An Integrated Experimental and Computational Approach for Characterizing the Kinetics and Mechanism of Triadimefon?Racemization

Article abstract of DOI:10.1002/chir.22622

Enantiomers of chiral molecules commonly exhibit differing pharmacokinetics and toxicities, which can introduce significant uncertainty when evaluating biological and environmental fates and potential risks to humans and the environment. However, racemization (the irreversible transformation of one enantiomer into the racemic mixture) and enantiomerization (the reversible conversion of one enantiomer into the other) are poorly understood. To better understand these processes, we investigated the chiral fungicide, triadimefon, which undergoes racemization in soils, water, and organic solvents. Nuclear magnetic resonance (NMR) and gas chromatography / mass spectrometry (GC/MS) techniques were used to measure the rates of enantiomerization and racemization, deuterium isotope effects, and activation energies for triadimefon in H₂O and D₂O. From these results we were able to determine that: 1) the alpha-carbonyl carbon of triadimefon is the reaction site; 2) cleavage of the C-H (C-D) bond is the rate-determining step; 3) the reaction is base-catalyzed; and 4) the reaction likely involves a symmetrical intermediate. The B3LYP/6–311?+?G** level of theory was used to compute optimized geometries, harmonic vibrational frequencies, nature population analysis, and intrinsic reaction coordinates for triadimefon in water and three racemization pathways were hypothesized. This work provides an initial step in developing predictive, structure-based models that are needed to identify compounds of concern that may undergo racemization. Chirality 28:633–641, 2016.

Full text of DOI:10.1002/chir.22622

CHIRALITY (2016)
An Integrated Experimental and Computational Approach for
Characterizing the Kinetics and Mechanism of Triadimefon
Racemization
QIANYI CHENG, QUINCY TENG, SATORI A. MARCHITTI, CALEB M. DILLINGHAM AND JOHN F. KENNEKE2*
1
2
3
1
1
Student Services Authority Contractor, National Exposure Research Laboratory, U.S. Environmental Protection Agency, Athens, Georgia, USA
2
National Exposure Research Laboratory, U.S. Environmental Protection Agency, Athens, Georgia, USA
3
ORISE Fellow, National Exposure Research Laboratory, U.S. Environmental Protection Agency, Athens, Georgia, USA
ABSTRACT
Enantiomers of chiral molecules commonly exhibit differing pharmacokinetics
and toxicities, which can introduce significant uncertainty when evaluating biological and envi-
ronmental fates and potential risks to humans and the environment. However, racemization
(
the irreversible transformation of one enantiomer into the racemic mixture) and
enantiomerization (the reversible conversion of one enantiomer into the other) are poorly under-
stood. To better understand these processes, we investigated the chiral fungicide, triadimefon,
which undergoes racemization in soils, water, and organic solvents. Nuclear magnetic resonance
(
NMR) and gas chromatography / mass spectrometry (GC/MS) techniques were used to mea-
sure the rates of enantiomerization and racemization, deuterium isotope effects, and activation
energies for triadimefon in H O and D O. From these results we were able to determine that:
2
2
1
) the alpha-carbonyl carbon of triadimefon is the reaction site; 2) cleavage of the C-H (C-D)
bond is the rate-determining step; 3) the reaction is base-catalyzed; and 4) the reaction likely in-
volves a symmetrical intermediate. The B3LYP/6–311 + G** level of theory was used to compute
optimized geometries, harmonic vibrational frequencies, nature population analysis, and intrin-
sic reaction coordinates for triadimefon in water and three racemization pathways were hypoth-
esized. This work provides an initial step in developing predictive, structure-based models that
are needed to identify compounds of concern that may undergo racemization. Chirality
0
0:000–000, 2016. © 2016 Wiley Periodicals, Inc.
KEY WORDS: chirality; conazoles; enantiomerization; environmental chemicals; pesticides; risk
assessment; stereochemistry
Chiral molecules possess at least two nonsuperimposable
mirror images (enantiomers) and may have additional stereo-
isomers depending on the number of asymmetric centers. As
a result of this “mixture” a chiral molecule in a chiral environ-
ment (e.g., biological system containing enzymes) will typi-
cally exhibit more varied and complex interactions than an
achiral molecule. This is illustrated by the fact that enantio-
mers commonly exhibit different environmental fates, phar-
macokinetics, and toxicities. Ignoring these differences can
introduce significant uncertainty in understanding and
modeling the physiological and environmental fate of chiral
chemicals and evaluating their risk to human health and the
environment. While it is common practice to evaluate the im-
pact of chirality on the efficacy and safety of pharmaceuticals,
this is typically not the case for chemicals of environmental
concern (e.g., pesticides). There is an even greater dearth
of stereoisomer-specific information regarding unintentional
exposures (outside the scope of product use) such as chiral
implement a “chiral switch” where the racemic mixture is re-
placed with the most active stereoisomer(s). To that end, the
U.S. Environmental Protection Agency (U.S. EPA) has issued
an interim policy for stereoisomeric pesticides to determine
whether the enriched mixture(s) are of greater concern envi-
ronmentally and from a human health perspective than the ra-
4
cemic mixture. The European Union has revised regulations
so that plant protection products containing significant pro-
portions of inactive stereoisomers become a candidate for chi-
5
ral switch.
The scientific literature contains many reports on the
stereoselective transformation of chiral compounds under bi-
ological and environmental conditions; in many cases one en-
antiomer is preferentially degraded or metabolized. Far less
information, however, exists on the biological and environ-
mental racemization (and enantiomerization) of chiral com-
pounds. Racemization is the macroscopic statistical process
of the irreversible transformation of one enantiomer into the
racemic mixture, while enantiomerization refers to the micro-
scopic and molecular process of the reversible conversion of
1
pharmaceuticals released into the environment and humans
2
exposed to chiral pesticides.
6
Approximately half of all pharmaceuticals are chiral com-
one enantiomer into the other. Racemization has been found
pounds, with a significant number produced as pure enantio-
3
mers. More than 30% of pesticides are chiral; however, in
*
9
Correspondence to: J. F. Kenneke, U.S. Environmental Protection Agency,
60 College Station Road, Athens, Georgia 30605. E-mail: kenneke.
most cases the racemic material is used even though the pes-
ticidal activity typically resides in one enantiomer (or several
stereoisomers when there is more than one chiral center). In
an effort to reduce the unnecessary environmental burden of
inactive stereoisomers, there has been a movement to
john@epa.gov
Received for publication 19 May 2016; Accepted 24 June 2016
DOI: 10.1002/chir.22622
Published online in Wiley Online Library
(wileyonlinelibrary.com).
©
2016 Wiley Periodicals, Inc.

CHENG ET AL.
7
8
to occur with pharmaceuticals and pesticides and is a point
of emerging concern. Due to differences in stereospecific tox-
icity and efficacy, it is critical to assess the potential for a com-
pound to undergo racemization. In terms of use and
application, if racemization occurs quickly it would not make
sense to use a chiral switch or develop enantiopure pharma-
ceuticals and pesticides. Racemization can also have a pro-
found impact on sample preparation, storage, and analysis—
especially if it occurs unknowingly. For these reasons, when
evaluating the exposure and effects of chiral compounds it
is imperative to consider configurational instability under en-
vironmental and biological conditions.
the fact that triadimefon has been shown to undergo varying
degrees of racemization in soils, water, and organic sol-
vents.1
2,13,16,17
Thus, the degree of racemization could impact
the distribution of triadimenol stereoisomers that are formed
from the metabolism or environmental transformation of
triadimefon.
A variety of mechanisms have been proposed to account
for the racemization of chiral compounds including: keto-
enol tautomerization, resonance-stabilized carbanion inter-
mediates, and base catalysis involving a transition state with
the proton halfway between the base and the chiral car-
18
bon. In the case of triadimefon, several crystallographic
8,19–21
8
Recent studies have shown that some chiral, 1,2,4-triazole
studies
and laboratory studies have been conducted
8
fungicides can undergo racemization. Triazole fungicides
and racemization was hypothesized to be related to the acid-
ity of the α-hydrogen; however, detailed kinetic studies ex-
amining microscopic and macroscopic rate constants were
not conducted nor was a mechanism of racemization
developed.
have both medical and agricultural applications and some of
9
these have been detected in surface and waste waters and
10
in human and ecological biomonitoring studies.
Triadimefon [(RS)-1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-
,2,4-triazol-1-yl)butan-2-one] (Fig. 1) is a chiral systemic
1
Here we combine experimental and computational studies
for the first time to characterize the kinetics and mechanism
for the racemization of triadimefon under physiological and
environmentally relevant conditions. By delineating the kinet-
ics and mechanism of racemization, this work provides an ini-
tial step in developing predictive, structure-based models that
are needed to identify quickly both pharmaceutical and
nonpharmaceutical compounds that may undergo racemiza-
tion. Such models will not only aid in evaluating chemicals
for chiral switch and enantiopure applications, but also pro-
vide a means for improving human health and ecological risk
assessment by identifying chemicals that may require special-
ized testing.
broad-spectrum 1,2,4-triazole fungicide that is used on orna-
mentals and food crops. It has been shown that R-triadimefon
is 2-fold more toxic than the racemate to blackfly larvae and
11
under biological conditions, the prochiral carbonyl of both R-
12
and S-triadimefon undergoes stereoselective reduction to
1
3
yield four stereoisomers of triadimenol.
As with
triadimefon, triadimenol is a registered fungicide and studies
have shown that the 1S,2R-triadimenol stereoisomer (pro-
duced from S-triadimefon) exhibits 1000-fold greater fungi-
cidal activity14 and greater inhibition of sterol synthesis
1
5
than the other stereoisomers. A potential complicating fac-
tor in assessing the risk of triadimefon and triadimenol is
Fig. 1. Structures of two low-energy R-triadimefon conformers (A and B), and their possible enol and enolate products. The chiral center is marked with *
Chirality DOI 10.1002/chir

KINETICS AND MECHANISM OF TRIADIMEFON RACEMIZATION
k ¼ Aeꢁ
Ea=RT
(2)
MATERIALS AND METHODS
Chemicals
Rearranging (2) yields:
Triadimefon [(RS)-1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-
-yl)butan-2-one] (99.4% purity) was obtained from the U.S. EPA National
ꢀ ꢁ
Ea
1
T
1
lnðkÞ ¼ ꢁ R
þ lnðAÞ
(3)
Pesticide Standard Repository (Ft. Meade, MD) and was separated (Chi-
ral Technologies, West Chester, PA) into its two enantiomers using pre-
RT where k is the rate constant of racemization (k
the pre-exponential factor, R (1.9872 × 10 kcal mol
universal gas constant, and T is the absolute temperature. Least-squares
linear regression was applied to Arrhenius plots of ln(kracH) and ln(kracD
racH
and kracD), A is
1
3
ꢁ3
ꢁ1
ꢁ1
parative supercritical fluid chromatography as previously described.
K ) is the
All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO)
and were reagent grade or better.
)
as a function of 1/T yielding a slope of ꢁE
a
/R. The Gibbs energy of
activation ΔG was calculated according to the Eyring equation:
‡
H/D Substitution Rates
S and R triadimefon (2 mg) was added to 0.5 mL 100% D
,2-dimethyl-2-silapentane-5-sulfonate sodium salt (DSS; added as a ma-
trix internal standard) in a 2-mL microcentrifuge tube containing a 3.2-
mm stainless-steel bead. The tube was shaken using a tissuelyser
Qiagen, Valencia, CA) at 20 Hz for 3 min. Subsequently, the sample
was centrifuged at 10,000 g for 2 min and 230 μL of the sample was trans-
ferred into a 3-mm nuclear magnetic resonance (NMR) tube for NMR
analysis.
2
O with 20 μM
ꢀ
ꢁ
kih
kBT
‡
2
ΔG ¼ ꢁRT ln
(4)
where k
i
is the measured reaction rate constant kdeut, kracH , and kracD, T
is the absolute temperature, h (6.6261 × 10
constant, and k
ꢁ
34
2
ꢁ1
(
m
K
kg s ) is Planck’s
) is the Boltzmann
ꢁ
23
2
ꢁ2
ꢁ1
B
(1.3806 × 10
m
kg s
constant.
All NMR spectra were acquired on an Agilent (Palo Alto, CA) Inova
00 MHz NMR spectrometer using a 3-mm triple-resonance probe. A se-
ries of presaturation spectra measured at 25°C, 35°C, 45°C, and 55°C
were collected with 2-sec presaturation time, 2-sec acquisition time and
88 transients (or scans), with the exception that the spectra measured
Theoretical Methods
We applied one of the most commonly used hybrid functionals of den-
sity functional theory (DFT), the B3LYP functional,
basis set which is helpful for anion-involved computations. B3LYP/6–
6
2
3–25
with 6–311 + G**
2
3
11 + G** has consistently provided good results for organic mole-
at 35°C were collected with 96 transients. Peak identities were assigned
2
6,27
based on Carbonell’s result.22 The integrated signal at 7.30 ppm corre-
cules.
Optimized geometries, harmonic vibrational frequencies, na-
2
8–31
32–
ture population analysis,
and intrinsic reaction coordinates (IRC)
were computed with the GAUSSIAN 09 package, which generates ap-
proximate numerical solutions of the governing quantum-model
sponding to the α-hydrogen located on the chiral carbon of triadimefon
was monitored to evaluate the rate of H/D exchange (deuteration). The
concentration of triadimefon in each sample was calculated using the
peak area at 7.30 ppm relative to the DSS peak at 0.0 ppm. Least-squares
linear regression of the natural logarithm of the signal normalized to the
DSS peak area as a function of time was used to calculate pseudo-first-
order rate constants of H/D substitution (kdeut) for each experimental
3
5
3
6
equation.
RESULTS AND DISCUSSION
EXPERIMENTAL: Site of Deuterium Exchange
Full-scan mass spectra of MTBE extracts from studies conducted with
1
8
temperature.
triadimefon and D
ments containing the α-carbonyl carbon of triadimefon. H-NMR studies
conducted with triadimefon and D O exhibited a continual decrease in
the singlet at 7.30 ppm indicating deuterium exchange with the labile α-
hydrogen of triadimefon. Both studies confirm that the α-carbonyl carbon
of triadimefon is the site of reaction.
2
O exhibited an increase of 1 m/z for molecular frag-
Racemization Rates
A set of 7–10 samples containing 500 μL of pH buffer (pH 1 to pH 9)
were placed in a thermostatically controlled heating block. After 10 min,
μL of concentrated triadimefon enantiomer stock (in acetonitrile) was
added to each sample and vortexed to yield a final enantiomer concen-
tration of 35 μM. At selected times, 500 μL of methyl-t-butyl ether
1
2
5
(
MTBE) was added to individual sample tubes and vortexed; the MTBE
H/D Exchange and Racemization
layer was then removed and transferred to a gas chromatography (GC)
vial containing a micro-insert. MTBE extracts were analyzed by gas
chromatography mass spectrometry (GC/MS) (Hewlett-Packard
The activation energies (E
R-and S-triadimefon were 21.2 and 22.6 kcal mol in D
) measured for racemization of
a
ꢁ1
O
2
ꢁ
1
6
890/5973) equipped with a BGB 172 chiral capillary column (BGB
and 21.4 and 21.4 kcal mol in H O (Table 1). The similarity
2
Analytik, Switzerland) as previously described.13 Enantiomers of
of these values suggests that R- and S-triadimefon have the
same mechanism of racemization.
triadimefon were detected and quantified using selected ion monitoring
(
(
SIM) at m/z 181, 208, and 210. The triadimefon enantiomeric excess
ee) was calculated as:
Since racemization and deuteration can proceed at different
rates, it is critical to measure racemization (chiral GC/MS)
1
½
TFNaꢀ ꢁ ½TFNbꢀ
and H/D exchange ( H-NMR) separately. As shown in
t
t
ee ¼
(1)
½
TFNaꢀ þ ½TFNbꢀ
Table 1, the ratio of kdeut to kracD for S- and R-triadimefon
range from 0.71 to 0.93 and suggests that deuteration occurs
with complete racemization and that both deuteration and ra-
cemization share a common mechanism. These results fur-
ther suggest that the reaction mechanism involves a
symmetrical intermediate such as a neutral enol, carbanion,
or enolate (carbanion resonance structure).37
t
t
where [TFN
]
a t
is the concentration of one of the triadimefon enantio-
mers and [TFN
]
b t
is the concentration of the other triadimefon enantio-
mer at time t. Least-squares linear regression was applied to the natural
logarithm of ee as a function of t to calculate pseudo-first-order rate con-
stants of racemization (krac) for each experimental pH.18 Additional stud-
ies were conducted with 100 mM phosphate buffer prepared with either
2 2
H O (pH 7.4) or 99 + % D O (pD 7.8) and were maintained at four selected
temperatures ranging from 25°C to 55°C. Mass spectra were collected in
SIM mode as described above, including m/z 182, 209, and 211. Pseudo-
pH Effects
Studies conducted between pH 1 and pH 5 did not exhibit
any appreciable racemization over a 24-h period. For studies
conducted from pH 6 to pH 9 (Table 1), the rate of racemiza-
tion (kracH) increased linearly with increasing pH for both R-
and S-triadimefon (r = 0.995 and 0.996) and supports a gen-
eral base-catalyzed mechanism of racemization. In base-
first-order rate constants of racemization in H
2 2
O (kracH) and D O (kracD)
were calculated using eq. 1 as described for krac
.
Thermodynamic Properties
2
a
Activation energies (E ) were calculated for the racemization reaction
according to the Arrhenius equation:
Chirality DOI 10.1002/chir

CHENG ET AL.
ꢁ
3
ꢁ1
ꢁ3
ꢁ1
TABLE 1. Rate constants for deuteration (kdeut, × 10 min ), rate constant for racemization [in D
2
2
O (kracD, × 10 min ) and
ꢁ
3
ꢁ1
‡
ꢁ1
H
O (kracH, × 10 min )], with the corresponding Gibbs energies of activation (ΔG , in kcal mol ) of triadimefon enantiomers at
different reaction temperatures (in °C) and pH (pD)
‡
‡
‡
Temp
pH or pD
k
deut
k
racD
k
racH
kdeut/kracD
kdeut/kracH
ΔG (kdeut
)
ΔG (kracD
)
ΔG (kracH
)
S-Triadimefon
25
25
25
35
45
55
25
25
6.0
7.0
7.8
7.8
7.8
7.8
8.0
9.0
—
—
—
1.7
6.0
—
—
—
—
—
—
—
0.1
1.0
5.0
14.7
45.4
135.3
7.4
64.0
21.4
—
—
0.93
0.91
—
—
—
—
—
—
—
—
—
23.7
23.8
—
—
—
—
—
—
—
25.4
24.0
23.1
23.2
23.3
23.3
22.8
21.6
—
a
1.8
6.5
19.8
59.8
—
—
22.6
2.7
2.2
2.3
2.3
—
23.7
23.7
23.8
23.8
—
a
a
a
—
—
—
—
E
a
R-Triadimefon
25
25
25
35
45
55
25
25
6.0
7.0
7.8
7.8
7.8
7.8
8.0
9.0
—
—
—
1. 9
—
—
—
—
—
—
—
—
0.1
1.3
8.1
33.2
78.6
239.4
11.0
138.5
21.4
—
—
0.71
—
—
—
—
—
—
—
—
—
—
23.8
—
—
—
—
—
—
—
—
25.4
23.9
22.8
22.7
22.9
23.4
22.6
21.1
—
a
a
a
a
2.7
9.0
30.1
68.8
—
—
21.2
3.0
3.7
2.6
3.5
—
23.4
23.5
23.5
23.7
—
—
—
—
—
E
a
Measured at pH 7.4 and corrected to pH 7.8 using univariate linear model developed from ln(kracH ) as a function of pH and Arrhenius plot described in text.
catalyzed racemization, proton abstraction to form a carban-
ion is likely the rate determining step.
Conformer A Reaction With One Water
3
8
The reaction pathway of R-triadimefon conformer A with
one water molecule is shown in Figure 2, where H is ab-
1
Isotope Effects
stracted by water, then replaced by a hydrogen atom from wa-
ter. The optimized geometries of the six reactant complexes,
transition states, and product complexes for the one water re-
action pathway are listed in Table S2.
Comparing the rates of triadimefon racemization in H O
2
and D O under various conditions (k
/kracD; Table 1) re-
2
racH
veals a significant isotope effect ranging from 2.2 to 3.7 and
is within the range of isotope effects that have been reported
Initially, water binds to conformer A, forming a six-
membered ring with two hydrogen bonds in reactant com-
plex 1 (RC11w: O ···H and H ···O with lengths 2.38 Å and
3
8,39
for the α-hydrogen of ketones.
These results suggest that
cleavage of the C-H (C-D) bond is the rate-determining step
for the racemization of triadimefon in water.
a
1
b
2
1
.99 Å), respectively. One OꢁH bond of water is slightly lon-
ger than the other (0.97 and 0.96 Å) since the longer one is
involved in hydrogen bonding with conformer A. There are
no large bond length changes in the rest of conformer A.
τH1C1N1C8 changes from 6.3° to 3.1° (in the free rotation
range). In the transition state (TS11w), as the reaction pro-
ceeds, the C ꢁO bond length changes significantly from
Computational: R-Triadimefon Conformers
For chiral molecules, enantiomers possess identical physi-
cal and chemical properties as illustrated by the similar
values of E we measured for R- and S-triadimefon in H O
and D O (Table 1). Thus, our computational approach for R-
triadimefon is also applicable to S-triadimefon.
Two low-energy conformations (A and B) of R-triadimefon
Fig. 1) were computed in this study, and the optimized geome-
a
2
2
2
2
1
.21 Å to 1.27 Å, and the H from water migrates much closer
b
to O (1.17 Å) and away from O (1.27 Å). Meanwhile, H ꢁO
2
a
1
a
(
shortens to 1.18 Å, C ꢁH elongates to 1.49 Å in the six-
1
1
tries for the two conformers are shown in Supporting Table S1.
In conformer B there are two hydrogen bonds near the chi-
ral center (H ···N = 2.42 Å and H ···O = 2.68 Å), while there
membered ring. The H is not only pulled away from C ,
1
1
but also out of the triazole plane, resulting in τH C N C
1
1
1 8
1
2
8
2
3
4.4°. In product complex 1(PC11w), C ꢁO elongates to
2
2
is only one hydrogen bond (H ···O = 2.51 Å) in A. This differ-
1
2
ꢁ
1
1.35 Å, which is more like a single bond. H bonds to the
b
ence may explain why B is approximately 3 kcal mol lower
in energy than A (Fig. 1).
O in an enol form while C ꢁC shortens to a length in the
2
1
2
double bond range (1.35 Å). H is abstracted from C and
1
1
Racemization Mechanism
the distance between the two is over 4.0 Å. H ꢁO now has
1
a
normal water bond length of 0.97 Å, and H bonds to N
4
Based on our experimental observations (the α-carbonyl car-
bon of triadimefon is the site of reaction), cleavage of the C-H
C-D) bond is the rate-determining step, the reaction is base-
catalyzed, and the reaction likely involves a symmetrical inter-
mediate along with our computed geometries, as well as the
charge distributions for conformer A and B (Fig. S2), we hypoth-
esize three racemization pathways: conformer A reaction with
one water molecule, conformer A reaction with two water mole-
cules, and conformer B reaction with hydroxide ion.
Chirality DOI 10.1002/chir
1
(
2.39 Å) forming a long hydrogen bond, which lowers the en-
ergy of PC11w. The newly formed water (H O H ) easily
(
1
a
c
breaks from the product complex leaving the enol (Fig. 1).
-O -C -C and the triazole ring are co-planer forming a
new ring with one hydrogen bond (H ···N = 1.77 Å).
H
b
2
2
1
b
2
After the enol forms in the solution, a water molecule at-
tacks the enol product from the right side of the triazole ring
in reactant complex 2 (RC21w). The H
···C
is 4.16 Å, O
ꢁH
e
1
d
f

KINETICS AND MECHANISM OF TRIADIMEFON RACEMIZATION
Fig. 2. Potential energy surface for the reaction of R-triadimefon conformer A with one water molecule. Energies in black are relative energies compared to the
separated reactants: conformer A and water molecule; energies in red are relative energies compared to reactant complex RC11W; and energies in blue are relative
ꢁ1
energies compared to reactant complex RC21W. All energies are in kcal mol
is 0.96 Å slightly shorter than O ꢁH (0.98 Å), and now
8.75 below RC21w, driving the reaction to form the final prod-
uct complex.
2
b
C ꢁO elongates to 1.35 Å. As the reaction proceeds, the wa-
2
2
ter molecule, six-membered ring, and the t-butyl group rotate
The reaction enthalpy, entropy, and Gibbs energy for reac-
tion RC11w ! TS1 ! PC1 are shown in Table S3, which
along the C ꢁN bond. The water moves to the left side of the
1
1
1w
1w
triazole ring in the transition state 2 (TS21w), forming a new
indicate that this part of the reaction is endothermic and would
not occur spontaneously. However, after the enol is formed,
the reaction RC21w ! PC21w, which leads to the formation
of S-triadimefon, is exothermic and spontaneous. These com-
putational results help explain why the enol could not be easily
captured and lead to the formation of the other enantiomer.
six-membered ring (C -O -H -O -H -C ) with bond length
2
2
b
d
e
1
changes: 1.35 to 1.28 Å in C ꢁO , 0.98 to 1.28 Å in O ꢁH ,
2
2
2
b
1
1
.90 to 1.16 Å in H ꢁO , 0.97 to 1.18 Å in O ꢁH , 4.16 to
b
d
d
e
.52 Å in H ꢁC , and 1.35 to 1.45 Å in C ꢁC . C-C is more
e
1
1
2
like a single bond in TS2 , while C-O is more like a double
1
w
bond. In the product complex 2 (PC21w), C ꢁC elongates
1
2
Conformer A Reaction With Two Water Molecules
to 1.56 Å while C ꢁO continuously shortens to 1.21 Å. H
2
2
b
bonds much more tightly to O forming a normal water bond
Because both the activation and local barriers for the one
water racemization pathway are high, a reaction pathway in-
volving two water molecules was also examined in this study
(Fig. 3). The local barriers for transition state 1 (TS12w,
d
(
0.97 Å) while the O ꢁH length elongates to 1.99 Å. H
2
b
e
transfers to C1 (1.09 Å) in PC2 , forming a H O···S-
triadimefon complex.
1w
2
ꢁ
ꢁ1
1
For this reaction pathway (Fig. 2), it is favorable for RC1
40.99 kcal mol , Table S4) and transition state 2 (TS2
,
1
w
2w
ꢁ1
ꢁ1
ꢁ1
to form, since it is ꢁ7.49 kcal mol lower in energy than the
35.59 kcal mol ) are 3.05 kcal mol and 4.00 kcal mol
separated conformer A plus one water molecule. However,
lower respectively, and more favorable than for the one water
pathway. The activation barriers are reduced to
ꢁ
1
the 36.55 kcal mol activation barrier is much higher than
our experimentally calculated results (Table 1), and the local
ꢁ
1
ꢁ1
23.11 kcal mol for TS12w, and 24.80 kcal mol for TS22w
,
ꢁ
1
barrier is even higher (44.0 kcal mol ).
which are much lower than those in our one water pathway
(36.55 and 40.84 kcal mol , respectively), and in much better
agreement with the experiment calculated E (21.7 kcal mol
ꢁ
1
PC11w is higher in energy compared to the separated reac-
ꢁ
1
ꢁ1
tants, but by only 1.9 kcal mol . An enol is formed after the
,
a
ꢁ
1
water leaves PC11w, which lies 4.1 kcal mol above con-
average of R- and S-triadimefon racemization). When two wa-
ter molecules bond to the chiral center an eight-membered
ring in the reactant complex 1 (RC12w) is formed, where
the ring strain may be reduced more than that in the six-
membered ring of RC11w. Thus, the total energy of RC12w
ꢁ
1
former A. RC21w is 1.26 kcal mol lower in energy than
the separated conformer A and water reactants, and
2
.84 kcal mol-1 lower than the separated enol + water, which
ꢁ1
leads to a high local barrier (40.63 kcal mol ) and activation
barrier (40.84 kcal mol ). PC21w lies 7.49 kcal mol below
conformer A + H O, 11.59 kcal mol below enol + H O, and
ꢁ1
ꢁ1
ꢁ1
is 17.88 kcal mol well below the separated conformer A
ꢁ
1
2
2
and two water molecules. After passing over TS12w, the
Chirality DOI 10.1002/chir

CHENG ET AL.
Fig. 3. Potential energy surface for the reaction of R-triadimefon conformer A with two water molecules. Energies in black are relative energies compared to the
separated reactants conformer A and water molecules; energies in red are relative energies compared to reactant complex RC12W; and energies in blue are relative
ꢁ1
energies compared to reactant complex RC22W. All energies are in kcal mol
product complex
1
(PC12w
)
is formed, which is
reaction. Instead, the oxygen from a hydroxide ion bonds to
ꢁ
1
6
.38 kcal mol lower in energy than separated reactants
the most positively charged C (+0.606 in Fig. S2) in reactant
2
ꢁ
1
but 11.50 kcal mol higher than RC12w. Similar to the one
water reaction pathway, the enol forms after two water mole-
cules leave PC12w, then two waters tightly bond to the enol
complex (RCOH , Fig. 4). The optimized geometries of reac-
tant complexes, transition states, and product complexes for
this pathway are shown in Table S7.
ꢁ
1
forming the reactant complex 2 (RC22w, 14.89 kcal mol
Table S5) lower in energy than separated enol and two wa-
ters). The final product complex 2 (PC22w) again is favorably
RCOH
,
TS
,
and PCOH are 55.06, 36.80, and
OH
ꢁ
1
(
62.57 kcal mol lower in energy, respectively, than the sepa-
rated triadimefon conformer B and hydroxide ion, and PC
OH
ꢁ
1
ꢁ1
generated from TS22w since it is 17.88 kcal mol lower in en-
ergy than conformer A plus two waters, 21.98 kcal mol (Ta-
is 7.52 kcal mol lower than RCOH, indicating that this reac-
ꢁ
1
tion is favorable. The local barrier for this pathway is
ꢁ
1
ꢁ1
ble S5) lower than enol plus two waters, and 7.09 kcal mol
lower than RC22w
18.26 kcal mol , which is much lower than the pathways in-
.
volving water as the primary reactant and comparable to our
ꢁ
1
Similar to the triadimefon plus one water reaction pathway,
the reaction enthalpy, entropy, and Gibbs energy for reaction
RC12w ! TS1 ! PC1 (Table S4) indicate that this part
experimental result (21.7 kcal mol ). Thus, it appears the hy-
droxide ion-based pathway is the most favorable for
explaining triadimefon racemization.
2w
2w
of the reaction is endothermic and would not occur spontane-
ously; the reaction RC22w ! TS22w ! PC22w is exothermic
and spontaneous.
The reaction enthalpy, entropy, and Gibbs energy for the
reaction of triadimefon conformer B (TB) with hydroxide
ion to form an enolate ion plus water (TB
ꢁ
ꢁ
The optimized geometries of the six reactant complexes,
transition states, and product complexes for the two water re-
action pathway are listed in Table S6. Changes are similar to
previous reaction pathway.
+OH !enolate +H O) are shown in Table S8, which indi-
2
cates that this part of the reaction is exothermic and will take
place spontaneously even at low temperature.
The relative energies, enthalpies, entropies, and Gibbs en-
ergies of activation for all five transition states are summa-
rized in Table 2. Different from TSOH, the Gibbs energy of
activation for TS11w, TS21w, TS12w, and TS22w are all posi-
Conformer B Reaction With Hydroxide Ion
While both the one and two water pathways have higher re-
action barriers than our experimental result (21.7 kcal mol
ꢁ1
‡
ꢁ1
,
tive; ΔG (TS12w) is 43.1 kcal mol , the lowest among the
four, which indicates that this reaction will take place very
quickly. However, all four energies are much higher than
average E of R-and S-triadimefon), and we have shown the
a
reaction rates vary with pH, a reaction may take place be-
tween conformer B and hydroxide ion (Fig. 4). Differing from
the previous one and two water molecule pathways, no large
ring structure is formed when hydroxide is involved in the
Chirality DOI 10.1002/chir
ꢁ
1
the experimentally determined value 24.5 kcal mol (aver-
age from R-and S-triadimefon). For the triadimefon + OH
ꢁ
pathway, all these activation energies are negative, which

KINETICS AND MECHANISM OF TRIADIMEFON RACEMIZATION
Fig. 4. Potential energy surface for the reaction path of conformer B plus hydroxide ion. Energies in black are relative energies compared to separated reactants
ꢁ
1
conformer B and hydroxide ion, energies in red are the relative energies compared to (RCOH). All energies are in kcal mol
ꢁ
1
TABLE 2. Activation energy (ΔE, in kcal mol ), standard en-
uncertainty when evaluating potential risks to humans and
the environment. Triadimefon is a chiral systemic 1,2,4-
triazole fungicide; R-triadimefon is 2-fold more toxic than
the racemate, and depending on the biological or environ-
mental condition, triadimefon undergoes varying degrees of
stereoselective reduction to yield triadimenol stereoisomers
with differing toxicities. Because the degree of racemization
can impact the distribution of triadimenol stereoisomers that
are formed, we sought to better understand triadimefon race-
mization and enantiomerization reactions. We utilized both
experimental and computational methods to determine the
reaction site, rate-determining step, and most probable reac-
tion pathways. Collectively, our results indicate that the reac-
tion site is the alpha-carbonyl carbon and the rate-
determining step is the cleavage of the C-H (C-D) bond. In ad-
dition, we have shown that the reaction is base-catalyzed and
likely involves a symmetrical intermediate. This work pro-
vides an initial step in developing predictive, structure-based
models for identifying chiral compounds of concern and
should improve our understanding of the potential impact
these compounds may have on human health and the
environment.
‡
ꢁ1
thalpy of activation (ΔH , in kcal mol ), standard entropy of
‡
ꢁ1 ꢁ1
activation (ΔS , in cal mol
K
), and ordered Gibbs energies
‡
ꢁ1
of activation (ΔG , in kcal mol ) with respect to separated
reactants at 298.18 K
‡
‡
‡
ΔE kcal
ΔH kcal
ΔS kcal
ΔG kcal
mol-1
mol-1
mol-1
mol-1
TS11w
TS21w
TS12w
TS22w
TSOH
36.55
40.84
23.11
24.80
ꢁ36.80
33.18
37.68
19.93
22.30
ꢁ37.11
ꢁ41.11
ꢁ45.08
ꢁ77.80
ꢁ79.71
ꢁ36.61
45.43
51.12
43.13
46.07
ꢁ26.19
indicates that triadimefon racemization takes place easily and
quickly. In this case the rate-determining step will be from the
reactant complex to the transition state (after the reactant
complex RCOH, which lies 55.1 kcal mol below separated
B plus OH ). The Gibbs energy is 15.3 kcal mol , lower than
experimental predicted value. These all indicate that the
triadimefon reactions with water and OH may take place si-
multaneously. With increasing pH, the reaction favors the ba-
sic mechanism, so the reaction goes faster.
ꢁ
1
ꢁ
ꢁ1
ꢁ
ACKNOWLEDGMENTS
This research received funding from the U.S. Environmental
Protection Agency (U.S. EPA) and by an appointment to the
Postdoctoral Research Program at the Ecosystems Research
Division, administered by the Oak Ridge Institute for Science
Chirality DOI 10.1002/chir
CONCLUSION
Enantiomers of chiral compounds can undergo interconver-
sion leading to markedly different toxicities, which leads to

CHENG ET AL.
1
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which is supported by the Office of Science of the U.S. De-
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