Synthesis of adducts with amino acids as potential dosimeters for the biomonitoring of humans exposed to toluenediisocyanate

Article abstract of DOI:10.1021/tx010053+

Toluenediisocyanates (TDI) are important intermediates in the chemical industry. Among the main damages after low levels of TDI exposure are lung sensitization and asthma. Protein adducts of TDI might be involved in the etiology of sensitization reactions. Blood protein adducts are used as dosimeters for modifications of macromolecules in the target organs where the disease develops. The functional groups of cysteine, tyrosine, serine, lysine, tryptophan, histidine, and N-terminal amino acids are potential reaction sites for isocyanates. Especially the N-terminal amino acids, valine, and aspartic acid of hemoglobin and albumin, respectively, are reactive toward electrophilic xenobiotics. To develop methods for the quantitation of protein adducts of 2,4- and 2,6-TDI, we reacted 3-nitro-4-methylphenyl isocyanate (1a) with single amino acids and reduced the nitro group using catalytic hydrogenation or ammonium formate with palladium on carbon yielding N-[(3-amino-4-methylphenyl)carbamoyl]valine (2a), N-[(3-amino-4-methylphenyl)carbamoyl]aspartic acid (8a), N^α-acetyl-N^ε-[ (3-amino-4-methylphenyl)-carbamoyl]lysine (12a), and Nα-acetyl-O-[(3-amino-4-methylphenyl)carbamoyl]serine (15a). The same reactions were performed with 5-nitro-2-methylphenyl isocyanate (1b) and 3-nitro-2-methylphenyl isocyanate (1c). The valine adducts were boiled in acid to obtain the corresponding hydantoins: 3-(3-amino-4-methylphenyl)-5-isopropylimidazoline-2,4-dione (5a), 3-(5-amino-2-methylphenyl)-5-isopropylimidazoline-2,4-dione (5b), and 3-(3-amino-2-methylphenyl)-5-isopropylimidazoline-2,4-dione (5c). A method for the detection of N-terminal adducts with valine in biological samples was developed. The tripeptide adduct N-[(3-amino-4-methylphenyl)-carbamoyl]valyl-glycyl-glycine (19a) was hydrolyzed with acid in the presence of globin and the internal standard N-[(3-amino-4-methylphenyl-d₆)carbamoyl]valyl-glycl-glycine (19d). The released hydantoins were determined by LC/MS/MS and after derivatization with pentafluoropropionic anhydride by GC/MS. The determination limit was 0.16 pmol/sample. The same N-terminal adduct with valine was found in globin of a TDI-worker and in two women with polyurethane covered breast implants.

Full text of DOI:10.1021/tx010053+

Chem. Res. Toxicol. 2001, 14, 1573-1583
1573
Syn th esis of Ad d u cts w ith Am in o Acid s a s P oten tia l
Dosim eter s for th e Biom on itor in g of Hu m a n s Exp osed to
Tolu en ed iisocya n a te
Gabriele Sabbioni,* Renate Hartley, and Siegfried Schneider
Walther-Straub-Institut fu¨r Pharmakologie und Toxikologie, Ludwig-Maximilians-Universita¨t
Mu¨nchen, Nussbaumstrasse 26, D-80336 Mu¨nchen, Germany
Received March 5, 2001
Toluenediisocyanates (TDI) are important intermediates in the chemical industry. Among
the main damages after low levels of TDI exposure are lung sensitization and asthma. Protein
adducts of TDI might be involved in the etiology of sensitization reactions. Blood protein adducts
are used as dosimeters for modifications of macromolecules in the target organs where the
disease develops. The functional groups of cysteine, tyrosine, serine, lysine, tryptophan,
histidine, and N-terminal amino acids are potential reaction sites for isocyanates. Especially
the N-terminal amino acids, valine, and aspartic acid of hemoglobin and albumin, respectively,
are reactive toward electrophilic xenobiotics. To develop methods for the quantitation of protein
adducts of 2,4- and 2,6-TDI, we reacted 3-nitro-4-methylphenyl isocyanate (1a ) with single
amino acids and reduced the nitro group using catalytic hydrogenation or ammonium formate
with palladium on carbon yielding N-[(3-amino-4-methylphenyl)carbamoyl]valine (2a ), N-[(3-
amino-4-methylphenyl)carbamoyl]aspartic acid (8a ), NR-acetyl-Nꢀ-[(3-amino-4-methylphenyl)-
carbamoyl]lysine (12a ), and NR-acetyl-O-[(3-amino-4-methylphenyl)carbamoyl]serine (15a ). The
same reactions were performed with 5-nitro-2-methylphenyl isocyanate (1b) and 3-nitro-2-
methylphenyl isocyanate (1c). The valine adducts were boiled in acid to obtain the corresponding
hydantoins: 3-(3-amino-4-methylphenyl)-5-isopropylimidazoline-2,4-dione (5a ), 3-(5-amino-2-
methylphenyl)-5-isopropylimidazoline-2,4-dione (5b), and 3-(3-amino-2-methylphenyl)-5-iso-
propylimidazoline-2,4-dione (5c). A method for the detection of N-terminal adducts with valine
in biological samples was developed. The tripeptide adduct N-[(3-amino-4-methylphenyl)-
carbamoyl]valyl-glycyl-glycine (19a ) was hydrolyzed with acid in the presence of globin and
the internal standard N-[(3-amino-4-methylphenyl-d6)carbamoyl]valyl-glycyl-glycine (19d ). The
released hydantoins were determined by LC/MS/MS and after derivatization with pentafluo-
ropropionic anhydride by GC/MS. The determination limit was 0.16 pmol/sample. The same
N-terminal adduct with valine was found in globin of a TDI-worker and in two women with
polyurethane covered breast implants.
occupational carcinogen for humans (8). Isocyanates and
arylamines can bind with proteins and/or DNA (Figure
1) and lead to cytotoxic and genotoxic effects (4-7). For
arylamines, it has been shown that blood protein adducts
are a dosimeter for the adducts in the target organ (9-
11). Protein adducts of isocyanates might be involved in
the etiology of sensitization reactions (12). An established
method to biomonitor exposed people is the determination
of blood protein adducts. Such adducts are a marker of
exposure and possibly a dosimeter for modifications of
macromolecules in the target organs where the disease
develops. To improve the risk assessment for workers
exposed to TDI, it is important to develop dosimeters to
establish if the toxic reactive intermediate is TDI or a
metabolite of TDA.
Arylamines are metabolized to highly reactive N-
hydroxyarylamines (9, 10) by mixed mono-oxygenases.
N-Hydroxyarylamines can be further metabolized to
N-(sulfonyloxy)arylamines, N-acetoxy-arylamines, or N-
hydroxyarylamine N-glucuronides. These highly reactive
intermediates which bind covalently to biomolecules are
responsible for the genotoxic and cytotoxic effects of this
class of compounds. In exposed animals, arylamines such
In tr od u ction
Toluenediisocyanate (TDI)1 is the most important
isocyanate product after methylene diphenyl diisocyanate
(MDI) in industry. TDI is an intermediate in the manu-
facturing of polyurethanes, dyes, pigments, and adhe-
sives. Higher concentrations of isocyanates cause respi-
ratory irritation. Among, the main damages after low
levels of isocyanates exposures are lung sensitization and
asthma (1-6). The sensitization properties of TDI are
well documented (2-6). One of the corresponding aro-
matic amine of TDI, 2,4-toluendiamine (2,4-TDA), is
carcinogenic in animal experiments (7, 8). Commercial-
grade TDI has been classified by the International
Agency for Research on Cancer (IARC) as a carcinogen
in animals, with ”sufficient evidence” and as a potential
* To whom correspondence should be addressed. Phone and Fax:
(089) 51607265. E-mail: gabriele.sabbioni@t-online.de.
1 Abbreviations: TDI, toluenediisocyanates; MDI, methylene diphen-
yl diisocyanate; 24TDA, 2,4-toluenediamine; Pd/C, palladium 10% on
carbon; DEPT, distortionless enhancement by polarisation transfer;
CH-COSY, CH correlation method; ESI-MS, electrospray ionization-
mass spectrometry; TBME, tert-butyl methyl ether; PFPA, pentafluoro
propionic anhydride; PU, polyurethane.
10.1021/tx010053+ CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/20/2001

1574 Chem. Res. Toxicol., Vol. 14, No. 12, 2001
Sabbioni et al.
F igu r e 2. Reactions of amino acids with 3-nitro-4-methylphen-
ylisocyanate.
Virtually all functional groups on proteins can react
with isocyanates (28), but under physiological conditions,
the potential sites of reaction are restricted: (i) the
R-amino-group of the N-terminal amino acids, (ii) the
sulfhydryl group of cysteine (29, 30), (iii) the hydroxyl
groups of tyrosine (31) and especially serine (32), (iv) the
ꢀ-amino-group of lysine, and (v) the imidazole ring of
histidine. Although the pKa of lysine is around 10.5,
especially reactive lysines have been located in proteins
(33, 34). In vivo adducts of lysine have been found with
acetaldehyde (35), glycated proteins (36), and aflatoxin
B1 (37). Carbamylation of cysteine has been noticed as
an artifact in peptide mapping of hemoglobins in the
presence of urea (38). Carbamylated hemoglobin is
formed by the reaction of hemoglobin with cyanate, a
product of in vivo urea dissociation. It is found in high
levels in patients with renal failure and may be useful
in their clinical evaluation (39).
The main goal of the present study is to synthesize
typical TDI protein adducts in order to assess the
exposure to isocyanates. According to the present litera-
ture, it is not likely that both isocyanate groups of one
molecule would react at the same time with proteins.
Therefore, in the present work we investigated the
reaction products resulting from the reaction of one
isocyanate group (Figure 2). To develop methods for the
quantification of protein adducts of 2,4- and 2,6-TDI,
amino acids were reacted with monoisocyanate deriva-
tives of TDI. 3-Nitro-4-methylphenyl isocyanate (1a ) was
reacted with single amino acids: L-valine (2), L-aspartic
acid (6), NR-acetyl-L-lysine (10), N-acetyl-DL-serine (13),
and N-acetyl-L-cysteine (16). The resulting N-[(3-nitro-
4-methylphenyl)carbamoyl]valine (3a), N-[(3-nitro-4-meth-
ylphenyl)carbamoyl]aspartic acid (7a ), NR-acetyl-Nꢀ-[(3-
nitro-4-methylphenyl)carbamoyl]lysine (11a ), N-acetyl-
O-[(3-nitro-4-methylphenyl)carbamoyl]serine (14a ), and
N-acetyl-S-[3-nitro-4-methylphenyl-carbamoyl]cysteine
(17a ) were reduced to the corresponding amino deriva-
tives. As shown for other environmental pollutants,
isocyanates might react with the N-terminal amino acids
of hemoglobin and albumin, this would be L-valine and
L-aspartic acid, respectively. Therefore, an adduct with
a tripeptide containing L-valine as the N-terminal amino
acid was synthesized to mimic the release of the formed
F igu r e 1. Putative DNA adducts and protein-adducts of 2,4-
toluendiamine and 2,4-toluenediisocyanate.
as 4-aminobiphenyl (11), a human bladder carcinogen,
is known to form adducts with DNA as well as with tissue
proteins and the blood proteins albumin and hemoglobin
in a dose-dependent manner. In contrast, isocyanates do
not need any further activation to react with biomolecules
(Figure 1).
Arylamine specific adducts are of the sulfinic acid
amide type (13). Mild base hydrolysis of such adducts
releases the parent aromatic amine (14). At a dose of 50
mg of 2,4-TDA/kg body weight, ca. 0.82 nmol was co-
valently bound to hemoglobin (15). The determination of
such hydrolyzable hemoglobin adducts is well-established
(14). 2,4-TDA binds covalently with DNA (16). The
chemical structure of TDI adducts with DNA (17) and
plasma proteins found in vivo is unknown (17), since for
the quantification thereof harsh hydrolyses conditions
were used in order to determine the released parent
aromatic amine (18-20). The distribution after oral
dosage or inhalation has been studied in rats with
radiolabeled TDI (21, 22). Recently, Day et al. (23) found
carbamylation products with hemoglobin isolated from
guinea pigs exposed by inhalation to 2,4-TDI. For all
adducts, one of the two original isocyanato groups had
been hydrolyzed to the amine. In addition, Day et al.
found an amine-nitroso adduct on the R chain of hemo-
globin. These results indicate that at least one of the
isocyanato moieties (or a masked derivative) of 2,4-TDI
survived passage through the lung, into the serum, and
through the erythrocyte membrane to form adducts with
hemoglobin that were stable to dialysis, gel filtration, and
reversed-phase HPLC separation under acidic conditions.
Carbamylation of the N-terminal valine of hemoglobin
with methyl isocyanate in rats and rabbits has been
demonstrated in vitro and in vivo by gas chromatography
(24, 25). Recently, Mraz and Bouskova described the
reaction of TDI with single amino acids (26). None of the
reaction products were isolated and characterized by
NMR or MS. Sabbioni et al. (27) found an N-terminal
valine adduct with hemoglobin after chronic exposure of
rats to MDI.

Adducts of Amino Acids with Toluenediisocyanate
Chem. Res. Toxicol., Vol. 14, No. 12, 2001 1575
H-C(6)); 8.27 (s, Ar-NH); 7.71 (dd, J ) 8.3, 2.5, H-C(4)); 7.40
(d, J ) 8.3, H-C(3)); 7.21 (d, J ) 8.6, NHCH); 4.17 (dd, J )
8.6, 4.6, NHCH); 2.32 (s, Me); 2.13 (mc, Me2CH); 0.93 (d, J )
7.4, MeCH); 0.91 (d, J ) 7.4, MeCH). 13C NMR (DMSO-d6): δ
173.7 (COOH); 154.9 (NHCONH); 146.1 (C, C(5)); 139.3 (C,
C(1)); 133.3 (C, C(2)); 130.9 (CH, C(3)); 115.8 (CH, C(4)); 112.6
(CH, C(6)); 57.5 (NHCH); 30.4 (Me2CH); 19.2 (Me); 18.2 (Ar-
Me); 17.6 (Me). ESI-MS: 296 [M + H]+, 278 [(M - H2O) + H]+,
118 [Val + H]+.
N-[(3-Nit r o-2-m et h ylp h en yl)ca r b a m oyl]va lin e (3c). 3c
was obtained according to general procedure 1, with L-valine
(365 mg, 3.12 mmol). White solid 3c (660 mg, 72%), mp 167 °C.
1H NMR (DMSO-d6): δ 8.28 (s, Ar-NH); 8.12 (dd, J ) 8.2, 1.2,
H-C(6)); 7.48 (d, J ) 8.2, 1.2, H-C(4)); 7.35 (t, J ) 8.2, H-C(5));
7.03 (d, J ) 8.7, NHCH); 4.15 (dd, J ) 8.7, 4.8, NHCH); 2.25 (s,
Me-Ar); 2.10 (mc, Me2CH); 0.94 (d, J ) 7.3, MeCH); 0.92 (d, J )
7.3, MeCH). 13C NMR (DMSO-d6): δ 173.4 (COOH); 154.9
(NHCONH); 150.9 (C, C(3)); 139.6 (C, C(1)); 126.5 (CH, C(5));
124.5 (CH, C(6)); 120.7 (C, C(2)); 117.1 (CH, C(4)); 57.3 (NHCH);
30.3 (Me2CH); 19.1 (Me); 17.5 (Me); 13.2 (Me-Ar). ESI-MS: 296
[M + H]+, 278 [(M - H2O) + H]+, 118 [Val + H]+.
hydantoin from a protein. The same reactions were
performed with 2-methyl-5-nitrophenyl isocyanate (1b)
and 2-methyl-3-nitrophenyl isocyanate (1c).
Ma ter ia ls a n d Meth od s
Ch em ica ls. L-Aspartic acid, L-glutamic acid, BF3-MeOH,
ammonium formate, Palladium 10% on carbon (Pd/C), dry
dioxane, 4-fluoroaniline, NaHCO3 (analytical grade), 37% HCl
TraceSelect, formic acid, triphosgene and DMSO-d6 were pur-
chased from Fluka (Neu-Ulm, Germany); N-acetyl-L-cysteine
from Merck (Darmstadt, Germany); NR-acetyl-L-lysine and
N-acetyl-DL-serine from Sigma (Deisenhofen, Germany); L-valine
from Serva (Heidelberg, Germany); Celite Hyflo Super Cel by
J ohns Manville from Roth (Karlsruhe, Germany); tert-butyl
methyl ether (TBME) picograde from Promochem (Wesel,
Germany); 4-methyl-3-nitrophenyl isocyanate (1a ), 2-methyl-
5-nitrophenyl isocyanate (1b), 2-methyl-3-nitrophenyl isocyan-
ate (1c), and 2-fluoro-nitrophenyl isocyanate from Lancaster
(Mu¨hlheim am Main, Germany).
In str u m en ta tion . NMR Spectra: Bruker-AC-250 or Bruker
-DPX-400 (CH-COSY-experiments) instrument; DMSO-d6 as
solvent and as internal standard (1H: 2.50 ppm; 13C: 39.43
ppm); δ in ppm, J in Hz; C-substitution determined using the
distortionless enhancement by polarization transfer (DEPT)
method or the CH correlation method (CH-COSY). The raw
NMR data were processed with the program MestRe-C (Mag-
netic Resonance Companion, J . C. Cobas, J . Cruces, and F. J .
Sordina, Departamente de Quimica Organica, Universidad de
Santiago de Compostela, 15706 Santjago de Compostela, Spain).
mp, melting points, apparatus by Dr. Tottoli (510) from Bu¨chi,
Glasapparatefabrik Flawil, Switzerland, uncorrected. Positive
electrospray ionization-mass spectrometry (ESI-MS): quadru-
pole ion trap tandem mass spectrometer (LCQ Duo, Thermo-
quest). The samples were dissolved in methanol and introduced
with a syringe pump into the MS (flow ) 25 µL/min). The
parameters were set as follows: capillary temperature of 220
°C, sheath gas flow of 20 (arbitrary units) and auxiliary gas flow
of 0, full-scan MS mode. MS/MS spectra were obtained by ion
trap collision-induced dissociation. HPLC analyses were per-
formed on a Hewlett-Packard 1100 system with a quaternary
HPLC pump and a photodiode array detector. GC/MS: Hewlett-
Packard GC (HP 5890II) equipped with an autosampler (HP7236)
and interfaced to MS (HP 5989A); m/z (rel.%).
Ur ea s fr om Isocya n a tes a n d Am in o Acid s. Gen er a l
P r oced u r e 1. To a solution of amino acid (2.0 mmol) in 0.25 M
NaHCO3 (40 mL) was added the isocyanate (4.0 mmol), and the
solution was stirred at 80 °C for 3 h. After cooling the solution
with ice, the precipitate was filtered off and washed with 0.25
M NaHCO3 (5 mL). The filtrate was acidified to pH 1-2 with 2
M HCl, extracted with ethyl acetate (3 × 50 mL). The organic
phase was extracted with 0.5 M NaHCO3 (3 × 50 mL). The
water phase was made acidic to pH 1-2 with 2 M HCl and
reextracted with ethyl acetate (3 × 50 mL). The organic extract
was dried (MgSO4), filtered, and evaporated.
N-[(3-Nit r o-4-m et h ylp h en yl)ca r b a m oyl]a sp a r t ic Acid
(7a ). 7a was obtained according to general procedure 1, with
L-aspartic acid (413 mg, 3.10 mmol). Yellowish solid (316 mg,
1
33%), mp 152-153 °C. H NMR (DMSO-d6): δ 12.65 (s, broad,
2H, COOH); 9.26 (s, Ar-NH); 8.24 (d, J ) 2.2, H-C(2)); 7.44
(dd, J ) 8.3, 2.2, H-C(6)); 7.39 (d, J ) 8.3, H-C(5)); 6.67 (d, J
) 8.4, NHCH); 4.51 (mc, NHCHCH2); 2.81 (dd, J ) 16.9, 5.6,
1H, NHCHCH2); 2.70 (dd, J ) 16.9, 4.8, 1H, NHCHCH2); 2.42
(s, Me). 13C NMR (DMSO-d6): δ 172.9; 172.3 (COOH); 154.5
(NHCONH); 148.7 (C, C(3)); 139.2 (C, C(1)); 133.0 (CH, C(5));
124.9 (C, C(4)); 122.3 (CH, C(6)); 112.5 (CH, C(2)); 48.8 (NH-
CHCH2); 36.6 (NHCHCH2); 19.0 (Me). ESI-MS: 312 [M + H]+,
134 [Asp + H ]+.
N R-Ace t yl-N ꢀ-[(3-n it r o-4-m e t h ylp h e n yl)ca r b a m oyl]-
lysin e (11a ). 11a was obtained according to general procedure
1, with NR-acetyl-L-lysine (566 mg, 3.01 mmol). Crystallization
from ethyl acetate/n-heptane yielded 11a (383 mg, 35%). White
solid, mp 149 °C. 1H NMR (DMSO-d6): δ 12.52 (s, COOH); 8.80
(s, Ar-NH); 8.23 (d, J ) 2.2, H-C(2)); 8.12 (d, J ) 7.9,
AcNHCH); 7.43 (dd, J ) 8.5, 2.2, H-C(6)); 7.29 (d, J ) 8.5,
H-C(5)); 6.26 (t, J ) 5.4, NHCH2); 4.14 (mc, AcNHCH); 3.06
(mc, NHCH2); 2.39 (s, Me); 1.84 (s, Ac); 1.55-1.80 (m, CH2);
1.25-1.45 (m, 2 CH2). 13C NMR (DMSO-d6): δ 173.7 (COOH);
169.3 (MeCO); 154.9 (NHCONH); 148.8 (C, C(3)); 139.5 (C, C(1));
132.7 (CH, C(5)); 124.4 (C, C(4)); 122.2 (CH, C(6)); 112.5 (CH,
C(2)); 51.8 (AcNHCH); 39.1 (CH2(ꢀ)); 30.7 (CH2(â)); 29.2 (CH2-
(δ)); 22.8 (CH2(γ)); 20.6 (Me); 18.8 (Ar-Me). ESI-MS: 367 [M +
H]+, 349 [(M - H2O) + H]+, 215 [(M - toluidine-NO2) + H]+, 173.
N r-Ace t yl-N E-[(5-n it r o-2-m e t h ylp h e n yl)ca r b a m oyl]-
lysin e (11b). 11b was obtained according to general procedure
1, with NR-acetyl-L-lysine (565 mg, 3.00 mmol). Beige solid 11b
(561 mg, 51%), mp 154-156 °C. 1H NMR (DMSO-d6): δ 8.93
(d, J ) 2.5, H-C(6)); 8.10 (d, J ) 7.8, AcNHCH); 7.97 (s, Ar-
NH); 7.69 (dd, J ) 8.3, 2.5, H-C(4)); 7.41 (d, J ) 8.3, H-C(3));
6.88 (t, J ) 5.4, NHCH2); 4.15 (mc, AcNHCH); 3.10 (mc, NHCH2);
2.28 (s, Me); 1.83 (s, Ac); 1.80-1.55 (m, CH2); 1.41-1.25 (m, 2
CH2). 13C NMR (DMSO-d6): δ 173.8 (COOH); 169.2 (MeCO);
154.9 (NHCONH); 146.0 (C, C(5)); 139.4 (C, C(1)); 133.3 (C,
C(2)); 130.8 (CH, C(3)); 115.6 (CH, C(4)); 112.7 (CH, C(6)); 51.8
(AcNHCH); 39.1 (CH2(ꢀ)); 30.8 (CH2(â)); 29.2 (CH2(δ)); 22.9 (CH2-
(γ)); 22.3 (Me); 18.0 (Me). ESI-MS: 367 [M + H]+, 349 [(M -
H2O) + H]+, 215 [(M - toluidine-NO2) + H]+.
N-[(3-Nitr o-4-m eth ylp h en yl)ca r ba m oyl]va lin e (3a ). 3a
was obtained according to general procedure 1, with L-valine
(372 mg, 3.18 mmol). Crystallization from EtOH/H2O yielded
3a (465 mg, 50%). White solid, mp 136-138 °C. 1H NMR
(DMSO-d6): δ 9.01 (s, NH-Ar); 8.25 (d, J ) 2.2, H-C(2)); 7.42
(dd, J ) 8.4, 2.2, H-C(6)); 7.35 (d, J ) 8.4, H-C(5)); 6.53 (d, J
) 8.6, NHCH); 4.12 (dd, J ) 8.6, 4.7, NHCH); 2.42 (s, Me); 2.10
(mc, Me2CH); 0.92 (d, J ) 6.8, MeCH); 0.87 (d, J ) 6.8, MeCH).
13C NMR (DMSO-d6): δ 173.5 (COOH); 154.7 (NHCONH); 148.7
(C, C(3)); 139.1 (C, C(1)); 132.9 (CH, C(5)); 124.7 (C, C(4)); 122.1
(CH, C(6)); 112.3 (CH, C(2)); 57.2 (NHCH); 30.1 (Me2CH); 19.1
(Me); 18.9 (Ar-Me); 17.4 (Me). ESI-MS: 296 [M + H]+, 278 [(M
- H2O) + H]+, 118 [Val + H]+.
N r-Ace t yl-N E-[(3-n it r o-2-m e t h ylp h e n yl)ca r b a m oyl]-
lysin e (11c). 11c was obtained according to general procedure
1, with NR-acetyl-L-lysine (568 mg, 3.02 mmol). Crystallization
from H2O yielded 11c (729 mg, 66%). White solid, mp 149 °C.
1H NMR (DMSO-d6): δ 12.54 (s, COOH); 8.13 (d, J ) 7.7,
AcNHCH); 8.08 (dd, J ) 8.0, 1.0, H-C(6)); 8.04 (s, Ar-NH);
7.48 (dd, J ) 8.0, 1.0, H-C(4)); 7.34 (t, J ) 8.0, H-C(5)); 6.67
(t, J ) 5.5, NHCH2); 4.18 (mc, AcNHCH); 3.10 (mc, NHCH2);
2.24 (s, Me); 1.86 (s, Ac); 1.75-1.50 (m, CH2); 1.47-1.22 (m, 2
N-[(5-Nitr o-2-m eth ylp h en yl)ca r ba m oyl]va lin e (3b). 3b
was obtained according to general procedure 1, with L-valine
(367 mg, 3.13 mmol). White solid 3b (570 mg, 62%), mp 124 °C.
1H NMR (DMSO-d6): δ 12.7 (s, COOH); 8.98 (d, J ) 2.5,

1576 Chem. Res. Toxicol., Vol. 14, No. 12, 2001
Sabbioni et al.
CH2). 13C NMR (DMSO-d6): δ 173.8 (COOH); 169.3 (MeCO);
155.1 (NHCONH); 151.0 (C, C(3)); 140.0 (C, C(1)); 126.5 (CH,
C(5)); 125.2 (CH, C(6)); 121.2 (C, C(2)); 117.1 (CH, C(4)); 51.8
(AcNHCH); 38.8 (CH2(ꢀ)); 30.8 (CH2(â)); 29.3 (CH2(δ)); 22.9 (CH2-
(γ)); 22.3 (Me); 13.3 (Ar-Me). ESI-MS: 367 [M + H]+, 349 [(M -
H2O) + H]+, 215 [(M - toluidine-NO2) + H]+.
Ca r ba m a tes fr om Isocya n a tes a n d N-Acetyl-DL-ser in e.
Gen er a l P r oced u r e 2. To a solution of N-acetyl-DL-serine (0.5
mmol) in dry pyridine (3 mL) was added the isocyanate (0.55
mmol) , and the solution was stirred at 80 °C for 3 h. The solvent
was evaporated. The residue was taken up in 0.5 M NaHCO3
(15 mL). The insoluble residue was separated by filtration. The
water phase was washed with ethyl acetate (2 × 15 mL) acidified
with HCl to pH 1.5 and extracted with ethyl acetate (3 × 30
mL). The organic phase was dried over MgSO4 and the solvent
was evaporated at reduced pressure.
N -Ac e t y l-O -[(3-n it r o -4-m e t h y lp h e n y l)c a r b a m o y l]-
ser in e (14a ). 14a was obtained according to general procedure
2, with N-acetyl-DL-serine (500 mg, 3.40 mmol). Crystallization
from ethyl acetate/n-heptane yielded 14a (774 mg, 70%). White
solid, mp 174-175 °C. 1H NMR (DMSO-d6): δ 10.11 (s, Ar-
NH); 8.22 (d, J ) 7.7, AcNH); 8.19 (d, J ) 1.8, H-C(2)); 7.50
(dd, J ) 8.2, 1.8, H-C(6)); 7.04 (d, J ) 8.2, H-C(5)); 4.54 (mc,
CHCH2O); 4.44 (dd, J ) 11.0, 4.5, 1H, CHCH2O); 4.22 (dd, J )
11.0, 6.6, 1H, CHCH2O); 2.45 (s, Me); 1.86 (s, Ac). 13C NMR
(DMSO-d6): δ 170.9 (COOH); 169.4 (MeCO); 153.0 (NHCOO);
148.6 (C, C(3)); 138.0 (C, C(1)); 133.1 (CH, C(5)); 126.3 (C, C(4));
122.8 (CH, C(2)); 113.2 (CH, C(6)); 63.6 (CHCH2O); 51.4
(CHCH2O); 22.3 (Me); 19.0 (Me). ESI-MS: 326 [M + H]+, 130
[(M - toluidine-NO2COO) + H]+.
N -Ac e t y l-O -[(5-n it r o -2-m e t h y lp h e n y l)c a r b a m o y l]-
ser in e (14b). 14b was obtained according to general procedure
2, with N-acetyl-DL-serine (500 mg, 3.40 mmol). White solid 14b
(830 mg, 75%), mp 177 °C. 1H NMR (DMSO-d6): δ 9.34 (s, Ar-
NH); 8.34 (d, J ) 2.5, H-C(6)); 8.33 (d, J ) 7.0, AcNH); 7.91
(dd, J ) 8.4, 2.5, H-C(4)); 7.47 (d, J ) 8.4, H-C(3)); 4.59 (mc,
CHCH2O); 4.46 (dd, J ) 11.1, 4.6, 1H, CHCH2O); 4.24 (dd, J )
11.1, 6.8, 1H, CHCH2O); 2.34 (s, Me); 1.89 (s, Ac). 13C NMR
(DMSO-d6): 170.9 (COOH); 169.4 (MeCO); 153.8 (NHCOO);
145.8 (C, C(5)); 139.0 (C, C(1)); 137.4 (C, C(2)); 131.3 (CH, C(3));
119.0 (CH, C(4)); 118.0 (CH, C(6)); 63.7 (CHCH2O); 51.4
(CHCH2O); 22.3 (Me); 18.0 (Me). ESI-MS: 326 [M + H]+, 130
[(M - toluidine-NO2COO) + H]+.
N -Ac e t y l-O -[(3-n it r o -2-m e t h y lp h e n y l)c a r b a m o y l]-
ser in e (14c). 14c was obtained according to general procedure
2, with N-acetyl-DL-serine (500 mg, 3.40 mmol). White solid 14c
(894 mg, 81%), mp 140 °C. 1H NMR (DMSO-d6): δ 9.41 (s, Ar-
NH); 8.33 (d, J ) 7.9, AcNH); 7.69 (d, J ) 8.1, 1.0, H-C(6) or
H-C(4)); 7.63 (d, J ) 8.1, 1.0, H-C(4) or H-C(6)); 7.41 (t, J )
8.1, H-C(5)); 4.57 (mc, CHCH2O); 4.43 (dd, J ) 11.1, 4.6, 1H,
CHCH2O); 4.21 (dd, J ) 11.1, 6.8, 1H, CHCH2O); 2.25 (s, Me);
1.88 (s, Ac). 13C NMR (DMSO-d6): δ 171.0 (COOH); 169.5
(MeCO); 154.0 (NHCOO); 150.9 (C, C(3)); 138.2 (C, C(1)); 129.7
(CH, C(6)); 126.7 (CH, C(5)); 126.4 (C, C(2)); 120.5 (CH, C(4));
63.8 (CHCH2O); 51.5 (CHCH2O); 22.4 (Me); 13.7 (Ar-Me). ESI-
MS: 326 [M + H]+, 130 [(M - toluidine-NO2COO) + H]+.
Ca r ba m ic Acid S-Ester s. Gen er a l P r oced u r e 3. To a
solution of N-acetyl-L-cysteine (163 mg, 1.0 mmol) in 0.25 M
NaHCO3 (10 mL) was added the isocyanate (2.0 mmol). The
mixture was treated by ultrasound during 3 min in 10 min
intervals at 25 °C for 2 h. The reaction mixture was cooled on
ice. The precipitate was separated by filtration. The pH of the
filtrate was adjusted to 2.5 and extracted with ethyl acetate (3
× 30 mL). The combined organic phases were extracted with
0.6 M NaHCO3 (3 × 50 mL). The pH of the basic water solution
was then adjusted to 2.5 and extracted with ethyl acetate (3 ×
40 mL). The extract was dried (MgSO4) and evaporated, and
the residue was recrystallized with EtOH/H2O.
188 °C. 1H NMR (DMSO-d6): δ 12.5 (br. s, COOH); 10.76 (s,
Ar-NH); 8.33 (d, J ) 8.2, AcNH); 8.25 (d, J ) 2.3, H-C(2));
7.64 (dd, J ) 8.5, 2.3, H-C(6)); 7.45 (d, J ) 8.5, H-C(5)); 6.29
(mc, AcNH); 4.16 (mc, NHCHCH2); 3.42 (dd, J ) 13.8, 5.0, 1H,
CHCH2S); 3.07 (dd, J ) 13.8, 8.7, 1H, CHCH2S); 2.45 (s, Me);
1.85 (s, Me). 13C NMR (DMSO-d6): δ 171.7 (COOH); 169.2
(MeCO); 164.6 (NHCOS); 148.5 (C, C(3)); 137.4 (C, C(1)); 133.3
(CH, C(5)); 127.3 (C, C(4)); 123.3 (CH, C(6)); 114.0 (CH, C(2));
51.9 (CHCH2S); 30.9 (CHCH2S); 22.2 (Me); 19.1 (Me). ESI-MS:
342 [M + H]+, 164 [N-AcCys + H]+.
Red u ction of Nitr o-Com p ou n d s. Gen er a l P r oced u r e 4.
The nitro compound (0.3 mmol) and Pd/C (30 mg) in dry MeOH
(6 mL) was stirred under a hydrogen atmosphere at room
temperature overnight. After 20 h the reaction mixture was
filtered over Celite to remove the Pd/C and washed twice with
MeOH (3 mL each). The solvent was evaporated under reduced
pressure yielding the desired product.
Red u ction of Nitr o-Com p ou n d s. Gen er a l P r oced u r e 5.
Pd/C (50 mg) and ammonium formate (540 mg, 8.56 mmol) were
added under nitrogen at room temperature to a solution of the
nitro compound (0.3 mmol) in dry MeOH (10 mL). After 3 h the
reaction mixture was filtered over Celite. The solvent was
evaporated. To eliminate ammonium formate, the residue was
dried overnight at 0.01 Torr and 30 °C.
N-[(3-Am in o-4-m eth ylp h en yl)ca r ba m oyl]va lin e (4a ). 4a
was obtained according to general procedure 4 with 3a (280 mg,
1.0 mmol). Crystallization from EtOH yielded 4a (138 mg, 51%).
White solid, mp 114-115 °C. 1H NMR (DMSO-d6): δ 8.35 (s,
Ar-NH); 6.71 (d, J ) 2.0, H-C(2)); 6.71 (d, J ) 8.0, H-C(5));
6.48 (dd, J ) 8.0, 2.0, H-C(6)); 6.28 (d, J ) 8.4, NHCH); 4.04
(dd, J ) 8.4, 4.5, NHCH); 2.06 (mc, Me2CH); 1.95 (s, Me-Ar);
0.89 (d, J ) 6.8, MeCH); 0.84 (d, J ) 6.8, MeCH). 13C NMR
(DMSO-d6): δ 174.0 (COOH); 155.0 (NHCONH); 146.4 (C, C(3));
138.8 (C, C(1)); 129.7 (CH, C(5)); 114.2 (C, C(4)); 106.0 (CH,
C(6)); 103.6 (CH, C(2)); 57.4 (NHCH); 30.4 (MeCH); 19.2
(MeCH); 17.6 (MeCH); 16.8 (Me-Ar). ESI-MS: 266 [M + H]+,
248 [(M - H2O) + H]+, 118 [Val + H]+.
N-[(5-Am in o-2-m eth ylp h en yl)ca r ba m oyl]va lin e (4b). 4b
was obtained according to general procedure 5 with 3b (280 mg,
1.0 mmol). Crystallization from ethyl acetate/n-heptane yielded
4b (117 mg, 44%). Beige solid, mp 205 °C. 1H NMR (DMSO-d6):
δ 7.76 (s, Ar-NH); 7.14 (d, J ) 2.2, H-C(6)); 6.72 (d, J ) 8.0,
H-C(3)); 6.67 (d, J ) 8.5, NHCH); 6.11 (dd, J ) 8.0, 2.2,
H-C(4)); 3.94 (dd, J ) 8.5, 4.3, NHCH); 2.08 (mc, Me2CH); 2.03
(s, Me); 0.87 (d, J ) 7.0, MeCH); 0.84 (d, J ) 7.0, MeCH). 13C
NMR (DMSO-d6): δ 174.1 (COOH); 155.2 (NHCONH); 146.7 (C,
C(5)); 138.3 (C, C(1)); 130.2 (CH, C(3)); 113.7 (C, C(2)); 108.2
(CH, C(4)); 106.8 (CH, C(6)); 57.6 (NHCH); 30.5 (Me2CH); 19.3
(Me); 17.7 (Me); 17.1 (Ar-Me). ESI-MS: 266 [M + H]+, 248 [(M
- H2O) + H]+, 118 [Val + H]+.
N-[(3-Am in o-2-m eth ylp h en yl)ca r ba m oyl]va lin e (4c). 4c
was obtained according to general procedure 4 with 4c (280 mg,
1
1.0 mmol). White solid 4c (238 mg, 90%), mp 194 °C. H NMR
(DMSO-d6): δ 12.6 (s, COOH); 7.75 (s, Ar-NH); 6.92 (dd, J )
7.9, 1.0, H-C(6)); 6.76 (t, J ) 7.9, H-C(5)); 6.35 (dd, J ) 7.9,
1.0, H-C(4)); 6.61 (d, J ) 8.8, NHCH); 4.15 (dd, J ) 8.8, 4.9,
NHCH); 2.12 (s, Me-Ar); 2.12 (mc, Me2CH); 0.95 (d, J ) 6.7,
MeCH); 0.90 (d, J ) 6.7, MeCH). 13C NMR (DMSO-d6): δ 173.6
(COOH); 154.8 (NHCONH); 146.8 (C, C(3)); 137.8 (C, C(1));
125.4 (CH, C(5)); 112.0 (C, C(2)); 110.7 (CH, C(6)); 109.5 (CH,
C(4)); 57.3 (NHCH); 30.3 (Me2CH); 19.1 (Me); 17.6 (Me); 11.1
(Me). ESI-MS: 266 [M + H]+, 248 [(M - H2O) + H]+, 118 [Val
+ H]+.
N-[(5-Am in o-2-flu or op h en yl)ca r ba m oyl]va lin e (4e). 4e
was obtained according to general procedure 1 and 5 starting
with 1 mmol of valine. White solid 118 mg (44%). 1H NMR
(DMSO-d6): δ 8.83 (s, NH); 7.15 (dd, J ) 2.8, 7.3, H-C(6)); 6.84
(d, J ) 8.2, NH-CH); 6.54 (dd, J ) 8.6, 11.3, H-C(3)); 5.82 (ddd,
J ) 2.8, 3.9, 8.6, H-C(4)); 4.10 (dd, J ) 8.1, 4.2, CH-COOH);
2.03 (mc, MeCH); 1.92 (s, Me-Ar); 0.83 (dd, J ) 6.9, Me-CH);
0.79 (d, J ) 6.9, Me-CH). 13C NMR (DMSO-d6): δ 172.8 (COOH);
154.8 (MeCO); 145.0 (C, C(5)); 144.3 (d, J ) 228.5 (C, C(2)); 128.3
N -Ace t yl-S -[3-n it r o-4-m e t h ylp h e n yl-ca r b a m oyl]cys-
tein e (17a ). 17a was obtained according to general procedure
3, with N-acetyl-L-cysteine (1.0 mmol). Crystallization from
EtOH/H2O yielded 17a (205 mg, 60%). White solid, mp 187-

Adducts of Amino Acids with Toluenediisocyanate
Chem. Res. Toxicol., Vol. 14, No. 12, 2001 1577
(d, J ) 11.1, C, C(1)); 114.3 (d, J ) 19.5, CH, C(3)); 106.0 (d, J
) 6.5, CH, C(4)); 105.8 (C, C(6)); 58.2 (NHCH); 30.6 (Me2CH);
19.5 (MeCH); 17.7 (MeCH). ESI-MS: 270 [M + H]+, 252 [(M -
H2O) + H]+, 118 [Val + H]+.
N -Ac e t y l-O -[(5-a m in o -2-m e t h y lp h e n y l)c a r b a m o y l]-
ser in e (15b). 15b was obtained according to general procedure
4 with 14b (150 mg, 0.46 mmol). Beige solid 15b (113 mg, 85%),
1
mp 136 °C. H NMR (DMSO-d6): δ 8.45 (s, Ar-NH); 7.68 (d, J
) 7.6, AcNH); 6.77 (d, J ) 8.0, H-C(3)); 6.62 (d, J ) 2.2,
H-C(6)); 6.27 (dd, J ) 8.0, 2.2, H-C(4)); 4.36 (dd, J ) 10.3,
3.7, 1H, CHCH2O); 4.24 (mc, CHCH2O); 4.10 (dd, J ) 10.3, 6.3,
1H, CHCH2O); 2.00 (s, Me); 1.85 (s, Me). 13C NMR (DMSO-d6):
δ 171.6 (COOH); 168.8 (MeCO); 154.3 (NHCOO); 146.7 (C, C(5));
136.6 (C, C(1)); 130.2 (CH, C(3)); 118.2 (C, C(2)); 110.8 (CH,
C(4)); 110.5 (CH, C(6)); 65.1 (CHCH2O); 53.6 (CHCH2O); 22.8
(Me); 16.8 (Me). ESI-MS: 296 [M + H]+, 130 [(M - toluidine-
NH-COO) + H]+.
N-[(3-Am in o-4-m eth ylp h en yl)ca r ba m oyl]a sp a r tic Acid
(8a ). 8a was obtained according to general procedure 4 with
7a (300 mg, 0.96 mmol). Beige solid 8a (239 mg, 88%), mp 110
°C. 1H NMR (DMSO-d6): δ 8.59 (s, Ar-NH); 6.72 (d, J ) 2.1,
H-C(2)); 6.72 (d, J ) 8.0, H-C(5)); 6.48 (dd, J ) 8.0, 2.1,
H-C(6)); 6.38 (d, J ) 5.2, NHCH); 4.03 (mc, NHCH); 2.53-2.49
(m, NHCHCH2); 1.95 (s, Me).13C NMR (DMSO-d6): δ 174.2
(COOH); 172.9 (COOH); 154.4 (NHCONH); 146.4 (C, C(3)); 138.9
(C, C(1)); 129.7 (CH, C(5)); 114.0 (C, C(4)); 106.1 (CH, C(6));
103.7 (CH, C(2)); 49.4 (NHCH); 41.2 (NHCHCH2); 16.7 (Me).
ESI-MS: 282 [M + H]+, 134 [Asp + H]+.
N r-Ace t yl-N E-[(3-a m in o-4-m e t h ylp h e n yl)ca r b a m oyl]-
lysin e (12a ). 12a was obtained according to general procedure
4 with 11a (120 mg, 0.33 mmol). Crystallization from MeOH/
ethyl acetate yielded, 12a (60 mg, 52%). Beige solid, mp 89 °C.
1H NMR (DMSO-d6): δ 8.14 (s, Ar-NH); 7.99 (d, J ) 7.7,
AcNH); 6.73 (d, J ) 1.9, H-C(2)); 6.73 (d, J ) 7.9, H-C(5));
6.48 (dd, J ) 7.9 1.9, H-C(6)); 6.14 (t, J ) 5.4, NHCH2); 4.11
(mc, NHCH); 3.02 (mc, NHCH2); 1.95 (s, Me); 1.84 (s, Me); 1.77-
1.45 (m, CH2); 1.45-1.20 (m, 2 CH2). 13C NMR (DMSO-d6): δ
174.1 (COOH); 169.1 (MeCO); 155.3 (NHCONH); 146.4 (C, C(3));
139.0 (C, C(1)); 129.7 (CH, C(5)); 114.0 (C, C(4)); 106.2 (CH,
C(6)); 103.8 (CH, C(2)); 52.3 (NHCH); 39.2 (CH2(ꢀ)); 31.1 (CH2-
(â)); 29.6 (CH2(δ)); 22.9 (Me); 22.4 (CH2(γ)); 16.7 (Me). ESI-MS:
337 [M + H]+, 319 [(M - H2O) + H]+, 215 [(M - toluenediamine)
+ H]+, 189 [AcLys + H]+, 123 [toluenediamine + H]+.
N r-Ace t yl-N E-[(5-a m in o-2-m e t h ylp h e n yl)ca r b a m oyl]-
lysin e (12b). 12b was obtained according to general procedure
4 with 11b (90 mg, 0.246 mmol). Beige solid 12b (61 mg, 74%),
mp 130-132 °C. 1H NMR (DMSO-d6): δ 8.07 (d, J ) 7.7,
AcNHCH); 7.33 (s, Ar-NH); 7.12 (d, J ) 2.2, H-C(6)); 6.73 (d,
J ) 8.0, H-C(3)); 6.47 (t, J ) 5.4, CH2NH); 6.11 (dd, J ) 2.2,
8.0, H-C(4)); 4.15 (mc, NHCH); 3.05 (mc, NHCHCH2); 2.00 (s,
Me); 1.85 (s, Me); 1.77-1.45 (m, CH2); 1.40-1.20 (m, 2 CH2).
13C NMR (DMSO-d6): δ 173.9 (COOH); 169.3 (MeCO); 155.4
(NHCONH); 146.7 (C, C(5)); 138.4 (C, C(1)); 130.1 (CH, C(3));
113.9 (C, C(2)); 108.2 (CH, C(4)); 107.0 (CH, C(6)); 52.0 (NHCH);
39.1 (CH2(ꢀ)); 30.9 (CH2(â)); 29.5 (CH2(δ)); 23.0 (Me); 22.3 (CH2-
(γ)); 17.0 (Me). ESI-MS: 337 [M + H]+, 319 [(M - H2O) + H]+,
215 [(M - toluenediamine) + H]+, 189 [AcLys + H]+, 123
[toluenediamine + H]+.
N r-Ace t yl-N E-[(3-a m in o-2-m e t h ylp h e n yl)ca r b a m oyl]-
lysin e (12c). 12c was obtained according to general procedure
5 with 11c (70 mg, 0.193 mmol). Beige solid 12c (61 mg, 92%),
mp >174 °C decomp. 1H NMR (DMSO-d6): δ 8.07 (d, J ) 7.6,
AcNHCH); 7.50 (s, Ar-NH); 6.86 (d, J ) 7.8, H-C(6)); 6.75 (t,
J ) 7.8, H-C(5)); 6.34 (d, J ) 7.8, H-C(4)); 6.28 (t, J ) 5.4,
NHCH2); 3.05 (mc, NHCH); 1.90 (s, Me); 1.84 (s, Me); 1.77-1.45
(m, CH2); 1.45-1.20 (m, 2 CH2). 13C NMR (DMSO-d6): δ 174.7
(COOH); 168.5 (MeCO); 155.8 (NHCONH); 146.7 (C, C(3)); 138.2
(C, C(1)); 125.2 (CH, C(5)); 112.5 (C, C(2)); 111.2 (CH, C(6));
109.3 (CH, C(4)); 53.4 (NHCH); 39.1 (CH2(ꢀ)); 32.0 (CH2(â)); 29.8
(CH2(δ)); 22.8 (Me); 22.7 (CH2(γ)); 11.3 (Me). ESI-MS: 337 [M
+ H]+, 319 [(M - H2O) + H]+, 215 [(M - toluenediamine) +
H]+, 189 [AcLys + H]+, 123 [toluenediamine + H]+.
N -Ac e t y l-O -[(3-a m in o -2-m e t h y lp h e n y l)c a r b a m o y l]-
ser in e (15c). 15c was obtained according to general procedure
4 with 14c (150 mg, 0.46 mmol). Beige solid 15c (109 mg, 83%),
1
mp 212 °C. H NMR (DMSO-d6): δ 8.76 (s, Ar-NH); 8.29 (d, J
) 7.9, AcNH); 6.82 (t, J ) 7.9, H-C(5)); 6.47 (d, J ) 7.9, H-C(4),
H-C(6)); 4.53 (mc, CHCH2O); 4.34 (dd, J ) 11.0, 4.6, CHCH2O);
4.14 (dd, J ) 11.0, 6.9, 1H, CHCH2O); 1.89 (s, broad, 6H, 2 Me).
13C NMR (DMSO-d6): δ 171.1 (COOH); 169.4 (MeCO); 154.1
(NHCOO); 147.1 (C, C(3)); 136.2 (C, C(1)); 125.4 (CH, C(5));
116.6 (C, C(2)); 113.9 (CH, C(6)); 111.4 (CH, C(4)); 63.2
(CHCH2O); 51.6 (CHCH2O); 22.4 (Me); 11.8 (Me). ESI-MS: 296
[M + H]+, 130 [(M - toluidine-NH-COO) + H]+.
Nr-Acet yl-S-[3-a m in o-4-m et h ylp h en yl-ca r b a m oyl]cys-
tein e (18a ). 18a was obtained according to general procedure
4 with 17a (90 mg, 0.26 mmol). Yellowish solid (74 mg, 90%),
1
mp >230 °C decomp. H NMR (DMSO-d6): δ 9.93 (s, Ar-NH);
8.23 (d, J ) 8.0, AcNH); 6.85 (d, J ) 1.8, H-C(2)); 6.80 (d, J )
8.1, H-C(5)); 6.54 (dd, J ) 8.1, 1.8, H-C(6)); 4.35 (mc, NH-
CHCH2); 3.36 (dd, J ) 13.7, 5.0, 1H, CHCH2S); 3.00 (dd, J )
13.7, 8.7, 1H, CHCH2S); 1.98 (s, Me); 1.85 (s, Me). 13C NMR
(DMSO-d6): δ 171.9 (COOH); 169.3 (MeCO); 163.2 (NHCOS);
146.7 (C, C(3)); 137.3 (C, C(1)); 129.8 (CH, C(5)); 116.6 (C, C(4));
107.3 (CH, C(6)); 105.0 (CH, C(2)); 52.3 (CHCH2S); 30.6
(CHCH2S); 22.4 (Me); 16.9 (Me). ESI-MS: 312 [M + H]+, 164
[N-AcCys + H]+.
N-[(3-Am in o-4-m et h ylp h en yl)ca r b a m oyl]va lyl-glycyl-
glycin e (19a ). 19a was obtained according to general procedure
1 and 4 starting with 1 mmol of valyl-glycyl-glycine. Crystal-
lization from ethyl acetate yielded 19a (95 mg, 25%). Beige solid,
mp >300 °C. 1H NMR (DMSO-d6): 8.34 (s, NH); 8.32 (“t”,
NHCH2); 8.06 (“t”, NHCH2); 6.72 (d, J ) 8.2, H-C(5)); 6.68 (d,
J ) 2.0, H-C(2)); 6.48 (dd, J ) 8.2, 2.0, H-C(6)); 6.31 (d, J )
8.4, NHCH), 4.07 (dd, J ) 8.4, 5.6, NHCH), 3.73 (mc, 4H, CH2-
(Gly); 1.97 (mc, Me2CH), 1.95 (s, Me-Ar); 0.89 (d, J ) 6.8, MeCH);
0.84 (d, J ) 6.8, MeCH). 13C NMR (DMSO-d6): δ 172.2, 171.2,
169.0 (2NHCO, COOH); 155.2 (NHCONH); 146.6 (C, C(3)); 138.8
(C, C(1)); 129.8 (CH, C(5)); 114.3 (C, C(4)); 106.0 (CH, C(6));
103.6 (CH, C(2)); 57.8 (NHCH); 41.7 (CH2NH); 41.0 (CH2NH);
30.8 (Me2CH); 19.3 (MeCH); 17.7 (MeCH); 16.8 (Ar-Me). ESI-
MS: 380 [M + H]+, 248 [(M - glycyl-glycine) + H]+.
N-[(3-Am in o-4-m eth ylp h en yl-d 6)ca r ba m oyl]va lyl-glycyl-
glycin e (19d ). 2,4-Dinitrotoluene-d6 was synthesized according
to Sabbioni and Beyerbach (40). 4-Amino-2-nitrotoluene-d6 was
synthesized according to the procedure described for the syn-
thesis of 4-amino-2-nitrotoluene (41). 4-Amino-2-nitro-toluene-
d6 (1 mg, 6.32 µmol) in dry dioxane (0.5 mL) was reacted with
triphosgene (2 mg) for 2 h at 80 °C, then valyl-glycyl-glycine
(10 mg) in 0.25 M NaHCO3 (1 mL) was added. After 2 h at 80
°C, 4 mL of water was added and the reaction mixture was
extracted with dichloromethane (1 × 3 mL) and ethyl acetate
(2 × 3 mL). The pH was then adjusted to pH 2. The water phase
was extracted with ethyl acetate (2 × 4 mL). The organic
extracts were dried over a pipet filled with dry Na2SO4. The
filtrate was evaporated at the speed vac. The residue was taken
up in dry MeOH (1 mL). Pd/C (2 mg) was added and the reaction
mixture was placed under a hydrogen atmosphere following
general procedure 4. After 8 h at room temperature the reaction
N -Ac e t y l-O -[(3-a m in o -4-m e t h y lp h e n y l)c a r b a m o y l]-
ser in e (15a ). 15a was obtained according to general procedure
5 with 14a (100 mg, 0.31 mmol). Beige solid 15a (70 mg, 77%),
1
mp 148 °C. H NMR (DMSO-d6): δ 9.22 (s, Ar-NH); 7.58 (d, J
) 7.0, AcNH); 6.81 (d, J ) 1.8, H-C(2)); 6.74 (d, J ) 8.2,
H-C(5)); 6.52 (dd, J ) 8.2, 1.8, H-C(6)); 4.36 (dd, J ) 9.8, 2.8,
1H, CHCH2O); 4.13 (mc, CHCH2O); 4.07 (dd, J ) 9.8, 6.3, 1H,
CHCH2O); 1.96 (s, Me); 1.86 (s, Me). 13C NMR (DMSO-d6): δ
171.4 (COOH); 168.6 (MeCO); 153.5 (NHCOO); 146.5 (C, C(3));
137.6 (C, C(1)); 129.6 (CH, C(5)); 115.1 (C, C(4)); 107.8 (CH,
C(6)); 105.4 (CH, C(2)); 65.2 (CHCH2O); 53.8 (CHCH2O); 22.9
(Me); 16.8 (Me). ESI-MS: 296 [M + H]+, 130 [(M - toluidine-
NH-COO) + H]+.
mixture was filtered over
a pipet filled with Celite. The
concentration of the filtrate was determined by HPLC. HPLC

1578 Chem. Res. Toxicol., Vol. 14, No. 12, 2001
Sabbioni et al.
analyses were performed on a Lichrospher RP18 (125 mm × 4
mm, 5 µm) column with a 20 min, 20 to 80% MeOH gradient in
0.1% formic acid [flow rate 1 mL/min, tR (N-[(3-amino-4-
methylphenyl-d6)carbamoyl]valyl-glycyl-glycine) ) 6.3 min, tR
(N-[(3-nitro-4-methylphenyl-d6)carbamoyl]valyl-glycyl-glycine) )
13.4 min]. The yield was determined by HPLC with calibration
line obtained with standard solutions of the undeuterated
corresponding compound 19a . The purity and identity of the
compound were checked by HPLC with a photodiode array
detector [UV (0.1% formic acid/MeOH) λmax 244 nm] in com-
parison to the corresponding undeuterated compound 19a . Yield
472 µg (19.3%) 19d . ESI-MS: 386 [M + H]+ (100), 385 (79), 384
(28), 254 (3), 253 (4) [M-glycyl-glycine]+, 252 (3).
294 (20), 274 (28), 175 (27), 147 (18), 72 (100), 55 (34). NCI-MS:
374 (21), 373 (100, M-HF).
3-(3-Am in o-4-m eth ylp h en yl-d 6)-5-isop r op ylim id a zolin e-
2,4-d ion e (5d ). 5d was obtained according to general procedure
6 with 19b (900 ng). The identity of the sample was confirmed
by positive ESI-MS and by GC-MS after derivatization with
PFPA: ESI-MS: 254 [M + H]+ (66), 253 (100), 252 (86), 251
(47). ESI-MS: MS/MS (40% collision energy): 253 f 251 (6),
210 (6), 209 [M-isopropyl]+ (11), 208 (6), 155 (11), 154 [CD3(NH2)-
C6D3-NCO]+ (60), 153 (100), 152 (56). GC-MS of the PFPA de-
rivative: NCI-MS: 379 (58, M-HF), 378 (100), 377 (87), 376 (45).
3-(5-Am in o-2-flu or op h en yl)-5-isop r op ylim id a zolin e-2,4-
d ion e (5e). 5e was obtained according to general procedure 6
with (100 mg, 0.38 mmol). Slightly rosa solid (61 mg, 65%), mp
174 °C. 1H NMR (DMSO-d6): δ 8.70 (s, 1H, NH); 7.8-7.4 (m,
3H, aromatic-H); 4.24 (d, J ) 2.7, NHCHCH); 2.25-2.00 (m,
1H, CHCHMe2); 2.13 (s, Me); 1.02 (d, J ) 6.9, MeCH); 0.90 (d,
J ) 6.8, MeCH). 13C NMR (DMSO-d6): δ 172.2 (CO); 155.7 (d,
J ) 249.2, C, C(2)); 154.9 (NCONH); 130.4 (C, C(5)); 124.7 (CH,
C(4), d, J ) 8.8); 124.0(CH, C(6)); 120.2 (C, C(1), d, J ) 14.6);
117.4 (CH, C(3), d, J ) 21.5); 61.9 (NHCHCH); 29.9 (Me2CH);
18.3 (Me); 15.8 (Me). EI-MS: 252 (6), 251 (51), 153 (20), 152
(100), 72 (21). PFPA-derivative: 398 (8), 397 (47), 352 (15), 355
(100), 298 (30), 257 (16), 179 (18), 151 (18), 72 (56). NCI: 378
(18), 377 (100).
Hyd a n toin s. Gen er a l P r oced u r e 6. The urea derivatives
(4a -c,e) (0.2 mmol) were dissolved in 6 M HCl (10 mL) and
stirred for 2 h at 80-90 °C. The solvent was evaporated, the
residue was dissolved in EtOH and filtrated. Then H2O was
added dropwise to the filtrate until precipitation occurred. After
cooling, the precipitate was filtered off and dried.
3-(3-Am in o-4-m et h ylp h en yl)-5-isop r op ylim id a zolin e-
2,4-d ion e (5a ). 5a was obtained according to general procedure
6 with 4a (100 mg, 0.38 mmol). Crystallization from EtOH
yielded 5a (65 mg, 70%). White solid, mp 191 °C. 1H NMR
(DMSO-d6): δ 8.39 (s, 1H, NH); 6.97 (d, J ) 7.9, H-C(5)); 6.50
(d, J ) 2.1, H-C(2)); 6.34 (dd, J ) 7.9, 2.1, H-C(6)); 5.03 (s,
broad, NH2); 4.09 (dd, J ) 3.3, 1.2, CHCHMe2); 2.17-2.07 (m,
Me2CH); 2.07 (s, Me); 1.01 (d, J ) 7.1, MeCH); 0.87 (d, J ) 6.7,
MeCH). 13C NMR (DMSO-d6): δ 172.8 (CO); 156.3 (NCONH);
146.9 (C, C(3)); 130.3 (C, C(1)); 129.7 (CH, C(5)); 120.8 (C, C(4));
114.1 (CH, C(6)); 112.0 (CH, C(2)); 61.1 (CHCHMe2); 29.9
(Me2CH); 18.4(Me); 17.0 (Me-Ar); 15.6 (Me). EI-MS: 248 (13),
247 (84, M+), 149 (25), 148 (100), 147 (21), 106 (11). PFPA-
derivative: EI-MS: 394 (14), 393 (84, M+), 351 (74), 295 (19),
294 (21), 274 (21), 175 (65), 147 (22), 132 (14), 72 (100), 55 (18).
NCI-MS: 374 (21), 373 (100). ESI-MS: 248 [M + H]+. MS/MS
248 (40% collision energy) f 149 (100%).
1-(3-Am in o-4-m eth ylp h en yl)-2,5-d ioxoim id a zolid in e-4-
a cetic a cid m eth ylester (9a ). 9a was obtained according to
general procedure 6 with 8a (150 mg, 0.53 mmol). The residue
was then derivatized with a 10% BF3-MeOH (2 mL) solution at
room temperature. After 1 h, water (1 mL) was added and the
reaction mixture was evaporated to dryness. The residue was
taken up in water (5 mL) and extracted with ethyl acetate (3 ×
5 mL). Evaporation of the solvents yielded a beige solid (78 mg,
1
50%), mp 199 °C. H NMR (DMSO-d6): δ 8.31 (s, NH); 6.97 (d,
J ) 8.0, H-C(5)); 6.54 (d, J ) 2.0, H-C(2)); 6.39 (dd, J ) 8.0,
2.0, H-C(6)); 5.05 (s, broad, NH2); 4.43 (dt, J ) 1.2, 4.9,
NHCHCH2); 3.64 (s, OMe); 2.92 (dd, J ) 17.0, 5.4, 1H,
NHCHCH2); 2.81 (dd, J ) 17.0, 4.6, 1H, NHCHCH2); 2.07 (s,
Me). 13C NMR (DMSO-d6): δ 172.6 (COOMe); 169.9 (NCOCH);
156.2 (NCONH); 146.8 (C, C(3)); 130.6 (CH, C(5)); 129.7 (C,
C(1)); 120.8 (C, C(4)); 114.3 (CH, C(6)); 112.1 (CH, C(2)); 52.6
(OMe); 51.7 (CH); 35.1 (CH2); 17.1 (Me). ESI-MS: 278 [M + H]+,
246 [(M - MeOH) + H]+ , 204 [(M - CH2COOMe) + H]+. EI-
MS: 278 (17), 277 (100, M+), 218 (19), 149 (31), 148 (66), 147
(24), 122 (21), 106 (21), 74 (13).
3-(5-Am in o-2-m et h ylp h en yl)-5-isop r op ylim id a zolin e-
2,4-d ion e (5b). 5b was obtained according to general procedure
6 with 4b (100 mg, 0.38 mmol). Beige solid 5b (70 mg, 75%),
mp 176-177 °C. 1H NMR (DMSO-d6): δ 8.39 and 8.36 (s, 1H,
NH, diast.); 6.948b and 6.939a (d, J ) 8.2, 1H, H-C(3)); 6.566
(dd, J ) 8.2, 2.2, 1H, H-C(4)); 6.344a and 6.256b (d, J ) 2.2,
H-C(6)); 4.172b and 4.137a (d, J ) 3.3, NHCHCH); 2.25-2.08
(m, 1H, CHCHMe2); 1.91b and 1.90a (s, Me); 1.019 (d, J ) 7.0,
MeCH); 0.906 (d, J ) 6.8, MeCH). Ratio isomers ) a/b ) 1.2/1.
13C NMR (DMSO-d6): δ 172.89 and 172.79 (CO); 156.3 (NCONH);
147.34 and 147.28 (C, C(5)); 131.42 and 131.07 (C, C(1)); 130.6
and 130.5 (CH, C(3)); 122.21 and 121.71 (C, C(2)); 114.60 and
114.43 (CH, C(4)); 114.06 and 113.60 (CH, C(6)); 61.72 and 61.18
(NHCHCH); 29.97 and 29.57 (CHCHMe2); 18.62 and 18.33 (Me);
16.49 and 16.20 (Ar-Me); 16.10 and 15.70 (Me). EI-MS: 248 (15),
247 (95, M+), 149 (29), 148 (100), 147 (27), 120 (14), 106 (34),
72 (16), 55(14). PFPA-derivative: 394 (18), 393 (99, M+), 352
(15), 351 (100), 253 (13), 295 (38), 294 (46), 175 (15), 72 (63), 57
(36), 55 (20). NCI-MS: 374 (21), 373 (100).
GC/MS An a lysis a n d Ca libr a tion Lin e of th e Hyd a n toin
(5a ). 19a (0, 0.66, 1.32, 6.59, and 13.2 pmol), 19d (6.59 pmol)
in MeOH (10 µL), and 4-fluoroaniline (1 µg) in MeOH (10 µL)
were added to 2 M HCl (4 mL), and the mixture was heated for
2 h at 100 °C in a centrifuge tube with a Teflon-lined screw
cap. The experiments were performed in triplicate. The hydroly-
sate was adjusted to pH 9 with NaHCO3, and then extracted
with tert-butyl methyl ether (TBME) (6 mL). The organic layer
was passed through a pipet filled with anhydrous Na2SO4 (1
g). The Na2SO4 was rinsed with TBME (1.5 mL). The dried
organic phase was collected in a tapered tube. Decane (10 µL)
was added as a keeper to the organic phase prior to evaporation
with the speed evacuator. The residue was taken up in ethyl
acetate (2 × 100 µL) and derivatized with pentafluoropropionic
anhydride (PFPA) (5 µL). After 20 min at room temperature,
the derivatization was stopped by adding a methanol solution
(10 µL)). After evaporation to dryness, the residue was taken
up in ethyl acetate (10 µL). The extracts were analyzed on a
fused silica capillary column [ZB-5 (Phenomenex, Aschaffen-
burg, Germany), 0.25 mm i.d., 15 m long, 0.5 µm film thickness]
attached to a methyl-deactivated tubing precolumn (Supelco,
0.25 mm, 1 m long) with a Hewlett-Packard chromatograph
(model 5890II) coupled to a mass spectrometer as a detector (HP
5989A). An aliquot (1 µL) was injected splitless (inactivated liner
with glass wool) at 80 °C. The transfer line temperature and
the injector temperature were set at 280 °C. The oven temper-
ature was kept for 1 min at 70 °C and then increased at a rate
3-(3-Am in o-2-m et h ylp h en yl)-5-isop r op ylim id a zolin e-
2,4-d ion e (5c). 5c was obtained according to general procedure
6 with 4c (100 mg, 0.38 mmol). White solid 5c (61 mg, 65%),
mp 160 °C. 1H NMR (DMSO-d6): δ 8.374b and 8.342a (s, 1H,
NH); 6.95 (t, J ) 7.8, H-C(5)); 6.69 (d, J ) 7.8, H-C(4)); 6.37a
and 6.23b (d, J ) 7.8 H-C(6)); 5.03 (s, broad, NH2); 4.19b and
4.14a (d, J ) 2.7, NHCHCH); 2.17-2.07 (m, Me2CH); 1.80 and
1.78 (s, Me); 1.03 and 1.02 (d, J ) 6.8, MeCH); 0.92 and 0.89 (d,
J ) 6.6, MeCH). Ratio isomer a/isomer b ) 1.2/1 13C NMR
(DMSO-d6): δ 172.96 and 172.88 (CO); 156.38 and 156.32
(NCONH); 147.64 and 147.52 (C, C(3)); 131.54 and 131.44 (C,
C(1)); 126.10 and 126.03 (CH, C(5)); 119.68 and 119.36 (C, C(2));
116.49 and 115.99 (CH, C(6)); 114.3 (CH, C(4)); 61.82 and 61.34
(CHCHMe2); 29.96 and 29.58 (Me2CH); 18.68 and 18.40(Me);
16.24 and 15.77 (Me); 11.9 (Ar-Me). EI-MS: 248 (15), 247 (100,
M+), 149 (24), 148 (61), 147 (60), 120 (11), 106 (23), 72 (11). EI-
MS: PFPA derivative: 394 (15), 393 (79, M+), 351 (39), 295 (33),

Adducts of Amino Acids with Toluenediisocyanate
Chem. Res. Toxicol., Vol. 14, No. 12, 2001 1579
of 50 °C/min to 300 °C. The compounds were identified and
quantified using negative chemical ionization mass spectrom-
etry, with methane as the reactant gas (1.5 Torr, ion source
temperature of 250 °C, electron energy of 200 eV) and SIM of
m/z 373 and 377 (dwell time of 150 ms). The derivatized internal
standard 5d and 5a eluted at 6.27 min. With a shorter column
and lower film thickness [Rtx-5MS (Restek, Bellefonte, PA), 0.25
mm i.d., 12 m long, 0.25 µm film thickness], the retention time
of 5d was 5 min. The calibration line was generated from the
amount (expressed in picomoles) of 19a (x) and the peak ratio
(y) of 5a and 5d : y ) 0.22x, r2 ) 0.99.
F igu r e 3. Hydrolysis of N-terminal-valine adduct 19a in the
presence of the internal standard 19d .
LC/MS/MS An a lysis a n d Ca libr a tion Lin e of th e Hy-
d a n toin (5a ). The same work-up procedure described for the
determination by GC/MS was applied for the LC/MS/MS method.
After extraction with TBME 100 µL of 0.1% formic acid was
used as a keeper instead of decane. The extracts were evapo-
rated at the speed vac to a volume of 60-80 µL. The residue
volume was increased to 85 µL by adding further 0.1% formic
acid. Then 20 µL of MeOH were added and the solution was
ultrasonicated for 5 min. An aliquot (10-25 µL) was injected
on a C18 reversed-phase column [150 × 1 mm, 3 µm, Luna C18-
(2) Column by Phenomenex, Aschaffenburg, Germany] and
analyzed with a linear gradient from 20 to 72% MeOH in 15
min and with isocratic conditions at 72% MeOH for 4 min and
a flow of 50 µL/min. The flow of 50 µL/min ()1/5 of total flow)
was produced using a high-pressure micro-splitter (UpChurch
Scientific, Oak Harbor, WA). To obtain this flow, a second
column Hypersil BDS 18, 125 × 2 mm, 3 µm) with a similar
back pressure had to be attached to the second channel (waste)
of the splitter. The detector was a quadrupole ion trap tandem
mass spectrometer (LCQ Duo, Thermoquest). The MS param-
eters were optimized in the electrospray ionization mode (ESI).
Positive ions were detected. Parameter optimization was carried
out with a 4 pg/µL solution and a flow rate of 50 µL/min. The
signal abundance of m/z 248 [M + H]+ was maximized with the
autotune program. The other parameters were set as follows:
capillary temperature of 220 °C, sheath gas flow of 80 (arbitrary
units) and auxiliary gas flow of 0. For maximum yield of the
daughter ions the collision energy was set at 40% and the
following masses were chosen: m/z 253 (isolation width ( 1.25
amu) f 153 and 248 (isolation width ( 0.75 amu) f 149. The
calibration line was linear over the range (0, 0.66, 1.32, 6.59,
and 13.2 pmol) (19a ). The calibration line was generated from
the amount (expressed in picomoles) of 19a (x) and the peak
ratio (y) of 5a and 5d : y ) 0.29x, r2 ) 0.99.
negative charge at the carboxylate moiety, and the
presence of the NR-acyl group, the racemization of C(R)
is facilitated (42). However, for the present study, the
enantiomeric purity of the products was not important.
The reaction of 1a -c with functional groups other than
the R-amino group was done under various conditions.
The synthesis of carbamoyl-adducts from 1a with thiol
groups was performed with N-acetyl-cysteine and 1a at
30 °C in 0.25 M NaHCO3. The carbamic acid S-ester 17a
was achieved with an acceptable yield of 60%. N-Acetyl-
D,L-serine did not react with 1a -c in the hydrogen-
carbonate buffer system. The lack of reactivity of the
functional groups of these amino acids under the given
conditions raises questions about the physiological rel-
evance of these types of adducts, although the reaction
with serine is supposed to deactivate cholinesterase in
in vitro reactions and in exposed workers (28, 32).
Therefore, the reaction of N-acetyl-D,L-serine with 1a -c
was performed in pyridine. The carbamate 14a -c was
obtained in good yield (53-77%).
The nitro group was reduced using two methods: (i)
catalytic hydrogenation with hydrogen on Pd/C, and (ii)
ammonium formate and Pd/C in methanol. With both
methods the reduced compounds are obtained (4a -c,
8a -c, 12a -c, 15a -c), except for 18a . The carbamic
S-ester 17a has to be reduced with catalytic hydrogena-
tion to obtain the product 18a .
Hydantoin formation was achieved by heating the
adducts 4a -c in 6 M HCl at 90 °C. The hydantoins (5a -
c) were obtained in ca. 70% yield. The same hydantoin
5a was generated from the tripeptide adduct N-[(3-
amino-4-methylphenyl)carbamoyl]valyl-glycyl-glycine
(19a ). The hydantoin 9a of the aspartic acid adduct
8a was synthesized in the same way. For GC/MS
analysis, 9a was derivatized with BF3-MeOH. The purity
of the products was determined by GC/MS and EI mode
on a fused silica column Rtx5-MS (Restek) 12 m × 0.25
mm internal diameter with a film thickness of 0.25 µm.
Sp ectr oscop ic Ch a r a cter iza tion of th e P r od u cts.
An a lysis of Hu m a n Sa m p les by GC/MS a n d LC/MS/MS.
Globin was isolated as previously described in ref 27. The
procedures described above were followed 50-100 mg of globin
were used.
Resu lts a n d Discu ssion
Syn th esis of Isocya n a te Ad d u cts. The urea deriva-
tives of 1a -c with the free amino group of L-valine,
L-aspartic acid, and NR-acetyl-L-lysine were synthesized
according to Figure 2. The compounds 3a -c, and 7a -c
were obtained by adding 1a -c to the corresponding
amino acid in 0.5 M NaHCO3 at 80 °C. The products were
obtained in yields ranging from 33 to 75% and were fully
characterized. The yields were lower than similar reac-
tions in organic solvents because of the concurring hydrol-
ysis of the isocyanate in aqueous solution to the unstable
carbamic acid, which decarboxylates and then reacts with
another molecule of 1a -c to the symmetric substituted
urea. At a more basic pH than the carbonate buffer
system or in organic solvents such as pyridine, possibly
the yields of the wished products would be higher. For
the amino acids with functional groups (10, 13, 16), the
R-amino group was protected with an acetyl group. We
did not investigate the optical purity of the products.
Under the given reaction conditions, the presence of the
1
All products were characterized with MS, H NMR, and
13C NMR (Figure 3). EI-MS was performed with the
hydantoins 5a -c and 9a . The molecular ion m/z 247 is
present in all three hydantoins 5a -c. The base peak
corresponds to the mass of the isocyanates 1a -c. All the
other compounds were analyzed using ESI-MS with
positive ionization. The [M + H]+ is the base peak for all
compounds. Another major fragment is after loss of water
or after the loss of the amino acid.
In the 1H NMR spectra, the signals of the aromatic
protons of the arylamine moiety are assigned according
to the chemical shifts estimated with the increment rules
(43, 44). In the following the 1H NMR and 13C NMR shifts
are discussed. The three protons of the aromatic ring can
be assigned unambiguously with the coupling constants,

1580 Chem. Res. Toxicol., Vol. 14, No. 12, 2001
Sabbioni et al.
except for the products resulting from 1c. For the
compounds synthesized from 1a the 1,3 coupling constant
between H-C(2) and H-C(6) amounts to ca. 2 Hz. The
vicinal coupling constants are all ca. 8.0 Hz. The chemical
shift of H-C(2) is lower than H-C(6) and H-C(5). The
protons of the amino acids were assigned according to
the literature (45). In all cases, the protons represented
an ABXY-spectrum except for the valine-adducts with an
AXY-spectrum. The N-H protons of the arylamine
moiety were found between 8.8 and 9.3 ppm for the urea
compounds 2a , 7a , and 11a , and above 10 ppm for the
carbamate 14a and for the carbamate S-ester 17a . The
amino protons of the amino acids were found at 6.2-6.7
when attached to the carbamoyl group and at 8.3-8.6
ppm when attached to the N-acetyl-group. In the com-
pounds with the reduced nitro compound (4a , 8a , 12a ,
15a , 18a ): H-C(6), H-C(2), and H-C(5) are shifted with
1.0, 1.5, and 0.5 ppm, respectively, to higher field in
comparison to the parent nitro derivatives. The chemical
shifts of these compounds are not predicted correctly with
the substituent increments or with the 1H NMR predictor
by ACD version 4.5 (Advanced Chemistry Development
Inc., Toronto, Canada). However, the signals can be
assigned with the coupling constants. In the 13C NMR of
the nitro-derivatives (3a , 7a , 11a , 14a , 17a ), the order
of the chemical shifts is the same for all compounds: C(3)
> C(1) > C(5) > C(4) > C(6) > C(2). In combination with
a DEPT experiment C(4) and C(6), which chemical shifts
are only 2 ppm apart, can be distinguished. In the nitro-
reduced compounds (4a , 8a , 12a , 15a , 18a ), the C(2) and
C(6) are only 2.4 ppm apart. For an unambiguous
assignment in these cases, a CH-COSY experiment was
performed with 4a . According to the increment rules (46)
and to the predicted 13C NMR by ACD, the following
signal order was elucidated: C(3) > C(1) > C(5) > C(4)
> C(6) > C(2). The same interpretations were applied
for the interpretation of NMR of adducts of 1b and 1c.
8 Hz for the precursor compounds 4a and 8a . In the case
of the hydantoins, this coupling constant is ca. 1 Hz for
5a and 9a . This can be explained with the change of the
dihedral angle CHNH to near 90°. Semiempirical (AM1,
HyperChem 3.5) showed a dihedral angle H-C-N-H of
104°. Compared to the protons of the aliphatic NH group
in 4a and 8a at ca. 6.3 ppm, the corresponding NH signal
shifts 2 ppm downfield to ca. 8.3 ppm for the hydantoins
5a and 9a . In the 13C NMR, the shift difference between
the hydantoins and the corresponding precursor com-
pounds are more pronounced: C(2) and C(6) shift by ca.
8.0 ppm to lower filed, C(3) and C(5) do not change and
C(4) is shifted by 7.0 ppm to lower field. In the hydantoins
5b-c resulting from the isocyanates 1b-c, most signals
for the hydrogen or the carbons are doubled, with a shift
difference of 0-0.15 ppm and 0-0.5 ppm, respectively.
The largest differences are noted for the H-C(6) with 0.14
ppm and C(6) with 0.5 ppm in ortho position to the
hydantoin ring. The NMR of the hydantoin 5e synthe-
sized from 2-fluoro-5-methyl-phenylisocyanate and L-
valine did show the expected amount of signals. These
results suggest the presence of rotamers for the hydan-
toins 5b-c. Force field calculations (MM+, HyperChem
3.5) were performed with the compounds 5a and 5b. The
most stable conformer was found by starting the force
field calculations with different rotamers. The most stable
rotamer of 5a has torsion angles of 45° and 50° between
the two rings. For 5b these torsion angles are 93° and
94°. The molecule was then rotated in 10° steps between
the two rings. The resulting torsion angles between the
two ring systems were kept restraint. The rest of the
molecule was optimized without any further restraints.
The most unstable rotamer was with both ring systems
in the same plane. The maximum energy difference
between two rotamers was 19 kcal/mol for 5b and 6 kcal/
mol for 5a . Similar rotamers have been found in hydan-
toins obtained from 2-methylphenyl isocyanate (46-50).
Differences were only noted in the 13C NMR-spectra. The
1H NMR spectra were the same for both rotamers.
An a lysis of Isocya n a t e Ad d u ct s a n d Biologica l
Ap p lica tion s. According to the present literature the
measurement of N-terminal adducts with blood proteins
is the most promising to biomonitor exposure to isocy-
anates. Therefore, experiments were performed for the
detection of such adducts. The hydantoins 5a -c were
first analyzed by GC-MS and EI detection. The detection
limit was investigated without derivatization by measur-
ing the major single ions, e.g., m/z 247, and 148.
Unfortunately, after a few injections the peak shape of
the chromatogram deteriorated drastically. Therefore,
quantification without derivatization is not possible. For
low level detection (<40 pmol) the hydantoins 5a -c were
derivatized with pentafluoropropionic anhydride. This
enables the analysis of the compounds by GC-MS with
negative chemical ionization (NCI). Only one major
fragment is seen in the NCI mode, [M - HF]-. To apply
the method for the analysis of human samples we
synthesized the N-terminal adduct of 1a with the tri-
peptide valyl-glycyl-glycine, N-[(3-amino-4-methylphen-
yl)carbamoyl]valyl-glycyl-glycine (19a ). As an internal
standard we used N-[(3-amino-4-methylphenyl-d6)car-
bamoyl]valyl-glycyl-glycine (19d ). The internal standard
19d and 0-13.2 pmol of 19a were added to 2 M HCl and
globin of an unexposed worker (Figure 4). After 2 h at
100 °C, the solution was neutralized extracted with
TBME, derivatized, with pentafluoropropionic anhydride
1
In the H and 13C NMR of the nitro derivatives (3b,
11b, 14b) the signal order correspond to the predicted
order: H-C(6) > H-C(4) > H-C(3), and C(5) > C(1) > C(2)
> C(3) > C(4) > C(6). In the nitro-reduced compounds
(4b, 12b, 15b), the signal order of the aromatic protons
was assigned with the help of the coupling constants
H-C(6) > H-C(3) > H-C(4), which does not correspond to
the predicted order. For the 13C NMR, the signals could
be assigned from the predicted order: C(5) > C(1) > C(3)
> C(2) > C(4) > C(6). However, the observed signals of
C(4) and C(6) are approximately one ppm. The assign-
ment of these signals was confirmed with a CH-COSY
experiment performed with 4b. For the adducts of 1c (3c,
11c, 14c), the signal order of the 1H and 13C NMR
corresponded to the prediction: H-C(6) > H-C(4) >
H-C(5) and C(3) > C(1) > C(5) > C(6) > C(5) > C(4). The
assignments of the carbons C(5) and C(6) which are only
ca. 2 ppm apart were confirmed with a CH-COSY
experiment. For the reduced derivatives (4c, 12c, 15c)
the predicted and observed order was H-C(6) > H-C(5)
> H-C(4) except for 15c and C(3) > C(1) > C(5) > C(2) >
C(6) > C(4). However, the carbons in C(6) and C(2) were
observed at 12.0 and 8.5 ppm lower field than predicted.
The spectroscopic properties of the hydantoins need to
be discussed separately. In the hydantoin 5a , H-C(2) and
H-C(6) are shifted with ca. 0.2 ppm to higher field and
H-C(5) 0.28 ppm to lower field in comparison to the
precursor compounds 4a and 8a . The vicinal coupling
constant of the amino acid NH with the R-CH is around

Adducts of Amino Acids with Toluenediisocyanate
Chem. Res. Toxicol., Vol. 14, No. 12, 2001 1581
F igu r e 4. Synthesized products with carbon numbers for the
NMR characterization.
F igu r e 6. LC/MS/MS chromatogram of a globin extract from
patient B: prior to PU-covered-breast implant surgery.
F igu r e 5. LC/MS/MS chromatogram of a globin extract from
a worker exposed to TDI. Adduct 5a was quantified against 5d .
(PFPA), evaporated to dryness, and analyzed by GC-MS
in the NCI mode. For the quantification of the PFPA-
derivatized internal standard 5d (d-4-TDA-Val-Hyd-
PFPA) and of the PFPA-derivatized 5a (4-TDA-Val-Hyd-
PFPA) the single ions 377 and 373 were monitored. A
calibration curve was obtained which is linear over the
range of 0-13.2 pmol. After ca. 10 injections the glass
wool in the liner had to be replaced, since the sensitivity
dropped. As an alternative internal standard, we suggest
the valine adduct 4e of 2-fluoro-5-methyl-phenylisocy-
anate. The obtained calibration curve is linear; however,
a background level of 1 pmol 5a is seen taking 4e as
internal standard.
A LC/MS/MS method was developed. The same work-
up described for the GC/MS approach was followed. The
derivatization step with PFPA is not necessary in this
case. The hydrolysate was analyzed on a RP18 column
with 1 mm diameter. The MS/MS ions of m/z 248 f 149
and of m/z 253 f 153 were monitored for 5a and for the
product 5d of the internal standard 19d , respectively
(Figures 5-7). The calibration line for the method was
linear from 0 to 13.2 pmol. The determination limit was
0.16 pmol/sample. Under the present conditions, not more
than one-quarter of the hydrolysate can be injected on
the column because of ion suppression effects. By inject-
ing larger amounts the size of the peaks decreases. The
method was applied to the analysis of a few biological
samples. Blood of control workers and of a worker
exposed to TDI was obtained and blood from women with
polyurethane (PU)-covered breast implants were avail-
F igu r e 7. LC/MS/MS chromatogram of a globin extract from
patient B: 113 days after PU-covered-breast implant surgery.
able. After isolation of globin the samples were hydro-
lyzed in the presence of the internal standard 19b. The
highest level was found in the worker with 7.1 pmol of
5a /100 mg of globin (Figure 5). The possible isomers 5b,c
of TDI would elute at 12. 5 and 13 min with no base peak
separation. Unfortunately, these peaks are covered by
coeluting impurities. The presence of the adduct 5a was
confirmed by GC/MS, tR ) 6.25 min. The PFPA deriva-
tives of the possible products 5b and 5c could not be
detected by GC-MS at, tR ) 6.15 min. In control workers
no adduct could be found. For the breast implant patient
A [corresponds to patient A in Sepai et al. (20)] no adduct
(Figure 6) was found in the first days after surgery (day

1582 Chem. Res. Toxicol., Vol. 14, No. 12, 2001
Sabbioni et al.
not investigated. After 30 days of surgery, 54 pmol of 2,4-
TDA/mg of albumin were released. Therefore, binding to
albumin is ca. 100 times larger than with globin.
Con clu sion s
The synthesized adducts can be used for the analysis
of biological samples obtained from animals or human
exposed to TDI. The analysis of with the N-terminal
valine of hemoglobin was applied successfully for the
analysis of human samples. The N-terminal amino acid
analysis of albumin will be more difficult. After acid
hydrolysis, the released hydantoin has to be extracted
at acidic pH. For GC/MS analysis, the carboxylic acid has
to be esterified with diazomethane or with BF3/MeOH.
To obtain a better response in the NCI-mode, we suggest
to derivatize the carboxylic acid with pentafluoroethanol/
BF3. The quantification of adducts with other amino acids
will be more difficult. The proteins have to be digested
to the single amino acids as demonstrated for albumin
adducts of aflatoxin G1 (57). The major problem consists
of the enrichment of the modified amino acid. In addition
the synthesized adducts might be used to quantify and
to test the specificity of the TDI-antibodies present in
exposed workers.
F igu r e 8. GC/MS chromatogram of a globin extract from
patient B: prior to PU-covered-breast implant surgery.
Note Ad d ed a fter ASAP P ostin g
This article was inadvertently released ASAP on 11/
20/01 before final corrections were made. For 5b, ratio
of isomers was changed to 1.2/1. The corrected version
was posted 12/10/01.
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