Agilent Cat. 861753-902) previously equilibrated
with 80% solvent A (water, Milli-Q)/20% solvent B
(acetonitrile with 0.1% formic acid) at a flow rate of
1.5 ml/min. The absorbance at 280 nm is monitored.
The solvent program is: 0–10 min, 20% B; 10–15 min,
20–38% B; 15–25 min, 38–65% B; 25–35 min, 65–100%
B. Leukotriene D4 elutes at about 28 min. The yield is
70%. The reaction was also carried out on a 2-fold
larger scale (10 mg of labeled leukotriene C4) with
Acknowledgements
This work was funded by grants from the National
Institutes of Health (HL36235 and HL50040).
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J Label Compd Radiopharm 2007; 50: 729–733
DOI: 10.1002.jlcr