Novel glutathione conjugates of phenyl isocyanate identified by ultra-performance liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance

Article abstract of DOI:10.1002/jms.3306

Phenyl isocyanate is a highly reactive compound that is used as a reagent in organic synthesis and in the production of polyurethanes. The potential for extensive occupational exposure to this compoundmakes it important to elucidate its reactivity towards different nucleophiles and potential targets in the body. In vitro reactions between glutathione and phenyl isocyanate were studied. Three adducts of glutathione with phenyl isocyanate were identified using ultra-performance liquid chromatography/ electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR). Mass spectrometric data for these adducts have not previously been reported. Nucleophilic attack on phenyl isocyanate occurred via either the cysteinyl thiol group or the glutamic acid α-amino group of glutathione. In addition, a double adduct was formed by the reaction of both these moieties. NMR analysis confirmed the proposed structure of the double adduct, which has not previously been described. These results suggest that phenyl isocyanate may react with free cysteines, the α-amino group and also with lysine residues whose side chain contains a primary amine. Copyright

Full text of DOI:10.1002/jms.3306

Research article
Received: 28 June 2013
Revised: 31 October 2013
Accepted: 4 November 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3306
Novel glutathione conjugates of phenyl
isocyanate identified by ultra-performance
liquid chromatography/electrospray
ionization mass spectrometry and
nuclear magnetic resonance
Tove Johansson Mali’n,* Sandra Lindberg and Crister Åstot
Phenyl isocyanate is a highly reactive compound that is used as a reagent in organic synthesis and in the production of polyure-
thanes. The potential for extensive occupational exposure to this compound makes it important to elucidate its reactivity towards
different nucleophiles and potential targets in the body. In vitro reactions between glutathione and phenyl isocyanate were
studied. Three adducts of glutathione with phenyl isocyanate were identified using ultra-performance liquid chromatography/
electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR). Mass spectrometric data for these adducts
have not previously been reported. Nucleophilic attack on phenyl isocyanate occurred via either the cysteinyl thiol group or
the glutamic acid α-amino group of glutathione. In addition, a double adduct was formed by the reaction of both these moieties.
NMR analysis confirmed the proposed structure of the double adduct, which has not previously been described. These results
suggest that phenyl isocyanate may react with free cysteines, the α-amino group and also with lysine residues whose side chain
contains a primary amine. Copyright © 2014 John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: glutathione; trapping; isocyanate; amino group; thiol group
analyzed in urine and plasma in an effort to measure the expo-
Introduction
sure of 11 workers to phenyl isocyanate.[6] Studies of reactivity
Isocyanates are highly reactive compounds that contain the
(ÀN = C = O) functional group. In general, they are used in
organic synthesis and as starting materials for the manufacture
of polyurethanes. Polyurethanes are polymers with diverse uses
in products such as rubber modifiers, paints, inks, insulating
materials, varnishes, foams and lacquers.[1] Methyl isocyanate
has been used in the production of carbamate pesticides. Follow-
ing the Bhopal accident of 1984, in which thousands of people
died due to a large-scale release of methyl isocyanate, numerous
studies have been performed to investigate the toxicity of this
compound.[2]
Phenyl isocyanate is another isocyanate that is used as a
reagent in organic synthesis and in the production of polyure-
thanes.[3] Its chemical structure is shown in Fig. 1. To our knowl-
edge, its toxicity has not been examined in as much detail as
that of methyl isocyanate. However, the clinical symptoms of
phenyl isocyanate exposure are similar to those described for
methyl isocyanate, including respiratory and ocular irritation
(with subsequent tissue damage) and delayed death.[4] In rats,
phenyl isocyanate has been shown to cause an asthma-like
syndrome.[5] The capacity of phenyl isocyanate to cause contact
sensitization in mice has been evaluated in the mouse ear swell-
ing test. The results indicated that phenyl isocyanate is a potent
inducer of humoral and cellular immune responses.[3] Aniline,
the hydrolysis product of phenyl isocyanate, has previously been
often focus on reactions between a reactive electron-deficient
(electrophilic) species and an electron-rich (nucleophilic) species.
Glutathione (GSH, γ-L-glutamyl-L-cysteinyl-glycine) is a tripeptide
that is both the most abundant cellular thiol and the most
abundant low molecular weight peptide in eukaryotic cells. It acts
as a nucleophilic scavenger, protecting the cells by functioning as
an antioxidant and by detoxifying electrophilic species. Elec-
trophiles may react directly with the nucleophilic thiol group of
glutathione and the conjugation may also be catalyzed by
glutathione S-transferase enzymes. Under certain conditions,
the GSH pool in the cells may become depleted, making other
endogenous thiol-containing molecules more susceptible to
attack by electrophiles. This may lead to the formation of covalent
bonds between reactive species and biological macromolecules
such as proteins, DNA or enzymes, and can have toxic effects.
GSH is widely used as a model nucleophile in studies of reactive
molecules.[7,8] For example, in the pharmaceutical industry, GSH is
used to trap reactive drug metabolites as their GSH conjugates,
which are generally more stable and detectable than the original
*
Correspondence to: Tove Johansson Mali’n, Swedish Defense Research Agency,
SE-901 82 Umeå, Sweden. E-mail: tove.johansson.malin@foi.se
Swedish Defense Research Agency, SE-901 82 Umeå, Sweden
J. Mass Spectrom. 2014, 49, 68–79
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Novel glutathione conjugates of phenyl isocyanate
546 → 378 (CE 15 V), the dwell time was set to 10 ms. Data were
processed using the MassLynx (version 4.1) and MetaboLynx
softwares (Waters, Milford, MA, USA).
Figure 1. Chemical structure of phenyl isocyanate.
Glutathione conjugation reactions
metabolite.[9] The respiratory tract is a major route for isocyanate
exposure and isocyanates are known to cause sensitization of the
respiratory tract and occupational asthma. The lower respiratory
tract contains high levels of glutathione, and it has been shown that
methyl isocyanate forms conjugates with glutathione.[10–12] We
therefore decided to explore the ability of phenyl isocyanate to
form conjugates with glutathione.
The aim of this study was to elucidate the reactivity of the
cysteinyl thiol moiety and the glutamic acid α-amino group of
glutathione towards phenyl isocyanate in vitro and to identify
the resulting glutathione conjugates by ultra-performance liquid
chromatography/electrospray mass spectrometry (UPLC/ESI-MS)
and nuclear magnetic resonance (NMR).
Phosphate buffer (0.1 M, pH 7.4) was prepared from potassium
dihydrogen phosphate and dipotassium hydrogen phosphate. A
fresh 5 mM glutathione solution was then prepared in the
phosphate buffer. Phenyl isocyanate was added to the glutathi-
one solution to a final concentration of 50 μM. The mixture was
vortexed for 10 s and then left to react at 37 °C for 2 h. A precip-
itate formed during the reaction, and so the samples were
filtered prior to dilution 1:100 (v/v) with 0.4% formic acid in water.
The samples were then injected into the UPLC-ESI/MS system.
Glutathione conjugation time study
A fresh 5 mM glutathione solution was then prepared in the phos-
phate buffer (0.1M) and phenyl isocyanate was added to a final
concentration of 50μM. The mixture was vortexed for 10 s, diluted
1:10 (v/v) with 0.1M phosphate buffer and put in the autosampler
(20 °C). Samples were injected every minute during 60min.
Experimental procedures
Materials
Synthesis of 5-[[2-(carboxymethylamino)-2-oxo-1-
(phenylcarbamoylsulfanylmethyl)-ethyl]amino]-
5-oxo-2-(phenylcarbamoylamino)pentanoic acid
(3) for characterization by NMR
L-glutathione (reduced form), phenyl isocyanate and potassium
dihydrogen phosphate were obtained from Sigma–Aldrich (St. Louis,
MO, USA). Formic acid was obtained from Honeywell Riedel-de Haën
(Seelze, Germany). Dipotassium hydrogen phosphate and acetoni-
trile were obtained from Merck (Darmstadt, Germany). S-acetyl-L-
glutathione was obtained from abcr GmbH & Co. KG (Karlsruhe,
Germany). All solvents were of analytical grade and the water used
in the experiments was obtained from a water purification system
(Milli-Q, Merck Millipore, Billerica, MA, USA).
In a 5 ml vial, 2-amino-5-[[1-(carboxymethylamino)-1-oxo-3-sulfanyl-
2-propanyl]amino]-5-oxopentanoic acid (GSH) (50 mg, 0.163 mmol)
was dissolved in 1 ml DMF and a minimal amount of water. Neat
phenyl isocyanate (194 mg, 1.627 mmol, 10 mol.eq.) was then added
drop wise at room temperature over a period of two days. The
reaction mixture was stirred at room temperature for another 48 h
before being quenched with water. The precipitate formed (1,3-
diphenyl-urea) was filtered off and the filtrate was concentrated
under reduced pressure prior to purification by preparative HPLC.
The purest fractions with the desired product were pooled and the
solvent removed in vacuo to afford 3 as a white solid.
UPLC-ESI/MS method
The samples were analyzed by liquid chromatography (Waters
ACQUITY UPLC, Milford, MA, USA), on a 2.1× 50 mm, 1.7 μm C18-
column (Waters Acquity UPLC BEH), at a flow rate of 600 μl/min
(the column temperature was set at 60 °C). Mobile phase A
consisted of 0.1% (v/v) formic acid in Milli-Q water and mobile
phase B consisted of 0.1% (v/v) formic acid in acetonitrile. The
solvent gradient was linear, increasing from 5% B to 95% B over
5.5min. The initial mobile phase composition was then restored
over 0.5min and this condition was maintained for a further
0.5 min. For the time study, the following UPLC conditions were
used: 35% B for 0.8 min, increasing to 95% B over 0.1min and the
initial mobile phase conditions were restored over 0.1min. For the
response study, the solvent gradient was linear, increasing from
5% B to 95% B over 0.7min. The initial mobile phase composition
was then restored over 0.1 min and this condition was maintained
for a further 0.1 min. The UPLC system was coupled to a triple quad-
rupole mass spectrometer (Waters Xevo TQ, Milford, MA, USA)
interfaced with an electrospray ionization source. A capillary volt-
age of 3.5kV was used and the cone voltage was set to 30 V. GSH
adducts were detected using full scan analyses acquired in positive
ionization mode. For the acquisition of MS/MS spectra, a collision
energy (CE) of 15 V was used and argon was employed as collision
gas. The injection volume was 5 μl for full scan mode detection and
10 μl for product ion detection. For the time study, the following
multiple reaction monitoring transitions were used: 213 → 94
(CE 30 V), 427 → 259 (CE 30 V), 427 → 298 (CE 30 V),
Preparative HPLC
The crude mixture from the synthesis was purified by liquid chro-
matography (Waters AutoPurification HPLC, Milford, MA, USA), on
a 10 × 50 mm, 5 μm C18-column (Waters SunFire), at a flow rate of
25 ml/min. Mobile phase A consisted of 0.1% (v/v) formic acid in
water and mobile phase B consisted of 0.1% formic acid in aceto-
nitrile. The solvent gradient was linear, increasing from 5% B to
70% B over 3.33 min and then from 70% B to 95% B over 7 s. This
composition was maintained for 3.67 min, after which the initial
mobile phase composition was restored over 6 s; the resulting
conditions were maintained for 0.87 min. The HPLC system was
coupled to a photodiode array detector (Waters 2998 PDA).
NMR
1H NMR (500 MHz) and 13C NMR (100 MHz) spectra were recorded
on an Avance 500 instrument from Bruker (Karlsruhe, Germany),
using methanol-d4 as the solvent. Chemical shifts are reported
in ppm using the residual solvent peak as an internal standard.
The residual 1H peak for methanol occurs at δH 3.31 and the
corresponding 13C peak occurs at δC 49.00.
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T. Johansson Mali’n, S. Lindberg and C. Åstot
1H NMR (500 MHz, methanol-d4) δ = 7.47 (dd, J = 1.0, 8.5 Hz,
2 H), 7.35 (dd, J = 1.1, 8.6 Hz, 2 H), 7.30 – 7.19 (m, 4 H), 7.04 (quin,
J = 1.0 Hz, 1 H), 6.96 (tt, J = 1.1, 7.4 Hz, 1 H), 4.67 (dd, J = 4.7,
8.6 Hz, 1 H), 4.36 (dd, J = 4.9, 8.2 Hz, 1 H), 3.94 – 3.80 (m, 2 H),
3.53 (dd, J = 4.7, 14.2 Hz, 1 H), 3.24 – 3.13 (m, 1 H), 2.49 – 2.36
(m, 2 H), 2.29 – 2.20 (m, 1 H), 2.07 – 1.95 (m, 1 H); 13C NMR
(126 MHz, methanol-d4) d 175.5, 172.8, 157.9, 140.9, 140.2,
130.1, 129.9, 125.1, 123.6, 120.7, 55.1, 48.0, 33.3, 32.3, 29.6, 9.4
detected in the LC-MS full scan analysis: the extracted ion chro-
matograms are shown in Fig. 2. The first adduct eluted at
1.38 min (1, m/z 427), the second at 1.58 min (2, m/z 427) and
the third at 2.41 min (3, m/z 546).
The glutathione adducts detected at m/z 427 (1 and 2) corre-
spond to an addition of 307 Da to phenyl isocyanate, implying
that these adducts were formed by the equimolar reaction of
phenyl isocyanate with glutathione.
The product ion spectrum of the first adduct (m/z 427,
[M + H]+), which eluted after 1.38 min (1), is shown in Fig. 3.
Proposed precursor ions, neutral losses and fragment ions are
shown in Table 1.
Results
The base peak (m/z 298) was the y-type ion formed by the
characteristic neutral loss of anhydroglutamic acid (129 Da) and
In this study, the in vitro reaction between phenyl isocyanate and
glutathione was investigated. Three putative GSH adducts were
Figure 2. Extracted ion chromatograms of the [M + H]+ ions of 1 (m/z 427), 2 (m/z 427) and 3 (m/z 546).
Figure 3. CID (CE 15 V) spectrum of the [M + H]+ ion of the first eluting glutathione adduct 1 (m/z 427).
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Novel glutathione conjugates of phenyl isocyanate
Table 1. Proposed precursor ions, neutral losses and fragment ions for the MS/MS fragmentations of adduct 1 (CE 15 V)
Precursor ion
Neutral loss
Fragment ion
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T. Johansson Mali’n, S. Lindberg and C. Åstot
together with the second y-ion at m/z 76 identified adduct 1 as
the product arising from a nucleophilic attack of the cysteinyl
thiol moiety of glutathione on phenyl isocyanate. The prominent
m/z 298 ion corresponds to the fragmentation pattern of a short
peptide, protonated at the α-amino terminal. In a singly charged,
α-linkage peptide, the fragmentation is initiated by the proton
transfer from the α-amino group to the C-terminal fragment with
the release of a cyclic neutral loss.[13,14] For adduct 1, the proton
transfer to the nearby peptide nitrogen atom producing the m/z
298 y-ion will be energetically favourable. From the m/z 298 ion,
two major fragments ions were formed: the m/z 179 ion by the
subsequent loss of phenyl isocyanate and the m/z 195 fragment
ion, presumably produced by a cyclization reaction producing an
aldoxime ion (Scheme 1). The m/z 195 ion is characteristic of the
adducts where phenyl isocyanate has been conjugated to the
cysteine moiety of glutathione (adduct 1 and 3).
The product ion spectrum of the second adduct (m/z 427,
[M + H]+), which eluted after 1.58 min (2), is shown in Fig. 4. The
product ion spectrum of adduct 2 contained more ions than
the spectrum of adduct 1, and y- and b-ion series with about
equal abundances were observed. A peptide with multiple sites
of equal basicity will produce a heterogeneous population of pro-
tonated species upon ionization promoting a high complexity of
fragmentation after collision activation. Thus, the adduct was
tentatively identified as the product arising from a nucleophilic
attack of the basic glutamic acid α-amino moiety of glutathione
on phenyl isocyanate to form a urea derivative. In agreement
with the ‘mobile proton theory’, the conversion of the N-terminal
to a non-basic urea group promotes the mobility of the proton in
the single charged ion with a reduction of the activation energies
for a number of fragmentation reactions along the peptide
backbone.[15,16]
Proposed precursor ions, neutral losses and fragment ions are
shown in Table 2.
The ions at m/z 76 and 179 and b-ion series containing the ions
of m/z 352 and 249 confirmed a modification on the N-terminal
of the molecule. The structure of b-ions has been shown to
include the interaction of the C-terminal carbonyl with a carbonyl
group of the polypeptide backbone creating an oxazolone
structure.[17,18] In adduct 2, the urea carbonyl group offers an
alternative reaction site out of the polypeptide chain. The
involvement of the urea carbonyl group in the formation of the
m/z 352 b-ion was confirmed by the subsequent loss of aniline,
thereby generating the base peak product ion of m/z 259
(Scheme 2). Fragmentation initiated by protonation of the urea
group gave rise to the protonated aniline at m/z 94, loss of aniline
at m/z 334 and the loss of phenyl isocyanate to reform charged
GSH was detected as the ion m/z 308.
The third glutathione adduct (m/z 546, [M + H]+), eluting at
2.41 min (3), corresponds to the reaction of two phenyl isocya-
nate molecules with one glutathione molecule and was tenta-
tively identified as the product arising from a nucleophilic
attack of both the cysteinyl thiol group and the glutamic α-amino
moiety of glutathione. The product ion spectrum of adduct 3 is
shown in Fig. 5; proposed precursor ions, neutral losses and
fragment ions are shown in Table 3.
The product ion spectrum of the glutathione adduct 3
contained a mixture of specific fragments from both 1 and 2
and a number of new fragments in the high mass region. The ma-
jor fragmentation reaction is proposed to be the formation of a
cyclic b-type fragment ion, involving the carbonyl group of the
urea moiety (m/z 471) followed by the loss of aniline (m/z 378).
The mechanism is analogous to the reactions of adduct 2
Scheme 1. Proposed formation of the fragment ion m/z 195.
Figure 4. CID (CE 15 V) spectrum of the [M + H]+ ion of the second eluting glutathione adduct 2 (m/z 427).
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Novel glutathione conjugates of phenyl isocyanate
Table 2. Proposed precursor ions, neutral losses and fragment ions for the MS/MS fragmentations of adduct 2 (CE 15 V)
Precursor ion
Neutral loss
Fragment ion
(Continues)
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T. Johansson Mali’n, S. Lindberg and C. Åstot
Table 2. (Continued)
Precursor ion
Neutral loss
Fragment ion
Scheme 2. Proposed formation of the fragment ions m/z 352 and m/z 259.
(Scheme 2). The fragmentation pattern of 3 also indicated the for-
mation of an m/z 471 b-ion with a proposed oxazolone structure
based on the loss of the phenyl thiocarbamide moiety producing
the m/z 318 product ion. The subsequent loss of aniline from the
m/z 318 ion, producing the m/z 225 ion, was best explained by
the m/z 318 b-type ion involving the urea carbonyl group. A
fragment was detected at m/z 427 resulting from loss of one phenyl
isocyanate (119Da) from the adduct. Since this fragment was signif-
icant for adduct 2 and not for adduct 1, the fragment is proposed
to be arising from the loss of the phenyl isocyanate that was
bound to the glutamic acid α-amino moiety. The proposed
structures of the glutathione adducts 1, 2 and 3 are shown
in Fig. 6.
The peak response areas of the extracted ion chromatograms
for the glutathione adducts 1 and 3 did not differ significantly.
However, the peak response area for adduct 2 was approximately
three times smaller than those for 1 and 3. Adducts 2 and 3, with
the N-terminal converted to phenyl urea groups, are likely to
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Novel glutathione conjugates of phenyl isocyanate
Figure 5. CID (CE 15 V) spectrum of the [M + H]+ ion of the third eluting glutathione adduct 3 (m/z 546).
display a lower ionization efficiency based on the lower degree of
protonation in solution. The peak response areas of the acetyl an-
alogues of adduct 1 and 2 were determined in a response study
and were found to differ by a factor of 2 (Supplemental data).
Thus, the levels of adduct 2 and 3 relative to adduct 1 were
underestimated based on the peak response.
within 1 min. The response (peak height) of the three glutathione
conjugates and the hydrolysis product was measured during
60 min, detected every minute by UPLC-ESI/MS (data not shown).
No significant difference in response was observed between the
analysis performed after 1 min and the data acquired at the
following time points.
The chromatogram of the full scan analysis also contained a
species that eluted after 2.77 min and had an m/z of 213 (data
not shown). This species is proposed to be 1,3-diphenyl-urea,
formed by the hydrolysis of phenyl isocyanate (with the release
of carbon dioxide) to produce aniline, followed by reaction with
a second phenyl isocyanate molecule.[19] This suggestion is
supported by its product ion spectrum (data not shown) contai-
ning fragments at m/z 120 (corresponding to a neutral loss of
93 Da to produce phenyl isocyanate), 94 and 77. The peak
response area of the extracted ion chromatogram of 1,3-
diphenyl-urea is approximately the same as that for glutathione
adduct 2. This is significant because phenyl isocyanate hydrolysis
competes with glutathione adduct formation.
Discussion
In a previous study by Fleischel et al., two glutathione conjugates
resulting from reactions of phenyl isocyanate via the cysteinyl
thiol group and the glutamic acid α-amino group were identified
by NMR.[20] In this work, we have demonstrated that phenyl
isocyanate forms three different adducts with glutathione.
Adduct formation occurred via nucleophilic attacks involving
either the cysteinyl thiol group or the glutamic acid α-amino
group of GSH. In addition, one adduct was formed by reactions
involving both of these reactive groups. The adducts were iden-
tified by UPLC-ESI/MS and the structure of the double adduct
was confirmed by organic synthesis of a reference material with
a structure confirmed by NMR. The CID MS/MS fragmentation of
the three adduct ions were assessed and the three distinct
patterns could be interpreted in terms of protonation sites and
ion structures. The CID MS/MS spectra of adduct 1 and 2 were
characterized by abundant y- and b-ions, respectively. In addi-
tion, a number of fragments produced by reactions in the phenyl
urea and the phenyl thiocarbamide groups took place. For
adduct 1, the formation of the cyclic m/z 195 product ion
provided a diagnostic transition specific for the phenyl thiocar-
bamide moiety. Another interesting example was the interactions
of the urea carbonyl group of adduct 2 in the formation of the
m/z 352 b-type fragment, stabilized by the high electron density
in the urea group. The confirmation of this structure was the sub-
sequent loss of an aniline group which would not take place from
the oxazolone b-ion. The CID MS/MS spectrum of adduct 3 was
complex and involved the b-type of peptide fragmentation and
a high degree of adduct-specific fragments. Analogous to adduct
2, the b-type fragmentation with a loss of glycine (m/z 471),
To verify its structure, the proposed glutathione adduct 3 was
1
synthesized by organic synthesis. A higher shift H NMR spec-
1
trum of the synthetic standard is shown in Fig. 7. The H NMR
spectrum indicates that the product is pure, having five signals
representing a total of ten protons in the aromatic region. This
indicates that two nonequivalent phenyl isocyanate groups
were added to the GSH backbone. This information, together
with an increased number of signals in the aromatic and car-
bonyl region of 13C NMR, confirmed the proposed structure of
the of the reference material. Additional NMR spectra and
chemical shifts both for the synthetic standard of 3 and reduced
glutathione are available in the supplemental data. The reten-
tion time of the glutathione conjugate (3) formed in vitro
matched the data of the synthetic standard (EIC are available
in the supplemental data). In addition, the product spectra of
the adduct 3 and the synthetic standard were almost identical
(supplemental data).
A time study of the reaction between phenyl isocyanate and
glutathione was performed. A new UPLC method was developed
to separate the three conjugates and the hydrolysis product
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T. Johansson Mali’n, S. Lindberg and C. Åstot
Table 3. Proposed precursor ions, neutral losses and fragment ions for the MS/MS fragmentations of adduct 3 (CE 15 V)
Precursor ion
Neutral loss
Fragment ion
(Continues)
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Novel glutathione conjugates of phenyl isocyanate
Table 3. (Continued)
Precursor ion
Neutral loss
Fragment ion
Figure 6. Proposed structures of the glutathione adduct 1 (C17H23N4O7S), 2 (C17H23N4O7S) and 3 (C24H28N5O8S). The protonated elemental composi-
tions are shown in brackets.
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T. Johansson Mali’n, S. Lindberg and C. Åstot
Figure 7. 1H NMR of the synthetic standard of 3 showing the aromatic region. Since the NMR analysis was done with methanol-d4 as solvent, the het-
eroatom protons are not visible.
followed by a loss of aniline (m/z 378), was detected in the
spectra of adduct 3. However, the formation of the m/z 318 frag-
ment ion was proposed to take place by the loss of N-phenyl
thiocarbamic acid (153 Da) from the proposed m/z 471 oxazolone
b-ion. The data indicated that both of the two ion isomers are
produced and a rapid conversion between the ion structures
may occur. The isomerization of the structure element was
supported by the subsequent fragmentation of the m/z 318 ion.
The oxazolone ring is believed to open to allow the loss of CO
alternative reaction site out of the polypeptide chain which
increases the complexity of the fragmentation. Regarding the dou-
ble adduct, the product ion spectrum of this adduct (3) contained a
mixture of specific fragments from both adduct 1 and adduct 2,
and a number of new fragments in the high mass region.
The peak response areas for the glutathione adducts did not
differ significantly, but the peak response area for adduct 2 was
approximately three times smaller than those for 1 and 3. The
determination of response factors of the acetylated analogues
of adduct 1 and 2 indicated that the difference in concentration
probably is below a factor of 1.5. Adduct 3 is expected to have a
response factor close to adduct 2 and based on the peak
response area, this adduct was produced in the highest levels.
Based on the peak response area of 3, reaction of both the
nucleophilic sites of glutathione with phenyl isocyanate is also
proposed to occur to a significant extent. Conjugation between
glutathione and phenyl isocyanate does not seem to hinder the
subsequent nucleophilic attack of the initial adduct on a second
phenyl isocyanate molecule. This is presumably because phenyl
isocyanate is a highly reactive and relatively small molecule,
and the first reaction between glutathione and phenyl isocyanate
does not introduce enough steric hindrance to block the reaction
with a second phenyl isocyanate molecule.
In the time study, no significant difference was found between
the analysis performed after 1 min and the data acquired at
following time points. Thus, the reactions of phenyl isocyanate
and glutathione seem completed within 1 min and are too rapid
to be monitored by the current technique. In an aqueous solu-
tion, phenyl isocyanate is prone to hydrolysis with the formation
of 1,3-diphenylurea.[19] However, 1,3-diphenylurea was not
detected as a dominant component in the conditions used in this
study. As discussed in the introduction, phenyl isocyanate is used
in the production of various materials. Consequently, there is a
significant risk of extensive human exposure to this compound,
from the b-ions and a weak m/z 290 ion was detected.[17]
A
recyclization of the open chain m/z 318 ion with the urea
carbonyl group would finally explain the loss of aniline detected
as the m/z 225 ion.
To our knowledge, no adducts resulting from conjugation at
the glutamic α-amino group of glutathione have been identified
using LC-MS. Instead, many publications describe adducts
resulting from conjugation at the cysteinyl thiol group of gluta-
thione. In order to detect glutathione conjugates, screening using
neutral loss scanning of 129 Da (anhydroglutamic acid/pyroglu-
tamic acid) in positive ion mode and negative precursor ion scan-
ning at m/z 272 (deprotonated γ-glutamyl-dehydroalanyl-glycine)
is used.[21,22] Once GSH adducts are discovered in the screening,
product ion spectra are utilized to elucidate the structures. We
conclude that for the adducts identified in this study, the product
ion spectrum for the adduct resulting from conjugation at the thiol
group of glutathione was less complex, and it was easier to
elucidate the fragmentation of this adduct, compared to the
product ion spectra for the double adduct and the adduct resulting
from conjugation at the α-amino group of glutathione. This may
be explained by the fact that adduct 2 turned out to be a pep-
tide with multiple sites of equal basicity and will produce a
number of protonated species with about equal abundances
upon ionization promoting a high complexity of CID fragmenta-
tion. Also, for adduct 2, the urea carbonyl group offers an
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Novel glutathione conjugates of phenyl isocyanate
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making it important to determine its reactivity towards potential
targets in the body. This study describes a chemical, non-
enzymatic reaction between glutathione and phenyl isocyanate
at physiologically relevant conditions. In an in vivo situation,
glutathione S-transferase enzymes will be present and promote
the conjugation at the thiol group of glutathione. Therefore,
further investigations will be required to determine the relevance
of the GSH adducts identified in this work in vivo. It has been
proposed that the reaction of GSH with isocyanates may be
reversible.[23] If so, the electrophilic parent isocyanate could be
released from the conjugate at some stage, enabling it to react
with alternative nucleophilic functional groups such as those
found in proteins and DNA. It would therefore be interesting to
study the reactions of phenyl isocyanate with various biologically
important macromolecules. Phenyl isocyanate reacted with both
the cysteinyl thiol and the glutamic acid α-amine of glutathione.
These results suggest that phenyl isocyanate may react with free
cysteines, the α-amino group and also with lysine residues whose
side chain contains a primary amine.
In conclusion, three different adducts formed by the reaction
of glutathione with phenyl isocyanate have been identified. To
our knowledge, these compounds have not previously been
analyzed by mass spectrometry. Both the cysteinyl thiol group and
the glutamic acid α-amino group of glutathione were found to at-
tack phenyl isocyanate. To a significant extent, both of these reactive
groups attacked phenyl isocyanate, forming the double adduct 3.
Conjugation occurred to a greater extent on the cysteinyl thiol
moiety than on the glutamic acid α-amino group. NMR analysis
confirmed the proposed structure of the glutathione adduct formed
by conjugation with two phenyl isocyanate molecules. To our
knowledge, this double adduct has not previously been described.
[18] A. G. Harrison. To b or not to b: the ongoing saga of peptide b ions.
Mass Spectrom. Rev., 2009, 28, 640–654
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methyl and phenyl isocyanates. J. Chem. Soc. Perkin Trans. 2, 1985,
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Acknowledgements
Professor Calle Nilsson is gratefully acknowledged for valuable
input to the manuscript. This study was financially supported by
the Swedish Ministry of Defence.
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Additional supporting information may be found in the online
version of this article at the publisher’s web site.
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