Mechanism of aqueous decomposition of trichloroethylene oxide

Article abstract of DOI:10.1021/ja9914240

The aqueous decomposition of trichloroethylene (TCE) oxide is shown to involve both pH-independent and hydronium ion-dependent regions. C-C bond scission is a major reaction at all pH values. Disappearance of TCE oxide is the rate-determining step for the formation of CO under the conditions studied. The product distribution of CO and three carboxylic acids (HCO₂H, Cl₂CHCO₂H, and glyoxylic acid) did not change considerably over the pH range of -1.5-14, in general, even though the hydrolysis mechanism changes from hydronium ion-dependent to pH-independent. Mechanisms for the hydronium ion-dependent and pH-independent hydrolysis of TCE oxide were elucidated on the basis of the results of H₂¹⁸O and H incorporation and identification of products of the reaction of TCE oxide with lysine in both H₂¹⁶O and H₂¹⁸O. In the pH-independent hydrolysis, a zwitterionic intermediate could be formed and undergo an intramolecular rearrangement (Cl^- shift) to generate dichloroacetyl chloride, which would subsequently decompose to Cl₂CHCO₂H. The zwitterionic intermediate could also hydrolyze at the less sterically hindered methylene to give a glycol anion, which would dehydrohalogenate to form an oxoacetyl chloride intermediate. The oxoacetyl chloride could hydrolyze to generate either glyoxylic acid, as a final product, or an anionic intermediate, which could go through a concerted mechanism to generate CO, HCO₂H, and chloride. A mechanism proposed for the hydronium ion-dependent hydrolysis is very similar to that for the pH-independent hydrolysis except for the first step, which involves hydronium ion attack on TCE oxide to form a TCE-oxide cation intermediate. The lysine amide adducts were characterized by HPLC and mass spectrometry as those resulting from reaction with the postulated acyl chlorides.

Full text of DOI:10.1021/ja9914240

11656
J. Am. Chem. Soc. 1999, 121, 11656-11663
Mechanism of Aqueous Decomposition of Trichloroethylene Oxide
Hongliang Cai and F. Peter Guengerich*
Contribution from the Department of Biochemistry and Center in Molecular Toxicology,
Vanderbilt UniVersity, NashVille, Tennessee 37232-0146
ReceiVed April 30, 1999
Abstract: The aqueous decomposition of trichloroethylene (TCE) oxide is shown to involve both pH-independent
and hydronium ion-dependent regions. C-C bond scission is a major reaction at all pH values. Disappearance
of TCE oxide is the rate-determining step for the formation of CO under the conditions studied. The product
distribution of CO and three carboxylic acids (HCO2H, Cl2CHCO2H, and glyoxylic acid) did not change
considerably over the pH range of -1.5-14, in general, even though the hydrolysis mechanism changes from
hydronium ion-dependent to pH-independent. Mechanisms for the hydronium ion-dependent and pH-independent
1
8
hydrolysis of TCE oxide were elucidated on the basis of the results of H2 O and H incorporation and
1
6
18
identification of products of the reaction of TCE oxide with lysine in both H2 O and H2 O. In the pH-
independent hydrolysis, a zwitterionic intermediate could be formed and undergo an intramolecular
-
rearrangement (Cl shift) to generate dichloroacetyl chloride, which would subsequently decompose to
Cl2CHCO2H. The zwitterionic intermediate could also hydrolyze at the less sterically hindered methylene to
give a glycol anion, which would dehydrohalogenate to form an oxoacetyl chloride intermediate. The oxoacetyl
chloride could hydrolyze to generate either glyoxylic acid, as a final product, or an anionic intermediate,
which could go through a concerted mechanism to generate CO, HCO2H, and chloride. A mechanism proposed
for the hydronium ion-dependent hydrolysis is very similar to that for the pH-independent hydrolysis except
for the first step, which involves hydronium ion attack on TCE oxide to form a TCE-oxide cation intermediate.
The lysine amide adducts were characterized by HPLC and mass spectrometry as those resulting from reaction
with the postulated acyl chlorides.
Introduction
However, the hydrolytic reactions are more complex and, with
polyhalogenated alkene epoxides, involve extensive C-C scis-
sion, which is poorly understood but may be highly relevant to
issues of the associated biology and toxicology. Trichloro-
ethylene (TCE) is an extensively used industrial solvent and
Oxiranes, also called “epoxides,” are versatile intermediates
in organic synthesis.
1-11
They are easily prepared from a variety
of starting materials, and the inherent polarity and strain of the
three-membered ring makes them reactive toward a large
number of reagents, such as electrophiles, nucleophiles, acids,
bases, reducing agents, and some oxidizing agents.
Halogenated epoxides are rather rare but nonetheless an
important part of the diverse oxirane family. The mechanisms
of their thermal decomposition have been studied in organic
1
4
common water pollutant. The compound is of biological
interest because oxidative metabolism of TCE leads to the
1
5-20
covalent binding of TCE to proteins and possibly DNA
as
2
1
well as to inactivation of cytochrome P450. Furthermore,
human liver microsomes oxidize TCE to products that alkylate
2
1
protein and, to a much lesser extent, DNA. Studies of the
microsomal oxidation of TCE suggest that a number of
potentially reactive electrophiles are formed during oxidation
metabolism of TCE. TCE oxide (1) is one of the products1
and has often been postulated to be responsible for the covalent
1
2,13
solvents and shown to be dominated by halide migration.
*
Correspondence author. Telephone: (615) 322-2261. Fax: (615) 322-
3,21
3
141. Email: guengerich@toxicology.mc.vanderbilt.edu.
(
1) Smith, J. G. Synthesis 1984, 629-656.
(2) Costantino, P.; Crotti, P.; Ferretti, M.; Macchia, F. J. Org. Chem.
1
7,18
binding of TCE to proteins and possibly DNA.
Currently,
1
982, 47, 2917-2923.
3) Borhan, B.; Nazarian, S.; Stocking, E. M.; Hammock, B. D.; Kurth,
M. J. J. Org. Chem. 1994, 59, 4316-4318.
4) Torii, S.; Uneyama, K.; Tanaka, H.; Yamanaka, T.; Yasuda, T.; Ono,
(
it is not known whether TCE oxide reacts directly with
nucleophiles, or whether decomposition products of TCE oxide
or other P450 oxidation products react.
(13) Uehleke, H.; Tabarelli-Poplawski, S.; Bonse, G.; Henschler, D. Arch.
Toxicol. 1977, 37, 95-105.
(14) Seltzer, R. J. Chem. Eng. News 1975, 41, 1-43.
(15) Uehleke, H.; Poplawski-Tabarelli, S. Arch. Toxicol. 1977, 37, 289-
294.
(
M.; Kohmoto, Y. J. Org. Chem. 1981, 46, 3312-3315.
(
5) Layer, R. W. J. Org. Chem. 1981, 46, 5224-5225.
(6) Barton, D. H. R.; Motherwell, R. S. H.; Motherwell, W. B. J. Chem.
Soc., Perkin Trans. 1 1981, 2363-2367.
7) Rastetter, W. H.; Chancellor, T.; Richard, T. J. J. Org. Chem. 1982,
7, 1509-1512.
8) Diez-Barra, E.; Pardo, M. C.; Elguero, J. J. Org. Chem. 1982, 47,
409-4412.
9) Griesbaum, K.; Lie, G. O.; Raupp, E. Chem. Ber. 1981, 114, 3273-
280.
10) Adam, W.; Hadjiarapoglou, L.; Peters, K.; Sauter, M. J. Am. Chem.
Soc. 1993, 115, 8603-8608.
11) Rao, A. S.; Paknikar, S. K.; Kirtane, J. G. Tetrahedron 1983, 39,
323-2367.
12) McDonald, R. N. In Mechanisms of Molecular Migrations; Thya-
garajan, B. S., Ed.; Wiley: New York, 1969; pp 67-107.
(
4
4
3
(
(16) Bolt, H. M.; Filser, J. G. EnViron. Health Perspect. 1977, 21, 107-
112.
(
(17) Van Duuren, B. L.; Banerjee, S. Cancer Res. 1976, 36, 2419-2422.
(18) Banerjee, S.; Van Duuren, B. L. Cancer Res. 1978, 38, 776-780.
(19) Mazzullo, M.; Bartoli, S.; Bonora, B.; Colacci, A.; Lattanzi, G.;
Niero, A.; Silingardi, P.; Grilli, S. Res. Commun. Chem. Pathol. Pharmacol.
1992, 76, 192-208.
(20) Stott, W. T.; Quast, J. F.; Watanabe, P. G. Toxicol. Appl. Pharmacol.
1982, 62, 137-151.
(21) Miller, R. E.; Guengerich, F. P. Cancer Res. 1983, 43, 1145-1152.
(
(
2
(
1
0.1021/ja9914240 CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/01/1999

Trichloroethylene Oxide Hydrolysis
J. Am. Chem. Soc., Vol. 121, No. 50, 1999 11657
MA). Samples were either prepared in D O, CDCl , or CD OD (100.0
atom % D grade, Aldrich Chemical Co., Milwaukee, WI). Mass
spectrometry was done with a HP 5890A gas chromatograph (Hewlett-
Packard Company, Wilmington, DE) and a Finnigan MAT INCOS 50
mass spectrometer (Finnigan Mat, San Jose, CA) for CO (electron
impact mode) or performed with a Waters 2690 separations module
2
4
Scheme 1. Two Proposed Mechanisms (This Laboratory )
for the Formation of CO and HCO2H under Basic
Conditions
2
3
3
(Alliance) (Waters Corporation, Milford, MA) and a Finnigan MAT
TSQ/SSQ 7000 series mass spectrometer for carboxylic acids (elec-
trospray mode unless noted otherwise).
Syntheses. TCE oxide (1). The epoxide was synthesized by using
2
8
a general two-phase method for acid-sensitive epoxides. m-Chloro-
perbenzoic acid (mCPBA, 25 g) was dissolved in 200 mL of CH Cl
The CH Cl solution was washed with 0.10 M sodium phosphate buffer
pH 7.4, 3 × 200 mL) and 200 mL of the same buffer was added.
TCE (5 g) was added dropwise, and the mixture was stirred at room
temperature for 2 h. The organic layer was washed with 0.5 M Na
3 × 200 mL), washed with ice-cold 6 N NaOH (3 × 100 mL), and
dried over anhydrous Na SO . The dried solution was carefully
concentrated under a stream of N
to ∼10 mL. The product, TCE oxide
yield 10% based on TCE), was purified by distillation (∼40 °C, 130
mmHg). The H NMR spectrum showed two singlets at δ 6.52 and
2
2
.
2
2
(
In 1978 Van Duuren et al. synthesized TCE oxide and
reported its hydrolysis and thermolysis rates.22 Cl2CHCO2H was
2 2 3
S O
(
the only product reported to be formed in the hydrolysis and
thermolysis of TCE oxide. The mechanism for the thermal
rearrangement of TCE oxide was proposed to proceed through
an R-carbonyl carbonium ion intermediate.22 However, a mech-
anism for hydrolysis of TCE oxide was not considered.
Henschler et al. later reported that TCE oxide rearranged to
2
4
2
(
1
22,24
5.31, corresponding to TCE and TCE oxide, respectively.
TCE oxide
accounted for 20% of the volume. The decoupled 13C NMR spectrum
Cl2CHCO2H or hydrolyzed to CO, HCO2H, and to a much lesser
showed two singlets for TCE oxide at 73.7 (CHCl) and 85.7 ppm
extent, glyoxylic acid.23 Mechanisms were also proposed to
(CCl ), in addition to the two singlets for TCE at 116.6 (CHCl) and
2
24
1
23.9 ppm (CCl
2
).
account for the formation of glyoxylic acid, CO, and HCO2H
in aqueous solutions. Our laboratory subsequently examined the
2
2
The same procedure was used to prepare [ H]TCE oxide from H-
13
TCE (Cambridge Isotopes, Cambridge, MA). The C NMR spectrum
showed two peaks for [ H]TCE oxide at 73.7 (t, C HCl) and 85.7 ppm
) in addition to the two peaks for [ H]TCE at 116.6 (t, C HCl)
).
N -Formyl-N -CBZ-L-lysine. Acetic anhydride (1.5 mL) was added
2
4
rearrangement of TCE oxide under a variety of conditions. In
the absence or presence of cytochrome P450, TCE oxide did
2
2
2
2
(
s, CCl
2
24
not rearrange to form chloral, in contrast to the proposals of
Byington and Leibman25 and Henschler et al. Hydrolysis of
TCE oxide yielded primarily glyoxylic acid and Cl2CHCO2H
and 123.9 ppm (s, CCl
2
23
r
E
ꢀ
2
dropwise to a mixture of 88% HCO H (7 mL) and N -CBZ-L-lysine
2
4
at low pH or CO and HCO2H at neutral or high pH.
(1.5 g). After 4 h (room temperature), the reaction mixture was
evaporated to dryness in vacuo. The product was further purified by
Mechanisms were proposed to account for the hydrolysis and
rearrangement of TCE oxide under acidic and basic conditions,
with two possible mechanisms for the observed C-C scission
postulated for neutral and basic conditions (Scheme 1). The first
involves nucleophilic attack by hydroxide ion and opening of
the oxirane ring; the second involves formation of a carbene
intermediate 5. Other work with the analogue vinylidene chloride
recrystallization from EtOH29 (yield 65%): 1H NMR (CD
OD) δ 1.34
), 1.60-1.90 (m, 2H, δ-CH ),
), 4.57 (t, 1H, R-CH), 4.99 (s, 2H, PhCH ), 7.20-
.30 (m, 5H, Ph), 8.13 (s, 1H, CHO).
3
2
(m, 2H, γ-CH ), 1.45 (m, 2H, â-CH
2
2
3
7
.07 (t, 2H, ꢀ-CH
2
2
r
R
ꢀ
N -Formyl-L-lysine. N -Formyl-N -CBZ-L-lysine (vide supra, 0.8
g) was hydrogenated over Pd-charcoal (0.15 g) in 8 mL of CH OH at
3
room temperature for 5 h. After removal of the catalyst (filtration) and
solvent (in vacuo), the product was purified by recrystallization from
2
6
epoxide also indicated extensive C-C bond scission. This
behavior is not observed with the more stable monochloro
epoxide, 2-chlorooxirane.27
EtOH, yield 55%: 1H NMR (D
29
2
O) δ 1.40 (m, 2H, γ-CH
2
), 1.67 (m,
2
H, â-CH
2
), 1.83 (m, 2H, δ-CH
2
), 2.95 (t, 2H, ꢀ-CH
2
), 3.10 (t, 1H,
+
To elucidate the mechanism of aqueous decomposition of
TCE oxide, we carried out an extensive study on this epoxide,
including pH profiles of its disappearance and the formation of
R-CH), 8.06 (s, 1H, CHO); MS, m/z 175.22 (MH ).
E
N -Dichloroacetyl-L-lysine. Cl CHCO H (0.11 mL) was added
2
2
R
3
dropwise to a solution of N -tert BOC-L-lysine in CH OH with 0.15 g
18
CO, product analyses over a wide pH range, H2 O incorpora-
NaOH at room temperature. After 2.5 h, concentrated HCl (80 µL)
was added to the reaction and the mixture was taken to dryness in
tion, H incorporation, and reaction of 1 with the model
16
18
2
vacuo. The residue was dissolved in a mixture of 1 mL of H
mL of CF CO H and evaporated to dryness again after 30 min. The
product was purified by recrystallization from EtOH,
2
O and 2
nucleophile lysine in both H2 O, H2 O, and H2O.
3
2
30,31
yield 60%:
), 1.88 (m,
), 3.70 (t, 1H, R-CH), 6.26 (s, 1H,
Experimental Section
1
H NMR (D
H, δ-CH
Cl
2
O) δ 1.38 (m, 2H, γ-CH
2
), 1.59 (m, 2H, â-CH
2
Chemicals. Solvents for HPLC and HPLC/MS were HPLC grade
2
2
), 3.22 (t, 2H, ꢀ-CH
2
(
(
EM Science, Gibbstown, NJ). Solvents for GC/MS were nanograde
Mallinckrodt Baker, Paris, KY). TCE was distilled to remove inhibitors
+
2
CH); MS, m/z 258.22 (MH ).
E
N -Acryloyl-L-lysine. Acryloyl chloride (0.12 mL) was added
prior to epoxide synthesis. Reagents for synthesis, kinetics, and product
analysis were ACS grade or better, unless otherwise specified.
R
dropwise to a solution of 0.3 g N -tert BOC-L-lysine in a mixture of 3
3
mL of aqueous 1 N NaOH and 3 mL CH CN. Two hours later, the
mixture was acidified to pH with 1 N HCl, and the mixture was
1
13
Spectroscopy. H and C NMR spectra were recorded on a Bruker
AM 400 spectrometer at 400.13 MHz and 27 °C (Bruker, Billerica,
extracted with EtAc (3 × 3 mL). The EtAc extracts were combined,
dried over anhydrous Na
residue was dissolved in H
2
SO
O/CF
mixture was taken to dryness, first under N
4
, and evaporated to dryness in vacuo. The
(
22) Kline, S. A.; Solomon, J. J.; Van Duuren, B. L. J. Org. Chem. 1978,
3, 3596-3600.
23) Henschler, D.; Hoos, W. R.; Fetz, H.; Dallmeier, E.; Metzler, M.
CO
2
H (1:1, v-v). After 30 min, the
and then in vacuo (yield
4
2
3
(
2
Biochem. Pharmacol. 1979, 28, 543-548.
(
24) Miller, R. E.; Guengerich, F. P. Biochemistry 1982, 21, 1090-1097.
25) Byington, K. H.; Leibman, K. C. Mol. Pharmacol. 1965, 1, 247-
(28) Iyer, R. S.; Harris, T. M. Chem. Res. Toxicol. 1993, 6, 313-316.
(29) Dalgliesh, C. E. J. Chem. Soc., Chem. Commun. 1952, Part I, 137-
(
54.
(
2
5
141.
26) Liebler, D. C.; Guengerich, F. P. Biochemistry 1983, 22, 5482-
(30) Bernardi, L.; De Castiglione, R.; Fregnan, G. B.; Glasser, A. H. J.
Pharm. Pharmacol. 1967, 19, 95-101.
(31) Morozova, E. A.; Zevail, M. A.; Zhukova, G. F. Zh. Obsh. Khim.
1970, 40, 1376-1379.
489.
(
27) Guengerich, F. P.; Crawford, W. M., Jr.; Watanabe, P. G.
Biochemistry 1979, 18, 5177-5182.

11658 J. Am. Chem. Soc., Vol. 121, No. 50, 1999
Cai and Guengerich
7
8%):30,31 1H NMR (CD
3
OD) δ 1.12 (m, 4H, γ-CH and â-CH
2
2
), 1.47
Incorporation of H
2
18O into glyoxylic acid derived from TCE was
(
m, 2H, δ-CH ), 2.80 (t, 2H, ꢀ-CH ), 3.50 (t, 1H, R-CH), 5.17 (dd, 1H,
2
2
measured in the following manner. TCE oxide (4 µL of a mixture of
CH
2
dCH), 5.75 (d, 2H, CH
2
dCH).
TCE oxide/TCE, 1:5, v-v) was injected into a tightly capped vial
E
ꢀ
18
N -Glyoxylyl-L-lysine. A solution of N -acryloyl-L-lysine (vide supra,
(Teflon liner) containing 100 µL H
2
O (95% atom excess) and 6 µL
was added to
0
.2 g) in CH
C. The reaction was purged with Ar for 5 min to remove dissolved
and 5 mL of (CH S was added, and the reaction mixture was stirred
at -78 °C for 2 h and then at room temperature for 12 h.
reaction mixture was taken to dryness in vacuo and the product was
3
OH was treated with a stream of O
3
for 45 min at -78
of diglyme. After 30 s of the reaction, 2 mg of NaBH
4
°
O
the reaction mixture to reduce the glyoxylic acid to glycolic acid and
minimize carbonyl exchange. In an alternate experiment, the reaction
3
)
3 2
32,33
16
The
was diluted 10-fold with unlabeled H
aldehyde oxygen to 16O, and then NaBH
solutions were subjected to HPLC/MS analysis. The relative amounts
2
O (H
4
2
O) to exchange the
was added. The reaction
1
purified by recrystallization from EtOH (yield 30%): H NMR (D
δ 1.45 (m, 2H, γ-CH ), 1.68 (m, 2H, â-CH ), 1.87 (m, 2H, δ-CH
.98 (t, 2H, ꢀ-CH ), 3.76 (t, 2H, R-CH), 5.12 (s, 1H, COCHO);
NMR (D O) 22.0 ppm (γ-CH ), 26.6 (δ-CH ), 30.5 (â-CH ), 53.9 (ꢀ-
2
O)
18
16
2
2
2
3
),
C
of O and O in glyoxylic acid were determined by monitoring the
1
2
2
signals at m/z 75, 77, 79, and 81. HPLC/MS conditions (Zorbax RX-
-
1
2
2
2
2
C18, 2.1 × 150 mm) were: flow 0.5 mL min , 50 mM NH
4 3
HCO
CH
2
), 55.3 (R-CH), 169.0 (COCHO), 174.7 (COCHO), 175.7 (COOH);
(pH 6.0), atmospheric pressure chemical ionization source, mass range
m/z 20-180. All results are presented as means ( SD of triplicate
determinations.
+
MS, m/z 203.22 (MH ).
3
Kinetics. TCE oxide was diluted into dry CH CN, and the
concentration was estimated using a colorimetric assay with 4-(4-
2 2 2
HCO H and Cl CHCO H analyses were done in the same general
manner as that described above with the same HPLC/MS conditions.
After 120 s reaction time, the reaction mixture was made basic by the
4
-1
-1 34
nitrobenzyl)pyridine, using ꢀ560 ) 3.0 × 10 M cm
. The kinetics
of the decomposition of TCE oxide were observed by diluting TCE
oxide stock solution into 1.0 mL of buffer (final concentration ∼1.0
mM) at 0 °C. Aliquots of 50 µL were withdrawn at various times, and
the concentration of residual TCE oxide was measured colorimetrically
at 520 nm following reaction with 4-(4-nitrobenzyl)pyridine.24
The kinetics of formation of CO were determined with an electro-
chemical sensor (ERMF-0503; Draeger, Pittsburgh, PA). The detector
was placed in a circuit containing the reaction vial and a peristaltic
addition of 4 µL Et
HCO H, the relative amounts of O and O were determined by
monitoring the signals at m/z 45, 47, and 49. For Cl CHCO H, the
3
N and used for HPLC/MS analysis. In the case of
18
16
2
2
2
18
16
relative amounts of O and O were quantitated using the signals at
m/z 127, 129, and 131.
CO analyses used the same general experimental procedure. After a
certain amount of time, 10 µL of the headspace of the reaction vial
was injected into the GC/electron impact (EI) MS system for analysis.
3
5
pump. TCE oxide stock solution was injected into a vial containing
1
.0 mL of buffer (final concentration ∼1.0 mM) at 0 °C. The readings
18
16
The relative amounts of O and O in CO were determined by
of the sensor were recorded at various times.
monitoring the signals at m/z 28 and 30. The GC/EI MS conditions
Product Analysis. CO was measured using the electrochemical
detector system described above. The gas volume of the circuit was
pumped through the system until it reached an equilibrium between
the gas and water phase. Subsequently, the reading of the sensor was
recorded. The amount of CO formed was calculated based on a standard
curve generated using measured volumes of pure CO gas.
(
Alltech AT-Mole Sieve column, 0.53 mm i.d. × 30 m) were:
isothermal temperature 40 °C, injector temperature 150 °C, septum
purge 4.9 mL min , total flow 49 mL min , purge-off time 0 s, purge-
on time 30 s, mass range m/z 20-50, multiplier voltage 1200 V.
-1
-1
Control experiments for 18O incorporation were done with the organic
acids. HCO
acid (0.1 mg) was added to 50 µL of H
case of glyoxylic acid, NaBH (1 mg) was added to the reaction mixture
3
after 30 s. For both HCO H and glycolic acid, 4 µL of Et N was added
2
2
H (1 µL, 88% in H O), glyoxylic acid (0.1 mg), or glycolic
HCO
solution was injected into 1.0 mL of buffer (final concentration ∼1.0
mM). After 5 min, the reaction solution was acidified with conc H SO
to pH ∼1), and the mixture was extracted with Et O (3 × 1.0 mL).
The Et O extracts were combined and extracted with 1.0 mL of 10
mM tetrabutylammonium hydrogen sulfate (TBAS) (pH 6.0) in H O.
2 2 2
H and Cl CHCO H were measured by HPLC. TCE oxide stock
18
2
O (95% atom excess). In the
4
2
4
2
(
2
after 2 min reaction time. The amounts of 18O incorporation into those
2
acids were determined by direct injection of the diluted reaction
mixtures (CH
of those acids after HPLC column separations was also quantified by
2
OH) into MS. The amount of 18O remaining with each
3
The final aqueous solution was subjected to HPLC analysis. For HPLC
analysis, a stock solution (200 mM) of the ion-pair reagent TBAS was
1
8
HPLC/MS, using the conditions described earlier. O-enriched
prepared and the pH was adjusted to 6.0 with solid Na
2 4
HPO . The
1
8
Cl
atom excess). The amount of 18O incorporation into Cl
determined by direct injection of the diluted reaction mixture (CH
into the mass spectrometer (direct loop injection). The amount of O
remaining with Cl
2
CHCO
2
H was synthesized by reacting Cl
2
CHCOCl with H
2
O (95%
H was
OH)
isocratic reverse phase solvent was prepared by diluting the 200 mM
36
2
CHCO
2
stock TBAS to give a final concentration of 10 mM in H
conditions (Econosphere C18, 4.6 × 150 mm) were: flow rate 1.5 mL
min , solvent: 10 mM TBAS in H O (pH 6.0), λ 210 nm.
2
O. HPLC
3
1
8
-
1
2
2
CHCO
2
H after HPLC column separations was also
Glyoxylic acid formation (from TCE oxide) was measured in
reactions of the type described above. After 5 min, the products were
derivatized with 2,4-dinitrophenylhydrazine and analyzed for glyoxylic
quantified by HPLC/MS.
2
2
H Incorporation into TCE Oxide Products. H incorporation into
2
4
TCE oxide products (HCO
done in the same general manner as that described above. After 3 min
of the reaction, 4 µL of Et N was added to the mixture. The relative
amounts of H and H were determined by monitoring the signals at
m/z 45 and 46 for HCO
128 for Cl CHCO H.
2 2 2
H, Cl CHCO H, and glyoxylic acid) was
acid by isocratic reversed-phase HPLC. HPLC conditions (Zorbax
-
1
C18, 6.2 × 80 mm) were: flow rate 1.0 mL min , solvent A: CH
3
CN/
H
2
O, 10/90, v-v, solvent B: CH CN/H O, 45/55, v-v, t )0 min:
00% A, t ) 3 min: 50% A, 50% B, t ) 5 min: 100% B, t ) 9 min:
00% B, t ) 12 min: 100% A, λ 360 nm.
3
2
3
2
1
1
2
H, 73 and 74 for glyoxylic acid, and 127 and
1
1
8
2
2
O Incorporation into TCE Oxide Products. Decomposition of
1
8
2
TCE oxide was carried out at room temperature in H
excess, Aldrich). The reaction was allowed to proceed for a certain
amount of time, depending on which products were analyzed, and Et
was added to neutralize the sample.
2
O (95% atom
Control experiments for H incorporation were done with the organic
acids as described for the 18O incorporation experiments.
3
N
1
H Incorporation into
2
H-TCE Oxide Products. H incorporation
1
2
into H-TCE oxide products was performed in the same general manner
as that described above for the opposite experiment (vide supra).
(
32) Rueppel, M. L.; Rapoport, H. J. Am. Chem. Soc. 1972, 94, 3877-
883.
33) Pappas, J. J.; Keaveney, W. P.; Berger, M.; Rush, R. V. J. Org.
Chem. 1968, 33, 787-792.
34) Barbin, A.; Br e´ sil, H.; Croisy, A.; Jacquignon, P.; Malaveille, C.;
Montesano, R.; Bartsch, H. Biochem. Biophys. Res. Commun. 1975, 67,
Reaction of TCE Oxide with Lysine. TCE oxide (2 µmol), in
3
(
CH
mL). After 30 min, the reaction mixture was evaporated to dryness. A
coupling buffer (CH CN/pyridine/Et N/H O, 10/5/2/3, v-v-v-v, 0.10
mL) was used to dissolve the residue, and then phenylisothiocyanate
PITC) (5 µL, 42 µmol) was added. After 15 min, the reaction mixture
was taken to dryness in vacuo. The residue was dissolved in 0.5 mL
of a solution of 0.1 M NH CH CO (pH 6.4)/CH CN/CH OH (5/4/1,
3
CN, was added to a solution of L-lysine (2.6 µmol) in H
2
O (0.10
(
3
3
2
5
96-603.
(
(
35) Dowideit, P.; Mertens, R.; von Sonntag, C. J. Am. Chem. Soc. 1996,
1
18, 11288-11292.
(
36) McCaskill, D.; Croteau, R. Anal.Biochem 1993, 215, 142-149.
4
3
2
3
3

Trichloroethylene Oxide Hydrolysis
J. Am. Chem. Soc., Vol. 121, No. 50, 1999 11659
v-v-v) for both HPLC/UV(at 254 nm) and HPLC/MS analyses.37
HPLC conditions (Zorbax XDB-C8, 2.1 × 150 mm) were: flow rate
-1
0
4 3 2 2
.3 mL min , solvent A: 20 mM NH CH CO in H O (pH 6.0), solvent
B: 40 mM NH
4 3 2 3 3
CH CO /CH CN/CH OH, pH 8.4, 5/4/1, v-v-v, t )
0
min: 95% A, 5% B, t ) 5 min: 85% A, 15% B, t ) 12.5 min: 50%
A, 50% B, t ) 16 min: 50% A, 50% B, t ) 17.5 min: 0% A, 100%
B, t ) 19 min: 0% A, 100% B, t ) 21.5 min: 95% A, 5% B, t ) 28
min: 95% A, 5% B. In control experiments, TCE oxide (2 µmol) was
added to 0.10 mL of H O. After 30 min, lysine (2.6 µmol) was added
2
to the reaction mixture. Analysis of the products was done as described
for the direct reaction with TCE oxide.
1
8
O Incorporation into Reaction Products of TCE Oxide with
Lysine. 1 O incorporation into reaction products of TCE oxide with
8
lysine was carried out in the same manner as that described above except
Figure 1. pH profiles for the hydrolysis of TCE oxide (1) and the
formation of CO in aqueous solutions at 0 °C. (A) TCE oxide
1
8
that H
2
O (95% atom excess) was used instead of unlabeled H
O). After derivatization with PITC, the residue was dissolved in
.5 mL of a solution of 10 mM NH CH CO in H O/ CH CN (3/7,
2
O
1
6
(H
2
disappearance, (B) CO formation. Buffers used include HClO
pH -0.49, -0.04, 0.63, and 0.99), H PO /NaOH (pH 1.17, 2.34, 3.25,
and 3.30), EDTA/HCl (pH 4.14, 5.41, 6.15, 7.34, 8.38, 9.37, and 10.36),
EDTA/NaOH (pH 11.62), NaH PO /NaOH (pH 6.50, 7.25, and 7.40),
CH CO H/NaOH (pH 3.37, 3.50, 4.30, and 4.51), 2-(N-morpholino)-
4
/NaOH
0
4
3
2
2
3
(
3
4
3
7
16
18
v-v) for HPLC/MS analyses. The relative amounts of O and
O
were determined by monitoring the signals at m/z 310 and 312 for
N-formyllysine, 396 and 398 for N-dichloroacetyllysine, and 338 and
2
4
3
2
3
40 for N-glyoxylyllysine, as well as by collecting full spectra. HPLC
conditions (Zorbax RX-C18, 2.1 × 150 mm) were: flow rate 0.2 mL
min , solvent A: 10 mM NH CH CO in H O (pH 4.0), B: 10 mM
NH CH CO in H O/CH CN, 3/7, v-v, t ) 0 min: 95% A, 5% B, t )
ethanesulfonic acid (MES)/NaOH (pH 5.30), 2-(N-cyclohexylamino)-
ethanesulfonic acid (CHES)/NaOH (pH 8.70), 3-(N-cyclohexylamino)-
-
1
4
3
2
2
1
-propanesulfonic acid (CAPS)/NaOH (pH 10.39 and 11.13), and
HClO /NaOH (pH 12.20 and 13.24). The ionic strength was 0.10 M
NaClO ) in all cases.
4
3
2
2
3
4
1
5
1
0 min: 85% A, 15% B, t ) 25 min: 50% A, 50% B, t ) 32 min:
0% A, 50% B, t ) 35 min: 0% A, 100% B, t ) 40 min: 0% A,
00% B, t ) 41.5: min 95% A, 5% B, t ) 45 min: 95% A, 5% B.
(
4
2
H Incorporation into Reaction Products of TCE Oxide with
2
Lysine. H incorporation into reaction products of TCE oxide with
lysine was performed in the same manner as described above, using
2
2 2
H O instead of H
O. The relative amounts of H and 2H were
18
1
determined by monitoring the signals at m/z 310 and 311 for
N-formyllysine.
Results
pH Dependence of Decomposition of TCE Oxide (1). The
decomposition of TCE oxide and the formation of CO both
showed clear first-order kinetics. At 23 °C the t1/2 of TCE oxide
is 12 s. To make careful comparisons, we did all kinetic assays
at 0 °C, where the t1/2 is ∼100 s. Most of the determinations
were in the pH range of -2-14 at 0 °C (ionic strength 0.10
M). The change in kobsd as a function of pH for the decomposi-
tion of TCE oxide is shown in Figure 1A with kH ) 3.3 ×
-
2
-1 -1
-3 -1
1
0
M
s
and kH O ) 6.9 × 10 s . The kobsd (kH O) for
2
2
-
3
-1
the formation of CO (Figure 1 B) was 8 × 10 s , identical
to that for TCE oxide hydrolysis.
Products of TCE Oxide Hydrolysis. Four products (CO,
HCO2H, Cl2CHCO2H, and glyoxylic acid) were observed, as
Figure 2. Products formed in the hydrolysis of TCE oxide (1) at
varying pH in aqueous solutions at 0 °C. (A) CO, (B) HCO H (C)
Cl CHCO H, (D) glyoxylic acid. Buffers are listed under Figure 1.
Results are presented as means ( range of duplicate experiments in
each case. When range values are not indicated, the range was within
the limits shown by the mean point.
2
4
before, and quantitated (Figure 2). The formation of HCHO
could not be detected by HPLC (as the 2,4-dinitrophenyl-
2
2
2
38
1
hydrazone) or H NMR product analyses. The sum of the four
products was in the range of 70-95% theoretically expected
from TCE oxide at all pH values. For instance, at neutral pH
H2 O Incorporation into TCE Oxide Products. 18O
incorporation into TCE oxide degradation products was quan-
titated either by HPLC/MS, in the case of these acids, or by
GC/MS in the case of CO (Table 1). CO was formed without
18
(7.3) 1.0 mM TCE oxide yielded 0.36 mM Cl2CHCO2H, 0.20
mM glyoxylic acid, 0.42 mM HCO2H, and 0.21 mM CO. The
amount of Cl2CHCO2H formed was higher at pH < 0 and almost
constant in the pH range 0-14. In the case of glyoxylic acid,
the amount formed was similar at all pH values (with two low
values probably due to buffer artifacts). CO formation was less
at low pH (-2 to 0) and constant in the pH range 0-9.5.
Between pH ∼9.5-11.5 the amount of CO increased (∼2-fold)
with increasing pH and was rather constant from pH 11.5 to
1
8
any O incorporation. About 90% of the HCO2H formed had
1
8
two O atoms incorporated into the product. In the case of
18
Cl2CHCO2H, 91% contained one O atom. The carbonyl group
18
of the glyoxylic acid can readily exchange with H2 O. To
reduce the amount of carbonyl exchange, NaBH4 was added to
1
8
18
the reaction mixture of TCE oxide in H2 O after 30 s. An H2 O
control experiment indicated that about 50% of standard
1
≈
4. The amount of HCO2H formed remained constant from pH
-1 to 11 and increased at pH > 12.
1
8
glyoxylic acid contained one O atom after 30 s (carbonyl
(37) Ng, L. T.; Pascaud, A.; Pascaud, M. Anal. Biochem. 1987, 167,
18
exchange). Control experiments for O incorporation done
4
7-52.
under the same conditions indicated that there was no 18
O
(
38) Brady, J. F.; Lee, M. J.; Li, M.; Ishizaki, H.; Yang, C. S. Mol.
Pharmacol. 1988, 33, 148-154.
incorporation into HCO2H, Cl2CHCO2H, or glycolic acid (the

11660 J. Am. Chem. Soc., Vol. 121, No. 50, 1999
Cai and Guengerich
Table 1. H, Cl CHCO
Glyoxylic Acid Formed in the Decomposition of TCE Oxide
H
2
18O Incorporation into CO, HCO
2
2
2
H, and
oxylyllysine accounted for 4, 12, 5, and 9% of the starting
material (TCE oxide), respectively. The relative amount of the
unknown compound at tR 7:40 (which is probably N -gly-
R
%
%
%
%
with
with
with
two
with
oxylyllysine) was estimated at 3%, based on the standard curve
1
8
18
18
three 18O
product
CO
method
no
O
one
O
O
ꢀ
for N -glyoxylyllysine.
a
18
GC/MS
99.4 ( 0.2 0.6 ( 0.2
99.2 0.8
99.5 ( 0.1 0.5 ( 0.1
O Incorporation into Reaction Products of TCE Oxide
b
GC/MS
18
with Lysine. In the reaction of 1 with lysine in H2 O, ∼90%
c
GC/MS
1
8
of the formyllysine formed had one O atom incorporated into
the product (Table 2). In the case of dichloroacetyllysine only
d
d
e
HCO2H
Cl2CHCO2H HPLC/MS
HOCH2CO2H HPLC/MS
HPLC/MS
2 ( 1
9 ( 1
1 ( 1
10 ( 1
91 ( 1
4 ( 1
88 ( 1
18
90 ( 2
9 ( 2
5 ( 1
3% contained one O atom. Most of the glyoxylyllysine (95%)
had no O incorporated. Control experiments done with the
HPLC/MSf
91 ( 2
18
a
18O (95% atom excess, 0.10 mL) was used. The data were
synthetic lysine adducts (formyllysine, dichloroacetyllysine, and
glyoxylyllysine) under the same conditions indicated that there
was no O incorporation into any of the lysine adducts.
H
2
obtained by the selected ion monitoring (SIM) method. The reaction
time was 45 min. The reaction solvent for TCE oxide hydrolysis was
.055 N KOH (in 95% atom excess H
5 min). Conditions are in footnote a except that the reaction time
was 2 min and total ion current data was collected for the analysis.
Et N was added to the reaction mixture after 2 min reaction time.
NaBH
was diluted with unlabeled H
to stand for 10 min, and then NaBH
b
18
18
0
4
2
O) (SIM method, reaction time
2
H Incorporation into Reaction Products of TCE Oxide
c
2
with Lysine. In the reaction of 1 with lysine in H2O (99%
enriched), ∼5% of the N-formyllysine contained H.
2
d
3
e
f
4
was added to the reaction mixture after 30 s. The sample
16
2
O (i.e., H
2
O, 10 vol) after 2 min, allowed
was added.
Discussion
4
pH Profiles. The pH rate profile in Figure 1 A indicated that,
reduced product of glyoxylic acid by NaBH4) after 2 min
reaction time. Glyoxylic acid, recovered as glycolic acid, mostly
contained two (90%) 18O atoms. In a separate experiment, TCE
in the pH range studied, the rate law for the hydrolysis of TCE
oxide (1) contains two terms: a pH-independent term, kH O (6.9
2
-3
-1
× 10 s ) and a hydronium ion-dependent term, kH (3.3 ×
18
-2
-1 -1 39,40
oxide was mixed with H2 O for 2 min and then diluted into a
10
M
s ).
16
1
0-fold excess of unlabeled H2O (H2 O) prior to the addition
+
of NaBH4 and mass spectral analysis. In this experiment, the
extent of labeling at the carboxylic acid oxygens can be
kobsd ) k + k [H ]
(1)
H O
H
2
18
determined, since any O incorporated into the aldehyde is
1
8
A hydroxide ion-dependent region can also be involved in
diluted out. One O atom (91%) was found to be incorporated
into the carboxylic oxygen in this experiment.
39,41,42
the decomposition of epoxides.
In the case of TCE oxide,
none was observed, possibly because of assay limitations. The
rate law for the formation of CO (Figure 1 B) contained only
H Incorporation into TCE Oxide Products. When analyses
2
similar to those described above were done with H2O and TCE
-
3
-1
2
a pH-independent term, kH2O (8 × 10 s ), in the pH range
0-14. The rate constants for pH < -1 or pH > 15 were not
amenable to measurement because of the response time (10-
25 s) of the electrochemical CO sensor.
oxide, <5% of each product showed incorporation of H. An
2
1
inverse experiment was done with [ H]TCE oxide and H2O
and the same results were obtained (i.e., no incorporation of
H).
1
Comparison of the pH-independent rate constant for the
formation of CO with that for the disappearance of TCE oxide
indicates that the disappearance of TCE oxide is the rate-limiting
step for CO formation under the conditions examined.
Product Analyses of TCE Oxide. Four major products (CO,
The amounts of Cl2CHCO2H formed in the decomposition
of TCE oxide and [ H]TCE oxide were compared to determine
if a hydrogen kinetic isotope effect existed. The differences in
yield were within (5% in the pH range 0.5-5.
2
Reaction of TCE Oxide with Lysine. Five lysine adducts
were detected by both HPLC/UV and HPLC/MS (Figure 3).
HCO2H, Cl2CHCO2H, and glyoxylic acid) are formed in the
R
ꢀ
ꢀ
21,23,24,43,44
N -Formyllysine, N -dichloroacetyllysine, and N -glyoxylyl-
hydrolysis of TCE oxide.
Their amounts were not
ꢀ
lysine were synthesized, and N -formyllysine was purchased as
dramatically affected by pH except at pH > 14 (Figure 2).
HCHO was not formed. The small breaks seen at certain pH
values (e.g., pH 3 in the case of Figure 2 B and C) are possibly
related to the buffer and not considered to be unusual. The lack
of a major pH effect on product distribution indicated that the
mechanism leading to the formation of each of the four final
products is not affected dramatically (<3×) by changing the
pH in the range of -1.5 to 14, even though the hydrolysis of
TCE oxide 1 is changed from hydronium ion-dependent to pH-
independent. At higher pH (pH > 14), the amount of HCO2H
increased.
standards for the reaction. The four standards eluted from the
column with similar tR values as the peaks (tR 9:18, 10:58, 11:
9, and 16:45) displayed in Figure 3. The control experiment
for the reaction indicated no formation of these five lysine
adducts, under the same conditions, when TCE oxide hydrolysis
preceded the addition of lysine (Figure 3).
1
The mass spectra of the products derived form the reaction
match those expected and experimentally determined for the
+
ꢀ
standard compounds. The MH multiplet for N -dichloroacetyl-
lysine showed a ratio of m:m + 2:m + 4 peaks near the
theoretical ratio of 9:4:1 for a dichloro compound (with
(39) Ross, A. M.; Pohl, T. M.; Piazza, K.; Thomas, M.; Fox, B.; Whalen,
13
correction for C natural abundance). The unknown compound
D. L. J. Am. Chem. Soc. 1982, 104, 1658-1665.
(40) Gillilan, R. E.; Pohl, T. M.; Whalen, D. L. J. Am. Chem. Soc. 1982,
104, 4482-4484.
R
at tR 7:40 is probably N -glyoxylyllysine, as judged by its mass
R
spectrum. The possibility that N -dichloroacetyllysine is formed
ꢀ
(41) Pocker, Y.; Ronald, B. P.; Anderson, K. W. J. Am. Chem. Soc. 1988,
and eluted with the N isomer cannot be excluded on the basis
1
10, 6492-6497.
42) Rogers, D. Z.; Bruice, T. C. J. Am. Chem. Soc. 1979, 101, 4713-
4719.
of the available evidence.
(
The relative amounts of lysine adducts formed in the reaction
were estimated based on the standard curves obtained using the
(43) McKinney, L. L.; Uhing, E. H.; White, J. L.; Picken, J. C., Jr. J.
Agric. Food Chem. 1955, 3, 413-419.
R
four authentic lysine adducts mentioned previously. N -Formyl-
(44) Fox, B. G.; Borneman, J. G.; Wackett, L. P.; Lipscomb, J. D.
ꢀ
ꢀ
ꢀ
lysine, N -formyllysine, N -dichloroacetyllysine, and N -gly-
Biochemistry 1990, 29, 6419-6427.

Trichloroethylene Oxide Hydrolysis
J. Am. Chem. Soc., Vol. 121, No. 50, 1999 11661
Figure 3. HPLC/MS traces and mass spectra of the PITC derivatives of the reaction of lysine with TCE oxide (1). The control experiment for this
reaction is also shown. The structure assignments for the PITC derivatives are indicated in the inserts showing the mass spectra. As indicated in
R
the text, the material eluting at t
R
7:40 is probably N -glyoxylyllysine.
Table 2.
from Trichloroethylene Oxide
H
2
18O Incorporation into Lysine Conjugates Derived
Scheme 2. Mechanism of pH-Independent Hydrolysis of
TCE Oxide (1) in Aqueous Solution at 0 °C
producta
% with no 18
O
% with one 18
O
N-formyllysine
N-dichloroacetyllysine
N-glyoxylyllysine
10 ( 2
97 ( 1
95 ( 2
90 ( 2
3 ( 1
5 ( 2
a
Isotopic content was determined by HPLC/MS in all cases.
Mechanism of pH-Independent Hydrolysis of TCE Oxide.
The dominant mechanism for hydrolysis of TCE oxide (1) is
displayed in Scheme 2. In the formation of Cl2CHCO2H
(Scheme 2), a zwitterionic intermediate 9 could be formed (from
8
which could be formed by cleavage of the C-O bond of TCE
-
oxide) and undergo an intramolecular rearrangement (Cl shift)
to generate dichloroacetyl chloride 12 which would subsequently
decompose to Cl2CHCO2H.1
2,13,22-24,41,45
The zwitterion inter-
mediate 9 could also hydrolyze at the less sterically hindered
and more electronically deficient carbon of the zwitterion to
give a glycol anion 2, which would dehydrohalogenate to form
a reactive oxoacetyl chloride (glyoxyl chloride) intermediate
1
0. The oxoacetyl chloride 10 could hydrolyze to generate either
23,24
glyoxylic acid
or an anionic intermediate 11 which could
generate HCO2H, CO, and chloride through a concerted
mechanism.46 The decomposition of the anionic intermediate
11 could also go through stepwise mechanisms to form HCO2H
and CO as shown in Scheme 3, which cannot be ruled out by
the available evidence. In Scheme 3, as demonstrated in pathway
A, the anionic intermediate 11 could go through a C-C bond
cleavage to form HCO2H and formyl chloride anion, the latter
of which would lose chloride to afford CO. In pathway B the
anionic intermediate 11 could eliminate chloride first to generate
an intermediate 13, then with the loss of CO, the intermediate
H2 O Incorporation. Without any further 18O exchange the
18
Cl2CHCO2H, HCO2H, CO, and glyoxylic acid formed in the
hydrolysis of TCE oxide in the presence of H2 O should
theoretically contain one, two, none, and two O atoms,
respectively. (Less than 5% O was found incorporated into
the other standard carboxylic acids in control experiments.) The
results from the H2 O experiment (Table 1) showed that
Cl2CHCO2H, HCO2H, CO, and glyoxylic acid contained ∼91%
18
1
3 would generate HCO2H.
18
The mechanism postulated in Scheme 2 is supported by the
following results.
1
8
(
45) Gold, B.; Leuschen, T.; Brunk, G.; Gingell, R. Chem.-Biol. Interact.
981, 35, 159-176.
46) Czarnowski, J. J. Chem. Soc., Perkin Trans. 2 1991, 9, 1459-1462.
1
8
1
(

1
1662 J. Am. Chem. Soc., Vol. 121, No. 50, 1999
Cai and Guengerich
Scheme 5. Possible Mechanism for the Formation of
Scheme 3. Possible Mechanism for the Formation of
HCO2H and CO from Oxoacetyl Chloride (10) through
Stepwise Pathways in Aqueous Solution at 0 °C
Cl2CHCO2H Involving a Hydride Shift
Scheme 6. Possible Mechanism for the Formation of
Cl2CHCO2H, Involving Hydrolysis of the Zwitterionic
Intermediate 9 at the More Substituted Carbon Site
Scheme 4. Mechanism of Reaction of TCE Oxide (1) with
Lysine in Aqueous Solution
Schemes 2 and 4. The results also argue against formyl chloride
4
as an intermediate in the formation of N-formyllysine. Formyl
3
5
chloride yields CO at pH <13. However, the hydrolysis of
TCE oxide in H2 O yielded CO without O incorporation
1
8
18
1
8
(Table 1) and N-formyllysine with complete O incorporation.
ꢀ
Control experiments done with the synthetic lysine adducts (N -
formyllysine, dichloroacetyllysine, and glyoxylyllysine) under
the same conditions indicated that there was no O incorpora-
tion into any of the lysine adducts.
1
8
one, ∼90% two, <1%, and 90% two 18O atom(s), respectively,
consistent with the mechanism proposed in Scheme 2. The
labeling pattern for glyoxylic acid indicates that one oxygen
from H2O is incorporated into the carboxylate. A second oxygen
Hydronium Ion-Dependent Hydrolysis of TCE Oxide. The
mechanism proposed for hydronium ion-dependent hydrolysis
of TCE oxide is very similar to that for the pH-independent
hydrolysis except for the first step, which involves hydronium
ion attacking TCE oxide to form a TCE oxide cation intermedi-
ate. The mechanism appears to be very similar to that of Scheme
2, the pH-independent reaction, in that the product distribution
is not dramatically changed (Figure 2).
(from H2O) appears to be incorporated into aldehyde. Thus, only
one of the two carboxylic acid oxygen atoms originated in TCE
oxide.
H Incorporation Experiments. No deuterium incorporation
2
from H2O into Cl2CHCO2H, HCO2H, or glyoxylic acid was
found to occur during the hydrolysis of TCE oxide. Further, no
protium from H2O was incorporated into the products derived
Other Possible Mechanisms for Hydrolysis of TCE Oxide.
One possible mechanism proposed for the formation of Cl2-
2
from [ H]TCE oxide, consistent with Scheme 2 in which the H
CHCO2H involves a hydride shift (Scheme 5). In general,
of TCE oxide is found in the carboxylic acid products.
Reaction of TCE Oxide with Lysine. The reactive acyl
halides 10 and 12 should be potentially reactive with nucleo-
R-chloroepoxide rearrangements involve halogen migration.12
Product analysis showed that the amount of Cl2CHCO2H formed
2
ꢀ
in the hydrolysis of [ H]TCE oxide was within (5% of the
philes such as lysine (Scheme 4). N -Dichloroacetyllysine (and
R
R
Cl2CHCO2H formed from TCE oxide. A significant kinetic
deuterium isotope effect on a hydride shift might have been
expected if a hydride shift were involved, which would lower
the yield of Cl2CHCO2H in favor of other products.
most probably N -dichloroacetyllysine) (total 5% yield), N -
ꢀ
and N -glyoxylyllysine (total yield 12%, under the assumption
that the unknown compound at tR 7:40 is probably N -
glyoxylyllysine), and N - and N -formyllysine (total yield16%)
were detected by both HPLC/UV and HPLC/MS measurements.
It was recently reported that an intermediate species, dichloro-
R
R
ꢀ
Another possible mechanism proposed for the formation of
Cl2CHCO2H is displayed in Scheme 6. The zwitterion inter-
mediate 9 could hydrolyze at the more sterically hindered carbon
site to give a dichloroacetyl chloride hydrate anion 15 which
would dehydrohalogenate to form Cl2CHCO2H. However, if this
pathway is dominant, dichloroacetyllysine would contain g50%
acetyl chloride 12, was identified in the photooxidation of TCE
on TiO2 using infrared spectroscopy.47 This reported observation
also suggests the possible existence of dichloroacetyl chloride
1
2 in our system, which was trapped by lysine, in the reaction
1
8
18
ꢀ
R
one O atom in the O incorporation study with lysine.
Therefore Scheme 6 is inconsistent with the experimental results
presented previously and could not be the dominant pathway
for the formation of Cl2CHCO2H.
of TCE oxide, to form N -dichloroacetyllysine and probably N -
dichloroacetyllysine.
1
8
O Incorporation into Reaction Products of TCE Oxide
with Lysine. The results of 1 O incorporation experiments
8
1
8
One possible mechanism for the formation of CO and HCO2H
could involve initial cleavage of C-C bond of TCE oxide. In
this mechanism, hydrolysis of TCE oxide could give an ether
intermediate. This unstable ether intermediate could react with
H2O to generate formyl chloride 4 and chlorodihydroxymethane,
showed that ∼90% of the N-formyllysine contained one
O
atom (Table 2). The N-dichloroacetyllysine and N-glyoxylyl-
lysine contained <5% 18O. These results are consistent with
(47) Fan, J.; Yates, J. T., Jr. J. Am. Chem. Soc. 1996, 118, 4686-4692.

Trichloroethylene Oxide Hydrolysis
J. Am. Chem. Soc., Vol. 121, No. 50, 1999 11663
Scheme 7. Mechanism Proposed by Henschler et al.23 to
Account for Formation of CO and HCO2H
pH-independent and hydronium ion-dependent regions (Figure
1 A). C-C bond scission in epoxides is associated with one
carbon being at the oxidation state equivalent to that bearing
two leaving groups (Cl). The formation of CO mainly involves
a pH-independent region (Figure 1 B). Comparison of the rates
and pH profile for the formation of CO (Figure 1 B) with the
disappearance of TCE oxide (Figure 1 A) implies that the
disappearance of TCE oxide is the rate-determining step for
the formation of CO under the conditions studied. The similar
product distribution of CO and these carboxylic acids (HCO2H,
Cl2CHCO2H, and glyoxylic acid) over the pH range of -1.5 to
which could form CO35 and HCO2H. However, in ab initio
molecular orbital studies on the structure and stability of
ethylene oxide cation radical, it has been concluded that the
1
4 (Figure 2) indicates that the mechanism leading to the
4
8,49
formation of each of the four major products is not dramatically
affected by pH even though the hydrolysis of TCE oxide is
changed from being hydronium ion-dependent to pH-inde-
pendent. The mechanisms for the hydronium ion-dependent and
pH-independent hydrolysis (Scheme 2) of TCE oxide were
C-O bonds are relatively weak and easily broken.
The
fission of the C-C bond requires more energy (105-120 kJ
-1
mol ). In the case of TCE oxide, the scission of the C-C bond
will most likely require more energy than that of C-O bond,
and we do not favor this pathway.
18
developed based on three experimental results: H2 O incor-
poration (Table 1), H incorporation, and reaction of TCE oxide
Several proposed mechanisms for the formation of CO and
23,24
HCO2H in the previous literature
are inconsistent with our
1
6
18
with lysine in H2 O (Figure 3) and in H2 O. In the pH-
independent hydrolysis (Scheme 2), a zwitterionic intermediate
experiment results. A mechanism proposed by Henschler et al.
2
3
2
2
(
Scheme 7) predicts 100% H incorporation from H2O into
9
could be formed (from TCE oxide) and undergo an intramo-
HCO2H in the hydrolysis of TCE oxide carried out in the
-
2
lecular rearrangement (Cl shift) to generate dichloroacetyl
presence of H2O. This possibility is excluded by the results
chloride 12 which would subsequently decompose to Cl2-
presented here. Another problem with the results presented in
this earlier paper is that the reported product distribution differs
1
2,13,22-24,41,45
CHCO2H.
The zwitterionic intermediate 9 could
24
also hydrolyze at the less sterically hindered carbon to give
glycol anion 2 which would dehydrohalogenate to form an
considerably from our own (Table 1). Further, the different
1
8
patterns of O incorporation into CO (Table 1) and N-
oxoacetyl chloride intermediate 10. The oxoacetyl chloride 10
formyllysine (vide supra) argue against a role of formyl chloride
could hydrolyze to generate either glyoxylic acid2
3,24
or an
4
.
anionic intermediate 11 which could go through a concerted
In 1982 this laboratory proposed a mechanism similar to that
4
6
mechansim to afford CO and HCO2H. Our mechanism
proposed for the hydronium ion-dependent hydrolysis is very
similar to that for the pH-independent hydrolysis except for the
first step, which involves hydronium ion attacking TCE oxide
to form a TCE oxide cation intermediate. Several mechanisms
of Scheme 2 to account for formation of Cl2CHCO2H and
glyoxylic acid and two possible mechanisms to account for the
formation of CO and HCO2H under basic conditions, based on
evidence available at that time (Scheme 1). Three pieces of new
evidence are inconsistent with Scheme 1. First, one would expect
base-catalyzed decomposition of TCE oxide at high pH.
However, only pH-independent hydrolysis was observed up to
pH ∼15. Second, formyl chloride 4 predominantly decays into
(Schemes 1 and 5-7) (either possible or proposed in previous
literature) are discussed but ruled out on the basis of evidence
presented in this study.
35
The hydrolytic mechanism is of interest in that it yields acyl
halides 10 and 12, which were shown to react with lysine. The
direct reaction of TCE oxide with nucleophiles would not be
expected to yield those adducts, in that all should contain
linkages of the type N-CH2-Cl, S-CH2-Cl, etc. Whether
lysine adducts are prominent in protein modification is currently
unknown and the subject of further investigation.
CO except at very high pH (∼14), at which HCO2H is formed.
2
2
Third, 50% H incorporation from H2O into HCO2H would be
expected in 2H2O in the carbene mechanism presented in
2
Scheme 1. However, no H incorporation was observed in this
study.
Conclusions
The decomposition of TCE oxide (1) is considered as a
paradigm for multi-halogenated epoxide hydrolysis and involves
Acknowledgment. This work was supported in part by U.
S. Public Health Service Grants R35 CA44353 and P30
ES00267.
(
(
48) Lopez, L.; Troisi, L. Tetrahedron Lett. 1989, 30, 3097-3100.
49) Bouma, W. J.; MacLeod, J. K.; Radom, L. J. Am. Chem. Soc. 1979,
1
01, 5540-5545.
JA9914240