Chemical Research in Toxicology
Rapid Report
7
using nonlinear regression as reported. Rate constants were
8
determined by the Monte Carlo simulation. The correspond-
ing first order or pseudo-first order T1/2 values were calculated
from T1/2 = ln(2)/k for each value of k with confidence
intervals of 95%. The T1/2 values for individual species were
calculated using the % total conversion (see Supporting
Information).
The overall fit of the 31P NMR data to the integrated rate
2
2
equations is good (average r = 0.887). The r values for C
(
(
0.771) and E (0.783) are lower than those for A (0.985), B
0.917), and D (0.979). This likely results in part from error
due to the pseudo-first-order approximation (k [D] ≈ k ′) used
5
5
in deriving the integrated rate equations. While the
concentration of D is always at least 10 times that of B, it
varies from 0 to 93 mol %, and thus, k ′ is an approximation of
5
the average value over time. At 0 h, the relative conversion of B
to C or E is expected to shift toward the formation of C and as
the concentration of D increases over time, the conversion of B
to E would increase. Furthermore, in phosphate buffer, a higher
percentage conversion to E is expected.
The T1/2 degradation rate of A (32.7 h) and formation rate of
D (33.0 h) (Supporting Information) are in agreement with
1
reported rates (11−58 h) summarized previously. Rate4s
derived from measurements of ethephon degradation,
9
,10
phosphate formation, or ethylene generation are all reported
as a measure of the rate of ethephon hydrolysis. This study
supports that approximation as the measured overall hydrolysis
rate of A does not differ from the formation rate of D. The
measured rate for the formation of D from A (T1/2 = 37 h) is
Figure 2. Time course spectra for low concentration products B, C,
and E formed from the degradation of A at pH 7.4.
acidic D O. The singlet peak at 18.76 ppm is thus in the
2
expected region for C at pH 7.4 and is assigned as such. The
doublets at −6.29 and 13.51 ppm have J couplings of 24 Hz and
1
0 times faster than the rate for the formation of B from A
(
T1/2 = 297 h) indicating that the overall rate of ethephon
1
integrate nearly equally at all time points. The spectra are H
hydrolysis is governed by the rate of phosphate formation (k3).
The rate of ethylene generation would also approximately equal
the measured rate of phosphate generation.
decoupled, and peak splitting results from P−P spin spin
coupling only. J = 24 Hz matches two bond P−P coupling of a
5
diphosphate. Two doublets indicate unequal phosphorus
2
-Hydroxyethylphosphonate (C) has been reported pre-
atoms corresponding to a phosphate (−6.29 ppm) and a
phosphonate (13.51 ppm). We therefore assign these peaks as
the phosphorus atoms in E. All peak assignments are
viously as a degradation product of A in buffered solutions4,
formed in 1% and 8% yield at pH 7.4 and 13.8, respectively,
suggesting either differential formation or degradation of B at
varying pH. Up to 10 mol % C forms from photolysis or soil
degradation of A and up to 3.7 mol % is formed from anaerobic
1
1
31
corroborated by H NMR and H coupled P NMR spectra
Supporting Information).
(
3
1
The transient P NMR peak at 28.11 ppm (designated B) is
11
degradation, likely by direct SN reaction at the β-carbon
2
of particular interest. The atom connectivity to phosphorus in
B is similar to that in monoalkyl-C. We have observed
monomethyl 2-hydroxyethylphosphonic acid (26.9 ppm) as an
intermediate in the hydrolysis of dimethyl 2-hydroxyethylphos-
phonic acid (35.87 ppm) to C (25.3 ppm) in refluxing HCl.
Thus, oxaphosphetane B is expected to have a chemical shift
downfield of the 18.76 ppm C signal. Although numerous
rather than via the intermediacy of B. To the best of our
knowledge, E is previously unreported as a degradation product
of A.
The BChE inhibitor forms spontaneously at neutral to
alkaline pH and is short-lived. The concentration−time curves
1
31
based on enzyme inhibition or P NMR analysis (this study)
are almost identical (Figure 3) indicating that the two methods
measure the same compound (the transient BChE inhibitor,
B). The results of our kinetic modeling suggest that in pH 7.4
carbonate buffer in the absence of BChE, the total conversion
mechanistic 31P NMR studies have proposed an oxaphosphe-
tane intermediate in the Wittig reaction, to our knowledge, only
6
one of these studies examined the phosphonate modification
of the Wittig reaction where the oxaphosphetane intermediates
3
1
to B is 10.7 mol % and that B degrades to D (6 mol %, k =
would be most similar to B. P NMR chemical shifts of 35 and
4
−1
−1
6
0
.139 h ), C (3.2 mol %, k = 0.0584 h ), and E (1.5 mol %,
k ′ = 0.0317 h ). In the presence of BChE, 3−5 mol % of A
was trapped as a BChE adduct over 168 h. The difference in
32 ppm were reported for these dialkyl oxaphosphetanes.
2
−1
Thus, a chemical shift at 28.11 ppm, slightly upfield of similar
dialkyl oxaphosphetanes, is as expected for 2-oxo-2-hydroxy-
5
1
1
,2-oxaphosphetane (B).
The reactions in Figure 1 were mathematically modeled
values for % conversion to B likely results from a 30−50%
efficiency for the BChE trap with the remainder of B degraded
to C, D, and E under the conditions of the trapping
experiments. Although the formation and degradation of B
depends on conditions, the high agreement (Figure 3) between1
the concentration−time curves generated from BChE trapping
using irreversible first order and pseudo-first-order kinetics for
k ≠ k ≠ k ≠ k ≠ k ′ and B = C = D = E = 0 to derive
1
2
3
4
5
0
0
0
0
integrated concentration−time rate equations (see Supporting
Information). These equations were fit simultaneously to the
2
31
assigned peak data sets by maximizing the sum of the r values
and direct P NMR monitoring of ethephon degradation (this
1
321
dx.doi.org/10.1021/tx4002429 | Chem. Res. Toxicol. 2013, 26, 1320−1322
Lantz, Stephen R.
