84 Chem. Res. Toxicol., Vol. 13, No. 2, 2000
Sabbioni et al.
many). Decane (>99%) and ethanol for spectroscopy were from
Sigma-Aldrich (Deisenhofen, Germany). Methanol for HPLC
analysis and toluene for residue analysis were from Promochem
(Weser, Germany). Heptafluorobutyric anhydride was from
Supelco (Deisenhofen, Germany) Valyl-glycyl-glycine was from
Bachem (Heidelberg, Germany). L-Valine was from Serva
(Heidelberg, Germany). AcMDA [N1-[4-(4-aminobenzyl)phenyl]-
acetamide] was synthesized according to ref 13. N1-[4-(4-Amino-
3,5-dideuteriobenzyl)-2,6-dideuteriophenyl]acetamide (Ac[2H4]-
MDA) was synthesized as described previously (42).
N-[4-[4-(Acet yla m in ob en zyl)p h en yl]ca r b a m oyl]va lin e
(AcMDA-Va l). L-Valine (117 mg, 1 mmol) was transformed
according to procedure 1. Crystallization from ethanol yielded
1
AcMDA-Val (138.0 mg, 36%) as white crystals. H NMR ([2H6]-
DMSO, 250 MHz): δ 0.86 (d, J ) 6.8 Hz, 3H, CHCH3), 0.90 (d,
J ) 6.8 Hz, 3H, CHCH3), 1.97 (s, 3H, COCH3), 2.08 (m, 1H,
CHCH3), 3.75 (s, 2H, CH2), 4.12 (dd, J ) 4.9, 8.7 Hz, 1H,
NHCH), 6.32 (d, J ) 8.7 Hz, 1H, NHCH), 7.03 (d, J ) 8.5 Hz,
2H), 7.08 (d, J ) 8.45 Hz, 2H), 7.27 (d, J ) 8.5 Hz, 2H), 7.47 (d,
J
) 8.45 Hz, 2H), 8.54 (s, 1H, Ph-NH), 9.94 (s, 1H, Ph-
NHCONH), 12.68 (s, 1H, COOH). 13C NMR ([2H6]DMSO, 63
MHz): δ 18.0 (CHCH3), 19.7 (CHCH3), 24.3 (COCH3), 30.7
(CHCH3), 40.4 (CH2), 58.0 (NHCH), 118.7 (CH), 120.2 (CH),
129.4 (CH), 129.6 (CH), 135.4 (C), 137.3 (C), 137.5 (C), 138.3
(C), 156.0 (NHCONH), 169.8 (COCH3), 174.7 (COOH). UV
(methanol): λmax ) 253 nm. HPLC/MS/MS [LCQ-Duo (Thermo-
quest, San J ose, CA), positive ESI]: 384 [M + H]+, MS of 384
) 241 [AcMDA + H]+, MS of 384 ) 366 (M - H2O), MS of 366
) 338. HPLC analyses were performed on a Lichrospher RP18
(125 mm × 4 mm, 5 µm) column with a 20 min, 30 to 80% MeOH
gradient in 50 mM ammonium formate [flow rate of 1 mL/min,
tR(AcMDA-Val) ) 11.2 min] or a 20 min, 30 to 80% MeOH
gradient in 0.01% trifluoroacetic acid [flow rate of 1 mL/min,
tR(AcMDA-Val) ) 14.3 min].
In str u m en ta tion . NMR spectra were recorded on a Bruker
AC 250 instrument with [2H6]DMSO as the solvent and as the
internal standard. The degree of substitution of the C atoms
was determined using the distortionless enhancement by po-
larization transfer (DEPT) method. The raw NMR data were
processed with the program MestRe-C (Magnetic Resonance
Companion, J . C. Cobas, J . Cruces, and F. J . Sordina, Depar-
tamente de Quimica Organica, Universidad de Santiago de
Compostela, 15706 Santjago de Compostela, Spain). HPLC
analyses were performed on a Hewlett-Packard 1100 system
with a quaternary HPLC pump and a photodiode array detector.
Gas chromatography/mass spectrometry (GC/MS) analyses were
carried out on a Hewlett-Packard gas chromatograph (HP
5890II) interfaced with a mass spectrometer (HP 5989A).
N-[4-[4-(Acetyla m in o-3,5-d id eu ter ioben zyl)-2,6-d id eu te-
r iop h en yl]ca r ba m oyl]va lin e (Ac[2H4]MDA-Va l). The deu-
terated adduct was synthesized according to the procedure given
above for the undeuterated compound. The purity and identity
of the compound were checked by HPLC with a photodiode array
detector and by HPLC/MS/MS (LCQ-Duo, positive ESI): 388
[M + H]+, MS of 388 ) 245 [Ac[2H4]MDA + H]+.
Isola tion of Globin . The animal experiments were per-
formed at the Fraunhofer-Institut in Hannover and have been
described previously (43). The animals were exposed for 3
months for 17 h per 5 days a week with MDI concentrations of
0, 0.26, 0.7, and 2.06 mg/m3. The rats were sacrificed 1 day after
their last day of exposure. The animals were anesthetized with
pentobarbital, and blood was taken by heart puncture. For the
analysis of protein adducts, 1-3 mL of blood was obtained, and
centrifuged for 5 min at 2000g. After removal of the plasma,
the red blood cells were washed three times with equal volumes
of a 0.9% sodium chloride solution. For complete hemolysis, the
erythrocytes were diluted with 4 volumes of water and left at
room temperature for 30 min. Cell debris was removed by
centrifugation (10 min at 4000g). Globin was precipitated from
the clear red supernatant by adding 4 volumes of cold 0.2% HCl
in acetone at 0 °C. The precipitate was resuspended and isolated
by centrifugation after the following washing sequence: ethanol/
water (8:2), ethanol, ethanol/diethyl ether (1:3), and diethyl
ether. Globin was dried over silica gel in a desiccator, and all
samples were stored at -20 °C.
Syn th esis of N1-[4-(4-Isocya n a toben zyl)p h en yl]a ceta -
m id e (AcMDI). Triphosgene (415 mg, 1.4 mmol) was added to
a solution of AcMDA (480 mg, 2.00 mmol) in dry dioxane (20
mL) and the mixture stirred for 3 h at 80 °C under nitrogen.
The reaction mixture was used without further cleanup for the
next steps. According to HPLC analyses, all AcMDA was
consumed under these conditions. HPLC analyses were per-
formed on a Lichrospher RP18 (125 mm × 4 mm, 5 µm) column
with a 20 min, 30 to 80% MeOH gradient in 50 mM ammonium
formate [flow rate of 1 mL/min, tR(AcMDA) ) 9.9 min, tR(AcMDI)
) 13.0 min] or with a 20 min, 30 to 80% MeOH gradient in
0.01% trifluoroacetic acid [flow rate of 1 mL/min, tR(AcMDA) )
6.4 min, tR(AcMDI) ) 13.2 min].
Syn t h esis of Ur ea s fr om AcMDI a n d Am in o Acid s.
P r oced u r e 1. Amino acid (1 mmol) or tripeptide (1 mmol) was
dissolved in 0.25 M NaHCO3 (20 mL). The solution was stirred,
and a hot solution (80 °C) of freshly prepared AcMDI (1 mmol)
in dioxane (10 mL) was added dropwise. NaHCO3 was added to
maintain the pH at ca. 8.0. After 3 h, the reaction mixture was
cooled with ice and the ice solution was filtered. The filtrate
was evaporated to a final volume of ca. 15 mL, carefully acidified
to pH 2 with 2 M HCl, and extracted with ethyl acetate (3 × 50
mL). The organic phase was extracted with a saturated NaHCO3
solution (3 × 20 mL). The pooled aqueous phases were acidified,
saturated with NaCl, and extracted with ethyl acetate (3 × 50
mL). After the sample had been dried over MgSO4, the ethyl
acetate was evaporated at reduced pressure. The residue was
recrystallized from ethanol after adding a few drops of water.
N-[4-[4-(Acet yla m in ob en zyl)p h en yl]ca r b a m oyl]va lyl-
glycyl-glycin e (AcMDA-Va l-Gly-Gly). Val-Gly-Gly (115.5 mg,
0.5 mmol) was transformed according to procedure 1. Crystal-
lization from ethanol yielded AcMDA-Val-Gly-Gly (85 mg, 34%)
1
as a white solid. H NMR ([2H6]DMSO, 200 MHz): δ 0.86 (d, J
) 6.8 Hz, 3H, CHCH3), 0.90 (d, J ) 6.8 Hz, 3H, CHCH3), 1.97
(s, 3H, COCH3), 2.08 (m, 1H, CHCH3), 3.75 (s, 6H, CH2), 4.06
(dd, J ) 5.5, 8.0 Hz, 1H, NHCH), 6.55 (d, J ) 8.0 Hz, 1H,
NHCH), 7.03 (d, J ) 8.5 Hz, 2H), 7.08 (d, J ) 8.45 Hz, 2H),
7.27 (d, J ) 8.5 Hz, 2H), 7.47 (d, J ) 8.45 Hz, 2H), 8.16 (t, J )
5.8 Hz, 1H, NHCH2), 8.29 (t, J ) 5.9 Hz, 1H, NHCH2), 8.90 (s,
1H, Ph-NH), 9.94 (s, 1H, Ph-NHCONH), 12.68 (s, 1H, COOH).
13C NMR ([2H6]DMSO, 50 MHz): δ 17.7 (CHCH3), 19.3 (CHCH3),
23.9 (COCH3), 30.6 (CHCH3), 40.2, 40.4 (CH2, CH2N), 41.7
(CH2N), 58.1 (NHCH), 117.5 (CH), 119.1 (CH), 128.6 (CH), 128.8
(CH), 133.9 (C), 136.1 (C), 137.3 (C), 138.4 (C), 155.3 (NH-
CONH), 168.0 169.1, 171.0, 172.1 (2NHCO, COOH, NHCOCH3).
UV (methanol): λmax ) 253 nm. HPLC/MS/MS (LCQ-Duo,
positive ESI): 498 [M + H]+, MS of 498 ) 366 (M - Gly-Gly),
MS of 366 ) 338. HPLC analyses were performed on
a
Lichrospher RP18 (125 mm × 4 mm, 5 µm) column with a 20
min, 30 to 80% MeOH gradient in 50 mM ammonium formate
[flow rate of 1 mL/min, tR(AcMDA-Val-Gly-Gly) ) 12.0 min] or
a 20 min, 30 to 80% MeOH gradient in 0.01% trifluoroacetic
acid [flow rate of 1 mL/min, tR(AcMDA-Val-Gly-Gly) ) 12.3 min].
3-[4-(4-Am in oben zyl)ph en yl]-5-isopr opyl-1,3-im idazolin e-
2,4-d ion e (MDA-Va l-Hyd ). AcMDA-Val (38 mg, 0.1 mmol) was
dissolved in 0.3 mL of methanol and the mixture heated for 90
min at 90 °C in 5 M HCl (5 mL). The reaction mixture was cooled
to room temperature and extracted with ethyl acetate (3 × 5
mL). The water phase was made basic by addition of NaHCO3
and extracted with ethyl acetate (3 × 6 mL). The ethyl acetate
extract was dried over Na2SO4, filtered, and evaporated to
dryness. A white solid was obtained, MDA-Val-Hyd (23 mg,
1
70%). H NMR ([2H6]DMSO, 250 MHz): δ 0.86 (d, J ) 6.7 Hz,
3H, CHCH3), 1.05 (d, J ) 6.7 Hz, 3H, CHCH3), 2.13 (m, 1H,
CHCH3), 3.78 (s, 2H, CH2), 4.11 (d, J ) 3.6 Hz, 1H, CHNH),
4.9 (s, 2H, NH2), 6.49 (d, J ) 8.2 Hz, 2H), 6.87 (d, J ) 8.2 Hz,
2H), 7.18 (d, J ) 8.3 Hz, 2H), 7.25(d, J ) 8.3 Hz, 2H), 8.52 (s,
1H, NH). 13C NMR ([2H6]DMSO, 63 MHz): δ 15.7 (CH3), 18.4
(CH3), 29.8 (CHCH3), 39.9 (CH2), 61.2 (CHNH), 113.9 (CH),