Synthesis, characterization, and solvolysis of mono- and bis-S-(glutathionyl) adducts of methylene-bis-(phenylisocyanate) (MDI)

Article abstract of DOI:10.1021/tx0255020

Bifunctional isocyanates are highly reactive compounds that undergo nucleophilic attack by a variety of functional groups available in the biological system. While the etiology of the respiratory disease caused by diisocyanates is not fully understood, a great deal of research has been performed to elucidate the chemical mechanisms involved in the direct and indirect effects of these compounds. Since adducts of isocyanates are found not only to proteins along the entire respiratory tree but also to proteins in the circulatory system, it is likely that a transport mechanism for the isocyanate from the respiratory to the circulatory system exists. The initial reaction of isocyanates with cellular thiols to form thiocarbamates, which are known to release the isocyanate under physiological conditions, is believed to provide a possible carrier mechanism for the isocyanate functional group. Previous work with aliphatic mono-isocyanates and the aromatic diisocyanate toluene diisocyanate has demonstrated the feasibility of this mechanism. Adding to this database, the products of the reaction of the highly water-insoluble, low vapor pressure, methylene-bis-(phenylisocyanate) (MDI) with glutathione were synthesized, and their chemical stability under various pH and buffer conditions was tested. Novel synthetic routes were developed for both the mono- and bis-S-(glutathionyl) adducts with MDI that yielded each compound in analytically pure form. Both compounds were found to be unstable under mild basic conditions (phosphate-buffered saline, pH 7.4, and NaHCO₃, pH 8.2), however to a different degree. Furthermore, a significant influence of the pH value (the rate of degradation increases with pH) and the concentration of free glutathione (increasing thiol stabilizes the adduct) on the stability was observed, indicating a base-catalyzed mechanism of the degradation/formation of the thiocarbamate bond. Unlike the monoadduct, which forms almost exclusively the polyurea upon degradation, a variety of products were formed upon degradation of the bis adduct. Though the disappearance of the bis adduct was complete as measured by HPLC, ¹H NMR spectra showed the existence of residual thiocarbamate bonds in the final mixture. In both cases, no evidence of the free methylene-bis-phenylamine (MDA) could be detected under the applicable conditions.

Full text of DOI:10.1021/tx0255020

Chem. Res. Toxicol. 2002, 15, 1235-1241
1235
Syn th esis, Ch a r a cter iza tion , a n d Solvolysis of Mon o- a n d
Bis-S-(glu ta th ion yl) Ad d u cts of
Meth ylen e-bis-(p h en ylisocya n a te) (MDI)
Martin Reisser, Brigitte F. Schmidt, and William E. Brown*
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213
Received J anuary 2, 2002
Bifunctional isocyanates are highly reactive compounds that undergo nucleophilic attack
by a variety of functional groups available in the biological system. While the etiology of the
respiratory disease caused by diisocyanates is not fully understood, a great deal of research
has been performed to elucidate the chemical mechanisms involved in the direct and indirect
effects of these compounds. Since adducts of isocyanates are found not only to proteins along
the entire respiratory tree but also to proteins in the circulatory system, it is likely that a
transport mechanism for the isocyanate from the respiratory to the circulatory system exists.
The initial reaction of isocyanates with cellular thiols to form thiocarbamates, which are known
to release the isocyanate under physiological conditions, is believed to provide a possible carrier
mechanism for the isocyanate functional group. Previous work with aliphatic mono-isocyanates
and the aromatic diisocyanate toluene diisocyanate has demonstrated the feasibility of this
mechanism. Adding to this database, the products of the reaction of the highly water-insoluble,
low vapor pressure, methylene-bis-(phenylisocyanate) (MDI) with glutathione were synthesized,
and their chemical stability under various pH and buffer conditions was tested. Novel synthetic
routes were developed for both the mono- and bis-S-(glutathionyl) adducts with MDI that
yielded each compound in analytically pure form. Both compounds were found to be unstable
under mild basic conditions (phosphate-buffered saline, pH 7.4, and NaHCO3, pH 8.2), however
to a different degree. Furthermore, a significant influence of the pH value (the rate of
degradation increases with pH) and the concentration of free glutathione (increasing thiol
stabilizes the adduct) on the stability was observed, indicating a base-catalyzed mechanism of
the degradation/formation of the thiocarbamate bond. Unlike the monoadduct, which forms
almost exclusively the polyurea upon degradation, a variety of products were formed upon
degradation of the bis adduct. Though the disappearance of the bis adduct was complete as
1
measured by HPLC, H NMR spectra showed the existence of residual thiocarbamate bonds
in the final mixture. In both cases, no evidence of the free methylene-bis-phenylamine (MDA)
could be detected under the applicable conditions.
responsiveness (NSBH), reactive airways dysfunction
syndrome (RADS), asthma, and alveolitis (hypersensitiv-
ity pneumonitis) (7). The mechanism for induction of
these various outcomes has been attributed to im-
munological and pharmacological mechanisms; however,
currently the precise mechanism or mechanisms are not
known. What is known is that the highly reactive nature
of the isocyanate functional group results in the modifi-
cation of a variety of proteins accessible either directly
or indirectly from the airways. These include albumin
(8), laminin (9), keratin (8), tubulin (10), hemoglobin (11),
and glutathione (6, 12, 13). Glutathione, a tripeptide of
γ-glutamyl-cysteinyl-glycine, has been hypothesized as
a molecule capable of ‘capturing’ the isocyanate through
formation of a reversible thioester on the cysteine side
chain and transporting the residue of isocyanate in an
inert form to a distal location where it can reverse
through the isocyanate functional group, which can
subsequently react with a nucleophile in the vicinity (13).
Formation of such exchangeable thioester conjugates
between cysteine and methyl isocyanate (MIC) (13) and
toluene diisocyanate (TDI) (6) has been demonstrated.
In tr od u ction
Diisocyanates are highly reactive, bifunctional com-
pounds used in the production of foams, coatings, and
various plastic polymers. The isocyanate functional group
is susceptible to nucleophilic attack at the carbonyl
carbon by a number of nucleophiles among which are the
hydroxyl, the amino, and the sulfhydryl groups that are
reactive in biological systems at the physiological pH of
7.4 (1, 2). The reaction of the isocyanate group with these
functional groups also competes with its hydrolysis to the
corresponding amine and the release of carbon dioxide.
Of the types of bonds formed with the hydroxyl (a
carbamate), the amino (a urea), and the sulfhydryl (a
thiocarbamate) groups, the former two are essentially
irreversible under physiological conditions while the
latter thiocarbamate bond has been shown to be revers-
ible under similar conditions (3-6).
Exposure to diisocyanates has been linked to a range
of clinical outcomes such as nonspecific bronchial hyper-
* Correspondence should be addressed to this author at the Depart-
ment of Biological Sciences, Carnegie Mellon University, 4400 Fifth
Ave., Pittsburgh, PA 15213.
10.1021/tx0255020 CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/05/2002

1236 Chem. Res. Toxicol., Vol. 15, No. 10, 2002
Reisser et al.
added, and stirring was continued for 12 h. The solution was
finally acidified with HCl to pH 2, and, after most of the dioxane
was evaporated, the product was collected on a Bu¨chner funnel.
Recrystallization from EtOH yielded 25.66 g (0.078 mol, 78%)
of 4-[4′-(tert-butyloxycarbonyl)aminobenzyl]benzoic acid (5) as
a pale yellow powder; mp decomposition from 180 °C on. IR
(KBr): 3327 (s), 2978 (w), 1699 (vs), 1526 (s), 1412 (m), 1161
Each of the thioester conjugates thus far studied
between glutathione and either MIC or TDI has demon-
strated variable lifetimes under physiological conditions.
To undergo significant exchange between the thioester
conjugates and some distal ‘target’ molecule, the thioester
must persist long enough to reach the ‘target’ molecule
intact while passing through the environment of the lung
and blood. The present study was undertaken to synthe-
size and characterize the conjugate between glutathione
and another of the commercially used diisocyanates, 4,4′-
methylene- bis-(phenylisocyanate) (MDI). Stability stud-
ies of the bis- and mono-adducts of glutathione with MDI
were performed and their lifetimes compared to those
reported for the TDI bis-glutathione adduct (6).
(s) cm-1
.
1H NMR (DMSO-d6, 300.13 MHz): 1.46 (s, 9 H,
3
C(CH3)3), 3.93 (s, 2 H, CH2), 7.11 (d, 2 H, J ) 8.1 Hz), 7.30 (d,
3
3
3
2 H, J ) 8.1 Hz), 7.38 (d, 2 H, J ) 8.1 Hz), 7.86 (d, 2 H, J )
8.1 Hz), 9.21 (s, 1 H, Ar-NH-Boc) ppm. C19H21NO4: calcd C
69.71, H 6.47, N 4.28 (327.38); found C 69.99, H 6.63, N 4.35.
4-{4′-[(ter t-Bu tyloxyca r bon yl)a m in o]ben zyl}p h en yliso-
cya n a te (6). A solution from 24.32 g (0.074 mol) of compound
5 and 8.27 g/11.4 mL (0.081 mol) of triethylamine in 300 mL of
dry acetone was brought to 0 °C. At this temperature, 8.87 g/7.8
mL (0.081 mol) of ethyl chloroformate in 30 mL of acetone was
slowly added. The solution was kept at this temperature for 2
h; then 6.5 g (0.1 mol) of sodium azide in 20 mL of water was
added. Stirring was continued for 1 h at 0 °C. After pouring the
solution into 800 mL of ice/water, the formed azide was
extracted with toluene (3 × 150 mL), and the toluene layer was
washed with water (3 × 100 mL) and dried over MgSO4 at -10
°C for 48 h. The drying agent was removed, and the solution of
the azide was slowly heated to 100 °C in a water bath. After 1
h at this temperature, the evolution of nitrogen was finished,
and the solvent was evaporated at 15 mbar. The residue was
recrystallized from hexane/toluene (3:1); yield 17.3 g (0.053 mol,
72%) of isocyanate 6 obtained as pale yellow crystals; mp 122-
123 °C. IR (KBr): 3387 (s), 2979 (w), 2279 (vs, br), 1594 (s),
Ma ter ia ls
Ch em ica l Syn th esis. All reactions, except those involving
water, were carried out in dried glassware under an argon
atmosphere. Dimethyl sulfoxide (DMSO), dimethylformamide
(DMF), and acetone (water content <0.005%) were purchased
from Aldrich Chemical Co. Inc. (Milwaukee, WI). THF was
distilled from Na/benzophenone ketyl prior to use. Column
chromatography was performed using reversed phase material
(STM Bulk-C18, Separation Methods Technologies Inc., Moore-
stown, NJ ). NMR spectra were recorded on a Bruker AC 300
operating at 300.1 MHz for 1H and at 75.5 MHz for 13C.
Chemical shifts are given relative to trimethylsilane (TMS) as
an internal standard unless otherwise noted (δTMS ) 0 ppm).
IR spectra were obtained on a ATI Mattson Infinity Series FTIR.
MS were carried out on a Finnigan Mat LCQ. Melting points
were determined on an Electrothermal Melting Point Apparatus
and are uncorrected. Elemental analyses were carried out by
Atlantic Microlab Inc. (Norcross, GA). MDI was a gift of Bayer
Corp. (Pittsburgh, PA).
1519 (s), 1412 (m), 1234 (m), 1157 (s) cm-1 1H NMR (DMSO-
.
d6, 300.13 MHz): 1.47 (s, 9 H, C(CH3)3), 3.83 (s, 2 H, CH2), 7.0-
7.4 (m, 8 H-aryl), 9.18 (s, 1 H, NH-Boc) ppm. 13C NMR (CDCl3,
125.77 MHz, (CDCl3) ) 77.0): 28.3 (C-(CH3)3), 40.6 (C-(CH3)3),
80.4 (CH2), 118.8, 124.6, 129.3, 129.9, 131.2, 135.2, 136.6, 139.0
(12 C-aryl), 152.8 (CdO) ppm. The NCO signal was not visible.
Meth ylen e-bis-(S-{[(4-p h en yl)a m in o]ca r bon yl}glu ta th i-
on e) (3). GSH (1.23 g; 4 mmol) (dried 24 h at 100 °C/0.1 mbar
prior to use) was dissolved in 25 mL of DMSO. The solution
was diluted with 25 mL of DMF and 25 mL of THF, and then
brought to -25 °C in a methanol/dry ice bath. At this temper-
ature, a solution of 0.25 g (1 mmol) of MDI in 2 mL of THF was
added under vigorous stirring. The mixture was held at this
temperature for 1 h, and then allowed to come to room
temperature within 6 h. After most of the THF was evaporated
at 15 mbar, the remaining solution was poured into 1.5 L of
water (pH 3, HCl) and the product allowed to precipitate. The
precipitate was collected on a Bu¨chner funnel, washed with
water, and dried at 0.1 mbar. A total of 675 mg (0.78 mmol,
78%) of compound 3 was obtained as an amorphous colorless
solid in 90-95% purity. Further purification was achieved by
column chromatography [RP-18, 250 g, solvent: H2O/CH3CN/
trifluoroacetic acid (TFA) 80:20:0.1, 200 mL, then 70:30:0.1, 500
mL]. IR (KBr): 3365, 3349 (s, br), 3061 (w), 1678 (s), 1658 (s),
C
19H20N2O3: calcd C 70.35, H 6.21, N 8.64 (324.38); found C
70.45, H 6.22, N 8.64.
N-(tert-Butyloxycarbonyl)-S-{[({4-[4′-(tert-butyloxycarbonyl)-
a m in o]ben zyl}p h en yl)a m in o]ca r bon yl}glu ta th ion e (8). To
a suspension of 1.02 g (2.5 mmol) of N-tert-butyloxycarbonyl
(Boc)-protected GSH 7 (dried 24 h at 100 °C/0.1 mbar prior to
use) in 20 mL of acetone was added 0.81 g (2.5 mmol) of the
Boc-protected MDI derivative 6 at room temperature. After 30
min, a clear solution was obtained. Stirring was continued for
12 h after which TLC (silica gel, solvent: n-BuOH/H2O/CH3-
COOH 3:1:1) showed complete consumption of the starting
material. The acetone was then removed at 15 mbar, and the
resulting colorless solid was purified by column chromatography
(RP-18, 250 g, H2O/CH3CN/TFA 60:40:0.01, 200 mL, then 50:
50:0.01, 500 mL). Yield: 1.83 g (1.72 mmol, 69%) of compound
8; mp 130 °C (dec). IR (KBr): 3500-3200 (m, br), 1695 (s), 1681
(s), 1667 (s), 1522 (s), 1516 (s), 1411 (m), 1245 (m), 1162 (s) cm-1
.
1H NMR (DMSO-d6, 300.13 MHz): 1.37 (s, 9 H, C-(CH3)3), 1.46
(s, 9 H, ArNH(CO)OC-(CH3)3), 1.81, 1.93 (mc, 2 H, CH2-CH2-
CH), 2.22 (mc, 2 H, CH2-CH2-CH), 3.01 (dd, 1 H, 2J ) 13.9
Hz, 3J ) 8.8 Hz), 3.34 (dd, 1 H, 2J ) 13.9 Hz, 3J ) 5.1 Hz), 3.76
(d, 2 H, 3J ) 6.6 Hz, CO-CH2-NH), 3.80 (s, 2 H, Ar-CH2-
Ar), 3.90 (mc, 1 H, CH2-CH2-CH), 4.48 (mc, 1 H, S-CH2-CH),
1
1649 (s), 1517 (s), 1410 (m), 1307 (m), 1233 (m) cm-1. H NMR
(DMSO-d6, 300.13 MHz): 2.05 (mc, 4 H, CH2-CH2-CH), 2.35
2
3
(mc, 4 H, CH2-CH2-CH), 3.07 (dd, 2 H, J ) 13.9 Hz, J ) 9.5
Hz, S-CHH), 3.35 (dd, 2 H, 2J ) 13.9 Hz, 3J ) 5.1 Hz, S-CHH),
3.77 (d, 4 H, 3J ) 5.9 Hz, CO-CH2-NH), 3.83 (s, 2 H, Ar-
CH2-Ar), 3.93 (mc, 2 H, CH2-CH2-CH), 4.51 (mc, 2 H, S-CH2-
CH). 13C NMR (DMSO-d6): 26.6, 31.4, 31.9, 40.7, 41.5, 52.5, 53.4
(13 C-aliphatic), 120.8, 129.6, 137.3, 137.7 (12 C-aryl), 165.0 (2
NH-CO-S), 171.1, 171.6, 171.7, 172.1 (4 NH-CO, 4 COOH)
ppm. MS (EIS) m/z (%) ) (865.9, MH+). C35H44N8O14S2 calcd
for C35H44N8O14S2‚H2O: C 46.66, H 5.37, N12.44 (864.90); found
C 47.00, H 5.26, N 12.44.
3
3
7.06 (d, 2 HAryl, J ) 8.1 Hz), 7.10 (d, 2 HAryl, J ) 8.1 Hz), 7.34
(d, 2 HAryl, J ) 8.1 Hz), 7.39 (d, 2 HAryl, J ) 8.1 Hz), 6.9-7.1
(1 H, NH-Boc), 8.09 (d, 1 H, J ) 8.1 Hz, SCH2CH-NH), 8.16
3
3
3
(t, 1 H, 3J ) 6.6 Hz, HOOCCH2-NH), 9.14 (s, 1 H, Ar-NH-
Boc), 10.17 (s, 1 H, NH-CO-S) ppm. 13C NMR (DMSO-d6):
26.7, 28.0 (C(CH3)3), 28.1 (C(CH3)3), 31.2, 31.8, 40.7, 52.2, 53.0,
78.0 (C(CH3)3), 78.7 (C(CH3)3), 118.2, 119.0, 128.7, 128.8, 134.8,
136.6, 136.7, 137.4 (12 C-aryl), 152.7 (CO-tBu), 155.5 (CO-tBu),
164.0 (NH-CO-S), 170.2, 170.8, 171.6, 173.7 (2 COOH, 2 NH-
4-{4′-[(ter t-Bu tyloxycar bon yl)am in o]ben zyl}ben zoic Acid
(5). 4-(4′-Aminobenzyl)benzoic acid (4) (23.82 g; 0.1 mol) was
dissolved in 800 mL of a water/dioxane (1:1) mixture which
contained 4.5 g of NaOH. To this solution was added 25 g/26.5
mL (0.115 mol) of di-tert-butyl dicarbonate (Boc2O) at room
temperature. After stirring for 5 h, additional Boc2O (4 mL) was
CO) ppm, Ar-CH2-Ar overlapped by DMSO-d6. C34H45N5O11
calcd for C34H45N5O11S‚H2O: C 54.46, H 6.32, N 9.34 (731.81);
found C 54.65, H 6.29, N 9.32.
S

Characterization of Bis-S-(glutathionyl)-MDI
Chem. Res. Toxicol., Vol. 15, No. 10, 2002 1237
S -({[4-(4′-Am i n o b e n z y l)p h e n y l]a m i n o }c a r b o n y l)-
glu ta th ion e (9). Bis-Boc protected monoadduct 8 (50 mg) was
dissolved in 0.5 mL of TFA at room temperature. After 15 min,
the excess TFA was removed at 0.1 mbar. Compound 9 was used
as obtained. IR (KBr): 3380, 3354 (m, br), 3069 (w), 2958 (w),
1652 (sh, vs), 1529 (s), 1513 (s), 1411 (m), 1308 (m), 1234 (m),
1200 (s), 1141 (m) cm-1. 1H NMR (DMSO-d6, 300.13 MHz): 2.14
(mc, 2 H, CH2-CH2-CH), 2.46 (mc, 2 H, CH2-CH2-CH), 3.12
literature, e.g., reactions in organic solvents with (5) or
without (15) catalysts (tertiary amines), reactions in
water at various pH values and temperatures (16, 17),
or reaction in water/solvent mixtures (13). In the specific
case of GSH (1) and MDI (2), none of these conditions
was found to be satisfying, either due to the sensitivity
of MDI and the formed thiocarbamate toward water, or
due to the nonsolubility of GSH in most organic solvents.
For example, no reaction could be observed in solvents
such as acetone, acetonitrile, or DMF due to the non-
solubility of GSH.
2
3
2
(dd, 1 H, J ) 13.9 Hz, J ) 8.8 Hz, S-CHH), 3.45 (dd, 1 H, J
) 13.9 Hz, 3J ) 5.0 Hz, S-CHH), 3.85 (d, 2 H, 3J ) 5.7 Hz,
CO-CH2-NH), 3.94 (s, 2 H, Ar-CH2-Ar), 3.98 (mc, 1 H, CH2-
3
CH2-CH), 4.61 (mc, 1 H, S-CH2-CH), 7.18 (d, 2 H-aryl, J )
3
8.5 Hz), 7.34 (s, 4 H-aryl), 7.49 (d, 2 H-aryl, J ) 8.5 Hz), 8.2-
Reaction in hydrogen carbonate solution according to
a protocol given by Day et al. for TDI (6) resulted in the
degradation of the desired thiocarbamate, although it was
initially formed under these conditions. Very recently the
synthesis of the bis adduct of MDI 3 in an acetonitrile/
water mixture has been published (18). The characteriza-
tion of the compound was done by solid-state carbon
NMR. The purity of the product was not given. In our
hands, however, this methodology yields, in addition to
the desired product, several other compounds as shown
by HPLC and proton NMR spectroscopy. Since these
byproducts have almost identical 1H NMR spectra to the
bis adduct, except for the aromatic region, it is most likely
that di- and oligomeres resulting from the partial hy-
drolysis of MDI as well as monoadduct 9 are formed.
To circumvent these problems, an anhydrous approach
was undertaken. DMSO is one of the very few organic
solvents that is able to solubilize GSH. DMSO, however,
catalyzes the reaction of nucleophiles with isocyanates
(19); therefore, the reaction of GSH with MDI in this
solvent at room-temperature involves both nucleophilic
groups of GSH (the thiol and the amino groups); thus,
thiocarbamate and urea bonds are formed. In principle,
this problem could be prevented by a protective group
strategy (13). This, however, leads to a complicated
synthesis for a relativly simple molecule. Fortunately,
kinetic control (low temperature, excess GSH) allows
specific reaction with the thiol group with little interfer-
ence from the amino group. A solvent mixture of DMSO/
DMF/THF, keeping GSH in solution and preventing
DMSO from freezing at the reaction temperature of -25
°C, was found to be suitable for this purpose (Scheme 1).
The product obtained under these conditions was esti-
8.4 (m, 2 H, NH-CO), 10.28 (s, 1 H, NH-CO-S) ppm. 13C NMR
(DMSO-d6): 26.6, 31.4, 31.9, 40.6, 41.5, 52.5, 53.5 (7 C-aliphatic),
120.1, 124.0, 129.7, 130.0, 130.5, 136.6, 137.9, 142.7 (12 C-aryl),
165.1 (NH-CO-S), 171.1, 171.6, 171.7, 172.2 (2 COOH, 2 NH-
CO) ppm. MS (EIS) m/z (%) ) (532.1, MH+). C24H29N5O7S
(531.5).
Bis-4-{[4′-(ter t-b u t yloxyca r b on yl)a m in o]b en zyl}p h en -
yl Ur ea (10). To a solution of 1.62 g (5 mmol) of the isocyanate
6 in 20 mL of acetonitrile was given 1 mL of water. Stirring
was continued for 12 h at 20 °C. Then the solution was diluted
with 20 mL of water and the precipitate isolated on a Bu¨chner
funnel. The product was recrystallized from methanol. Yield:
1.39 g (2.2 mmol, 86%) of a colorless powder; mp: decomposition
from 190 °C on. IR (KBr): 3325 (s), 2978 (m), 1698 (s), 1594
(m), 1505 (s), 1411 (m), 1313 (m), 1237 (s), 1162 (s) cm-1 1H
.
NMR (DMSO-d6, 300.13 MHz): 1.46 (s, 18 H, C(CH3)3), 3.78 (s,
4 H, CH2), 7.0-7.1 (m, 8 H-aryl), 7.3-7.4 (m, 8 H-aryl), 8.47 (s,
2 H, NH-Boc), 9.14 (2 H, NH-Ar) ppm. 13C NMR (DMSO-d6):
28.0 (CH3), 78.7 (C(CH3)3), 118.2, 128.6, 128.7, 134.8, 135.1,
137.3, 137.5 (24 C-aryl), 152.5 (NH-CO-NH), 152.7 (NH-CO-
t-Bu) ppm. C37H42N4O5: calcd C 71.36, H 6.80, N 9.00 (622.76);
found C 71.54, H 6.83, N 9.01.
HP LC a n a lyses were performed on a Beckman System Gold
equipped with a Phenomenex Bondclone C-18 300 × 3.9 mm
column. Effluents were monitored at 254 and 215 nm. A 1 mL/
min flow rate of the following mobile phase was used: solvent
A, H2O containing 0.1% (v/v) CF3COOH; solvent B, CH3CN/H2O
mixture (80/20, v/v) containing 0.1% (v/v) CF3COOH. 0-2 min
100% A; 2-21 min linear change to 100% B starting with 30%
B; 21-25 min 100% A.
Solvolysis Stu d ies. Compounds 3, 8, and 9 (5 × 10-3mmol
each) were dissolved separately in 10 mL (resulting concentra-
tion 0.5 mmol/L) of one or more of the following solutions: PBS
(0.2 mol/L, pH 6.5 or 7.4), NaHCO3 solution (0.1 mol/L, pH 8.2),
or NaHCO3/glutathione solution (0.1 mol/L NaHCO3, 10 mmol/L
glutathione, pH 8.2), and held at a constant temperature of 25
°C. At selected time points, samples were taken and im-
mediately frozen in liquid nitrogen until analyzed. A 20 µL
aliquot of each sample was analyzed by HPLC. The concentra-
tion of each compound was determined by comparison of the
integral of its chromatographic peak with the integral of this
peak at point zero (c ) 0.5 mmol/L). Rate constants and half-
times were calculated according to standard procedures (14).
Rea ction of MDI w ith GSH u n d er Qu a si-p h ysiologica l
Con d ition s. GSH (10 mmol) and NaHCO3 (20 mmol) were
dissolved in either 200 mL of water (CGSH ) 50 mmol/L, pH 7.0)
or 200 mL of 0.1 M NaHCO3 solution (CGSH ) 50 mmol/L, pH
8.2). After the addition of 25 mmol of MDI in 100 mL of toluene
(5-fold excess), the solutions were vigorously stirred with a
stirring bar. Samples of the aqueous phase were taken after 2,
5, and 24 h, diluted 1:100 with water, and analyzed by HPLC.
The formation of the GSH-MDI bis adduct could be shown
qualitatively by comparison of the resulting HPLC chromato-
grams with that of an authentic sample of compound 3.
1
mated by H NMR spectroscopy to be 90-95% pure, and
could be further purified by column chromatography.
Compound 3 is practically insoluble in water alone or
organic solvents (except DMSO); however, it is soluble
in buffer solutions at pH values >6 (PBS, HCO3-) and
water/acetonitrile/TFA mixtures.
Mon oa d d u ct. The preparation of this compound was
achieved by the reaction of N-Boc-protected GSH 7 (20)
with the mono-Boc-protected MDI derivative 6. The Boc-
protected MDI derivative 6 was prepared starting with
the literature known amino acid 4 (21). Reaction with
excess Boc2O under standard conditions resulted in good
yields of compound 5, which was converted in a straight-
forward manner to the isocyanate 6 under Curtius
conditions (22). The Boc-protected monoadduct 8 was
then obtained by direct reaction of the isocyanate 6 with
7 in acetone. Dissolution of this compound in TFA
resulted in the removal of the protective groups and
yielded the desired monoadduct 9 after evaporation of
excess TFA (Scheme 2).
Resu lts
Ch em ica l Syn th esis. Numerous instructions for the
reaction of thiols with isocyanates are given in the
Solvolysis Stu d ies. All investigated thiocarbamates
were found to degradate upon dissolution in PBS (pH 6.5

1238 Chem. Res. Toxicol., Vol. 15, No. 10, 2002
Reisser et al.
Sch em e 1. Syn th esis of Bis Ad d u ct 3 fr om Glu ta th ion e (1) a n d 4,4′-Meth ylen e-bis-(p h en ylisocya n a te) (2)
Sch em e 2. Syn th esis of Mon oa d d u cts 8 a n d 9
and 7.4) or hydrogen carbonate solution (pH 8.2). Each
reaction follows pseudo-first-order kinetics. For the dis-
sappearance of the bis adduct 3 at 25 °C at a physiological
pH of 7.4 (PBS), a kapp,unimolecular of 2 × 10-4 s-1 and a half-
life of t(1/2) ) 59 min were determined. This shows
compound 3 to be more labile than the literature known
bis adducts of glutathione with 2,4-TDI [kapp,unimolecular
)
4.7 × 10-5 s-1, t(1/2) ) 245 min] and 2,6-TDI [kapp,unimolecular
) 4.7 × 10-6 s-1, t(1/2) ) 2460 min] under the same
conditions (6). A marked increase in reaction rate was
-
observed in NaHCO3 solution (pH 8.2), leading to a
kapp,unimolecular ) 1.96 × 10-3 s-1 and a half-life of only 5.9
min. By comparison, at pH 6.5 (PBS) the disappearance
of compound 3 was significantly slower with a half-life
of 440 min (kapp,unimolecular ) 2.6 × 10-5 s-1). The kinetics
of these reactions are depicted in Figure 1. Despite its
lability at elevated pH values, compound 3 could be
stabilized strongly by the addition of glutathione to the
solution. A concentration of 10 mmol/L GSH, which can
be reached in the human epithelial lining fluid, for
example (23), was found to increase the half-life in
hydrogen carbonate solution (pH 8.2, 25 °C) by a factor
of 34 compared to the degradation in hydrogen carbonate
solution only. Thus, under these conditions kapp,unimolecular
was determined to be 5.8 × 10-5 s-1 [t(1/2) ) 200 min].
The kinetics of this reaction are shown in Figure 1. In
each of the above-described cases, several unstable
intermediates were observed during the course of the
reaction. Though not investigated in detail, the monoad-
duct 11 as the initial degradation product of 3 and the
amine monoadduct 9 as the hydrolysis product of 11 could
be identified among them. The monoadduct 11 appears
as a steady-state intermediate that disappears when the
bis adduct disappears. It has to be noted that the
concentration of 9 was found to be dependent on the pH
value. Based on the assumption of equal absorbance of 3
F igu r e 1. Time dependencies of the degradation of bis adduct
3 in PBS at pH values of 6.5 (filled triangles) and 7.4 (filled
squares), hydrogen carbonate at pH 8.2 (filled circles), and
hydrogen carbonate (0.1 mol/L, pH 8.2) containing GSH (10
mmol/L)(open circles). T ) 25 °C. The inset provides the log
relationship for the degradation of the bis adduct at pH 7.4.
and 9, the maximal concentration of 9 was determined
to be 3 × 10-5 mol/L at pH 6.5 (i.e., 6% of 3) and 1 ×
10-5 mol/L at pH 7.4 (i.e., 2% of 3). No free methylene-
bis-(phenylamine) (MDA) was detectable. Figure 2 shows
an example chromatogram of the degradation of 3 in PBS,
taken after 180 min.
The monoadducts 8 and 9 were found to be signifi-
cantly more stable than the bis adduct 3. For the
unprotected monoadduct 9 in PBS (pH 7.4) at 25 °C, a
kapp,unimolecular of 2.46 × 10-5 s-1 and a half-life t(1/2) ) 470
min were determined. This is very similar to the values
found for the Boc-protected monoadduct 8 which are
kapp,unimolecular ) 3.45 × 10-5 s-1 and t(1/2) ) 335 min (Figure
3). In both cases, as the exclusive product a colorless
precipitate was formed during the reaction. The degrada-

Characterization of Bis-S-(glutathionyl)-MDI
Chem. Res. Toxicol., Vol. 15, No. 10, 2002 1239
F igu r e 2. Example HPLC chromatogram of the degradation
of bis adduct 3 in PBS, pH 7.4. The sample was taken after 180
min. Peaks are labeled according to Schemes 1 and 2.
F igu r e 4. Example HPLC chromatogram of the formation of
3 taken after a reaction time of 2 h. (a) pH 7.0, (b) pH 8.2. Note
that the excess of GSH is not visible at 254 nm.
analysis of the aqueous phase shows the bis adduct 3 to
be the almost exclusive product at the begining of the
reaction (excess of GSH). Figure 4 shows example chro-
matograms taken after 2 h. With the ongoing consump-
tion of GSH, more and more degradation products of 3
were detectable. Samples taken after 24 h showed
complete disappearance of 3 in both cases. As expected,
the degradation reaction is faster at pH 8.2. The forma-
tion of di- and oligomeric ureas of MDI attached to GSH,
which should be soluble in the aqueous phase, could not
be established, although insoluble material, most likely
polyureas as hydrolysis products of excess MDI, was
formed in the toluene layer.
F igu r e 3. Time dependencies of the degradation of the
monoadducts 8 (circles) and 9 (squares) in PBS (pH 7.4) at
25 °C.
Discu ssion
Sch em e 3. Rever sible, Ba se-Ca ta lyzed F or m a tion /
Degr a d a tion of Th ioca r ba m a tes in Aqu eou s
Solu tion
The ability of isocyanates to cause modification at sites
in the body which have not been directly exposed has led
to the search for masked equivalents of these highly
reactive and hydrolysis-sensitive compounds. Glutathione
adducts with thiocarbamate bonds could represent these
masked equivalents since these compounds, which are
able to release the isocyanate at physiological pH (24),
may serve as a carrier (25, 26). Adducts of isocyanates
with GSH have since been made under physiological
conditions (6), though the possibility of the formation of
thiocarbamates in hydrogen carbonate solution has been
questioned (27). Even in vivo the formation of thiocar-
bamates from administered isocyanates or as metabolites
from drugs (28) could be successfully demonstrated in
several cases; e.g., Baillie and co-workers identified
MIC-glutathione and MIC-(N-acetylcysteine) adducts
in bile and urine of rats after exposure to MIC (29).
Several studies concerning the stability of these thiocar-
bamates in aqueous solution have been performed,
indicating a reversible, base-catalyzed degradation for
which a E1cb mechanism is most likely (25, 30).
Sch em e 4. F or m a tion of Ur ea 10 u p on Solu tion of
Isocya n a te 6 in Aqu eou s Aceton itr ile
tion product of 8 was identified as the urea 10 (Scheme
4) by comparison with an authentic sample of this
compound. No free amine (MDA as hydrolysis product
of 9, and mono-Boc-protected MDA as hydrolysis product
of 8, respectivly) was detectable in either case.
Rea ction of MDI w ith GSH in a Bip h a sic Solven t
System a t Neu tr a l p H. MDI was found to react with
GSH in a two-phase system (water/toluene) under neu-
tral (pH 7) and slightly basic (pH 8.2) conditions. HPLC
In the present study, the reaction products of MDI, the
most widely used isocyanate in the manufacturing of
polyurethanes, with GSH, the monoadduct 9 and the bis
adduct 3, have been synthesized and characterized
(Schemes 1 and 2). Based on their chromatographic
properties, the rapid formation of bis adduct 3 between

1240 Chem. Res. Toxicol., Vol. 15, No. 10, 2002
Reisser et al.
GSH and MDI under neutral and slightly basic conditions
in a two-phase system could be established. Furthermore,
it could be shown that (amine) monoadduct 9, as hy-
drolysis product of the initially formed monoadduct 11,
may not be formed in significant amounts due to the
rapid reaction of 11 with excess GSH (Scheme 3). From
a chemical point of view, these findings indicate the
likeliness of the formation of bis adduct 3 under physi-
ological conditions from inhaled MDI, since it was found
that GSH in epithelial lining fluid can reach high levels.
(23). In fact, the adduct of toluene diisocyanate with
glutathione has been implicated by the loss of glutathione
during in vivo inhalation experiments (12).
The degradation of all of the synthesized compounds
3, 8, and 9 was shown to be of pseudo-first-order. With a
half-life of 59 min (kapp,unimolecular ) 2 × 10-4 s-1) deter-
mined for the bis adduct 3 at pH 7.4 and T ) 25 °C, and
a t(1/2) ) 470 min (kapp,unimolecular of 2.46 × 10-5 s-1) for the
monoadduct 9, their stability is well within the range
expected for such compounds [compare to the literature
known 2,4-TDI-GSH bis adduct with t(1/2) ) 245 min and
kapp,unimolecular ) 4.7 × 10-5 s-1]. A first-order decay and
the strong dependence of the stability of bis adduct 3 on
the pH of the medium, established by solvolysis at pH
values of 6.5, 7.4, and 8.2, support a base-catalyzed E1cb
mechanism for the degradation of the thiocarbamate
moiety as proposed by Baillie (25, 29) and others (30).
Furthermore, the reversibility of this reaction, which
could be shown by a remarkable stabilization of 3 upon
the addition of excess glutathione, indicates the presence
of free isocyanate (Scheme 3) as demanded by this
mechanism. Additional support for this mechanism comes
from the formation of urea 10 during the degradation of
the Boc-protected monoadduct 8, which is certainly
formed in analogy to the preparative method from
released isocyanate 6 and its hydrolysis product (Scheme
4).
the mono and bis adducts of glutathione with MDI are
not equivalent.
Ack n ow led gm en t. We thank Virgil Simplaceanu for
his assistance and Dr. Chien Ho for the use of his 300
MHz NMR facilities. In addition, thanks are extended
to Mark Bier and the Center for Molecular Analysis for
the use of the ESI mass spectrometer, which was funded
by NSF Grant DBI-9729351.
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