Synthetic approaches to obtain amino acid adducts of 4,4′- methylenediphenyl diisocyanate

Article abstract of DOI:10.1021/tx300347e

4,4′-Methylenediphenyl diisocyanate (MDI) is the most important isocyanate used in the chemical industry. Lung sensitization and asthma are the main types of damage after exposure to MDI. Albumin adducts of MDI might be involved in the etiology of sensitization reactions. It is therefore necessary to have sensitive and specific biomarkers such as blood protein adducts to monitor people exposed to isocyanates. For the discovery of new isocyanate adducts with blood proteins present in vivo, new synthetic standards are needed. To achieve this, we developed five methods to obtain amino acid adducts of MDI. We synthesized and isolated MDI adducts of aspartic acid, glutamic acid, cysteine, and valine. The new adducts were characterized by LC-MS/MS and NMR. We synthesized the corresponding isotope-labeled MDI adducts to develop analytical methods using LC-MS/MS. Glutathione adducts of isocyanates are an important way of transportation of the reactive isocyanates to distant sites from the original site of exposure. Therefore, we used N-acetyl-cysteine adducts of MDI as reactants: N-acetyl-S-[[4-(4-aminobenzyl)phenyl]carbamoyl]-cysteine (MDI-AcCys) and N-acetyl-S-[[4-(4-acetylaminobenzyl)phenyl]carbamoyl]-cysteine (AcMDI-AcCys). MDI-AcCys or AcMDI-AcCys formed adducts with albumin, N _α-acetyl lysine, and valine. Isotope-labeled albumin adducts (= d₄-MDI-albumin) were synthesized from d₄-MDI-AcCys and albumin. d₄-MDI-albumin can be used as an internal standard to analyze biological samples. Such an internal standard will not correct only for the extraction recovery of the adducts but also for the potential variation of the enzymatic digestions used in the procedure to analyze albumin adducts of MDI. The synthetic procedures described in this manuscript will be applicable to the synthesis of amino acid adducts from other isocyanates.

Full text of DOI:10.1021/tx300347e

Article
pubs.acs.org/crt
Synthetic Approaches To Obtain Amino Acid Adducts of 4,4′-
Methylenediphenyl Diisocyanate
Gabriele Sabbioni,* Nagaraju Dongari, Siegfried Schneider, and Anoop Kumar
Global Department of Environmental Health Sciences, School of Public Health and Tropical Medicine, Tulane University, 1440
Canal Street, Suite 2100, New Orleans, Louisiana 70112, United States
S
* Supporting Information
ABSTRACT: 4,4′-Methylenediphenyl diisocyanate (MDI) is
the most important isocyanate used in the chemical industry.
Lung sensitization and asthma are the main types of damage
after exposure to MDI. Albumin adducts of MDI might be
involved in the etiology of sensitization reactions. It is
therefore necessary to have sensitive and specific biomarkers
such as blood protein adducts to monitor people exposed to
isocyanates. For the discovery of new isocyanate adducts with blood proteins present in vivo, new synthetic standards are needed.
To achieve this, we developed five methods to obtain amino acid adducts of MDI. We synthesized and isolated MDI adducts of
aspartic acid, glutamic acid, cysteine, and valine. The new adducts were characterized by LC-MS/MS and NMR. We synthesized
the corresponding isotope-labeled MDI adducts to develop analytical methods using LC-MS/MS. Glutathione adducts of
isocyanates are an important way of transportation of the reactive isocyanates to distant sites from the original site of exposure.
Therefore, we used N-acetyl-cysteine adducts of MDI as reactants: N-acetyl-S-[[4-(4-aminobenzyl)phenyl]carbamoyl]-cysteine
(MDI-AcCys) and N-acetyl-S-[[4-(4-acetylaminobenzyl)phenyl]carbamoyl]-cysteine (AcMDI-AcCys). MDI-AcCys or AcMDI-
AcCys formed adducts with albumin, Nα-acetyl lysine, and valine. Isotope-labeled albumin adducts (= d4-MDI-albumin) were
synthesized from d4-MDI-AcCys and albumin. d4-MDI-albumin can be used as an internal standard to analyze biological samples.
Such an internal standard will not correct only for the extraction recovery of the adducts but also for the potential variation of the
enzymatic digestions used in the procedure to analyze albumin adducts of MDI. The synthetic procedures described in this
manuscript will be applicable to the synthesis of amino acid adducts from other isocyanates.
Arylisocyanates lead to cytotoxic and genotoxic effect-
s.10,11Arylisocyanates react directly with biomolecules and/or
hydrolyze to arylamines (Figure 1). Protein adducts of
arylisocyanates are believed to be involved in the etiology of
sensitization reactions.12,13 Important vehicles for isocyanates
are their reaction products with glutathione.14,15 The
glutathione adducts release the isocyanate moiety to react
with other nucleophiles, for example, proteins. Therefore,
glutathione adducts are thought to be responsible for the
transport of isocyanate to reactive sites away from the site of
isocyanate uptake. Isocyanate-specific adducts of MDI with the
N-terminal valine of hemoglobin in rats16 and isocyanate-
specific adducts of MDI with lysine present in albumin have
been identified in rats and humans exposed to MDI.17,18
INTRODUCTION
■
Isocyanates are highly reactive compounds that have a variety of
commercial applications. Diisocyanates such as 4,4′-methyl-
enediphenyl diisocyanate (MDI) are used for manufacturing
polyurethane foam, elastomers, paints, adhesives, coatings,
insecticides, consolidation of loose rock zones in coal mining or
tunneling, and many other products.1 The high chemical
reactivity of diisocyanates makes them toxic. Adverse effects at
the cellular and subcellular level have been reported, such as
irritative and immunological reactions. Inhalation of diisocya-
nate vapors is associated with various pulmonary ailments, such
as eosinophilic airway inflammation, airway hyper-reactivity,
early and late-onset asthma, exogenous allergic alveolitis, and
direct toxic responses.2−6 Diisocyanates are of great concern for
occupational health, being considered one of the main causes of
occupational asthma.2,7,8 The steady rise in asthma over the
past decades points to the potential relationship between
isocyanates in consumer products and increasing prevalence of
asthma in the general population, especially children.9 The
prevalence and incidence of diisocyanate-induced disorders
depend on the degree of exposure. Occupational exposure to
diisocyanates may take place during their production and
application in the production of polyurethane foam and other
products containing monomeric or polymeric diisocyanates.
The main route of occupational exposure is through inhalation.
19
Albumin adducts have a half-life of 20−25 days. Therefore,
albumin adducts are a marker of exposure and a marker that is
related to the mechanism of sensitization caused by isocyanates.
Reaction products with other amino acids than lysine are
possible in albumin such as the N-terminal adducts with
aspartic acid (humans) and glutamic acids (rats), serine,
tyrosine, histidine, and tryptophan (Figure 1).
Received: August 3, 2012
Published: November 26, 2012
© 2012 American Chemical Society
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Figure 1. Putative reaction products of MDI with biomolecules.
obtained from Fisher Scientific (New Jersey). Pronase E (protease,
type XIV bacterial, from Streptomyces griseus) (#P5147), acylase I from
porcine kidney (#3010), ammonium formate (#17843), sodium
hydroxide, sodium phosphate monobasic monohydrate, human
serum albumin (# A1653), water for LC-MS/MS (Chromasolv;
#39253), triphosgene, trifluoroacetic acid, L-aspartic acid dimethyl
ester hydrochloride, anhydrous 1,4-dioxane (#296309), all of the N-α
Boc-protected amino acids, and all of the isotope-labeled amino acids
were purchased from Sigma-Aldrich (St. Louis, MO). S-(−)-2-
Isocyanatoglutaricacid diethyl ester and methyl-S-(−)-2-isocyanato 3-
methylbutyrate ester were purchased from TCI America (Portland,
OR). Coomassie plus protein assay reagent for protein determination
was acquired from Thermo Scientific (Rockford, IL). The Strata-X-33u
(8B-S100-FBJ) polymeric reversed-phase solid-phase extraction
cartridges (200 mg/3 mL) were purchased from Phenomenex Inc.
(Torrance CA). N1-[4-(4-Aminobenzyl)phenyl]acetamide
(AcMDA),24 tert-butyl [4-(4-aminobenzyl)phenyl]carbamate (BocM-
DA),17 tert-butyl [4-(4-isocyanatobenzyl)phenyl]carbamate (BocM-
DI),17 N-acetyl-4′-amino-4-nitrodiphenylmethane (4-AcNO2MDA),25
4,4′-methylene-bis(2,6-dideuteroaniline) (d4-MDA),26 N1-[4-(4-
amino-(3,5-dideuterobenzyl))-(2,6-dideuterophenyl)]acetamide (d4-
AcMDA),26 N-acetyl-O-[(4-methylphenyl)carbamoyl]-tyrosine
(4MPI-AcTyr),29 N-acetyl-O-[(4-methylphenyl)carbamoyl]-serine
(4MPI-AcSer),29 N2-acetyl-N6-[(4-methylphenyl)carbamoyl]-lysine
(4MPI-AcLys),29 and N-[(4-methylphenyl)carbamoyl]-aspartic acid
(4MPI-Asp)29 were synthesized previously in our laboratory.
Deacetylation with acylase28,29 of 4MPI-AcTyr, 4MPI-AcSer, and
4MPI-AcLys yielded N6-[(4-methylphenyl)carbamoyl]-lysine (4MPI-
Lys), O-[(4-methylphenyl)carbamoyl]-serine (4MPI-Ser), and O-[(4-
methylphenyl)carbamoyl]-tyrosine (4MPI-Tyr), respectively.
The following adducts of MDI and N-[4-(4-
isocyanatobenzyl)phenyl]acetamide (AcMDI) with amino
acids have been synthesized up to date. In vitro reactions of
MDI with cysteine and N-acetyl-cysteine yielded the bis-
thiocarbamate adduct, methylene-bis-(S-{[(4-phenyl)amino]-
carbonyl}cysteine),20 and methylene-bis-(S-{[(4-phenyl)-
amino]carbonyl}N-acetyl-cysteine),21,22 respectively. Gluta-
thione and MDI reacted in vitro to the monothiocarbamate,
S-({[4-(4′-aminobenzyl)phenyl]amino}carbonyl)-glutathione,
and to the bis-thiocarbamate product, methylene-bis-(S-{[(4-
phenyl)amino]carbonyl}glutathione).23 AcMDI reacted with
lysine and with valine to yield N6-[[4-[4-acetylaminobenzyl]-
phenyl]carbamoyl]-lysine (AcMDI-Lys)17 and N-[[4-[4-
acetylaminobenzyl]phenyl]carbamoyl]-valine (AcMDI-Val),16
respectively.
To identify further reaction products of MDI with proteins,
additional synthetic standards of MDI-amino acid adducts are
needed. Therefore, for the present work, we developed
methods to synthesize more amino acid adducts of MDI to
be used for the identification of new amino acid adducts in
proteins. In addition, we present a method to modify albumin
in vitro, which yields potentially the same products present in
vivo.
MATERIALS AND METHODS
■
Caution: The aromatic amines used in this work are potentially
carcinogenic. Isocyanates are strong irritants. Triphosgene is severely
irritating to the eyes and skin. Avoid contact and inhalation. All of these
compounds should be handled with protective clothing in a well-ventilated
fume hood.
Instrumentation and Analytical Methods. NMR spectra were
recorded on a Bruker AC 500 instrument with d6-DMSO as the
solvent and as the internal standard. The degree of substitution of the
C atoms was determined using the distortionless enhancement by
polarization transfer method. The raw NMR data were processed with
Chemicals. Methanol (A454-4) for sample preparation and
methanol (LC/MS Optima grade, A456-4) for LC-MS/MS were
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the program MestRe-C (Cobas, J. C., Cruces, J., and Sordina, F. J.
Magnetic Resonance Companion, Departamente de Quimica
Organica, Universidad de Santiago de Compostela, 15706 Santjago
de Compostela, Spain) or the ACD/NMR processor Academic
edition, Version 12.01 (Advanced Chemistry Development, Inc.,
Toronto, Canada). An API 4000Q Trap (ABSciex, Foster City, CA)
mass spectrometer interfaced to an HPLC (Shimadzu Prominence
20AD) was used in the LC-MS/MS analyses (software Analyst 1.4.2).
A UV-1800 Spectrophotometer from Shimadzu was used for protein
determination. HPLC monitoring of the reactions was performed with
an Agilent 1100 equipped with a photodiode detector: HPLC-column
= Lichrospher 100 RP18 (125 mm × 4 mm, 5 μm), flow rate of 1.0
mL/min, and λ = 250 nm. Method 1: solvent A, 0.1% formic acid;
solvent B, methanol. The analyses were performed with a linear
gradient of 15−80% B in 20 min. Method 2: solvent A, 10 mM
ammonium acetate; B, methanol. The analyses were done with a linear
gradient of 30−80% B in 20 min. The lipophilicity of the synthesized
products was estimated with ACD/LogD v8.02 (Advanced Chemistry
Development, Inc., Toronto, Canada, www.acdlabs.com) and/or
Marvin Beans 5.10.0 (ChemAxon, Budapest, Hungary, www.
chemaxon.com).
Analysis of Amino Acid Adducts Using LC-MS/MS. Shimadzu
Prominance 20AD interfaced to an API 4000Q Trap LC-MS/MS
(ABSciex, Foster City, CA) mass spectrometer system was used for all
of the quantitative analysis. The MS parameters were optimized in the
electrospray ionization mode (ESI). Parameter optimization was
carried out by infusing 100 pg/μL solution of analyte with the flow
rate of 10 μL/min in the negative ionization mode. MDI-Val, AcMDI-
Val, MDI-Glu, AcMDI-Glu, MDI-Asp, AcMDI-Asp, MDI-Lys, and
AcMDI-Lys were showing corresponding deprotonated [M − H]−
ions at m/z 340.1, 382.1, 370.1, 412.1, 356.1, 398.1, 369.1, and 411.1,
respectively. The quantitative optimization mode was used to optimize
the multiple reaction monitoring signals and set the maximum
declustering potential and collision energy parameters for the
compounds. For better resolution and sensitivity of the analyte,
quadrupole mass analyzers (Q1 and Q3) were set to 0.7 0.1 amu
resolution window. The mass spectrometer was operated in negative
(or positive) ionization mode and with an electrospray voltage −4500
V (+4500 V) and a source temperature of 500 °C. The same
parameters were used for positive ESI-MS analyses. The positive ESI-
MS data were only used for the characterization of the synthesized
MDI adducts. The negative ESI-MS runs were used for quantification
of the adducts. Nitrogen was used as the ion spray (GS1), drying
(GS2), and curtain gas at 40, 45, and 10 arbitrary units, respectively.
The declustering potential and collision energy were optimized for all
compounds. The entrance potential for all compounds was −10 V. All
data were processed using Analyst software 1.4.2 (Applied
Biosystems/MDS Sciex).
The MDI adducts were separated on a reversed phase C18 column
and analyzed with a LC-MS/MS Instrument. [Luna C18(2) (100 Å,
150 mm × 2.0 mm, 3 μm) (Phenomenex Inc., Torrance, CA)
protected by a C18 guard column (AJO-4287; 4 mm L × 3.0 mm i.d.),
using a gradient system with solvent A (5 mM ammonium formate)
and solvent B (methanol): 0.2 mL/min, 0−3 min: 10% B, 3−12 min,
10−90% B linear gradient, 12−15 min 90% B. LC-MS/MS analyses of
the standard compounds yielded the following peaks (Figure 5 in the
Supporting Information): MDI-Asp tR = 12.1 min (m/z 356.1 →
132.1, 88.1), MDI-Glu tR = 12.2 min (m/z 370.1 → 146.1, 128.1),
MDI-Lys tR = 13.2 min (m/z 369.1 → 145.0, 171.3), and MDI-Val tR =
14.3 (m/z 340.1 → 116.0). MDI-Lys and AcMDI-Lys was quantified
using the internal standards MDI-[13C615N2]Lys and d4-AcMDI-Lys as
published previously.17,18
Method 1: Synthesis of Amino Acid Adducts Using AcMDI.
Synthesis of Ureas from AcMDI and Amino Acids. General
Procedure 1. Triphosgene (367 mg, 1.25 mmol) was added to a
stirred solution of AcMDA (243 mg, 1 mmol) in dioxane (20 mL) and
then heated to 80 °C. The reaction was monitored by HPLC. After 3 h
of reaction time, the reaction mixture was added dropwise to a stirred
solution of an amino acid (1 mmol) or a peptide (1 mmol) in 0.25 M
NaHCO3 (20 mL). NaHCO3 was added to maintain the pH at ca. 8.0.
After 2 h of reaction time, the reaction mixture was cooled on ice. The
precipitate was filtered off and washed with water (20 mL) and ethanol
(1 mL). The filtrate was acidified with 2 M HCl to pH 2 and extracted
with ethyl acetate (3 × 50 mL). The organic layer was extracted twice
with saturated NaHCO3 solution (20 mL). The aqueous phase was
separated, acidified, and extracted with ethyl acetate (3 × 50 mL).
After it was dried over MgSO4, the organic phase was evaporated at
reduced pressure. The residue was recrystallized with ethanol/water.
N-[[4-(4-Acetylaminobenzyl)phenyl]carbamoyl]-aspartic Acid
(AcMDI-Asp). AcMDI-Asp was synthesized following general proce-
dure 1 with aspartic acid (200 mg, 1.50 mmol). Crystallization from
1
EtOH/H2O yielded a brown solid (104 mg, 17.3%). H NMR (d6-
DMSO): 12.6 (s, broad, 2 COOH), 9.82 (s, 1 H, NH-Ac), 8.74 (s, 1
H, NH-CO-NHCH), 7.46 (d, J = 8.4, 2H, Ar−H), 7.28 (d, J = 8.5, 2H,
Ar−H), 7.10 (d, J = 8.4, 2H, Ar−H), 7.05 (d, J = 8.5, 2H, Ar−H), 6.47
(d, J = 8.4, CONHCH), 4.49 (mc, CHCH2), 3.78 (s, Ar−CH2-Ar),
2.76 (dd, J = 16.8, 5.6, 1 H, CHCH2), 2.68 (dd, J = 16.8, 5.0, 1 H,
CHCH2), 2.01 (s, 3 H, Ph-NHCOMe). 13C NMR (d6-DMSO): 173.0
(CO), 172.1 (CO), 168.0 (CO), 154.7 (NHCONH), 138.1 (Ar C),
137.2 (Ar C), 136.2 (Ar C), 134.2 (Ar C), 128.8 (Ar CH), 128.7 (Ar
CH), 119.1 (Ar CH), 117.7 (Ar CH), 48.7 (CHCH2), 40.4 (Ar-CH2−
Ar), 36.8 (CHCH2), 23.9 (Ph-NHCOMe). ESI-MS (positive) m/z:
400.2 [M + H]+, MS/MS of 400.2 → 132.0. ESI-MS (negative) m/z:
398.1 [M − H]−, MS/MS of 398.0 → 132.0
N-[[4-[4-Acetylaminobenzyl]phenyl]carbamoyl]-glutamic Acid
(AcMDI-Glu). AcMDI-Glu was synthesized according to general
procedure 1 with L-glutamic acid (221 mg, 1.5 mmol). Crystallization
from EtOH/H2O yielded a beige solid (124 mg, 20.3%). 1H NMR (d6-
DMSO): 12.4 (s, broad, 2 H, COOH), 9.81 (s, 1 H, NH), 8.50 (s, 1 H,
NH), 7.46 (d, J = 8.4, 2 H, Ar−H), 7.27 (d, J = 8.5, 2 H, Ar−H), 7.09
(d, J = 8.4, 2 H, Ar−H), 7.05 (d, J = 8.5, 2 H, Ar−H), 6.39 (d, J = 8.0,
1 H, NHCH), 4.19 (dd, J = 8.0, 13.2, 1 H, CHCH2), 3.78 (s, 2 H, Ar−
CH2-Ar), 2.28 (mc, 2 H, CHCH2CH2), 2.00 (s, 3 H, PhNHCOCH3),
2.00 (mc, 1 H, CHCH2), 1.79 (mc, 1 H, CHCH2). 13C NMR (d6-
DMSO): 173.9 (CO), 173.6 (CO), 168.0 (CO), 154.8 (NHCONH),
138.1, 137.2 (Ar C), 136.2 (Ar C), 134.2 (Ar C), 128.8 (Ar CH), 128.7
(Ar CH), 119.1 (Ar CH), 117.8 (Ar CH), 51.5 (CHCH2CH2), 40.4
(Ar-CH2−Ar), 29.9 (CHCH2CH2), 27.2 (CHCH2CH2), 23.9
(PhNHCOCH3). ESI-MS (positive) m/z: 414.0 [M + H]+, 241.0.
ESI-MS (negative) m/z: 412.3 [M − H]−, MS/MS of 412.3 → 146.1,
128.0.
N-Acetyl-S-[[4-(4-acetylaminobenzyl)phenyl]carbamoyl]-cysteine
(AcMDI-AcCys). AcMDI-AcCys was synthesized according to general
procedure 1 with N-acetyl-L-cysteine (256 mg, 1.57 mmol).
Crystallization from ethanol yielded a yellow solid (175 mg, 26%).
1H NMR (d6-DMSO): 10.29 (s, NHCOS), 9.87 (s, NHCOCH3), 8.31
(d, J = 8.1, CHNH), 7.47 (d, J = 8.4, 2 H, Ar−H), 7.40 (d, J = 8.5, 2 H,
Ar−H), 7.13 (d, J = 8.3, 2 H, Ar−H), 7.11 (d, J = 8.3, 2 H, Ar−H),
4.35 (ddd, J = 8.5, 8.4, 5.0, NHCH), 3.81 (s, 2 H, Ar−CH2-Ar), 3.38
(dd, J = 13.8, 5.0, 1 H, SCH2), 3.05 (dd, J = 13.7, 8.8, 1 H, SCH2), 2.01
(s, 3 H, Ph-NHCOMe), 1.85 (s, 3 H, CHNHCOMe). 13C NMR (d6-
DMSO): 171.8 (COOH), 169.3, 168.0 (Ph-NHCOMe,
CHNHCOMe), 163.7 (NHCOS), 137.2 (Ar C), 136.7 (Ar CH),
136.5 (Ar C), 135.8 (Ar C), 128.9 (Ar CH), 128.7 (Ar CH), 119.1 (Ar
C), 52.1 (NHCH), 40.4 (Ar-CH2−Ar), 30.5 (SCH2), 23.8
(CHNHCOMe), 22.3 (Ph-NHCOMe). ESI-MS (positive) m/z:
430.0 [M + H]+, MS/MS of 430.0 → 132.2. ESI-MS (negative) m/
z: 428.2 [M − H]−, MS/MS of 428.2 → 162.1, 84.2.
N2-Acetyl-N6-[[4-(4-acetylaminobenzyl)phenyl]carbamoyl]-lysine
(AcMDI-AcLys). AcMDI-AcLys was synthesized according to general
procedure 1 with Nα-acetyl-L-lysine (231 mg, 1.23 mmol).
Crystallization from EtOH/H2O yielded a pale brown solid (65 mg,
11.6%). 1H NMR (d6-DMSO): 12.5 (s, COOH), 9.90 (s,
NHCOCH3), 8.35 (s, Ar−NH), 8.12 (d, J = 7.9, CHNH), 7.48 (d, J
= 8.1, 2 H, Ar−H), 7.28 (d, J = 8.0, 2 H, Ar−H), 7.13 (d, J = 8.1, 2 H,
Ar−H), 7.05 (d, J = 8.0, 2 H, Ar−H), 6.08 (t, J = 5.6, NHCH2), 4.12
(mc, NHCH), 3.78 (s, CH2), 3.06 (mc, NHCH2), 2.02 (s, COCH3),
1.85 (s, COCH3), 1.62 (mc, CH2), 1.35 (mc, 2 CH2). 13C NMR (d6-
DMSO): 173.7 (COOH), 169.2, 168.0 (NHCOCH3), 155.2
(NHCONH), 138.4 (Ar C), 137.1 (Ar C), 136.2 (Ar C), 133.8 (Ar
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C), 128.7 (Ar CH), 119.0 (Ar CH), 117.7 (Ar CH), 51.7 (NHCH),
39.8 (Ar-CH2−Ar), 38.7 (NHCH2CH2), 30.7 (CHCH2CH2), 29.4
(NHCH2CH2), 23.8 (COCH3), 22.8 (CHCH2CH2), 22.2
(CHNHCOCH3). ESI-MS (positive) m/z: 455.0 [M + H]+, MS/
MS of 453.0 → 241.0. ESI-MS (negative) m/z: 453.0 [M + H]−, MS/
MS of 453.0 → 187.0, 146.2.
Method 3: Synthesis of Amino Acid Adducts by Reaction of 2-
Isocyanato Amino Acid Esters with MDA. N-([4-(4-Aminobenzyl)-
phenyl]carbamoyl)-valine (MDI-Val). MDA (1237.5 mg, 6.25 mmol)
dissolved in dichloromethane (25 mL) was added slowly to methyl-S-
(−)-2-isocyanato 3-methylbutyrate ester (118.9 mg, 1.25 mmol)
dissolved in dichloromethane (2 mL). After it was stirred for 2 h at
room temperature, the reaction was stopped. According to HPLC
analyses, most of the isocyanate reacted with MDA yielding the MDI-
methyl-3-methylbutyrate ester (mono, 94%) and the dimer (6%). n-
Hexane (60 mL) was added to the reaction mixture and mixed well.
The solution was washed with acid (pH 2, 4 × 15 mL). The pH of the
aqueous phase was adjusted to 8 with NaHCO3 and extracted with
ethyl acetate (4 × 10 mL). The ethyl acetate layer was dried with
anhydrous sodium sulfate, filtrated, and concentrated with a
rotoevaporator to yield the crude MDI-methyl-3-methylbutyrate
ester. The crude ester was dissolved in tetrahydrofuran (2 mL). The
solution was diluted with water (16 mL), and 0.25 M NaOH (7 mL)
was added dropwise at 0 °C. After 2 h, the reaction mixture was
washed with ethyl acetate (3 × 25 mL). The organic phase was
discarded. The pH of the water phase was adjusted to pH 4 with 2 M
HCl (the log D = 2.39 of MDI-Val is highest at pH 4) and extracted
with ethyl acetate (3 × 20 mL). The organic extract was dried on
anhydrous sodium sulfate and concentrated with a rotoevaporator at
room temperature to avoid formation of the corresponding hydantoin,
3-[4-(4-aminobenzyl)phenyl]-5-(propan-2-yl)imidazolidine-2,4-dione
(MDI-Val-Hyd).16 The residue was recrystallization from ethyl acetate,
yielding yellowish crystals of MDI-Val (42.8 mg, yield 80.6%, HPLC
method 2, purity = 97.7%, tR = 8.15 min). 1H NMR (d6-DMSO): 8.46
(s, 1 H, NH-CO-NHCH), 7.25 (d, J = 8.4, 2 H), 7.01 (d, J = 8.4, 2 H),
6.83 (d, J = 8.1, 2 H), 6.48 (d, J = 8.1, 2 H), 6.27 (d, J = 8.7, 1 H,
NHCH), 4.12 (dd, 1 H, NCHCH), 3.66 (s, 2 H, Ar−CH2-Ar), 2.08
(mc, 2 H, CH(CH3)2), 0.92 (d, J = 6.9, 3H, CH3), 0.88 (d, J = 6.9, 3H,
CH3). 13C NMR (d6-DMSO): 173.5 (COOH), 154.9 (NHCONH),
146.3 (Ar C), 137.8 (Ar C), 134.9 (Ar C), 128.8 128.5 (Ar CH), 128.6
(Ar C), 117.5 (Ar CH), 113.9 (Ar CH), 57.1 (CHCHCH3), 40.4 Ar-
CH2−Ar), 30.1 (CH(CH3)2), 18.9 (CH3), 17.4 (CH3). ESI-MS
(positive) m/z: 342.2 [M + H]+, MS/MS of 342.2 → 118.3, 72.2. ESI-
MS (negative) m/z: 340.1 [M − H]−, MS/MS of 340.1 → 116.0.
N-([4-{4-Amino-3,5-dideuterobenzyl}2,6-dideuterophenyl]-
carbamoyl)-valine (d4-MDI-Val). d4-MDI-Val was synthesized accord-
ing to the synthesis of MDI-Val. Methyl-S-(−)-2-isocyanato 3-
methylbutyrate ester (1.51 mg, 10 μmol) in 3.5 mL of dichloro-
methane was added to d4-MDA (10.8 mg, 50 μmol) in dichloro-
methane (2 mL). After hydrolysis with NaOH, 2.5 mg (72%) of d4-
MDI-Val was obtained (HPLC method 2, tR = 8.3 min, 93% purity).
ESI-MS (positive) m/z: 346.1 [M + H]+, 118.1. ESI-MS (negative) m/
z: 344.0 [M − H]−, 116.0.
N-([4-(4-Aminobenzyl)phenyl]carbamoyl)-glutamic Acid (MDI-
Glu). MDA (495 mg, 2.5 mmol) dissolved in dichloromethane (10
mL) was added slowly to S-(−)-2-isocyanatoglutaricacid diethyl ester
(114 mg, 0.5 mmol) dissolved in dichloromethane (2 mL) at room
temperature. After it was stirred for 2 h, the reaction was stopped.
According to HPLC analysis, most of the isocyanate reacted with
MDA, yielding the MDI-glutaric acid diethyl ester (mono, 94%) and
dimer (6.0%). n-Hexane (60 mL) was added to the reaction mixture
and mixed well. The solution was washed with acid (pH 2, 4 × 15
mL). The pH of the aqueous phase was adjusted to 8 with NaHCO3
and extracted with ethyl acetate (4 × 10 mL). The ethyl acetate layer
was dried with anhydrous sodium sulfate, filtrated, and concentrated
with a rotoevaporator to yield the crude MDI-glutamic acid diethyl
ester. The crude ester was dissolved in tetrahydrofuran (2 mL). Water
(5 mL) was added, and the reaction mixture was cooled on ice to 0 °C.
NaOH (0.25 M, 6 mL) was added dropwise. After 2 h, the reaction
mixture was washed with ethyl acetate (3 × 25 mL). The pH of the
aqueous phase was adjusted to 4 with 2 M HCl (log D = 0.7 of MDI-
Glu at pH 4.0). While adjusting the pH of the reaction mixture from 8
to 4, a white solid precipitated. The white precipitate was collected
(HPLC analysis = MDI-Glu, 97.6% purity). The aqueous phase was
extracted with ethyl acetate (3 × 20 mL). The organic phase was dried
with anhydrous sodium sulfate, filtered, and concentrated in a
Method 2: Synthesis of Amino Acid Adducts Using BocMDI. N-
Acetyl-S-[[4-(4-aminobenzyl)phenyl]carbamoyl]-cysteine (MDI-
AcCys). Freshly prepared hot BocMDI in 1,4-dioxane was added
dropwise to a solution of N-acetyl-L-cysteine (50 mg, 0.226 mmol) in
0.25 M sodium bicarbonate (5 mL, pH 8.3). The reaction temperature
was kept at 25 °C. Ultrasonication was applied for 3 min for every 10
min interval. After 2 h, the reaction mixture was cooled with ice and
then filtered. The filtrate was evaporated to a final volume of
approximately 5.0 mL and then extracted with ethyl acetate (3 × 10
mL). The ethyl acetate phase was dried with anhydrous sodium sulfate,
filtered, and evaporated on a rotoevaporator. The crude product
(BocMDI-AcCys, purity = 81.7%, 28.8 mg) was treated with
trifluoroacetic acid (300 μL) for 20−25 min. After it was evaporated
under reduced pressure and dried with a high vacuum pump, MDI-
AcCys was recrystallized from ethyl acetate yielding yellow crystals
with a purity of 90% (HPLC method 2, tR = 6.5 min) (44.2 mg, yield
77%). 1H NMR (d6-DMSO): 8.27 (d, 1 H, J = 8.0, NH-CO-NHCH),
7.39 (d, J = 8.6, 2 H), 7.14 (d, J = 8.2, 2 H), 7.13 (d, J = 8.6, 2 H), 6.96
(d, J = 8.3, 2 H), 6.45 (d, J = 7.4, CONHCH), 4.42 (dt, J = 5.9, 8.6,
CHCH2), 3.83 (s, Ar−CH2-Ar), 2.76 (dd, J = 16.3, 4.8, 1 H, CHCH2),
2.68 (dd, J = 16.3, 5.8, 1 H, CHCH2), 1.84 (s, 3H, NHCOCH3). ESI-
MS (positive) m/z: 388.1 [M + H]+.
N-Acetyl-S-[(4-{4-amino-3,5-dideuterobenzyl}2,6-
dideuterophenyl)carbamoyl]-cysteine (d4-MDI-AcCys). Boc-d4-MDA
(2 mg, 66 μmol) was added to a solution of triphosgene (1.95 mg, 66
μmol) and triethylamine (667 μg, 66 μmol) in anhydrous 1,4-dioxane
(1 mL). The reaction mixture was stirred for 3 h at 80 °C under
nitrogen and then added slowly to a solution of N-acetyl-L-cysteine
(1.5 mg) in 0.25 M sodium bicarbonate (2.5 mL, pH 8.2) at room
temperature. After every 10 min, the mixture was sonicated for 3 min.
The reaction was monitored by HPLC. After 2 h, the reaction mixture
was cooled with ice and then filtered. The filtrate was evaporated to a
final volume of approximately 1 mL and then extracted with ethyl
acetate (3 × 2 mL). The ethyl acetate phase was dried with anhydrous
sodium sulfate, filtered, and evaporated on a rotoevaporator. The
crude product was treated with trifluoroacetic acid (100 μL) for 20−25
min. After it was evaporated under reduced pressure and dried with a
high vacuum pump, d4-MDI-AcCys was obtained with a purity of 84%
(HPLC method 2, tR = 6.8 min). ESI-MS (positive) m/z: 392.1 [M +
H]+, MS/MS of 392.1 → 164.1.
MDI-[13C515N]Val. L-[13C515N]Val (10 mg, 81.3 μmol) in 0.25 M
sodium bicarbonate (2.5 mL, pH 8.2) was reacted with BocMDI (81.3
μmol). After the final hydrolysis with trifluoroacetic acid, MDI-
[13C415N]Val (61%) and the corresponding hydantoin 3-[4-(4-
aminobenzyl)phenyl]-5-[(13C3)propan-2-yl](4,5-13C2,1-15N)-
imidazolidine-2,4-dione (MDI-[13C515N]Val-Hyd) (31%) were ob-
tained. MDI-[13C415N]Val was purified by HPLC (purity 90%, HPLC
method 2, tR = 8.0 min) with solvent A (10 mM ammonium acetate)
and solvent B (methanol): 20 min 30−80% B linear gradient. tR = 8.0
min for MDI-[13C515N]Val and 12.2 min for MDI-[13C515N]Val-Hyd.
ESI-MS (positive) m/z: [M + H]+ 348.3, 124.0 and [M + H]+ 330.2,
respectively.
MDI-[13C415N]Asp. L-[13C415N]Asp (10 mg, 72.5 μmol) dissolved in
0.25 M sodium bicarbonate (2 mL, pH 8.2) was reacted with freshly
prepared BocMDI (72.5 μmol) dissolved in dry 1,4-dioxane (1 mL).
After the final hydrolysis with trifluoroacetic acid and evaporation,
MDI-[13C415N]Asp (2.2 mg, 8% yield) was obtained (93% HPLC
purity, method 1, tR = 10.7 min). ESI-MS (negative) m/z: [M − H]−
361, MS/MS of 361.1 → 137.0. Acid hydrolysis (2 M HCl, 30 min,
100 °C) yielded [1-[4-(4-aminobenzyl)phenyl]-2,5-dioxo-
(4,5-13C2,3-15N)imidazolidin-4-yl](13C2)acetic acid (MDI
-[13C415N]Asp-Hyd). ESI-MS (negative) m/z: 343.0 [M − H]−,
MS/MS of 343.0 → 299.2, 254.0.
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rotoevaporator. After this was dried at the high vacuum pump, we
obtained yellowish white crystals of MDI-Glu (97.6% HPLC purity,
method 1, tR = 10.7 min): total yield (precipitate + extract) = 80.6%.
1H NMR (d6-DMSO): 8.59 (s, 1 H, NH-CO-NHCH), 7.25 (d, J = 8.4,
of MDA. The small amount of d4-MDI-Asp did not precipitate.
Therefore, the water phase (pH 4) was applied to a solid-phase
column (RP18). d4-MDI-Asp was eluted with methanol (6 mL). After
evaporation, d4-MDI-Asp was obtained with 93% purity (HPLC
method 1: tR = 11.0 min). ESI-MS (positive) m/z: 362.0 [M + H]+,
229.2, 134.1. ESI (negative) m/z: 360.0 [M − H]−, 132.2.
N-[(4-{4-Acetylamino-3,5-dideuterobenzyl}2,6-dideuterophenyl)-
carbamoyl]-aspartic Acid (d4-AcMDI-Asp). The procedure to
synthesize d4-MDI-Asp was applied. d4-AcMDA was taken instead of
d4-MDA. d4-AcMDI-Asp had the following MS properties. ESI-MS
(positive) m/z: 404.3 [M + H]+, MS/MS of 404.3 → 245.2, 134.2.
ESI-MS (negative) m/z: 402.0 [M − H]−, MS/MS of 402.0 → 132.0.
Method 4: Synthesis of Amino Adducts Using 4′-Isocyanate-4-
nitrodiphenyl Methane (NO2MDI). 4′-Amino-4-nitrodiphenylmethane
(NO2MDA). A 6 M HCl solution (46 mL) was added to a solution of
NO2AcMDA (3.66 mmol) in ethanol (95 mL). The reaction mixture
was kept under reflux for 3 h. A saturated Na2CO3 solution (50 mL)
was added carefully. The mixture was extracted with dichloromethane
(5 × 20 mL). The organic layer was dried over MgSO4, filtered, and
evaporated under reduced pressure. Crystallization from EtOH yielded
750 mg (90%) of NO2MDA as a yellow solid.
2 H), 7.01 (d, J = 8.4, 2 H), 6.82 (d, J = 8.3, 2 H), 6.48 (d, J = 8.3, 2
H), 6.44 (d, J = 7.5, 1 H, NHCH), 4.19 (mc, 1 H, CHCH2), 3.66 (s, 2
H, Ar−CH2-Ar), 2.28 (mc, 2 H, CHCH2CH2), 1.97 (mc, 1 H,
CHCH2), 1.81 (mc, 1 H, CHCH2). 13C NMR (d6-DMSO): 173.7
(COOH), 173.4 (COOH), 154.8 (NHCONH), 146.3 (Ar C), 137.9
(Ar C), 134.9 (Ar C), 128.8 (Ar CH), 128.4 (Ar CH), 128.6 (Ar C),
117.6 (Ar CH), 113.9 (Ar CH), 51.5 (CHCH2CH2), 40.4(Ar-CH2−
Ar), 29.9 (CHCH2CH2), 27.3 (CHCH2CH2). ESI-MS (positive) m/z:
372.1 [M + H]+, MS/MS of 372.1 → 148.2, 84.0. ESI-MS (negative)
m/z: 370.1 [M − H]−, MS/MS of 370.1 → 146.1, 128.1.
N-[(4-{4-Acetylamino-3,5-dideuterobenzyl}2,6-dideuterophenyl)-
carbamoyl]-glutamic Acid (d4-AcMDI-Glu). Synthesized from d4-
AcMDI (10 mg, 41 μmol) and S-(−)-2-isocyanatoglutaricacid diethyl
ester (9 mg, 41 μmol) yielded 15.2 mg (88%) of d4-AcMDI-Glu as a
gray solid. The HPLC purity was 97% (method 1, tR = 12.9). ESI-MS
(negative) m/z: 416.0 [M − H]−, MS/MS of 416.0 → 146.0.
N-([4-{4-Amino-3,5-dideuterobenzyl}2,6-dideuterophenyl]-
carbamoyl)-glutamic Acid (d4-MDI-Glu). S-(−)-2-Isocyanatoglutar-
icacid diethyl ester (2.3 mg, 0.01 mmol) in dichloromethane (1 mL)
was added slowly to d4-MDA (10 mg, 0.05 mmol) in dichloromethane
(2 mL) according to the procedure described for MDI-Glu. After
hydrolysis and workup, 2.8 mg (7.4% yield) with a purity of 93.1%
(HPLC method 1, tR = 10.7 min) was obtained. ESI-MS (negative) m/
z: 374.1 [M − H]−.
N-[[4-(4-Aminobenzyl)phenyl]carbamoyl]-aspartic Acid (MDI-
Asp). A mixture of L-aspartic acid dimethyl ester hydrochloride
(197.6 mg,1 mmol) and triethylamine (223.6 mg, 2.2 mmol) dissolved
in dichloromethane (4 mL) was added dropwise to triphosgene (109.5
mg, 0.37 mmol) dissolved in dichloromethane (2 mL) over the period
of 30 min. After 1 h, the reaction mixture was added slowly at room
temperature to MDA (990 mg, 5 mmol) dissolved in dichloromethane
(10 mL). After 2 h, the reaction was stopped based on the HPLC
results, indicating that most of the starting material reacted with MDA
and formed MDI-aspartic acid dimethyl ester (mono, 94%) and dimer
(5−6.0%). The workup procedure was the same as described in
section 1. The ethyl acetate phase dried the over anhydrous sodium
sulfate and concentrated on the rotoevaporator yielded a crude MDI-
aspartic acid dimethyl ester. The crude ester was taken into round-
bottom flask (50 mL), dissolved in tetrahydrofuran (2 mL), and mixed
well until it dissolved. Then, water (10 mL) was added, and the
solution was cooled to 0 °C with an ice-cold water bath. NaOH (7 mL,
0.25 M) was added dropwise. After 2 h, the reaction was stopped and
washed with ethyl acetate (3 × 25 mL). The pH of the aqueous phase
was adjusted to 4 with 2 M HCl (the log D of MDI-Asp is highest at
pH 4). While the pH of the reaction mixture was adjusted from 8 to 4,
a white solid precipitated. The mixture was centrifuged. The
supernatant was eliminated, and the remaining white solid was dried
under high vacuum overnight. White crystals of MDI-Asp were
obtained (HPLC method 1, purity 94.8%, tR = 10.9 min yield 85.9%).
1H NMR (d6-DMSO): 8.72 (s, 1 H, NH-CO-NHCH), 7.46 (d, J = 8.4,
2 H), 7.28 (d, J = 8.5, 2 H), 7.10 (d, J = 8.4, 2 H), 7.05 (d, J = 8.5, 2
H), 6.45 (d, J = 7.4, CONHCH), 4.42 (mc, CHCH2), 3.66 (s, Ar−
CH2-Ar), 2.76 (dd, J = 16.3, 4.8, 1 H, CHCH2), 2.68 (dd, J = 16.3, 5.8,
1 H, CHCH2). 13C NMR (d6-DMSO): 172.9 (COOH), 171.8
(COOH), 154.6 (NHCONH), 146.2 (Ar C), 137.9 (Ar C), 134.9 (Ar
C), 128.6 (Ar C), 128.8 (Ar CH), 128.4 (Ar CH), 117.6 (Ar CH),
114.0 (Ar CH), 48.8 (CHCH2), 39.6 (Ar-CH2−Ar), 37.4 (CHCH2).
ESI-MS (positive) m/z: [M + H]+ 358.3, MS/MS of 358.3 → 134.0,
74.1. ESI-MS (negative) m/z: 356.2 [M − H]−; MS/MS of 356.2 →
132.0, 88.0. Hydrolysis of MDI-Asp in 2 M HCl for 2 h yielded {1-[4-
(4-aminobenzyl)phenyl]-2,5-dioxoimidazolidin-4-yl}acetic acid (MDI-
Asp-Hyd). ESI-MS (negative) m/z: 338.0 [M − H]−, MS/MS of 338.0
→ 295.0, 251.2.
N-[[4-(4-Nitrobenzyl)phenyl]carbamoyl]valylglycyl-glycine
(NO2MDI-Val-GlyGly). NO2MDI-Val-GlyGly was obtained according
general procedure 1 using NO2MDA (360 mg, 1.58 mmol), phosgene
(325 mg, 1.1 mmol), and L-valine-glycine-glycine (235 mg, 1.1 mmol).
1
Yield (245 mg, 46%) colorless solid. H NMR (d6-DMSO): 8.65 (s,
Ar−NH), 8.35 (t, broad, NHCH2), 8.15−8.14 (m, Ar-H, Ar-H,
NHCH2, 3H), 7.47 (d, J = 8.8, 2 H), 7.30 (d, J = 8.5, 2 H), 7.12 (d, J =
8.5, 2 H), 6.35 (d, J = 8.5, NHCH), 4.00 (s, CH2), 3.77−3.75 (m, 5 H,
NHCH, 2 NHCH2), 1.98 (mc, CHMe2), 0.88 (d, J = 6.8, Me), 0.84 (d,
J = 6.8, Me). 13C NMR (d6-DMSO): 172.2, 171.3, 169.3 (COOH, 2
CONH), 155.3 (NHCONH), 150.2 (Ar C), 146.0 (Ar C), 138.9 (Ar
C), 132.6 (Ar C), 130.0 (Ar CH), 129.4 (Ar CH), 123.8 (Ar CH),
118.0 (Ar CH), 57.9 (NHCH), 41.8 (CH2), 40.7 (NHCH2), 40.0
(NHCH2), 31.1 (CHMe2), 19.5 (Me), 17.9 (Me). ESI-MS (positive)
m/z: 486.1 [M + H]+, 354.0.
N-([4-(4-Aminobenzyl)phenyl]carbamoyl)-valylglycyl-glycine
(MDI-Val-GlyGly). Pd−C and ammonium formate were added at room
temperature to a stirred solution of NO2MDI-Val-GlyGly in dry
methanol under nitrogen. After 3 h, the reaction mixture was filtered
over Celite. The solvent was evaporated in vacuum, and the residue
1
was dried. H NMR (d6-DMSO): 8.66 (s, NH), 8.36 (t, J = 5.9, 1 H,
NHCH), 8.07 (t, J = 5.5, 1 H, NHCH), 7.25 (d, J = 8.3, 2 H, Ar−H),
7.01 (d, J = 8.2, 2 H, Ar−H), 6.83 (d, J = 8.2, 2 H, Ar−H), 6.47 (d, J =
8.3, 2 H, Ar−H), 4.10 (mc, 1 H, NHCH), 3.80−3.69 (m, 4 H, 2 ×
NHCH2), 3.65 (s, CH2), 1.97 (mc, CHMe2), 0.89 (d, J = 6.8, Me),
0.85 (d, J = 6.8, Me). 13C NMR (d6-DMSO): 172.0 171.1 168.8 (CO),
155.1 (NHCONH), 146.5 (Ar C), 138.0 (Ar C), 134.9 (Ar C), 128.9
(Ar CH), 128.6 (Ar CH), 117.5 (Ar CH), 113.9 (Ar CH), 57.7 (CH),
41.6 (CH2), 40.9 (CH2), 39.8 (CH2), 30.8 (CH), 19.2 (CH3), 17.6
(CH3). ESI-MS (positive) m/z: 456.1 [M + H]+.
Method 5: Synthesis of Amino Acid Adducts Using MDI-AcCys
and AcMDI-AcCys. MDI-Val. L-Valine (6 mg, 51 μmol) and MDI-
AcCys (2 mg, 5.1 μmol) were stirred at 37 °C in 0.25 M sodium
phosphate (2 mL, pH 7.4). The product composition was analyzed by
HPLC. MDI-AcCys, MDI-Val, and the 1,3-bis({4-[4-aminobenzyl]-
phenyl}urea (MDI-MDA) eluted at 6.8, 8.6, and 14.5 min,
respectively. After 5 h, the ratio of the products was 1:16:8 for
MDI-AcCys, MDI-Val, and MDI-MDA, respectively. Pure MDI-Val
has been synthesized previously with method 3 (see above).
N2-Acetyl-N6-[[4-(4-aminobenzyl)phenyl]carbamoyl]-lysine (MDI-
AcLys). L-Nα-Acetyl-L-lysine (9.6 mg, 51 μmol) and MDI-AcCys (0.2
mg, 0.51 μmol) were stirred at 37 °C in 0.25 M sodium phosphate (2
mL, pH 7.4). After 1 h, no MDI-AcCys was present but mainly MDA
and MDI-AcLys in the ratio 1:1. Pure MDI-AcLys has been
17
synthesized previously with method 2.
AcMDI-AcLys. L-Nα-Acetyl-lysine (9.6 mg, 51 μmol) and AcMDI-
AcCys (219 μg, 0.51 μmol) were stirred at 37 °C in 0.25 M sodium
phosphate (2 mL, pH 7.4). After 30 min, no AcMDI-AcCys was
present but mainly AcMDA and AcMDI-AcLys in the ratio 1:1. The
N-([4-{4-Amino-3,5-dideuterobenzyl}2,6-dideuterophenyl]-
carbamoyl)-aspartic Acid (d4-MDI-Asp). The procedure used to
synthesize MDI-Asp was applied. d4-MDA (10 mg) was taken instead
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Figure 2. Methods to synthesize amino acid adducts of MDI. 1)PLE = pig liver esterase.
product composition was analyzed by HPLC: A, 0.1% formic acid; B,
methanol; linear gradient from 10 to 70% B in 19 min. Pure AcMDI-
AcLys has been synthesized with method 1; see above.
products were only partially converted to the deacetylated products
MDI-Val, MDI-Asp, and MDI-Glu, respectively.
Digestion of in Vitro-Modified Human Serum Albumin.
Unmodified albumin (5 mg), 100 μg of d4-MDI-albumin (modified
with d4-MDI-AcCys/albumin = 1/100), and MDI-[13C615N2]Lys
(13.25 pmol) in 50 mM ammonium bicarbonate buffer (2 mL, pH
8.9 adjusted with NaOH) were incubated for 15 h at 37 °C with
Pronase E (1.7 mg) following a procedure published previously for the
analysis of albumin from rats exposed to MDI.17 The digest was
acidified to pH 4.0 with 2 M hydrochloric acid and purified with solid-
phase extraction (Strata-X-33u, polymeric reversed phase columns,
200 mg). The columns were activated with 3 mL of methanol and then
equilibrated with 3 mL of 0.1% formic acid (pH 4.0). The samples
were applied on the columns and subsequently washed with 3 mL
fractions of 0, 10, and 20% methanol in 0.1% formic acid. d4-MDI-Lys
and MDI-[13C615N2]Lys were eluted with 6 mL of 80% methanol in
0.1% formic acid. The eluates were concentrated to approximately 1
mL in a speed evaporator at 45 °C and analyzed with LC-MS/MS.
The compounds were separated on a C-18 reversed phase column
(Luna C18(2), 100 Å, 150 mm × 2.0 mm, 3 μm) using a gradient
elution system with solvent A (10 mM ammonium formate) and
solvent B (methanol) at a flow rate of 0.2 mL/min: 0 (B 20%), 3 (B
20%), 16 (B 90%), and 20 min (B 90%). Quantitation of d4-MDI-Lys
was performed using the calibration line obtained from MDI-Lys and
MDI-[13C615N2]Lys as described previously.17,18 d4-MDI-Lys was
present at 9.93 0.42 pmol (4.2% relative standard deviation of 5
experiments) per mg modified albumin. The same procedure was used
for the digestion of albumin modified in vitro with MDI-AcCys and
AcMDI-AcCys, respectively. The quantitation of MDI-Lys (Figure 3)
and AcMDI-Lys was performed as described previously.17,18
In Vitro Modification of Human Serum Albumin with MDI-AcCys,
AcMDI-AcCys, and d4-MDI-AcCys. MDI-AcCys (0.15 μmol, 1.0 mg/
mL) dissolved in sodium phosphate buffer (50 mM, pH 7.4) was
added dropwise to a solution (3 mL) of albumin (10 mg, 0.15 μmol)
in sodium phosphate buffer (50 mM, pH 7.4) at 37 °C. After 16 h, the
reaction mixture was centrifuged to separate off the white precipitate.
The supernatant was washed with ethyl acetate (2 × 3 mL). Sodium
chloride was added to separate the organic and aqueous layers clearly.
The protein solution (3 mL) was transferred into a 15 cm dialysis tube
(Fisher brand, 11000−14000 molecular weight cutoff, 0.32 mL/cm,
0.2 mm thickness), which was presoaked in deionized water for 20
min. The dialysis was carried out against deionized water (2 L) at 4
°C. After 4 h, the dialysis was stopped. The concentration of the
obtained albumin solution (4 mL) was determined using the
Coomassie plus protein assay reagent (#23236) for protein
determination from Thermo Scientific (Rockford, IL). Using the
same method, albumin was modified with MDI-AcCys at two different
levels, 1:1 and 1:10 mol/mol, using the above procedure. Albumin was
modified with d4-MDI-AcCys in the same manner (100:1 mol/mol).
Deacetylation of AcMDI Derivatives. Pig liver esterase (PLE) (50
μL) was added to AcMDI-Lys (2.5 mg) dissolved in 50 mM sodium
phosphate buffer (pH 7.4) following a procedure by Schutze et al.25
After incubation at 37 °C in a shaking bath for 1 day, the deacetylated
product MDI-Lys was obtained. According to HPLC analyses, the
parent compound was converted to the final product. The same
reaction conditions were applied to the compounds AcMDI-Val,
AcMDI-Asp, and AcMDI-Glu. According to HPLC analyses, these
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Determination of the N-Terminal Adduct in Human Albumin
Modified in Vitro with MDI-AcCys at a Molar Ratio of 1:1. (a)
Control albumin, (b) in vitro-modified albumin (0.5 mg), and (c)
albumin (0.5 mg) with MDI-Asp (69.6 pmol) were hydrolyzed with 2
M HCl for 2 h in the presence of the internal standard MDI-
[13C415N]Asp (68.4 pmol). The digests were purified by solid-phase
extraction as described for the analysis of MDI-Lys18 and analyzed
with LC-MS/MS. The compounds were separated on a C-18 reversed
phase column (Luna C18(2), 100 Å, 150 mm × 2.0 mm, 3 μm), using
a gradient elution system with solvent A (10 mM ammonium formate)
and solvent B (methanol) at a flow rate of 0.2 mL/min: 0 (B 20%), 3
(B 20%), 16 (B 90%), and 20 min (B 90%). LC-MS/MS analyses
showed the formation of the corresponding hydantoin MDI-Asp-Hyd
(MS/MS of m/z 338 [M − H]− → 295.0, 251.2) with the same
retention time 12.9 min as the internal standard MDI-[13C415N]-Asp-
Hyd (MS/MS of m/z 343 [M − H]− → 299.2, 254.0. For quantitation
of experiments a and b, the peak ratio of MDI-Asp-Hyd/MDI-
[13C415N]-Asp-Hyd = 0.99 was taken from experiment c.
Degradation of MDI-AcCys and AcMDI-AcCys in Buffer Solutions.
MDI-AcCys (1.4 mg) was dissolved in 50 mM sodium phosphate (2.0
mL, pH 7.4) and stirred at 37 °C. The progress of the reaction was
monitored by HPLC. A 10 μL aliquot of the reaction mixture was
diluted with methanol (90 μL) and then analyzed by HPLC (25 μL)
using method 2. This was repeated after 1, 3, 5, and 20 h. The
logarithmic values of the obtained peak areas were plotted against the
time (r2 = 0.99).
respectively (Figure 2). The reaction of AcMDI with N-acetyl-
serine, Nα-acetyl-tyrosine, Nα-acetyl tryptophan, and Nα-acetyl
histidine was not successful with this method (data not shown).
This is partially in contrast to reactions of 4-methylphenyl
isocyanate (4MPI) published previously where adducts with N-
acetyl-serine and Nα-acetyl-tyrosine could be accomplished.27
For the synthesis of the nonacetylated products, the AcMDI
adducts obtained with method 1 were incubated with pig liver
esterase to yield the corresponding MDI-amino acid adducts.
This method worked for AcMDI-Lys but only partially for the
deacetylation of AcMDI-Val, AcMDI-Asp, and AcMDI-Glu.
Therefore, another approach was taken to synthesize MDI-Val,
MDI-Asp, and MDI-Glu.
We applied method 2 (Figure 2) starting from BocMDA, a
MDA derivative with one amino group protected with Boc.
BocMDI was generated from BocMDA and triphosgene. MDI-
Lys17 was obtained after reaction of BocMDI with Nα-Boc-Lys
and subsequent hydrolysis with trifluoroacetic acid. This
method was applied to the synthesis of the new cysteine
adduct MDI-AcCys. For the adducts with the N-terminal acids
(aspartic acid in human albumin, glutamic acid in rat albumin,
and valine in hemoglobin), the acidic conditions (TFA at room
temperature) used to cleave the Boc group yielded also partially
the corresponding hydantoins, for example, MDI-Val-Hyd (see
Figure 2). In this case, we had to purify the compounds with
HPLC. For a complete transformation to MDI-Val-Hyd,
The same approach was used to determine the stability kinetics of
AcMDI-AcCys. In this case, aliquots of 10 μL were measured after 0, 1,
4, 7, 22, and 26 h. The logarithmic values of the obtained peak areas
were plotted against the time (r2 = 0.96). AcMDI-AcCys decomposed
to AcMDA (tR = 9.7 min, identical with standard AcMDA, tR = 9.7
min) and to N-[4-({4-[({4-[4-acetamidobenzyl]phenyl}carbamoyl)-
16
boiling in 2 M HCl is necessary as shown previously.
Therefore, method 3 was applied to synthesize MDI-Val,
MDI-Asp, and MDI-Glu (Figure 2). The isocyanate function
was introduced on the α-amino group of the amino acids. 2-
Isocyanato aspartic acid dimethyl ester was synthesized from L-
aspartic acid dimethyl ester hydrochloride and triphosgene
following a method used for the synthesis of the corresponding
glutaric acid isocyanate.30 The isocyanates of valine and glutaric
acid, isocyanato 3-methylbutyrate ester, and 2-isocyanato
glutaric acid diethyl ester, respectively, were commercially
available. To avoid the reaction of both amino groups of MDA
with the amino acid isocyanate, 1-mol equiv of amino acid
isocyanate was added to 5 mol equiv of MDA. MDI-Glu, MDI-
Asp, and MDI-Val were obtained with more than 80% yield
(Figure 2). The corresponding deuterated standards were
synthesized following the same procedure but using d4-MDA
instead of MDA. d4-MDI-Glu, d4-MDI-Asp, and d4-MDI-Val
were obtained. Method 3 has great potential for the synthesis of
urea compounds deriving from amino acid adducts and
aromatic or aliphatic isocyanates.
Method 4 (Figure 2) was used to synthesize adducts of MDI,
which are not N-acetylated. For the analysis of N-terminal
adducts of isocyanates with hemoglobin, the isocyanate-
modified tripeptide AcMDI-Val-Gly-Gly, and the isotope-
labeled d4-AcMDI-Val-Gly-Gly were used to generate the
calibration line for the quantitation of the adducts present in
rats.16 However, in vivo the major adduct with the N-terminal
amino acidvalinewill be the nonacetylated MDI derivative
according to the results obtained for the albumin adducts MDI-
Lys and AcMDI-Lys.17 Therefore, for the present work, we
propose the synthesis of the nonacetylated product, MDI-Val-
Gly-Gly (Figure 2). 4-Amino-4-nitrodiphenylmethane
(NO2MDA) was reacted with triphosgene to obtain
NO2MDI, which was used for the reaction with Val-GlyGly
to obtain NO2MDI-Val-Gly-Gly. The nitro group of NO2MDI-
Val-Gly-Gly was reduced with ammonium formate and Pd on
carbon to yield MDI-Val-Gly-Gly.
amino]phenyl}methyl)phenyl]acetamide (AcMDI-AcMDA) (tR
=
18.1 min). After 26 h, AcMDA and AcMDI-AcMDA are the main
products formed in the ratio 3:4. ESI-MS (positive) of AcMDI-
AcMDA: m/z 507 [M + H]+; ESI-MS (negative) m/z: 505 [M − H]−.
The same experiment was performed with AcMDI-AcCys in 50 mM
ammonium bicarbonate buffer adjusted to pH 8.9. The half-life of the
products was 15 min. AcMDA eluting at tR = 8.9 min was the major
product after 3 h. AcMDI-AcMDA was a minor product
(AcMDA:AcMDI-AcMDA = 20:1).
RESULTS AND DISCUSSION
■
For the analysis of in vivo-modified proteins, especially albumin
and hemoglobin, we synthesized different potential reaction
products of MDI with amino acids. The most reactive centers
are the N-terminal acids such as valine in hemoglobin, glutamic
acid in rat albumin, aspartic acid in human albumin, the ε
amino groups of lysine, the hydroxyl group in serine and
tyrosine, the nitrogen in histidine and tryptophan, and sulfur of
cysteine. We applied several synthetic strategies (methods 1−5,
Figure 2). The adducts obtained in vivo can be acetylated on
the free amino group of MDA, yielding, for example, AcMDI-
Lys, as shown previously in rat albumin.17 Therefore, the
corresponding adducts derived from AcMDI had to be
synthesized.
For the first method, we synthesized AcMDI from AcMDA
and triphosgene (method 1). The α-amino groups of the amino
acids were protected with a Boc group or with an acetyl group,
except for valine, glutamic acid, and aspartic acid. The Boc
group can be cleaved with trifluoroacetic acid at room
temperature. The acetyl group can be cleaved enzymatically
with acylase. AcMDI was reacted with Nα-Boc-Lys and Val to
yield AcMDI-Lys17 and AcMDI-Val,16 respectively. The same
procedure was applied to the present work with glutamic acid,
aspartic acid, N-acetyl cysteine, and Nα-acetyl-lysine yielding
AcMDI-Glu, AcMDI-Asp, AcMDI-AcCys, and AcMDI-AcLys,
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All final products synthesized with methods 1−4 had to be
extracted from the water phase. To establish the pH to obtain
the best yields for the extractions into organic solvent, the log D
values of the adducts were calculated using software by
ACDlabs and/or Chemaxon. In most cases, the products were
most lipophilic (=highest log D − value) at pH 4, except for the
hydantoinsMDI-Asp-Hyd and MDI-Val-Hyd. The obtained
products were characterized with 1H NMR, 13C NMR, and ESI-
MS.
Important vehicles for isocyanates are their reaction products
with glutathione.14,15 The glutathione adducts release the
isocyanate moiety to react with other nucleophiles, for example,
proteins. Therefore, for method 5 (Figure 2), we investigated
the reactions of cysteine adducts of MDI (MDI-AcCys and
AcMDI-AcCys) with amino acids and then with albumin. MDI-
AcCys was reacted at pH 7.4 with valine and Nα-acetyl-lysine
to obtain MDI-Val and MDI-AcLys, respectively. A large excess
(100:1) of amino acid leads to less side products and shorter
reaction times as demonstrated with the synthesis of MDI-
AcLys. AcMDI-AcCys was used to synthesize AcMDI-AcLys at
pH 9. Nα-Acetyl-lysine was used in large excess, 100:1. The
HPLC profile of the reaction mixture did not change after 30
min. Therefore, for syntheses with MDI-AcCys derivatives, high
excess of amino acids should be used. These reactions were all
performed at room temperature. Therefore, because the
synthesis of AcMDI-AcLys and MDI-Val was successful, also
reactions with proteins should be successful under mild
conditions. The harsh reaction conditions described in methods
1−4 are not recommended for reactions with proteins.
The stability of MDI-AcCys and AcMDI-AcCys was studied
at pH 7.4. The half-life (=3.1 h) of MDI-AcCys was calculated
from the equation of the linear regression curve. The half-life of
AcMDI-AcCys was estimated to be 7.1 h. The main
decomposition products of AcMDI-AcCys were AcMDA and
AcMDI-AcMDA according to the performed HPLC analyses.
The same experiment was performed with AcMDI-AcCys at
pH 8.9. The half-life of the products was 15 min. AcMDA was
the major product.
Figure 3. Main reaction product obtained from albumin modified in
vitro with (A) MDI-AcCys at a molar ratio of 1:1 and with (B) d4-
MDI-AcCys at a molar ratio of 1:100 (d4-MDI-AcCys/albumin).
Albumin was digested with Pronase in the presence of the internal
standard MDI-[13C615N2]-Lys (13.25 pmol). The digest was purified
by solid phase extraction and analyzed with LC-MS/MS.
times. The variation coefficient was 4.2% (=relative standard
deviation).
Reactions with Albumin. Albumin was reacted at pH 7.4
using different molar ratios with MDI-AcCys and/or AcMDI-
AcCys. Albumin was digested with Pronase and analyzed with
LC-MS/MS (Figure 3). MDI-Lys and AcMDI-Lys were
quantified as described previously.17 In the experiments with
a molar ratio of 1:1 and 10:1 of MDI-AcCys to albumin, 4432
and 34324 pmol of MDI-Lys were found per mg of albumin. In
the experiments with a molar ratio of 1:1 and 10:1 of AcMDI-
AcCys to albumin, 3446 and 24757 pmol of AcMDI-Lys were
found per mg of albumin. Albumin was reacted with MDI-
AcCys at the ratio of 1:1 at pH 7.4 and at pH 9. The adduct
levels for MDI-Lys were 1.3 larger at pH 9 than at pH 7.4.
For the analysis of protein adducts of xenobiotics, enzymatic
digestions have been used.31 Ideally, all methods using
enzymatic digestion procedures should take into account the
variation of the digestion yields. Therefore, we synthesized
albumin adducts of d4-MDI, which can be used as internal
standard for the analysis of biological standards. For this
purpose, we synthesized first d4-MDI-AcCys. d4-MDI-AcCys
was reacted with albumin at pH 7.4 at a molar ratio of 1:100
(d4-MDI-AcCys/albumin). After enzymatic digestion of albu-
min with Pronase, we obtained 99.3 pmol of d4-MDI-Lys/mg
albumin (Figure 3). To test the precision of the method, we
repeated the digestion of d4-MDI-AcCys-modified albumin five
Albumin adducts of MDI have been in found in vivo17 and in
vitro32 with lysine. We investigated the presence of other
adducts such as the adduct with the N-terminal amino acid of
human albumin, which is aspartic acid. To achieve this, we
looked at proteins modified in vitro with MDI-AcCys. After
digestion of albumin with Pronase, we found only MDI-Lys.
We compared the digests of albumin to the LC-MS/MS
chromatograms of the synthetic standards (Figure 5 in the
Supporting Information). We monitored also the mass
fragment ions for the adducts that could not be synthesized
and characterized by NMR: MDI-His, MDI-Ser, MDI-Trp, and
MDI-Tyr, respectively. The following MS/MS transitions were
monitored: 378.2 → 154.2, 328.3 → 104.1, 427.3 → 203.2, and
404.4 → 180.1 for MDI-His, MDI-Ser, MDI-Trp, and MDI-
Tyr, respectively. These product ions correspond to the mass of
the parent amino acid. This is the main product ion seen in the
MS/MS analyses of the synthesized MDI-amino acid adducts.
According to the calculated lipophilic properties (calculated
with program by ChemAxon) of the compounds in a 10 mM
ammonium formate solution, the following elution time
increase was expected (=increasing lipophilicity): MDI-Asp,
MDI-His, MDI-Ser, MDI-Lys, MDI-Val, MDI-Trp, and MDI-
Tyr. Only MDI-Lys was found unambiguously in albumin
digested enzymatically with Pronase. This is contrast to results
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obtained with albumin modified in vitro with 4MPI (see below
and the Supporting Information). Other digestion procedures
as described for the analysis of nitrotyrosine33 might release
some of the other adducts. We applied acid hydrolysis to
release the N-terminal amino acid of albumin, a method that
has been successfully used for years for the analysis of N-
terminal adducts of environmental pollutants.34 The N-terminal
adduct with aspartic acid was detected in in vitro-modified
albumin after acid hydrolysis (Figure 4). The hydrolysis and the
literature.27 The α-amino acetyl group of the adducts 4MPI-
AcLys, 4MPI-AcSer, and 4MPI-AcTyr was cleaved with acylase
following a procedure applied successfully for aflatoxin adducts
of Nα-Ac-Lysine.28 The obtained products were compared to
the digest of 4MPI-modified albumin. 4MPI-Lys appears to be
the major product, since the relative ionization and dissociation
efficiencies of such different amino acid adducts are
comparable. However, the efficiency of Pronase to release
these different 4MPI adducts from albumin might be different.
Other adducts such as 4MPI-Asp, 4MPI-Ser, 4MPI-Lys, and
4MPI-Tyr were present (Figure 6 in the Supporting
Information). In control albumin, these adducts were not
found. Therefore, more products with amino acids could be
found in albumin modified with 4MPI than in albumin
modified with MDI derivatives. Possibly the larger MDI
adducts might hinder the digestion of albumin to the single
amino acids.
The methods developed for the synthesis of amino acid
adducts of MDI should be applicable to all aromatic and
aliphatic isocyanates. Blood protein adducts can be synthesized
under physiological conditions using MDI-AcCys, and AcMDI-
AcCys. This will enable us to optimize the synthesis of standard
antigens, which can be used in the immunoassays for the
quantitation of antibodies in exposed workers. The immuno-
logical results obtained in the past have been controversial and
topic of critical reviews.35,36 In future studies, it will be
important to produce antigens according to the adducts found
in vivo. Then, the immunological tests, the disease status, and
the albumin adduct levels can be compared to confirm the
relationship between the two biomarkers and the disease.
ASSOCIATED CONTENT
* Supporting Information
■
S
Figures of LC-MS/MS (negative ESI) analyses of the standard
compounds and reaction products. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: 504-988-2771. E-mail: gabriele.sabbioni@bluewin.
■
ch.
Funding
We are most grateful for the financial support of the Tulane
Cancer Center.
Notes
The authors declare no competing financial interest.
Figure 4. N-terminal adduct of MDI found in human albumin
modified in vitro with MDI-AcCys at a molar ratio of 1:1. (A) Control
albumin and (B) in vitro modified albumin (0.5 mg) were hydrolyzed
with 2 M HCl for 2 h in the presence of the internal standard MDI-
[13C415N]Asp (68.4 pmol). LC-MS/MS analyses showed the
formation of the corresponding hydantoin MDI-Asp-Hyd with the
same retention time as the internal standard MDI-[13C415N]-Asp-Hyd.
ABBREVIATIONS
■
4MPI, 4-methylphenyl isocyanate; 4MPI-AcLys, N2-acetyl-N6-
[(4-methylphenyl)carbamoyl]-lysine; 4MPI-AcSer, N-acetyl-O-
[(4-methylphenyl)carbamoyl]-serine; 4MPI-AcTyr, N-acetyl-O-
[(4-methylphenyl)carbamoyl]-tyrosine; 4MPI-Asp, N-[(4-
methylphenyl)carbamoyl]-aspartic acid; 4MPI-Lys, N6-[(4-
methylphenyl)carbamoyl]-lysine; 4MPI-Ser, O-[(4-
methylphenyl)carbamoyl]-serine; 4MPI-Tyr, O-[(4-
methylphenyl)carbamoyl]-tyrosine; AcMDA, N1-[4-(4-
aminobenzyl)phenyl]acetamide; AcMDI, N-[4-(4-
isocyanatobenzyl)phenyl]acetamide; AcMDI-AcCys, N-acetyl-
S-[[4-(4-acetylaminobenzyl)phenyl]carbamoyl]-cysteine;
AcMDI-Asp, N-[[4-(4-acetylaminobenzyl)phenyl]carbamoyl]-
aspartic acid; AcMDI-AcLys, N2-acetyl-N6-[[4-(4-
acetylaminobenzyl)phenyl]carbamoyl]-lysine; AcMDI-
workup were controlled using an isotope-labeled standard,
MDI-[13C415N]Asp. The levels of MDI-Asp-Hyd (5 pmol/mg)
are approximately 900 times lower than the MDI-Lys (4432
pmol/mg albumin) levels measured in the same samples after
enzymatic digestion with Pronase.
The reactions of MDI derivatives obtained with albumin in
vitro (see above) were compared with the results obtained with
4MPI. Albumin was reacted with 4MPI and then digested with
Pronase. The standards were obtained as described in the
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Respiratory and other hazards of isocyanates. Int. Arch. Occup. Environ.
Health 66, 141−152.
AcMDA, N-[4-({4-[({4-[4-acetamidobenzyl]phenyl}-
carbamoyl)amino]phenyl}methyl)phenyl]acetamide; AcMDI-
Glu, N-[[4-[4-acetylaminobenzyl]phenyl]carbamoyl]-glutamic
acid; AcMDI-Lys, N6-[[4-[4-acetylaminobenzyl]phenyl]-
carbamoyl]-lysine; AcMDI-Val, N-[[4-[4-acetylaminobenzyl]-
phenyl]carbamoyl]-valine; AcNO2MDA, N-acetyl-4′-amino-4-
nitrodiphenylmethane; BocMDA, tert-butyl [4-(4-
aminobenzyl)phenyl]carbamate; BocMDI, tert-butyl [4-(4-
isocyanatobenzyl)phenyl]carbamate; d4-AcMDA, N1-[4-(4-
amino-(3,5-dideuterobenzyl))-(2,6-dideuterophenyl)]-
acetamide; d4-AcMDI-Asp, N-[(4-{4-acetylamino-3,5-
dideuterobenzyl}2,6-dideuterophenyl)carbamoyl]-aspartic
acid; d4 -AcMDI-Glu, N-[(4-{4-acetylamino-3,5-
dideuterobenzyl}2,6-dideuterophenyl)carbamoyl]-glutamic
acid; d4-MDA, 4,4′-methylene-bis(2,6-dideuteroaniline); d4-
MDI-AcCys, N-acetyl-S-[(4-{4-amino-3,5-dideuterobenzyl}2,6-
dideuterophenyl)carbamoyl]-cysteine; d4-MDI-Asp, N-([4-{4-
amino-3,5-dideuterobenzyl}2,6-dideuterophenyl]carbamoyl)-
aspartic acid; d4 -MDI-Glu, N-([4-{4-amino-3,5-
dideuterobenzyl}2,6-dideuterophenyl]carbamoyl)-glutamic
acid; d4-MDI-Val, N-([4-{4-amino-3,5-dideuterobenzyl}2,6-
dideuterophenyl]carbamoyl)-valine; MDI, 4,4′-methylenedi-
phenyl diisocyanate; MDI-AcCys, N-acetyl-S-[[4-(4-
aminobenzyl)phenyl]carbamoyl]-cysteine; MDI-AcLys, N2-ace-
tyl-N6-[[4-(4-aminobenzyl)phenyl]carbamoyl]-lysine; MDI-
Asp, N-[[4-(4-aminobenzyl)phenyl]carbamoyl]-aspartic acid;
MDI-Asp-Hyd, {1-[4-(4-aminobenzyl)phenyl]-2,5-dioxoimida-
zolidin-4-yl}acetic acid; MDI-[13C515N]Asp-Hyd, [1-[4-(4-
aminobenzyl)phenyl]-2,5-dioxo(4,5-13C2,3-15N)imidazolidin-4-
yl](13C2)acetic acid; MDI-Lys, Nε-([4-(4-aminobenzyl)-
phenyl]carbamoyl)-lysine; MDI-Glu, N-([4-(4-aminobenzyl)-
phenyl]carbamoyl)-glutamic acid; MDI-MDA, 1,3-bis({4-(4-
aminobenzyl)methyl]phenyl}urea; MDI-Val, N-([4-(4-
aminobenzyl)phenyl]carbamoyl)-valine; MDI-Val-GlyGly, N-
([4-(4-aminobenzyl)phenyl]carbamoyl)-valylglycyl-glycine;
MDI-Val-Hyd, 3-[4-(4-aminobenzyl)phenyl]-5-(propan-2-yl)-
imidazolidine-2,4-dione; MDI-[13C515N]Val-Hyd, 3-[4-(4-
aminobenzyl)phenyl]-5-[(13C3)propan-2-yl](4,5-13C2,1-15N)-
imidazolidine-2,4-dione; NO2MDA, 4′-amino-4-nitrodiphenyl-
methane; NO2MDI-Val-GlyGly, N-[[4-(4-nitrobenzyl)phenyl]-
carbamoyl]valylglycyl-glycine; PLE, pig liver esterase
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