Characterization of the reactivity, regioselectivity, and stereoselectivity of the reactions of butadiene monoxide with valinamide and the N-terminal valine of mouse and rat hemoglobin

Article abstract of DOI:10.1021/tx990043x

Occupational exposure to 1,3-butadiene (BD) has been monitored by measuring the level of hemoglobin N-terminal valine adduct formation with the primary reactive metabolite, butadiene monoxide (BMO). However, mechanistic details concerning the relative reactivity, regioselectivity, and stereospecificity of BMO with the N-terminal valine of hemoglobin are lacking. In the studies presented here, L-valinamide was used as a model for the N-terminal valine of hemoglobin to compare the nucleophilic reactivity, regioselectivity, and stereoselectivity of the reaction both in aqueous solution and within a protein microenvironment. Four products produced by the reaction of L-valinamide with racemic BMO (two pairs of diastereomers produced by reactions at C-1 and C-2 of the epoxide moiety) were synthesized, purified, and characterized by ¹H NMR and GC/MS. These four reaction products were used as analytical standards for kinetic studies of the reaction of valinamide with BMO at physiological pH (7.4) and temperature (37 °C). The results show that the adducts formed by reaction at C-2 were formed at a ratio of approximately 2:1 compared to the adducts formed by reaction at C-1. The stereoisomers of each respective regioisomer were produced with similar rates of formation. The reaction of BMO with the N-terminal valine of hemoglobin was also studied in vitro using intact erythrocytes from Sprague- Dawley rats and B6C3F1 mice. After cleavage of the N-modified valine by the N-alkyl Edman degradation procedure using pentafluorophenyl-isothiocyanate (PFPITC), a novel procedure was developed that allowed GC/MS detection and quantitation of the four expected products by silylation of the PFPTH-valine- BMO derivatives. The hemoglobin results contrast with the valinamide results in that the reaction of BMO with the N-terminal valine residue in both rat and mouse hemoglobin produced mostly C-1 adducts. The rates obtained with rat hemoglobin were much slower than the rates obtained with mouse hemoglobin or with valinamide. These results, and the finding that the reaction with rat hemoglobin produced a higher ratio of C1:C2 adducts in comparison with the reaction with mouse hemoglobin, indicate the importance of measuring all four adducts when comparing the relative rates of adduct formation both with model compounds and among different species.

Full text of DOI:10.1021/tx990043x

Chem. Res. Toxicol. 1999, 12, 679-689
679
Ch a r a cter iza tion of th e Rea ctivity, Regioselectivity, a n d
Ster eoselectivity of th e Rea ction s of Bu ta d ien e
Mon oxid e w ith Va lin a m id e a n d th e N-Ter m in a l Va lin e of
Mou se a n d Ra t Hem oglobin
Thomas S. Moll and Adnan A. Elfarra*
Department of Comparative Biosciences and Center for Environmental Toxicology,
University of Wisconsin, Madison, Wisconsin 53706-1102
Received March 10, 1999
Occupational exposure to 1,3-butadiene (BD) has been monitored by measuring the level of
hemoglobin N-terminal valine adduct formation with the primary reactive metabolite, butadiene
monoxide (BMO). However, mechanistic details concerning the relative reactivity, regioselec-
tivity, and stereospecificity of BMO with the N-terminal valine of hemoglobin are lacking. In
the studies presented here, L-valinamide was used as a model for the N-terminal valine of
hemoglobin to compare the nucleophilic reactivity, regioselectivity, and stereoselectivity of the
reaction both in aqueous solution and within a protein microenvironment. Four products
produced by the reaction of L-valinamide with racemic BMO (two pairs of diastereomers
produced by reactions at C-1 and C-2 of the epoxide moiety) were synthesized, purified, and
1
characterized by H NMR and GC/MS. These four reaction products were used as analytical
standards for kinetic studies of the reaction of valinamide with BMO at physiological pH (7.4)
and temperature (37 °C). The results show that the adducts formed by reaction at C-2 were
formed at a ratio of approximately 2:1 compared to the adducts formed by reaction at C-1. The
stereoisomers of each respective regioisomer were produced with similar rates of formation.
The reaction of BMO with the N-terminal valine of hemoglobin was also studied in vitro using
intact erythrocytes from Sprague-Dawley rats and B6C3F1 mice. After cleavage of the
N-modified valine by the N-alkyl Edman degradation procedure using pentafluorophenyl-
isothiocyanate (PFPITC), a novel procedure was developed that allowed GC/MS detection and
quantitation of the four expected products by silylation of the PFPTH-valine-BMO derivatives.
The hemoglobin results contrast with the valinamide results in that the reaction of BMO with
the N-terminal valine residue in both rat and mouse hemoglobin produced mostly C-1 adducts.
The rates obtained with rat hemoglobin were much slower than the rates obtained with mouse
hemoglobin or with valinamide. These results, and the finding that the reaction with rat
hemoglobin produced a higher ratio of C1:C2 adducts in comparison with the reaction with
mouse hemoglobin, indicate the importance of measuring all four adducts when comparing
the relative rates of adduct formation both with model compounds and among different species.
tions with numerous cellular macromolecules, including
In tr od u ction
DNA (2) and proteins (3).
1,3-Butadiene (BD)1 is a carcinogenic and genotoxic
chemical used primarily as a chemical intermediate and
polymer component in the manufacture of synthetic
rubber and plastics, and is also listed by the EPA as a
hazardous air pollutant; environmental sources include
cigarette smoke and automobile exhaust. BD is bioacti-
vated by cytochrome P450-mediated oxidation to produce
racemic 1,2-epoxy-3-butene [butadiene monoxide (BMO)]
(reviewed in ref 1). This primary reactive metabolite has
been shown to undergo nucleophilic substitution reac-
Recently, several investigators have attempted to
develop highly specific and sensitive bioassays involving
the detection and quantitation of BMO adducts of the
N-terminal valine residue of hemoglobin after exposure
to BD. These assays are all based on a modified Edman
degradation procedure developed by To¨rnqvist et al. (4),
which has been termed the N-alkyl Edman method. The
derivatives of the alkylvaline adducts produced by this
method, the pentafluorophenylthiohydantoins (PFPTHs),
are quantitated using high-resolution GC/MS. Although
there has been great progress in the ability to detect and
quantitate specific adducts at low levels (<1 pmol of
adduct/g of globin) (5), very little data is available
concerning the regio- and stereoselective properties of the
reaction. Specifically, there is limited data concerning the
specific reactivity (i.e., kinetic rate constants) of BMO
with the N-terminal valine of hemoglobin (6-8). Relative
reactivities between species have been examined in a few
* To whom correspondence should be addressed: Department of
Comparative Biosciences, School of Veterinary Medicine, University
of WisconsinsMadison, 2015 Linden Dr. W., Madison, WI 53706-1102.
Phone: (608) 262-6518. Fax: (608) 263-3926. E-mail: elfarraa@
svm.vetmed.wisc.edu.
1
Abbreviations: PFPTH, pentafluorophenylthiohydantoin; BD, 1,3-
butadiene; BMO, butadiene monoxide; EI, electron-impact ionization;
BSTFA, bis(trimethylsilyl)trifluoroacetamide; TMCS, trimethylchlo-
rosilane; PFPITC, pentafluorophenyl isothiocyanate; Val-ME, valine
methyl ester; TMS, trimethylsilyl.
10.1021/tx990043x CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/30/1999

680 Chem. Res. Toxicol., Vol. 12, No. 8, 1999
Moll and Elfarra
human hemoglobin, and bis(trimethylsilyl)trifluoroacetamide
(BSTFA) containing 1% trimethylchlorosilane (TMCS) were
obtained from Sigma Chemical Co. (St. Louis, MO). PFPITC was
obtained from Fluka (Milwaukee, WI). Toluene and sodium
hydroxide were obtained from Mallinckrodt (Phillipsburg, NJ ).
HPLC-grade acetonitrile (ACN) was obtained from EM Science
(Gibbstown, NJ ). N-Hydroxypropylvaline (HPV; internal refer-
ence standard) was synthesized by the procedure of Cai et al.
(13). Whole blood (heparin anticoagulant) from Sprague-Dawley
rats and whole blood from B6C3F1 mice were obtained from
Harlan Bioproducts (Madison, WI). Ca u tion : BMO is a known
mutagen and carcinogen in laboratory animals and must be
handled using proper safety measures.
studies (9), but the interspecies regio- and stereospeci-
ficity of the reaction have not been characterized.
Since the epoxide itself may exist in different absolute
configurations [i.e., (R)- or (S)-enantiomers], the possibil-
ity that four reaction products (two regioisomers and
their respective diastereomers) could be formed from the
reaction of BMO with the N-terminal valine of hemoglo-
bin exists. Theoretically, the measurement and quanti-
tation of only one specific adduct is adequate as a
biomarker of internal dose given that the rate constant
for the formation of that adduct is known. However,
elucidation of the regio- and stereoselective properties of
this reaction is a critical step in our understanding of
the molecular mechanism of BD-induced carcinogenicity,
especially since it has been shown that multiple stereo-
chemical differences exist between species during the
biotransformation of BD and BMO metabolites (10).
Also lacking are studies that compare the absolute
nucleophilic character of the N-terminal valine of hemo-
globin toward BMO to the nucleophilicity of model
compounds. Protected amino acids, such as valine methyl
ester and valine methyl amide, have been used previously
(11, 12) as model compounds in studies in which the
intrinsic reactivities of epoxides with the N-terminal
valine of hemoglobin were examined. In the studies
presented here, valinamide (Val-CONH2) was used as the
model compound because (i) it behaves much like an
N-terminal valine (pKa2 of free valine ) 9.62; pKa2 of
valinamide ) 7.75; average pKa2 of N-terminal amine in
proteins ) 7.80) due to the absence of solvation effects,
(ii) the amide bond is relatively inert to most chemical
reactions (epoxides can react with the free carboxyl group
of valine), and (iii) valinamide is a much better nucleo-
phile than valine. Other beneficial qualities include the
chemical stability of the amide bond (esters are prone to
hydrolysis) as well as its commercial availability. Mea-
suring the particular reactivity of valinamide with BMO
under physiological conditions will allow the estimation
of the effect of protein tertiary structure on preliminary
association kinetics. These studies are important because
the ultimate reaction of BMO with the N-terminal valine
residues of hemoglobin may depend not only on the
inherent reactivity of BMO toward the valine amine
group but also on the accessibility of the two carbons of
the epoxide moiety of BMO to that amine group.
Meth od s. (1) Syn th esis a n d P u r ifica tion of Va lin a -
m id e-BMO Sta n d a r d s. L-Valinamide (50 mM) was placed in
a glass vial equipped with a Teflon septum and dissolved in 10
mL of 100 mM phosphate buffer (pH 7.4). BMO (100 mM) was
added, and the reaction mixture was incubated in a shaking
water bath at 37 °C for 24 h. The reaction was terminated by
extraction with 3 volumes (2 mL each) of toluene to remove
excess BMO and nonpolar byproducts. The mixture was acidified
with 0.1% TFA in water, and the valinamide-BMO adducts
were injected onto an Ultrasphere C18 (4.6 mm × 25 cm)
semipreparative HPLC column and eluted isocratically (H2O
containing 0.1% TFA) at a flow rate of 3 mL/min and monitored
at A210 (0.01 AUFS). Three main fractions eluting at 8.5 (P-1A),
9.5 (P-1B), and 12.6 min (P-2AB) were collected and lyophilized.
The products were redissolved in distilled H2O and relyophilized.
Yields were generally between 75 and 80%. The purity, deter-
mined by HPLC, was >97%.
(2) Ch a r a cter iza tion of Va lin a m id e-BMO Ad d u cts by
1H NMR. Purified valinamide-BMO adducts were dissolved (5
mg/mL) in D2O, and proton NMR spectra were obtained on a
Bruker spectrometer (Karslruhe, Germany) at 500 MHz at room
temperature. Chemical shifts are reported in parts per million
with the H2O peak as an internal standard; J values are
reported in hertz. P -1A: 1.01 (d, 3H, CδH3, J âδ ) 9.0), 1.06 (d,
3H, CγH3, J âγ ) 8.5), 2.23 (d, hep, 1H, CâH), 3.69 (m, 1H, C2H),
3.80 (d, 1H, CRH, J Râ ) 7.5), 3.88 (m, 2H, C1H2), 5.52 (d, 1H,
C4Hcis, J cis ) 10.0), 5.58 (d, 1H, C4Htrans, J trans ) 20.0), 5.61 (m,
1H, C3H). P -1B: 1.00 (d, 3H, CδH3, J âδ ) 9.0), 1.06 (d, 3H, CγH3,
J âγ ) 8.6), 2.30 (d, hep, 1H, CâH), 3.82 (d, 1H, CRH, J Râ ) 7.5),
3.86 (m, 1H, C2H), 3.89 (m, 2H, C1H2), 5.52 (d, 1H, C4Hcis, J cis
)
10.0), 5.57 (d, 1H, C4Htrans, J trans ) 20.0), 5.77 (m, 1H, C3H).
P -2A/P -2B: 1.03 (dd, 6H, CδH3, J âδ ) 9.0), 1.10 (dd, 6H, CγH3,
J âγ ) 8.5), 2.31 (d, hep, 2H, CâH), 3.06 (m, 2H, C1H2′′), 3.22 (m,
2H, C1H2′), 3.90 (d, 2H, CRH, J Râ ) 6.5), 4.50 (m, 2H, C2H), 5.32
(d, 2H, C4Hcis, J cis ) 13.5), 5.41 (d, 2H, C4Htrans, J trans ) 21.5),
5.86 (m, 2H, C3H).
(3) Ch a r a cter iza tion of Va lin a m id e-BMO Ad d u cts by
GC/MS. Valinamide-BMO adducts were dissolved in distilled
H2O, and aliquots were added to Teflon-capped vials along with
internal standard HPV and brought to dryness by N2. BSTFA
containing 1% TMCS as a catalyst was added (100 µL) and the
mixture heated in a dry bath at 100 °C for 15 min. The silylated
adducts were analyzed using a HP-6890 series mass selective
detector interfaced with an HP 6890 series gas chromatograph
(Hewlett-Packard, Palo Alto, CA). Separation of components was
accomplished using a 30 m × 0.25 mm i.d. HP-5MS (0.25 µm)
capillary column. The GC injection port temperature was
maintained at 280 °C, and the inlet pressure of the ultrapure
helium gas was maintained at 20 kPa during the run. All
injections were made in the splitless mode. The initial oven
setpoint was 100 °C with a hold for 1 min. A temperature
gradient of 20 °C/min was used until the temperature reached
280 °C, followed by a hold of 5 min. The mass spectrometer was
programmed for scanning mode with a scan range of 10-400
amu.
The primary objective of the current study was to
determine the nucleophilic reactivity and stereoselectivity
of valinamide (Val-CONH2) to BMO as a model for the
N-terminal valine of hemoglobin, and to compare the
model kinetic parameters with those derived from the
in vitro reaction with hemoglobin. Therefore, the contri-
butions to the observed rate constant from the formation
of all four regiospecific and diastereospecific products
were determined. The results obtained with valinamide
were compared with experimentally derived rate con-
stants from the in vitro reaction of BMO with the
N-terminal valine of hemoglobin. Species-specific param-
eters, including kinetic rate constants and regioselectiv-
ity, were examined using incubations of intact erythro-
cytes, from rats and mice, with BMO.
Exp er im en ta l P r oced u r es
(4) Kin etic An a lysis of Ad d u ct F or m a tion u n d er P h ysi-
ologica l Con d ition s. The reactions of valinamide and BMO
were carried out in 10 mL vials capped with Teflon septa.
Valinamide (final concentration of 50 mM) and BMO (final
Ma ter ia ls. Racemic BMO and trifluoroacetic acid (TFA) were
obtained from Aldrich Chemical Co. (Milwaukee, WI). L-Vali-
namide, L-valine methyl ester, heptane, acetone, 1-propanol,

Hemoglobin Adducts of Butadiene Monoxide
Chem. Res. Toxicol., Vol. 12, No. 8, 1999 681
concentration of 50-500 mM) were added to 0.1 M phosphate
buffer (pH 7.4, final volume of 5 mL), and the mixture was
placed in a shaking water bath (37 °C). Aliquots (100 µL) were
taken at timed intervals and extracted with 2 volumes (400 µL
each) of toluene. Internal standard (HPV) was added to 25 µL
aliquots of reaction mixture and brought to dryness under N2.
The reaction products were silylated and analyzed by GC/MS
as described above.
(5) Syn th esis a n d P u r ifica tion of Va lin e-BMO Ad d u cts.
L-Valine methyl ester (4 mmol, 675 mg) was placed in a glass
vial equipped with a Teflon septum. The compound was dis-
solved in distilled water (∼8 mL), and the solution was adjusted
to pH 7.8 with 2 M NaOH. BMO (12 mmol, 1 mL) was added,
and the mixture was incubated in a shaking water bath set at
37 °C for 24 h. The solution was extracted three times with 2
mL of toluene to remove excess BMO and byproducts. Aliquots
were diluted 2-fold with 2 M NaOH and placed in a dry bath
incubator set at 80 °C for 30 min to hydrolyze the methyl ester
moiety. The crude valine-BMO adduct mixture was adjusted
to pH 2.1 with dilute TFA, and the samples were injected onto
an Ultraprep C18 (21.2 mm × 15 cm) HPLC column at a flow
rate of 10 mL/min and monitored at A210 (0.01 AUFS). Mobile
phase A was 0.1% TFA in H2O; mobile phase B was 40% ACN
in H2O and 0.1% TFA. The samples were eluted isocratically
with 5% B. Valine-BMO adducts (retention times between 15
and 20 min) were collected and lyophilized. The products were
redissolved in distilled H2O and relyophilized. Yields were
typically ∼60-70%. Purities of the adducts were greater than
95% as determined by HPLC.
F igu r e 1. Reaction scheme of L-valine (or L-valinamide) with
BMO. The nucleophilic addition of the electrophile BMO to the
primary amine group of L-valine (or L-valinamide) can occur at
either the primary (C-1) or secondary (C-2) carbon of the epoxide
moiety, resulting in two regioisomers, each regioisomer consist-
ing of a pair of diastereomers.
pended in an equal volume of phosphate-buffered saline (0.1
M, pH 7.4). After addition of BMO (0.1-20 µL/mL, 1.21-242
µmol/mL), the mixture was placed in a shaking water bath at
37 °C and the reaction stopped at designated time points. After
being washed with cold saline, the cells were resuspended in
cold distilled water and stored overnight at -10 °C. The thawed
mixture was then centrifuged for 30 min at 30000g to remove
cell membranes, and the supernatant was added dropwise with
vigorous vortexing to cold acidified acetone (1% HCl) to pre-
cipitate globin. The sample was centrifuged for 15 min at 3000g,
and the pellets were washed once with cold acidified acetone,
once with cold acetone, and once with cold diethyl ether. The
globin (10-50 mg) was dried under a gentle stream of nitrogen
at 45 °C. The globin was then dissolved in 0.5 M NaHCO3 and
1-propanol (1 mL; 2:1 v/v), and the N-alkyl Edman procedure
and subsequent silylation with BSTFA/TCMS were performed
as described (vida infra).
(6) Syn th esis of P F P TH-Va lin e-BMO Ad d u cts. The
synthesis of PFPTH-valine-BMO adducts was carried out
according to the procedure of To¨rnqvist et al. (4). The valine-
BMO adducts (5 mg, 27 µmol) were dissolved in 200 µL of 0.5
M NaHCO3 and 1-propanol (2:1 v/v) in a glass vial equipped
with a Teflon septum. PFPITC was added (10 µL, 75 µmol), and
the mixture was allowed to react in a shaking water bath at 45
°C for 3 h. The samples were extracted (3 × 500 µL) with
heptane, and the combined extracts were dried under nitrogen
at 50 °C. The samples were then redissolved in 100 µL of toluene
and washed with 200 µL of 1 M HCl and 200 µL of distilled
water. The samples were diluted with ACN and analyzed by
GC/MS using an initial oven setpoint of 100 °C followed by a 1
min hold. The temperature was then increased at a rate of 30
°C/min to 250 °C, and then increased further at a rate of 10
°C/min to 280 °C.
Resu lts
The in vitro reaction between valinamide and racemic
BMO results in the formation of four stereoisomeric
adducts (two diastereomers of each regioisomer) as
depicted in the general scheme of Figure 1. Valinamide-
BMO adducts were synthesized, and the reaction prod-
ucts were purified by reverse phase HPLC. Three main
fractions, designated P-1A, P-1B, and P-2AB, were col-
lected at 8.5, 9.5, and 12.6 min, respectively (data not
shown). The products were then identified and character-
(7) Der iva tiza tion of P F P TH-Va lin e-BMO Ad d u cts
w ith BSTF A/TCMS. Aliquots of the PFPTH-valine-BMO
adducts were evaporated to dryness under nitrogen at 50 °C.
The samples were then silylated by the addition of 100 µL of
BSTFA, containing 1% TCMS as a catalyst, and incubation in
a dry bath heater at 100 °C for 15 min. The samples were
allowed to cool, then diluted with ACN, and analyzed by GC/
MS. The initial oven setpoint was 100 °C followed by a hold of
1 min. The temperature was increased at a rate of 35 °C/min to
250 °C, and then increased at a rate of 3 °C/min to 270 °C with
a 5 min hold. The final increase was carried out at a rate of 20
°C/min to 280 °C. The mass spectrometer was programmed to
monitor the major fragment at m/z 142 as well as a qualifying
ion at m/z 129. Quantitation of the valine-BMO adducts (0.5-
100 pmol/g of globin) was based on measurement of the internal
reference standard (HPV). The response factor for the adducts
in relation to HPV (mean ( standard deviation ) 0.97 ( 0.20)
was determined using mixtures of the internal reference
standard and the purified analytical standards.
1
ized by H NMR and GC/MS.
The 1H NMR spectra of P-1A (Figure 2A) and P-1B
(spectrum not shown; for chemical shifts and J values,
see Experimental Procedures) are very similar, indicative
of diastereomers, and the chemical shifts of the C-1 and
C-2 protons of the BMO moiety are consistent with the
branched form (attack at C-2 of the epoxide) of the
valinamide adduct. The only major difference in the
spectra is found in the chemical shifts of the C-2 proton,
which is 3.69 ppm in P-1A and 3.86 ppm in P-1B (∆ppm
) 0.17); the difference between the diastereomers is most
likely due to the proximity of the C-2 proton to the chiral
center of the valinamide moiety. The spectrum of the
P-2A/P-2B mixture (Figure 2B), upon integration, indi-
cates the presence of 28 protons (compared to 14 each
with P-1A and P-1B); the appearance of closely related
multiplets suggests a mixture of the other pair of dia-
stereomers. The downfield shift of the C-2 proton, which
(8) In Vitr o Rea ction of Mou se a n d Ra t Hem oglobin
w ith BMO. Whole blood with heparin as the anticoagulant was
obtained from freshly killed rats and mice and was processed
within 24 h. Intact erythrocytes were initially separated from
plasma proteins by centrifugation of whole mouse or rat blood
for 5 min at 1500g. After removal of the plasma fraction, the
red blood cells were washed three times with an equal volume
of isotonic saline (0.9% w/v). The red blood cells were resus-

682 Chem. Res. Toxicol., Vol. 12, No. 8, 1999
Moll and Elfarra
F igu r e 2. 1H NMR of L-valinamide-BMO adducts. Valinamide-BMO adducts were initially separated by reverse phase HPLC,
followed by lyophilization of the collected fractions. The samples were brought up in D2O at a concentration of ∼5 mg/mL and analyzed
1
on a Bruker spectrometer art 500 MHz at room temperature. (A) H NMR of one of the purified C-2 diastereomers [N-(2-hydroxy-
1
3-buten-1-yl)valine]. (B) H NMR of a pure mixture of the two C-1 diastereomers [N-(1-hydroxy-3-buten-2-yl)valine].
is explained by the deshielding effect of the hydroxyl
group, provides strong evidence that the linear form of
the adduct (attack at the terminal C-1) is present. Again,
the only major difference in the proton assignments is
found in the chemical shifts of the C-1 protons, which is
3.06 ppm for one diastereomer and 3.22 ppm for the other
(∆ppm ) 0.16). Similar to that for the P-1A and P-1B
spectra, this difference between the diastereomers is
directly related to the close proximity of these protons
to the chiral center of valinamide.
Further evidence for the identification of the reaction
products is provided by the results of the GC/MS experi-
ments (Figure 3). After derivatization by BSTFA/TCMS,
the four products were completely separated by GC and
the mass spectra determined by identification of the
characteristic fragment ions. The two peaks eluting at
8.31 and 8.61 min coincide with the P-1A and P-1B
products, respectively, and the two later eluting peaks
at 8.72 and 8.90 min represent the P-2A and P-2B
adducts, respectively. The molecular ion (M+) of all four

Hemoglobin Adducts of Butadiene Monoxide
Chem. Res. Toxicol., Vol. 12, No. 8, 1999 683
F igu r e 3. Total ion chromatogram and major ion fragmentation scheme of L-valinamide-BMO standards. (A) The peaks eluting at
8.31 and 8.61 min are the diastereomers arising from the reaction at C-2 of BMO, and the peaks eluting at 8.72 and 8.90 min are
the diastereomers obtained from reaction at C-1. (B) The m/z ratios of the characteristic fragment ions are listed above along with
the major ion fragmentation patterns for P-1A/P-1B and P-2A/P-2B.
products is m/z 330, which corresponds to two trimeth-
ylsilyl moieties per molecule. Theoretically, there are
three possible sites for derivatization (CONH2, NH, and
OH), but the proximity of the hydroxyl group to the
secondary amine group makes the simultaneous silyla-
tion of these two groups less likely due to steric hin-
drance. Since the hydroxyl group is significantly more
reactive to silylating compounds than amines, it is
postulated that the derivatization occurs primarily at the
OH and CONH2 groups.

684 Chem. Res. Toxicol., Vol. 12, No. 8, 1999
Moll and Elfarra
F igu r e 5. Kinetic rates of formation of valinamide-BMO
adducts. Valinamide-BMO adducts were analyzed by GC/MS
using N-(hydroxypropyl)valine as an internal standard and the
purified synthetic adducts as analytical reference standards. The
reaction of BMO (100 mM) and L-valinamide (50 mM) was
carried out under physiological conditions (0.1 M phosphate at
pH 7.4 and 37 °C).
F igu r e 4. Mass spectrum (EI-GC/MS) of valinamide-BMO-
TMS regioisomers. (A) Mass spectrum of one of the silylated
C-2 diastereomers [N-(2-hydroxy-3-buten-1-yl)valine] of the
purified valinamide-BMO adducts. (B) Mass spectrum of one
of the silylated C-1 diastereomers [N-(1-hydroxy-3-buten-2-yl)-
valine]. In each case, the mass spectra of the other diastereomer
of each regioisomer are identical (data not shown).
Despite the identical molecular weight and formula of
the regioisomers, the fragmentation patterns are strik-
ingly different (Figure 4). However, the diastereoisomers
of both regioisomers have identical spectra (data not
shown). The major similarities of P-1A and P-1B with
P-2A and P-2B are the molecular (m/z 330), the M+ - 15
(methyl group loss from TMS), and the 214 ions (frag-
mentation at the carbonyl group). The characteristic
fragments of P-1A/P-1B include the m/z 227 (TMSO-CH3
loss) and 143 (NH-valinamide loss) ions. With the P-2A/
P-2B adducts, the ion at m/z 201 (valinamide-C-1 loss)
is a major characteristic fragment.
(1)
The in vitro kinetics of valinamide-BMO adduct
formation were determined under physiological condi-
tions so the reaction rate constants could be correlated
with those obtained in vitro using hemoglobin. The four
reaction products under these conditions were monitored
by GC/MS as a function of time (Figure 5). Quantitation
of the adducts was based on the peak response ratio of
an internal reference standard (HPV) with the synthetic
adduct analytical standards. The data show that the level
of the products arising from attack at C-1 of BMO (P-2A
and P-2B) is significantly lower than the level of the
products arising from attack at C-2 (P-1A and P-1B). The
individual second-order rate constants are listed in
Figure 6 and were calculated using a formula which
corrects for the decrease in the concentration of BMO due
to spontaneous hydrolysis (eq 1; Figure 6). For BMO in
0.1 M phosphate buffer (pH 7.4) at 37 °C, the first-order
rate constant for hydrolysis (k′) that is used is 1.292 ×
10-5 s-1 (14). The rate constants for P-1A and P-1B are
significantly higher than the values obtained for P-2A
F igu r e 6. Rate constant parameters for the reaction of
valinamide with BMO. The second-order rate constants for
individual and total adduct formation are listed. The rate
constants (k) were determined according to the equation de-
scribed above using the specified constants. Values shown
represent the means ( standard deviations (n ) 3). The initial
BMO concentration represents the theoretical aqueous concen-
tration (i.e., not corrected for headspace redistribution).
and P-2B, and the extents of reaction indicate that the
C-2 products are produced at a molar ratio of nearly 2:1.
In addition, although the C-1 product rate constants are
similar to the rate constant for spontaneous hydrolysis
at neutral pH, the combined rate constants for adduct
formation (kP-1A + kP-1B + kP-2A + kP-2B) are nearly 5-fold
greater than k′.

Hemoglobin Adducts of Butadiene Monoxide
Chem. Res. Toxicol., Vol. 12, No. 8, 1999 685
F igu r e 7. Synthesis of valine-BMO adducts and derivatives. The valine-BMO adducts were synthesized using valine methyl
ester as the starting compound. The ester moiety was then hydrolyzed under basic conditions prior to purification. The adducts were
initially analyzed after silylation by EI-GC/MS. Consequently, the PFPTH-valine-BMO compounds were synthesized, and were
analyzed either with or without additional derivatization with BSTFA containing TCMS. For simplification, only the reaction occurring
at C-1 is shown.
To study the in vitro reaction of BMO with the
N-terminal valine of hemoglobin, the corresponding four
products of free L-valine and BMO were synthesized and
purified. However, since epoxides can also react with the
carboxyl group in free valine, valine methyl ester (Val-
ME) was used as the starting compound (Figure 7). The
reaction of Val-ME with BMO was allowed to occur under
slightly basic conditions (pH 7.8) at 37 °C for 24 h. Higher
temperatures resulted in decreased yields, apparently
due to the increased rate of spontaneous hydrolysis of
BMO. After extraction with toluene to remove excess
BMO and nonpolar byproducts, the mixture was adjusted
to pH 11, and the basic hydrolysis of the ester moiety
was carried out at 80 °C for 30 min. The valine-BMO
adducts were then purified on a preparative C18 reversed
phase column.
The synthesis of the PFPTH-valine-BMO adducts
was performed using the purified valine-BMO adducts
as the starting material, and was carried out essentially
by the procedure of To¨rnqvist et al. (4). The initial
characterization of the PFPTH-valine-BMO adducts
was accomplished by GC/MS in the electron-impact
ionization (EI-GC/MS) mode (Figure 8). PFPTH amino
acids are generally quite volatile at low temperatures,
and the presence of the fluorine atoms [compared to the
original phenyl isothiocyanate (PITC) used in the Edman
degradation procedure] enhances the sensitivity of the
compounds during mass selective detection. The molec-
ular ion of the four compounds is calculated at 394 amu
(Figure 8B,C), which corresponds to the peaks eluting
between 12 and 13 min (Figure 8A). The presence of only
two peaks in the total ion chromatogram indicated that
the diastereomers of the two regioisomers were not
resolved in this procedure, and was further evidenced by
the differences in the fragmentation patterns. The char-
acteristic fragment that supports the regioisomer assign-
ment is the M+ - 57 ion (337 amu; loss of CH2d
CHCHOH) found in the early eluting peak (12.22 min),
which could only be produced from the C-1 reaction
product since the carbon chain of this adduct is not
branched at the N-proximal carbon.
The presence of a hydroxyl group in the adducts, and
the results of the initial derivatization of these groups
with silylating compounds, led us to attempt the deriva-
tization of these groups as well in the PFPTH-valine-
BMO adducts. Although they have high thermal stability,
the silyl derivatives tend to be chemically unstable; all
samples were analyzed immediately after derivatization.
The four silylated derivatives (PFPTH-valine-BMO-
TMS) were efficiently separated in this procedure (Figure
9A). Surprisingly, the first and last eluting peaks rep-
resent the C-1 reaction products, while the middle peaks
comprise the C-2 compounds. The molecular ion of the
silylated derivatives is calculated and found to be 466
amu (Figure 9B,C). Again, the main characteristic frag-
ment in the C-1 compounds (Figure 9B) is the 129 amu
ion, which corresponds to the 57 amu ion produced by
the nonsilylated C-1 compound, although here it has a
trimethylsilyl group attached (72 amu). All four com-
pounds also produce a major fragment at 142 amu which
corresponds to the BMO adduct with a TMS group
attached at the hydroxyl moiety. Also produced is a minor
fragment at 225 amu corresponding to C6F5NCS. This
minor fragment is commonly found in the PFPTHs of
N-substituted valines (15). It was also found that the
silylation resulted in nearly a 10-fold increase in sensi-
tivity compared with those of the nonsilylated com-
pounds. In addition, measuring these adducts using

686 Chem. Res. Toxicol., Vol. 12, No. 8, 1999
Moll and Elfarra
F igu r e 8. Total ion chromatogram and mass spectrum of the
PFPTH-valine-BMO derivatives. (A) The column was main-
tained at 200 °C for 20 min, and the temperature was then
increased to 280 °C at a rate of 30 °C/min. The two peaks
represent the C-1 and C-2 regioisomers; the respective diaster-
eomers were not resolved under these conditions. (B) Mass
spectrum of the C-2 regioisomers. (C) Mass spectrum of the C-1
regioisomers. The respective diastereomers were not resolved
under the operating conditions. The internal standard PFPTH-
N-(hydroxylpropyl)valine elutes at 15.2 min (not shown).
F igu r e 9. Selected ion chromatograms and mass spectrum of
the PFPTH-valine-BMO-TMS adducts produced by the reac-
tion of hemoglobin with BMO. (A) Selected ion chromatogram
of the in vitro reaction of mouse whole blood with BMO. The
internal standard PFPTH-N-(hydroxylpropyl)valine-TMS elutes
at 15.8 min (not shown). (B) Mass spectrum of one of the C-2
silylated PFPTH-valine-BMO adducts. (C) Mass spectrum of
one of the diastereomers of the C-1 silylated PFPTH-valine-
BMO adducts. The mass spectrum of the other diastereomer of
each respective regioisomer is identical (data not shown).
selected ion monitoring at 129 and 142 decreased the
limit of detection from 0.5 nmol/g of globin to 0.5 pmol/g
of globin. This level is low enough to allow monitoring of
occupational exposure at levels less than 1 ppm BD (16,
17).
group of valinamide could be compared to that found in
vitro with hemoglobin. The rates of formation of the
various adducts were obtained from the second-order rate
constants for the respective reactions. The rate constants
were determined using the same relationship described
earlier for valinamide-BMO adducts. In this instance,
however, the initial nucleophile concentration pertains
to hemoglobin, which is 137 mg/mL (2.1 mM) in mouse
blood and 153 mg/mL (2.3 mM) in rat blood (19). Rate
velocities were determined from the initial linear portion
of the time course (Figure 10) and were used to determine
the kinetic rate constants.
The mouse and rat kinetic results, displayed in Figure
11, are strikingly different than those obtained using
valinamide as a model, especially with regard to the
regioselectivity of the products. It was noted previously
that the C-2 regioisomers are the predominant adducts
formed upon reaction of valinamide with BMO under
physiological conditions. With rat and mouse blood,
however, it is the C-1 diastereomers that are preferen-
tially formed. In addition, there is a significant difference
in the ratio of C-1 products to C-2 products between
species, with the rat (C-1:C-2 ) 26.2) showing much less
C-2 formation, relatively speaking, than the mouse
(C-1:C-2 ) 4.6). Species-specific differences in reactivity
were also seen, as evidenced by the second-order rate
constants for adduct formation. The total rate constant,
obtained by the addition of the individual rate constants,
was roughly twice as much with mouse hemoglobin (4.99
The racemization of amino acids upon reaction with
phenylthiohydantoins was previously observed during
the development of the Edman degradation procedure
(18). In our study, the products of the reactions of
PFPITC with the purified valine-BMO adducts were
rapidly racemized during the formation of the pentafluo-
rophenylthiohydantoins (unpublished observation). This
was evidenced by the observation that when purified
samples of either of the two diastereomers of the C-1
valine adducts of BMO were reacted with PFPITC and
silylated, both the first and last eluting peaks were
detected by the GC/MS analysis (Figure 9). Thus, al-
though the individual diastereomers of the two regio-
isomers could be detected and quantitated, whether they
arise directly from the reaction of the epoxide with valine
or during the racemization reaction with PFPITC cannot
be determined using this method. Therefore, the validity
of previously reported values of kinetic constants (6-8),
obtained by the quantitation of a single regioisomer,
should be interpreted with caution.
The reactions of BMO with mouse (B6C3F1) and rat
(Sprague-Dawley) erythrocytes were then studied under
physiological conditions [0.1 M phosphate (pH 7.4) at 37
°C] so the intrinsic reactivity of the N-terminal amine

Hemoglobin Adducts of Butadiene Monoxide
Chem. Res. Toxicol., Vol. 12, No. 8, 1999 687
F igu r e 11. Kinetic parameters (second-order rate constants)
from the in vitro reaction of mouse and rat whole blood with
BMO. Second-order rate constants for the individual and total
N-terminal valine adduct formation from in vitro experiments
with valinamide and rat and mouse whole blood. Values
represent the mean ( standard deviation (n ) 6).
F igu r e 10. Time course of the reaction of BMO with hemo-
globin using intact erythrocytes from Sprague-Dawley rats and
B6C3F1 mice. Erythrocytes from mice (A) and rats (B) were
incubated with BMO under physiological conditions (0.1 M
phosphate at pH 7.4 and 37 °C). Valine-BMO adducts were
analyzed by GC/MS using HPV as internal standard and the
purified synthetic adducts as analytical reference standards. The
plotted values are the mean ( standard deviation (n ) 6).
of the regioisomers were not resolved under the condi-
tions that were used. Further refinement of the GC/MS
procedure allowed Richardson et al. (16) to detect the four
expected products, although quantitation was based on
the sum of the areas of the two major peaks due to low
sensitivity. Unfortunately, very little information con-
cerning the reactivity and stereoselectivity of the N-
terminal valine of hemoglobin with BMO has been
reported utilizing these methods.
× 10-5 M-1 s-1) as with rat hemoglobin (2.45 × 10-5 M-1
s-1); as a comparison, the mouse rate constant was only
slightly lower than the value obtained with valinamide
(5.39 × 10-5 M-1 s-1).
Initial experiments used valinamide (C-terminal amide
of valine; Val-CONH2) as a model for the formation of
epoxide adducts with the N-terminal valine of hemoglo-
bin due to its favorable physical and chemical properties.
To determine both the particular reactivity of the nu-
cleophilic amine group of valine and the contribution of
protein tertiary structure on preliminary association
kinetics, the reaction of valinamide with BMO was
studied under physiological conditions (pH 7.4 at 37 °C).
The results indicated that the adducts formed by reaction
at C-2 [N-(1-hydroxy-3-buten-2-yl)valinamide] are formed
at an approximate ratio of 2:1 compared to the adducts
formed by reaction at C-1 [N-(2-hydroxy-3-buten-1-yl)-
valinamide]. The stereoisomers of each respective regio-
isomer were produced with similar rates. This is in stark
contrast to the results obtained with mouse and rat
erythrocytes, which demonstrated the preferential for-
mation of the C-1 adducts in in vitro studies of BMO-
hemoglobin adduct formation. The change in regioselec-
tivity in the reaction of BMO with valinamide and the
N-terminal valine could be due to steric hindrance in the
intact hemoglobin molecule. In the R-chain of hemoglobin,
for example, this steric hindrance could presumably be
exerted by a salt bridge from the N-terminal valine to
the C-terminal carboxylate group of arginine-141 of the
opposite R-chain (21). There are also regioselective forces
evident between the species that were studied. The
higher ratio of C-1 and C-2 regioisomers seen in mouse
blood could again be related to tertiary and/or quaternary
influences that are a direct result of amino acid differ-
Discu ssion
The formation of hemoglobin adducts upon exposure
to BMO was first studied by Sun et al. (20) using [14C]BD.
They showed that BMO could form adducts with mouse
and rat hemoglobin, and that adduct formation was
linearly related to administered doses up to 100 µmol of
[14C]BD per kilogram of body weight. In addition, re-
peated daily injections of BD within this range correlated
with a linear accumulation of hemoglobin adducts. In
1991, Osterman-Golkar et al. (3) measured the levels of
hemoglobin-BMO adducts in rats using the N-alkyl
Edman procedure and GC/MS of the thiohydantoin
derivatives. These experiments were carried out using
GC/MS in the negative ion chemical ionization mode
(NICI). Although the chemical ionization process can
enhance sensitivity due to the general absence of carbon-
carbon cleavage reactions, the resulting spectra provide
little structural information due to the reduced level of
fragmentation. Electron-impact ionization (EI) spectra
contain abundant ions related to the molecular weight
as well as numerous fragment ions which provide infor-
mation for the absolute identification of compounds, and
is especially suitable for the characterization of regio-
isomers. Modification of the GC/MS methodology employ-
ing EI-GC/MS allowed Albrecht et al. (14) to quantitate
valine-BMO regioisomer adducts, but the diastereomers

688 Chem. Res. Toxicol., Vol. 12, No. 8, 1999
Moll and Elfarra
ences in mouse and rat hemoglobin. Collectively, these
results strongly suggest that for accurate biomonitoring
of exposure to epoxides by the analysis of hemoglobin
adducts, quantitation of both regioisomers and their
diastereoisomers, when applicable, should be performed.
Of special interest, as well as specific relevance to the
current results, is the finding that distinct stereochemical
differences in the biotransformation of BD to BMO exist
between species. For example, the production of both (R)-
and (S)-BMO has been shown to occur in the metabolism
of BD by rat and mouse liver microsomes (10). The
oxidation of BD results in the formation of a chiral BMO
metabolites, which may play a critical role in subsequent
biotransformation pathways as well as during the non-
enzymatic formation of macromolecular adducts. Thus,
while racemic BMO has been employed in all of the
experiments described here, further insight into the
species-specific stereoselectivity and regioselectivity could
be gained by measuring relative C-1 and C-2 adduct
levels, as well as by monitoring configuration inversion,
using specific BMO enantiomers.
differences in BD and BMO metabolism may also con-
tribute to the observed difference in N-terminal valine
hemoglobin adducts in mice and rats in vivo (1).
In conclusion, the results in these studies accentuate
the caution that should be shown when measuring
hemoglobin adducts as a marker of chemical exposure.
Regioselectivity and stereoselectivity can vary greatly
between model compounds such as valinamide and
hemoglobin, as well as between species, as shown in the
results between mouse and rat. In addition, the racem-
ization of amino acids inherent in the alkyl Edman
degradation step should be taken into consideration when
measuring hemoglobin adducts, especially during quan-
titation. Finally, the finding that the reaction of BMO
with rat hemoglobin produced a higher C-1:C-2 adduct
ratio, in comparison with that from the reaction with
mouse hemoglobin, stresses the importance of measuring
all four adducts so the relative rates of adduct formation
are accurately compared both with model compounds and
among different species.
Ack n ow led gm en t. This research was made possible
by Grant ES06841 from the National Institute of Envi-
ronmental Health Sciences (NIEHS), NIH. The contents
of this manuscript are solely the responsibility of the
authors and do not necessarily represent the official
views of the NIEHS, NIH. T.S.M. was supported by an
NRSA Fellowship (NIEHS, ES05835).
Valine-BMO standards were also synthesized and
purified, which were used to identify and characterize
the in vitro reaction products by GC/MS, as well as in
the development of methods that would increase the
separation and sensitivity of detection. Silylation of the
PFPTH-valine-BMO adducts increased both the degree
of separation of the four products and also increased the
sensitivity nearly 10-fold over the nonsilylated products
when quantitated by total ion GC/MS. Together, these
studies allowed us to monitor the in vitro reaction of
BMO with mouse and rat hemoglobin. Indeed, single-ion
monitoring allowed the measurement of background
levels of BMO adducts in commercially available human
hemoglobin (0.7-0.9 pmol/g of globin; data not shown),
which could be important in the accurate estimation of
the internal dose associated with exposure and, ulti-
mately, in adequately assessing human risk to either
environmental or occupational BD exposure.
Several key observations were apparent when examin-
ing the differences in the kinetic parameters both be-
tween the in vitro studies using hemoglobin and valina-
mide and when interspecies comparisons were made. It
is of interest to note that the total rate constant for the
N-terminal valine in mouse hemoglobin, obtained by
summation of the individual rate constants, is only
slightly lower than the value derived with the model
compound valinamide. It should be noted, however, that
the values calculated in this report are related to the
molar concentration of globin, not hemoglobin, molecules,
since the specific reaction under study is that with the
N-terminal valine of each of the four globin chains per
hemoglobin. Hence, if the intact hemoglobin molar con-
centration was used instead of that of globin, the reported
rate constants would increase 4-fold.
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