Synthesis and mass spectrometric identification of the major amino acid adducts formed between sulphur mustard and haemoglobin in human blood

Article abstract of DOI:10.1007/s002040050372

As part of a program to develop methods for the verification of alleged exposure to sulphur mustard, we synthesized and characterized three amino acid adducts presumably formed by alkylation of haemoglobin: 4-(2-hydroxyethylthioethyl) -L-aspartate, 5-(2-hydroxy-ethylthioethyl)-L-gIutamate and N1- and N3-(2-hydroxyethylthioethyl)-L-histidine. Suitable derivatization methods for GC/MS analysis were developed for these adducts as well as for the cysteine and the N-terminal valine adduct. Incubation of human blood with [³⁵S]sulphur mustard in vitro followed by acidic hydrolysis of isolated globin and derivatization with Fmoc-Cl afforded three major radioactive peaks upon HPLC analysis, one of which coeluted with the synthetic Fmoc derivative of N1/N3-(2-hydroxyethylthioethyl)-L-histidine. After pronase digestion of globin the adducts of histidine, glutamic acid, aspartic acid, cysteine and N-terminal valine could be tentatively identified and quantitated. Final identification was obtained from GC/MS analysis. The most abundant adduct, N1/N3-(2-hydroxyethylthioethyl)-L-histidine, could not be sensitively analysed by GC/MS. A convenient LC-tandem MS procedure was developed for this compound, enabling the detection of exposure of human blood to 10 μM sulphur mustard in vitro.

Full text of DOI:10.1007/s002040050372

Arch Toxicol (1997) 71: 171–178
Springer-Verlag 1997
TOXICOKINETICS
Daan Noort · Albert G. Hulst · Hendrik C. Trap
Leo P.A. de Jong · Hendrik P. Benschop
Synthesis and mass spectrometric identification
of the major amino acid adducts formed between sulphur mustard
and haemoglobin in human blood
Received: 29 May 1996 / Accepted: 20 August 1996
Abstract As part of a program to develop methods for monitoring ·MTBSTFA N-methyl-N-(tert-butyldime-
the verification of alleged exposure to sulphur mustard, thylsilyl)trifluoroacetamide · NCI negative chemical
we synthesized and characterized three amino acid ad- ionization
ducts presumably formed by alkylation of haemoglobin:
4-(2-hydroxyethylthioethyl)-
ethylthioethyl)- -glutamate and N1- and N3-(2-hydro- LC tandem MS · Multiple reaction monitoring
xyethylthioethyl)- -histidine. Suitable derivatization
L
-aspartate, 5-(2-hydroxy- Key words Sulphur mustard ·Adducts ·Haemoglobin ·
L
L
methods for GC/MS analysis were developed for these
adducts as well as for the cysteine and the N-terminal Introduction
valine adduct. Incubation of human blood with
[35S]sulphur mustard in vitro followed by acidic hydro- Sulphur mustard [bis(2-chloroethyl) sulphide] is a strong
lysis of isolated globin and derivatization with Fmoc-Cl vesicant and primary carcinogen, which has been used
afforded three major radioactive peaks upon HPLC several times this century as a chemical warfare agent
analysis, one of which coeluted with the synthetic Fmoc (Papirmeister et al. 1991). The high reactivity of sulphur
derivative of N1/N3-(2-hydroxyethylthioethyl)- -histi- mustard in aqueous media is thought to be mediated by
L
dine. After pronase digestion of globin the adducts of intramolecular displacement of one of the chlorine
histidine, glutamic acid, aspartic acid, cysteine and N- atoms by the sulphur atom, giving rise to a highly re-
terminal valine could be tentatively identified and active episulphonium ion, which can react with e.g. nu-
quantitated. Final identification was obtained from GC/ cleophilic groups in proteins and DNA and with
MS analysis. The most abundant adduct, N1/N3-(2- phosphate. Subsequent hydrolysis of the second mustard
hydroxyethylthioethyl)- -histidine, could not be sensi- functionality will yield monoadducts (see Scheme 1).
L
tively analysed by GC/MS. A convenient LC-tandem The formation of diadducts has also been observed, e.g.
MS procedure was developed for this compound, en- for closely spaced 2′-deoxyguanosine residues in DNA
abling the detection of exposure of human blood to (Fidder et al. 1994).
10 lM sulphur mustard in vitro.
Abbreviations Boc N-tert-butyloxycarbonyl · DCTFA
1,3-dichlorotetrafluoroacetone · EI electron impact
ionization · Fmoc-Cl fluorenylmethyl chloroformate ·
HETE 2-hydroxyethylthioethyl · HFBA
Scheme 1
heptafluorobutyric anhydride · MRM multiple reaction
We are currently developing detection methods for
the retrospective confirmation of sulphur mustard ex-
posure. Since biological samples often become available
only days or weeks after exposure, these methods should
be based on the detection of stable adducts of the agent
to DNA or proteins. In this context we recently de-
scribed the synthesis, characterization and quantitation
of the adducts formed between sulphur mustard and
DNA (Fidder et al. 1994). For detection of the major
D. Noort(&), · H.C. Trap · L.P.A. de Jong · H.P. Benschop
Department of Chemical Toxicology,
TNO Prins Maurits Laboratory, P.O. Box 45,
2280 AA Rijswijk, The Netherlands
A.G. Hulst
Department of Analysis of Toxic and Explosive Substances,
TNO Prins Maurits Laboratory, P.O. Box 45,
2280 AA Rijswijk, The Netherlands

earlier by Fidder et al. (1996a). S-(2-hydroxyethylthioethyl)-
L-cy-
172
adduct, N7-(2-hydroxyethylthioethyl)-2′-deoxyguanosine,
we developed a sensitive ELISA which enabled the de-
tection of adducts in white blood cells after exposure of
human blood to sulphur mustard concentrations > 2 lM
(Van der Schans et al. 1994).
steine was synthesized as described by Grant and Kinsey (1946); the
spectroscopic data were identical to those given by Black et al.
(1992). Human blood was obtained from healthy volunteers, with
the consent of the donor and approval of the Netherlands Orga-
nization for Applied Scientific Research (TNO) Medical Ethical
Committee.
Since haemoglobin is much more abundant than
DNA and adducts to human haemoglobin have in vivo
life-spans of several months (Skipper and Tannenbaum,
1990), haemoglobin–sulphur mustard adducts might be
excellent biomarkers for exposure to sulphur mustard.
The alkylation of proteins by sulphur mustard was in-
vestigated in the 1940s and 1950s by various groups (for
a review see Wheeler 1962). For instance, Davis and
Ross (1947) showed that mustard gas could esterify
carboxyl groups in serum albumin and horse hae-
moglobin, and that alkylation had also occurred with
histidine residues. Douglas and Heard (1954) demon-
strated that sulphur mustard reacts with a-amino groups
of amino acids and peptides, whereas reaction with sul-
phydryl groups in serum proteins was established by
Banks et al. (1946). Sandelowsky et al. (1992) demon-
strated the presence of N1-(2-hydroxyethylthio-ethyl)-4-
methyl imidazole in urine, which is probably a de-
gradation product of histidine alkylated by sulphur
mustard. Recently we demonstrated that haemoglobin is
efficiently alkylated by sulphur mustard (Noort et al.
1996). In the same study we identified, using LC tandem
MS, various specific residues in haemoglobin which are
prone to alkylation by sulphur mustard. Furthermore,
we developed a sensitive GC/MS method for analysis of
the alkylated N-terminal valine, based on the modified
Edman degradation procedure (Fidder et al. 1996a).
We describe here the synthesis of the sulphur mustard
adducts of histidine, aspartic acid and glutamic acid. The
GC/MS analysis of these adducts and of the cysteine and
N-terminal valine adducts, together representing the
most likely formed adducts after alkylation of hae-
moglobin by sulphur mustard, is presented. The adducts
formed upon in vitro exposure of haemoglobin in human
blood were tentatively identified and quantitated using
the synthetic adducts as reference compounds. For the
histidine adduct, which is the most abundant adduct, a
convenient method of analysis is described, based on LC
tandem MS with multiple reaction monitoring (MRM).
Instrumentation
Fast protein liquid chromatography (FPLC) analyses were carried
out on a PepRPC 5/5 column (length 5 cm; i.d. 5 mm) using an
FPLC system equipped with two P-500 pumps, an LCC-501 Plus
controller and a UV-M II monitor (Pharmacia, Uppsala, Sweden).
HPLC with radiometric detection was performed with a Waters
HPLC system, using a PepRPC 5/5 column, coupled to a Radio-
metric Flo-one/Beta A-500 radiochromatography detector (Can-
berra Packard, Tilburg, The Netherlands). Thermospray LC-MS
spectra were recorded on a Nermag (Paris, France) R10-10C
quadrupole instrument operated in the positive mode and equipped
with a thermospray ion source (Nermag) coupled via a Vestec TSP
interface (Vestec, Houston, Tex., USA). The LC-system comprised
a reversed phase C18 column (250 × 5 mm, Lichrosorb 7 lm par-
ticles) with 0.1 M aqueous ammonium acetate and methanol in
varying ratios as eluent.
Electrospray LC-MS-MS spectra were recorded on a VG
Quattro II triple quadrupole mass spectrometer (Fisons, Altrinc-
ham, UK). The analyses were carried out with MRM at a collision
energy of 15 V, gas (Ar) cell pressure 0.3 Pa, and source tem-
perature 120 °C. The LC-system comprised a reversed phase C18
column (Lichrosorb, 5 lm particles; length 25 cm, i.d. 5 mm). GC-
NCI/MS analyses were performed on a Unicam Automass 150
coupled to a Carlo Erba 5300 gas chromatograph. A piece of un-
coated deactivated fused silica capillary, used as a retention gap
(length 0.5 m, i.d. 0.32 mm), was coupled with a press-fit all-glass
connector to a BP 1 column (length 12 m, i.d. 0.32 mm, film
thickness 1.0 lm; Scientific Glass Engineering Ltd., Ringwood,
Victoria, Australia). GC-EI/MS analyses were performed with a
VG70-250S mass spectrometer coupled to a HP 5890A gas chro-
matograph, which was equipped with a CP-Sil 8 CB fused silica
capillary column (length 50 m, i.d. 0.32 mm, film thickness
0.25 lm; Chrompack, Middelburg, The Netherlands). 1H-NMR
and 13C-NMR spectra were recorded on a Varian (Palo Alto,
Calif., USA) VXR 400S spectrometer operating at 400 MHz and
100.6 MHz, respectively. Chemical shifts are given in ppm relative
to tetramethylsilane.
Synthesis of 4-(HETE)-
L-aspartate
N-Boc-1-tert-butyl- -aspartate (0.95 mmol, 275 mg) was ester-
L
ified with thiodiglycol (13 mmol, 1.59 g) under the agency of
dicyclohexylcarbodiimide (1.0 mmol, 224 mg) and 4-dimethyl-
aminopyridine (0.58 mmol, 71 mg) in ethyl acetate (10 ml) at
0 °C, according to a procedure described by Neises and Steglich
(1979). After stirring for 30 min at 0 °C, the reaction mixture
was allowed to warm up to room temperature. After 16 h, TLC
analysis (eluent, dichloromethane/methanol, 95:5; v/v) showed
the conversion of starting material (Rf 0.0) into one major
product (Rf 0.5). Precipitated dicyclohexylurea was removed by
filtration and the filtrate was washed with 2% HCl, 2% NaHCO3
and water. The organic phase was dried (MgSO4) and con-
centrated under reduced pressure. The crude product was puri-
Materials and methods
Reagents
Thiodiglycol was purchased from Aldrich, Belgium. Hepta-
fluorobutyric anhydride (HFBA) was purchased from Fluka,
Switzerland. 1,3-Dichlorotetrafluoroacetone (DCTFA) and fluor-
enylmethyl chloroformate (Fmoc-Cl) were obtained from Janssen
Chimica, Belgium. Na-Boc-histidine methylester was purchased
fied
by
silica
gel
column
chromatography
(eluent,
dichloromethane with a linear gradient to dichloromethane/me-
thanol, 95:5; v/v in 90 min) affording a colourless oil (275 mg,
0.70 mmol, 74%). NMR spectroscopy gave satisfactory data,
which were in accordance with the proposed structure. Subse-
quently, the fully protected compound was dissolved in tri-
fluoroacetic acid (TFA; 10 ml). After 5 h at 20 °C, TLC analysis
(dichloromethane/methanol, 95:5; v/v in 90 min) demonstrated
complete conversion into baseline material. The solution was
from Bachem, Switzerland. Pronase E, N-Boc-1-tert-butyl-
L-glu-
tamate and N-Boc-1-tert-butyl- -aspartate were obtained from
L
Sigma Chemical Co., Mo., USA. Constant boiling 6 N HCl was
obtained from Pierce, The Netherlands. [35S]Sulphur mustard was
synthesized as described earlier (Noort et al. 1996). N-(2-
hydroxyethylthioethyl)-DL-valine was synthesized as described

173
concentrated under reduced pressure and the residue coevapo- The organic layer was dried (MgSO4, 100 mg) and concentrated
rated with toluene (3 × 5 ml) and water (2 × 5 ml), affording the under reduced pressure, giving a colourless oil. The free acid was
target compound as a clear oil with quantitative yield of 166 mg. dissolved in TFA/H2O (95:5, v/v; 1.0 ml) and left at room tem-
The compound was used without further purification; pur- perature. After 3 h, reversed-phase FPLC analysis showed com-
ity > 95%, according to 1H-NMR spectroscopy.
plete disappearance of starting material. The mixture was
1H-NMR (D2O): d 4.40 (m, 3H, CH + CH2OC(O)), 3.75 (t, 2H, concentrated under reduced pressure, coevaporated with toluene
CH2OH), 3.15 (m, 2H, CHCH2), 2.90–2.75 (2 × t, 2 × 2H, 2 × (2 × 1 ml) and D2O (2 × 1 ml), affording a colourless oil in both
CH2S).
13C-NMR (D2O): d 172.2 + 172.0 (2 × COO), 65.8 (CH2O- The purity checked by NMR spectroscopy was > 95%.
C(O)), 61.4 (CH2OH), 50.5 (CH), 35.1 (CH2CH), 34.7 + 30.8
1H-NMR (D2O) for N1-isomer: d 8.74 (bs, 1H, N = CH-N),
(CH2SCH2). Thermospray LC-MS: m/z 238 (MH+), 260 7.38 (bs, 1H, N-CH = C), 4.38 (t, 2H, NCH2; J = 6.5 Hz), 3.99 (t,
(MNa+).
cases. Yield, 5.1 mg (83%, N1-isomer); 5.4 mg (87%, N3-isomer).
1H, NCHCOO; J = 6.6 Hz), 3.67 (t, 2H, CH2OH; J = 6.1 Hz),
3.33 (m, 2H, NCHCH2), 3.00 (t, 2H, NCH2CH2S; J = 6.5 Hz), 2.64
(t, 2H, OCH2CH2S; J = 6.1 Hz).
13C-NMR (D2O): d 173.1 (COO), 136.9 (N = CH-N), 129.6
(CH = C-N), 120.0 (CH = C-N), 61.2 (CH2OH), 53.9 (NCHCH2),
47.0 (NCH2), 34.5 (SCH2CH2OH), 32.1 (NCH2CH2S), 25.5
(NCHCH2).
Synthesis of 5-(HETE)-L-glutamate.
5-(HETE)-
L
-glutamate was synthesized as described for 4-(HETE)-
starting with N-Boc-1-tert-butyl glutamate
L
-aspartate,
1H-NMR (D2O) for N3-isomer: d 8.74 (d, 1H, N = CH-N;
J = 1.2 Hz), 7.45 (bs, 1H, N-CH = C), 4.37 (t, 2H, NCH2;
J = 6.4 Hz), 4.05 (t, 1H, NCHCOO; J = 6.6 Hz), 3.68 (t, 2H,
CH2OH; J = 6.2 Hz), 3.30 (m, 2H, NCHCH2), 3.01 (t, 2H,
NCH2CH2S; J = 6.4 Hz), 2.66 (t, 2H, OCH2CH2S; J = 6.2 Hz).
13C-NMR (D2O): d 172.7 (COO), 136.1 (N = CH-N), 129.0
(CH = C-N), 121.4 (CH = C-N), 61.1 (CH2OH), 54.0 (NCHCH2),
49.6 (NCH2), 34.3 (SCH2CH2OH), 32.1 (NCH2CH2S), 26.6
(NCHCH2). Thermospray LC-MS: m/z 260 (MH+), 282 (MNa+)
for both isomers.
(3.58 mmol, 1.09 g). The crude end-product was crystallized from
dioxane/diethyl ether (7:45, v/v). Yield 270 mg (30% in two steps),
melting pt. 149–150 °C.
1H-NMR (Me2SO-d6): d 4.17 (t, 2H, COOCH2), 3.56 (t, 2H,
CH2OH), 3.34 (t, 1H, CH), 2.77 (t, 2H, CH2S), 2.63 (t, 2H, CH2S),
2.61–2.40 (m, 2H, CH2COO), 2.05–1.85 (m, 2H, CH2CH2COO).
13C-NMR (Me2SO-d6): d 172.3 and 170.0 (COO), 63.4 (COOCH2),
61.0 (CH2OH), 53.0 (CH), 34.2 (CH2S), 30.1 (CH2S), 30.0
(CH2COO), 26.2 (CH2CH2COO). Thermospray LC-MS: m/z 252
(MH+).
Derivatization of reference compounds
with DCTFA and HFBA
Synthesis of N1- and N3-(HETE)-L-histidine
Na-Boc- -histidine methyl ester (5 mmol, 1.34 g) was dissolved in
L
Reference compounds (5–20 lg) were derivatized with DCTFA
according to Husek et al. (1979), followed by acylation with HFBA
at 40 °C. The organic layer was extracted with 1 M Na2CO3 (1 ml),
1 M HCl (1 ml) and H2O (1 ml), respectively. The organic layer was
evaporated to dryness under a nitrogen stream, dissolved in n-
heptane (2.5 ml) and HFBA (125 ll) and heated at 70 °C for 5 min,
after which the sample was ready for analysis.
a mixture of acetonitrile/H2O (1:1, v/v; 6 ml). A solution of 2-(2-
tert-butyloxyethylthio)ethyl chloride (2.5 mmol, 491 mg; Noort et
al. 1995) in acetonitrile (2 ml) was added in five portions within 2 h
under vigorous stirring. The pH of the reaction mixture was
maintained at 7.5 by the addition of aqueous NaOH (0.5 M) with a
pH-stat equipment. After 1 h, a two-layer system resulted, prob-
ably because of the high ionic strength of the mixture. The mixture
was vigorously stirred for another 16 h, after which the consump-
tion of NaOH solution had ceased. Reversed phase FPLC analysis
showed the presence of a slower eluting compound and (excess)
Characteristic GC-NCI/MS data for the derivatized reference
compounds:
4-(HETE)-L
-aspartate: m/z 648/650 (M+Cl)), 613 (M).), 549
(M).-HF-CO2), 533 (M).-CO2-HCl), 513 (M).-HF-CO2-HCl), 477
(M).-HF-CO2-2 × HCl);
Na-Boc-
chloromethane (150 ml) and washed with aqueous acetic acid (4%,
50 ml) to remove excess Na-Boc- -histidine methyl ester. The or-
L-histidine methyl ester. The mixture was taken up in di-
5-(HETE)-
-glutamate: m/z 662/664. (M+Cl)), 627 (M).), 607
L
(M).-HF), 563 (M).-HF-CO2), 547 (M) -HCl-CO2);
S-(HETE)-
-cysteine: m/z 636/638 (M+Cl)), 601 (M).), 537
(M).-HF-CO2);
N-(HETE)-DL-valine: m/z 597 (M).), 577 (M).-HF), 557 (M).-2
× HF).
L
ganic layer was dried (MgSO4, 10 g) and concentrated. TLC ana-
lysis (methanol/dichloromethane, 4:96; v/v) showed two UV-
positive spots (Rf 0.45 and 0.40) in a ratio 1:1, presumably the N1-
and -N3 isomer of the desired compound.
The mixture of isomers was purified by silica gel column
chromatography (15 g of silica gel in a glass column with i.d. 3 cm;
eluent, dichloromethane with a gradient to 2% methanol). Total Derivatization of N1/N3-HETE-
L-histidine
yield, 488 mg (47%, based on 2-(2-tert-butyloxyethylthio)ethyl
chloride). During this chromatographic run a small quantity of
each individual isomer could be obtained. Yield of (pure) first
L
with methanol/HCl and HFBA
N1/N3-HETE- -histidine (0.1 mg) was dissolved in a solution of
L
eluting isomer, 111 mg; yield of (pure) more polar isomer, 170 mg. HCl in methanol (2 M, 200 ll). After 1 h at 80 °C, the mixture was
concentrated under reduced pressure using a SpeedVac apparatus
1H-NMR (CDCl3) signals: d 7.42 (d, 1H, N = CH-N, J = 1.4 Hz), and the residue was dissolved in ethyl acetate (100 ll). HFBA
(30 ll) was added and the mixture was heated at 110 °C. After
The first eluting isomer was identified as the N3-isomer. Specific
6.74 (d, 1H, N-CH = C, J = 1.4 Hz), 5.91 (bd, 1H, NH). The more
polar isomer was identified as the N1-isomer. Specific 1H-NMR 10 min the mixture was concentrated and the residue was dissolved
in ethyl acetate (100 ll). The two isomers gave comparable spectra.
However, derivatization of the N3 reference compound also gave
rise to the formation of an unsaturated derivative of the title
(CDCl3) signals: d 7.51 (d, 1H, N = CH-N, J = 1.0 Hz), 6.82 Hz
(bs, 1H, N-CH = C), 5.26 (bd, 1H, NH).
Subsequently,
Na-Boc-N1-(2-tert-butyloxyethylthioethyl)-L-
histidine methyl ester (10 mg, 0.024 mmol), or the corresponding compound in which a C3F7C(O)OCH2CH2SCH = CH- moiety is
attached to the imidazole ring.
N3 isomer, was dissolved in 0.2 M NaOH in methanol/H2O (9:1,
v/v; 0.5 ml). After 2.5 h at room temperature, reversed-phase FPLC
analysis showed the complete conversion of starting material into a
.
.
GC-EI/MS: m/z 665 (M+ ), 606 [M+ -C(O)OCH3], 452
.
.
[M+ )C3F7C(O)NH2], 381 [M+ -C3F7C(O)NHCHC(O)OCH3], 301
more polar compound. The mixture was neutralized with acetic [C3F7C(O)OC2H4SC2H4+], 241 [C3F7C(O)OC2H4+],. 169 (C3F7+);
.
for unsaturated derivative: m/z 663 (M+ ), 604 [M+ -C(O)OCH3],
acid and concentrated. The residue was taken up in di-
.
.
chloromethane (1 ml) and washed with 5% aqueous acetic acid (2 452 [M+ -C3F7C(O)NH2], 379 [M+ -C3F7C(O)NHCHC(O)OCH3].
GC-NCI/MS: m/z 645 (M).-HF).
ml). Phases were separated by centrifugation (3000 rpm; 10 min).

174
Derivatization of N1/N3-HETE-L-histidine
with N-methyl-N-(tert-butyldimethylsilyl)trifluoro-acetamide
Results and discussion
N1/N3-HETE-L-histidine was derivatized with N-methyl-N-
Synthesis of amino acid – sulphur mustard adducts
(tert-butyldimethylsilyl)trifluoro-acetamide (MTBSTFA) as de-
scribed by Cushnir et al. (1993). Characteristic GC-EI/MS signals
are: m/z 601 (M+), 545 [M+ - C(CH3)3].
The adducts of aspartic acid and glutamic acid to sul-
phur mustard were synthesized by dicyclohexyl carbo-
diimide mediated esterification (Neises and Steglich
1979) of suitably protected derivatives with thiodiglycol
(see Scheme 2). Deprotection with TFA gave the desired
reference compounds. It is anticipated that reaction of
sulphur mustard with histidine residues in proteins will
result in the formation of two isomeric adducts, as has
been observed for other alkylating agents (Calleman and
Wachtmeister 1979). Reaction of Na-Boc-histidine me-
thyl ester (see Scheme 2) with 2-(2-tert-butyloxy-
ethylthio)ethyl chloride (Noort et al. 1995) in H2O/
acetonitrile at pH 7.5 afforded a mixture of the fully
protected N1/N3-histidine adducts, which could be se-
parated by silica gel column chromatography. The
pH of the reaction mixture was kept constant by using
pH-stat equipment; by this method the formation of a
N1, N3-di-alkylated histidine derivative could not be
detected. By contrast, when the alkylation reaction was
carried out with manual pH adjustment, yields were low
( < 5%) and a considerable amount of the di-alkylated
histidine derivative resulted.
Exposure of human blood to [35S]sulphur mustard;
isolation and acidic/enzymatic hydrolysis of globin
Heparinized human blood (10 ml) was exposed to [35S]sulphur
mustard (5 mM; specific activity 1.6 mCi/mmol) by addition of a
solution of the agent in acetonitrile (1% end concentration). After
3 h of incubation at 37 °C, globin was isolated as described by
others (Bailey et al. 1988). Subsequently, a small sample of the
globin (10 mg) was hydrolysed in constant boiling 6 N HCl (2 ml)
under reflux at 110 °C. After 24 h the hydrolysis mixture was
concentrated under vacuum and coevaporated with H2O in order
to remove traces of HCl.
Otherwise, globin was digested with pronase E. Briefly, this
procedure comprised the addition of pronase E (substrate/enzyme
ratio 20:1, w/w) to a solution of the globin sample (3 mg protein/
ml) in phosphate buffered saline (0.12 M NaCl/2.7 mM KCl/
0.01 M Na2HPO4; pH 8.0) and incubation of the mixture at 37 °C
under shaking. After 16 h the pH was readjusted to 8.0, an addi-
tional aliquot of pronase E was added and the incubation was
continued for another 16 h. After the incubation, the mixture was
neutralized with 0.1 N HCl and concentrated.
Derivatization of reference compounds and globin
hydrolysates with Fmoc-Cl and HPLC analysis of the derivatives
The individual isomers were fully characterized by
NMR spectroscopy. According to Matthews and Ra-
To a solution of globin hydrolysate (0.5 mg) in borate buffer
(0.1 M boric acid, pH 7.8; 0.5 ml) was added a solution of Fmoc-Cl
in acetone (15 mM, 0.5 ml). The mixture was rapidly vortexed and,
after 40 s, extracted with n-pentane or light petroleum (3 × 2 ml).
The aqueous solution containing the Fmoc amino acid derivatives
was then ready for analysis. The same procedure was followed for
derivatization of the reference compounds N1/N3-(HETE)-
tidine, S-(HETE)- -cysteine, 4-(HETE)- -aspartate and 5-(HE-
TE)- -glutamate. Fmoc amino acids were analysed on a reversed
L-his-
L
L
L
phase PepRPC 5/5 column using gradient elution. A linear gradient
(0–25 ml) was applied from 0.1% TFA/25% acetonitrile to 0.1%
TFA/80% acetonitrile. The injection volume was 60 ll and the flow-
rate was 1.0 ml/min; detection was at 254 nm.
A radio-
chromatography detector was used when samples of protein treated
with [35S]sulphur mustard were analysed.
LC-tandem electrospray MS determination
of Na-Fmoc-N1/N3-(HETE)-L-histidine
Globin samples (2.5 mg) isolated from blood after exposure to 0,
0.01 or 0.1 mM sulphur mustard were hydrolyzed with constant
boiling 6 N HCl. The remaining mixtures of amino acids were
derivatized with Fmoc-Cl as described above (total volume of
sample after Fmoc derivatization, 1.7 ml). Na-Fmoc-N1/N3-
Scheme 2
(HETE)-L-histidine was analysed in the mixture of Fmoc-amino
acids by LC tandem MS using MRM. The protonated molecular
ion (m/z 482) was selected in the first mass spectrometer and sub-
sequently the most intense fragment ion (m/z 105) was detected in
the second mass spectrometer. A linear gradient was applied from
H2O/acetonitrile (80:20, v/v), containing 0.5% formic acid, to H2O/
acetonitrile (20:80, v/v), containing 0.5% formic acid, for 25 min
followed by H2O/acetonitrile (20:80, v/v), containing 0.5% formic
acid, for 5 min, with a flow of 1.0 ml/min. The eluent was directed
to the electrospray probe with a split ratio of 1/20. Injection volume
was 40 ll.
poport (1973), crossring coupling constants of the two
ring protons can be used to differentiate between 1,4-
and 1,5-disubstituted imidazoles. The first eluting isomer
was appointed as the N3-isomer (J = 1.4 Hz) and the
more polar isomer as the N1-isomer (J = 1.0 Hz).
Moreover, the N3-isomer showed a downfield shift
(0.7 ppm) of the a-amino proton compared to the a-
amino proton of the N1-isomer (5.2 ppm), which is in

175
accordance with data found for related compounds butyl chloroformate (Husek et al. 1979), in order to
(Jones and Hysert 1971). Deprotection was carried out preclude adsorption, is not possible for these derivatives,
in two steps: the methyl ester was saponified with NaOH no further attempts were undertaken. The histidine ad-
in a mixture of methanol/H2O (9:1, v/v). Subsequently, duct could be derivatized by esterification with metha-
the acid-labile groups were removed by treatment with nol/HCl followed by acylation with HFBA (see Fig. 2
TFA/H2O (95:5, v/v) giving N1- or N3-(HETE)-
L
-his- for EI-mass spectrum). The two isomers gave compar-
tidine in quantitative yield. The NMR spectra were in able spectra. Unfortunately, relatively drastic conditions
full accordance with the expected structure.
(T > 300 °C) had to be applied in order to elute the
histidine derivative from the column, leading to a severe
loss in sensitivity. The histidine adduct was also deri-
vatized with MTBSTFA. GC-EI/MS showed the pre-
sence of the desired tris-(tert-butyldimethylsilyl)
derivative. Unfortunately, for both types of derivatives
GC/MS analysis
of sulphur mustard – amino acid adducts
For derivatization of the adducts for GC/MS analysis, the detection limit was rather high (200 ng absolute
the mild procedure reported by Husek (1974) and Husek amount).
et al. (1979) was applied. After reaction of the reference
compounds with DCTFA (see Scheme 3) followed by
derivatization of the obtained oxazolidinone with Identification and quantitation of amino acids
HFBA, GC-NCI/MS analysis showed one major peak formed in globin after exposure of human blood
having the expected mass in each case (see Fig. 1 for the to sulphur mustard
S-(HETE)-
-cysteine derivative). Unfortunately, the
L
procedure could not be applied to the histidine adduct. When human blood was incubated with 1 and 5 mM
The derivatized compound was probably fully adsorbed [35S]sulphur mustard, in both cases 42% of the total
onto the stationary phase of the GC-column. Since de- radioactivity was present in plasma. In previous studies
rivatization of the polar imidazolyl function with iso- (Fidder et al. 1996a) we found an approximately pro-
portional amount of sulphur mustard bound to globin
after incubation of human blood with the agent (0.1 lM
to 5 mM), i.e. 25–30%, indicating a linear relationship
between exposure and adduct level. Thus, e.g. in the case
of 5 mM exposure, 25% of total radioactivity was bound
to globin, i.e. 9.0 nmol adduct/mg globin resulted
(140 mg globin/ml blood), indicating an average of one
modified chain/seven native globin chains.
In order to obtain suitable derivatives for reversed
phase HPLC analysis, the synthetic amino acid - sulphur
mustard adducts were derivatized with Fmoc-Cl (Ei-
narsson et al. 1983). Reproducible chromatograms were
obtained when globin hydrolysates were derivatized in
an analogous manner. HPLC analysis was performed
Scheme 3
Fig. 1 GC-NCI/MS analysis of S-(2-hydroxyethylthioethyl)-
steine after derivatization with 1,3-dichlorotetrafluoroacetone and
heptafluorobutyric anhydride. 1, GC/MS chromatogram; 2, mass Fig. 2 GC-EI/MS spectrum of N1-(2-hydroxyethylthioethyl)-
spectrum of the major peak
dine after derivatization with methanol/HCl and HFBA
L-cy-
L
-histi-

176
for an acidic hydrolysate of globin isolated from blood activity), respectively. It follows from these results that
that was exposed to 5 mM [35S]sulphur mustard, after 2.7 nmol histidine adduct is present per mg globin.
derivatization of the resulting mixture of amino acids These results confirm that the histidine adduct is the
with Fmoc-Cl, in order to obtain quantitative informa- most abundant adduct formed, as found after acidic
tion on the various amino acid adducts formed (Fig. 3). hydrolysis. The other (small) peaks coeluted with Fmoc
The chromatogram did not change significantly when derivatives of synthetic 4-(HETE)-
the acidic hydrolysis of the globin samples was con- (HETE)- -glutamate (peak 3; retention time 11.3 min),
tinued for another 24 h. After acidic hydrolysis and S-(HETE)- -cysteine (peak 4; retention time 12.0 min;
derivatization, 46% of the radioactivity was recovered. together with peak 3, 7% of activity), and N-(HETE)-
L
-aspartate, 5-
L
L
L
-
Peak 1 (retention time 1.4 min; 11% of total activity) is valine (peak 5; retention time 13.2 min, 3% of total ac-
probably [35S]thiodiglycol formed by hydrolysis of al- tivity). These data have to be considered with care
kylated glutamic acid and aspartic acid residues. Peak 2 however, since the possibility cannot be excluded that
(retention time 7.0 min; 34% of total activity) coelutes the base-labile glutamic/aspartic acid adducts are sus-
with synthetic Fmoc histidine-sulphur mustard adducts. ceptible to enzymatic cleavage. Moreover, we estab-
Peak 3 (retention time 13.8 min; 25% of total activity) lished that Fmoc derivatization of N-(HETE)-
did not coelute with any of the synthetic standards. It problematic.
L
-valine is
may be concluded that the histidine adduct is the most
abundant adduct, occurring at a level of 3.1 nmol/mg achieved by GC/MS analysis of acidic and enzymatic
globin (i.e. 34% of 9.0 nmol/mg globin). hydrolysates of globin, obtained from human blood
Final proof for the formation of the adducts was
It is anticipated that several sulphur mustard adducts after exposure to 1–10 mM of sulphur mustard. Thus,
are acid-labile, e.g. the adducts formed with glutamic the histidine adduct could be detected after derivatiza-
and aspartic acid. Moreover, we established that syn- tion of the acidic hydrolysate with MeOH/HCl and
thetic S-(HETE)-
-cysteine readily decomposes in re- HFBA. The cysteine adduct, the aspartic acid adduct
L
fluxing 6 N HCl. Therefore, enzymatic hydrolysis by and the glutamic acid adduct were detected in enzymatic
pronase E (Day et al. 1990) was applied. The recovery of digests after derivatization with DCTFA as described
the radioactivity after pronase E-mediated digestion and above. In addition, N-(HETE)- -valine could be de-
L
derivatization was quantitative. Two major peaks were termined with GC-NCI/MS after a modified Edman
detected in the HPLC chromatogram of the digest degradation of globin, as was recently reported by our
(Fig. 4) which coeluted with thiodiglycol (peak 1; re- group (Fidder et al. 1996a).
tention time 1.5 min, 15% of total activity analysed) and
the Fmoc derivatives of the histidine adducts of sulphur
mustard (peak 2; retention time 8.1 min, 30% of total
Fig. 3 HPLC analysis of acidic hydrolysate of globin isolated from
human blood that was exposed to 5 mM [35S]sulphur mustard, after
derivatization with Fmoc-Cl. A Detection of radioactivity; B UV Fig. 4 HPLC analysis of a pronase E digest of globin isolated from
detection (254 nm). Column, PepRPC 5/5 column (length 5 cm; i.d. human blood that was exposed to 5 mM [35S]sulphur mustard, after
5 mm; Pharmacia); temperature, 20 °C; injection volume, 60 ll derivatization with Fmoc-Cl. A detection of radioactivity; B UV
corresponding to 0.2 mg of globin hydrolysate. A linear gradient was detection (254 nm). All HPLC conditions were as given in the legend
applied from 0.1% trifluoroacetic acid (TFA)/25% acetonitrile to 0.1% to fig. 3. Peak 1, probably thiodiglycol; peak 2, N1/N3-(HETE)-
TFA/80% acetonitrile for 25 min with a flow rate of 1 ml/min. Peak 1, histidine; peak 3, 5-(HETE)- -glutamate + 4-(HETE)- -aspartate;
probably thiodiglycol; peak 2, N1/N3-(HETE)- -histidine; peak 3, peak 4, S-(HETE)- -cysteine; peak 5, N-(HETE)- -valine; peak 6,
unknown unknown
L-
L
L
L
L
L

177
LC-tandem electrospray MS analysis
of Na-Fmoc-N1/N3-(HETE)- -histidine
regarded with caution since no internal standard was
used. Nevertheless, there is a reasonable correlation with
the degree of alkylation extrapolated from the experi-
L
Since the histidine adduct was determined to be the most ments with 5 mM [35S] sulphur mustard, i.e. 60 and
abundant adduct, we tried to develop a sensitive and 6 pmol adduct/mg globin.
convenient method for analysis of this compound. Due
In conclusion, we have described the chemical
to the somewhat disappointing GC/MS results for the synthesis of the adducts of sulphur mustard to glutamic
histidine adduct, in terms of sensitivity, we directed our
attention toward LC-tandem MS using the MRM
technique, as has been reported for the analysis of al-
kylguanines in human urine (Cushnir et al. 1993; Fidder
et al. 1996b) and for deoxyguanosine adducts in DNA
(Ringden et al. 1995). With this technique, the first
quadrupole selects the protonated molecular ion and the
second quadrupole is adjusted to monitor the most in-
tense fragment ion formed. The major advantage of this
technique is the high selectivity due to the detection of a
specific fragment ion. In order to enable reversed phase
HPLC analysis, precolumn derivatization with Fmoc-Cl
was applied. Tandem mass spectrometric analysis of
synthetic Na-Fmoc-N1/N3-(HETE)-
-histidine (MH+,
L
m/z 482) yielded a major product ion at m/z 105 which
can be ascribed to [HOCH2CH2SCH2CH2]+ (see Fig. 5).
Sensitive and specific detection was achieved by
monitoring the m/z 482 → m/z 105 transition. The de-
tection level was 0.5 pmol/ml sample injected with an
absolute detection limit of 20 fmol. Unfortunately, the
isomers had identical retention times under various
elution conditions. Compared to GC/MS analysis of the
histidine adduct, sensitivity has significantly increased.
The adduct could be detected in Fmoc-derivatized acidic
hydrolysates of globin isolated from blood that had been
exposed to various concentrations of sulphur mustard
(Fig. 6). The results obtained were quantitated roughly
on the basis of comparison with a known amount of a
synthetic standard. The alkylation degree after exposure
to 0.1 and 0.01 mM sulphur mustard was determined to
be 100 and 10 pmol histidine adduct/mg globin, re-
spectively. However, these quantitative data have to be
Fig. 6 LC tandem MS analysis of Na-Fmoc-N1/N3-(HETE)-L-
histidine using the multiple reaction monitoring scanning mode for the
transition m/z 482 → m/z 105. Fmoc-derivatized hydrolysate of globin
from blood exposed to 10 lM sulphur mustard (A) and from blood
exposed to 100 lM sulphur mustard (B); synthetic reference (10 pmol/
ml; C). The LC system comprised a reversed phase C18 column
Fig. 5 Collision induced dissociation tandem MS spectrum of the
protonated molecular ion m/z 482 of synthetic Na-Fmoc-N1/N3- (length 25 cm; i.d. 5 mm), containing Lichrosorb 5 lm particles.
(HETE)-L-histidine at a collision energy of 20 V
Temperature, 20 °C; injection volume, 40 ll

178
phase high-performance liquid chromatography. J Chromatogr
282: 609–618
Farmer PB, Bailey E, Gorf SM, To¨rnqvist M, Osterman-Golkar S,
Kautiainen A, Lewis-Enright DP (1986) Monitoring human
exposure to ethylene oxide by the determination of haemoglo-
bin adducts using gas chromatography - mass spectrometry.
Carcinogenesis 7: 637–640
Fidder A, Moes GWH, Scheffer AG, Van der Schans GP, Baan
RA, De Jong LPA, Benschop HP (1994) Synthesis, character-
ization and quantitation of the major adducts formed between
sulfur mustard and DNA of calf thymus and human blood.
Chem Res Toxicol 7: 199–204
Fidder A, Noort D, De Jong AL, Trap HC, De Jong LPA, Ben-
schop HP (1996a) Monitoring of in vitro and in vivo exposure
to sulfur mustard by GC/MS determination of the N-terminal
valine adduct in hemoglobin after a modified Edman degrada-
tion. Chem Res Toxicol 9: 788–792
Fidder A, Noort D, Hulst AG, De Jong LPA, Benschop HP
(1996b) N7-(2-hydroxyethylthioethyl)-guanine: a novel urinary
metabolite following exposure to sulphur mustard. Arch Tox-
icol 70: 854–855
Grant WM, Kinsey VE (1946) Synthetic preparation of 2-chloro-
2′-hydroxydiethylsulfide, reaction with cysteine and valine and
measurement of reaction rate in aqueous media. J Am Chem
Soc 68: 2075–2076
acid, aspartic acid and histidine and have described their
GC/MS analysis. We demonstrated the histidine adduct
is the most abundant adduct formed. This is in agree-
ment with the results we obtained in an earlier study in
which we elucidated several specific histidine residues in
haemoglobin, which are prone to alkylation by sulphur
mustard (Noort et al. 1996). Due to the intrinsic lability
of the adducts to acid and base, quantitation of the other
adducts is complicated. For the analysis of the histidine
adduct, we described the use of LC-tandem electrospray
MS with MRM, employing the favourable chromato-
graphic properties of the corresponding Fmoc-deriva-
tive. This method circumvents the tedious ion-exchange
procedures normally used for the analysis of alkylated
histidines (Farmer et al. 1986), thereby minimizing
sample losses. Lower levels of exposure may be likely to
be detected when larger batches of globin are processed
or when samples are purified by semi-preparative HPLC
prior to analysis.
Acknowledgements This work was supported partly by grant no.
DAMD17-92-V-2005 from the US Army Medical Research and
Materiel Command, Fort Detrick, Frederick, Md., and partly by
the Directorate of Military Medical Services of the Ministry of
Defence, The Netherlands. We are grateful to Ad L. de Jong for
performing GC-EI/MS experiments, to Simon H. van Krimpen for
performing NMR spectroscopy and to Gerrit W.H. Moes for
preparing [35S]sulphur mustard.
Husek
P (1974) Derivatization of amino acids with 1,3-di-
chlorotetrafluoroacetone and its use in gas chromatography. J
Chromatogr 91: 475–481
Husek P, Felt V, Matucha M (1979) Cyclic amino acid derivatives
in gas chromatography. J Chromatogr 180: 53–68
Jones JB, Hysert DW (1971) Alkylations of the side-chain nu-
cleophiles of cysteine, methionine, histidine and lysine deriva-
tives with allyl bromide, 1-bromo-2-butyne, and 2-
bromoacetophenone. Can J Chem 49: 3012–3019
Matthews HR, Rapoport H (1973) Differentiation of 1,4- and 1,5-
disubstituted imidazoles. J Am Chem Soc 95: 2297–2303
Neises B, Steglich W (1979) Einfaches Verfahren zur Veresterung
von Carbonsa¨uren. Angew Chem 90: 556–557
References
Noort D, Jacobs EH, Fidder A, De Jong LPA, Drijfhout JW,
Benschop HP (1995) Solid-phase synthesis of peptide haptens
containing a cysteine-sulfur mustard adduct. Int J Pep Prot Res
45: 497–500
Noort D, Verheij ER, Hulst AG, De Jong LPA, Benschop HP
(1996) Characterization of sulphur mustard induced structural
modifications in human hemoglobin by LC-tandem mass
spectrometry. Chem Res Toxicol 9: 781–787
Papirmeister B, Feister AJ, Robinson SI, Ford RD (1991) Medical
defense against mustard gas. Toxic mechanisms and pharma-
cological implications. CRC Press, Boca Raton, Fla.
Ringden D, Turesky RJ, Vouros P (1995) Determination of in vitro
formed DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,
5-b]pyridine using capillary liquid chromatography/electrospray
ionization/tandem mass spectrometry. Chem Res Toxicol 8:
1005–1013
Sandelowsky I, Simon GA, Bel P, Barak R, Vincze A (1992) N1-(2-
hydroxyethythioethyl-4-methyl-imidazole (4-met-1-imid-thio-
diglycol) in plasma and urine: a novel metabolite following
dermal exposure to sulphur mustard. Arch Toxicol 66: 296–297
Skipper PL, Tannenbaum SR (1990) Protein adducts in the mole-
cular dosimetry of chemical carcinogens. Carcinogenesis 11:
507–518
Bailey E, Brooks AGF, Dollery CT, Farmer PB, Passingham BJ,
Sleightholm MA, Yates DW (1988) Hydroxyethylvaline adduct
formation in haemoglobin as a biological monitor of cigarette
smoke intake. Arch Toxicol 62: 247–253
Banks TE, Boursnell JC, Francis GE, Hopwood FL, Wormall A
(1946) Studies on mustard gas (b, b′-dichlorodiethyl sulphide)
and some related compounds. 4. Their action on proteins stu-
died with the aid of radioactive sulphur. Biochem J 40: 745–756
Black RM, Brewster K, Clarke RJ, Harrison JM (1992) The
chemistry of 1,1’-thiobis(2-chloroethane) (sulphur mustard).
Part II. The synthesis of some conjugates with cysteine, N-
acetylcysteine and N-acetylcysteine methyl ester. Phosphorus
Sulphur Silicon Relat Elem 71: 49–58
Calleman CJ, Wachtmeister CA (1979) Synthesis of Np- and Ns-(2-
hydroxyethyl)-L-histidines. Acta Chem Scand B 33: 277–280
Cushnir JR, Naylor S, Lamb JH, Farmer PB (1993) Tandem mass
spectrometric approaches for the analysis of alkylguanines in
human urine. Org Mass Spectrom 28: 552–558
Davis SB, Ross WF (1947) The reaction of mustard gas with
proteins. J Am Chem Soc 69: 1177–1181
Day BW, Skipper PL, Wishnock JS, Coghlin J, Hammond SK,
Gann P, Tannenbaum SR (1990) Identification of an in vivo
chrysene diol epoxide adduct in human haemoglobin. Chem
Res Toxicol 3: 340–343
Douglas DE, Heard, RDH (1954) Observations on the reaction of
b,b′-dichlorodiethyl sulphide with glycine and with glycylgly-
cine. Can J Chem 32: 221–226
Einarsson S, Josefsson B, Lagerkvist S (1983) Determination of
amino acids with 9-fluorenylmethylchloroformate and reversed-
Van der Schans GP, Scheffer AG, Mars-Groenendijk RH, Fidder
A, Benschop HP, Baan RA (1994) Immunochemical detection
of adducts of sulfur mustard to DNA of calf thymus and hu-
man white blood cells. Chem Res Toxicol 7: 408–413
Wheeler GP (1962) Studies related to the mechanisms of action of
cytotoxic alkylating agents: a review. Cancer Res 22: 651–688