A mechanistic study of thioester hydrolysis with heavy atom kinetic isotope effects

Article abstract of DOI:10.1021/jo502472m

The carbonyl-C, carbonyl-O, and leaving-S kinetic isotope effects (KIEs) were determined for the hydrolysis of formylthiocholine. Under acidic conditions, ¹³k_obs = 1.0312, ¹⁸k_obs = 0.997, and ³⁴k_obs = 0.995; for neutral conditions, ¹³k_obs = 1.022, ¹⁸k_obs = 1.010, and ³⁴k_obs = 0.996; and for alkaline conditions, ¹³k_obs = 1.0263, ¹⁸k_obs = 0.992, and ³⁴k_obs = 1.000. The observed KIEs provided helpful insights into a qualitative description of the bond orders in the transition state structure.

Full text of DOI:10.1021/jo502472m

Note
pubs.acs.org/joc
A Mechanistic Study of Thioester Hydrolysis with Heavy Atom Kinetic
Isotope Effects
John F. Marlier,‡ Emily J. Fogle,‡ Richard L. Redman,‡ Anthony D. Stillman,† Matthew A. Denison,†
and Lori I. Robins*,†
†University of Washington-Bothell, Bothell, Washington 98011, United States
‡California Polytechnic State University, San Luis Obispo, California 93407, United States
S
* Supporting Information
ABSTRACT: The carbonyl-C, carbonyl-O, and leaving-S kinetic
isotope effects (KIEs) were determined for the hydrolysis of
13
18
formylthiocholine. Under acidic conditions,
k
obs
13
= 1.0312,
k
=
obs
obs
18
34
34
0.997, and
k
obs
= 0.995; for neutral conditions,
k
= 1.022,
k
= 1.010, and
k
obs
= 0.996; and for alkaline conditions,
obs
13
k
= 1.0263, 18kobs = 0.992, and 34kobs = 1.000. The observed KIEs provided helpful insights into a qualitative description of the
obs
bond orders in the transition state structure.
hioesters are energy-rich compounds important in both
Torganic chemistry and biochemistry. In both disciplines,
thioesters commonly utilize this stored chemical energy as acyl
group donors. The high reactivity of thioesters toward
nucleophiles is partly due to lack of resonance stabilization in
the ground state. Sulfur contains a 3p orbital, and this leads to
very poor overlap with the 2p orbitals of the carbonyl and less
resonance stabilization in the ground state.1
The mechanism of alkyl thioester hydrolysis has been
investigated by both kinetic and solvent kinetic isotope effects
(KIE) for over five decades.2−5 Kinetic studies indicated that
there were two distinct types of thioesters. The first type
includes thioesters, which possess an activated carbonyl group
due to the presence of electron-withdrawing substituents on the
α-carbon.6,7 These thioesters display three distinct regions in
the pH-rate profile: hydroxide ion catalysis above pH 7,
uncatalyzed hydrolysis between pH 2−7, and inhibition by
hydronium ion below pH 2. The second type of thioester lacks
an activated carbonyl (such as FTC) and shows a similar profile
as those described above, except the hydrolysis is catalyzed by
hydronium ion below pH 2.8−10 See the Supporting
Information for a full FTC pH-rate profile.
A recent multiple KIE and positional isotope exchange (PIX)
investigation from our laboratories aided in assignment of the
rate-determining step and establishment of a qualitative
structure of the transition state for hydrolysis of FTC under
acidic, neutral, and alkaline conditions.10 Results for acidic and
neutral hydrolyses were in agreement with the commonly
accepted stepwise mechanism containing a tetrahedral inter-
mediate. However, in alkaline conditions, a slightly larger than
expected formyl-H KIE (Dkobs = 0.88) indicated that either the
stepwise or the concerted mechanisms were possibilities.
D
Nomenclature such as kobs is defined in the Supporting
Information.
In the present KIE study, we report the carbonyl-C,
carbonyl-O, and leaving-S KIEs on the acidic, neutral, and
alkaline hydrolysis of FTC. Details for measuring isotopic
composition and calculating the KIEs are given in the
Supporting Information. The results of the KIE experiments
are summarized in Table 1.
Three general mechanisms have been proposed for the
hydrolysis of acyl groups, including thioesters.11 These general
mechanisms are shown in eq 1 for the reaction of a negatively
charged nucleophile with FTC. The stepwise mechanism (top
pathway) involves tetrahedral intermediates and is the most
common mechanistic type. The concerted or associative
mechanism (middle pathway) has been proposed for the
alkaline hydrolysis of oxoesters with very good leaving
groups.12,13 The dissociative mechanism (bottom pathway) is
proposed for the hydrolysis of acid chlorides.14
Acidic Hydrolysis. The acid-catalyzed hydrolysis of alkyl
thioformates is first-order in hydronium ion.8 A PIX experiment
from our laboratories gave a ratio for the rate of hydrolysis to
that for exchange of 18O into the carbonyl of FTC of kh/kex >
25.10 The ratio may be higher than 25, but this is limited by the
NMR methodology. Nevertheless, the result indicates that
hydrolysis is much faster than exchange, assuming rapid proton
transfers.15,16 This further implies that formation of the
Received: October 29, 2014
Published: December 29, 2014
© 2014 American Chemical Society
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The Journal of Organic Chemistry
Note
Table 1. Leaving-S, Carbonyl-C, and Carbonyl-O KIEs on the Hydrolysis of FTC under Acidic, Neutral, and Alkaline
Conditions
reaction conditions
leaving-S KIE
carbonyl-C KIE
carbonyl-O KIE
0.20 M HCl (μ = 0.20)
neutral H2O (μ = 0.20)
0.0050 M NaOH (μ = 0.20)
0.995 0.002
0.996 0.003
1.000 0.001
1.0312 0.0009
1.022 0.001
0.997 0.002
1.010 0.004
0.992 0.003
1.0263 0.0008
18
tetrahedral intermediate is rate-determining. The formyl-H KIE
the nonbridging
k
obs
trended toward the inverse direction as
D
is kobs = 0.80. The magnitude of this inverse KIE is in
the transition state became tighter. Presumably, this was also
due to the newly created bending modes to oxygen becoming
dominant and to the large radius of the sulfur atom.
agreement with a transition state containing considerable sp3
character.17 These data are consistent with the mechanism
shown in eq 2.
Neutral Hydrolysis. A stepwise mechanism involving at
least two water molecules in the transition state has been
proposed for neutral hydrolysis (eq 3).26 Our previous PIX and
KIE experiments are consistent with this prevailing mecha-
nism.10 The PIX experiment again indicated that formation of
the tetrahedral intermediate is rate-determining (kh/kex > 25).
The formyl-H KIE (Dkobs = 0.75) is a slightly more inverse KIE
than in acid-catalyzed hydrolysis and is in agreement with a
The carbonyl-C and carbonyl-O KIEs measured in the
present study (Table 1) are similar to those for the acidic
18
hydrolysis of methyl formate (13kobs = 1.028 and
k
obs
=
transition state containing even more sp3 character than under
acidic conditions. The solvent nucleophile-O KIE is
0.9945).18 Theoretical calculations for the carbonyl-C KIE
predict a very gradual decrease in the KIE on going from early
transition states (little sp3 character) to late transition states
(more sp3 character).17 This decrease is so gradual that the
carbonyl-C has not been widely used in determination of
transition state bonding. Published KIEs on aryl carbonate
hydrolysis agree with this prediction.19 The carbonyl-O KIE for
acidic hydrolysis is essentially unity, and interpretation of this
effect is complex. At least three factors affect the observed KIE.
First, there is loss of the π bond to the carbonyl-O, resulting in
a normal contribution to the observed KIE. Second, the
carbonyl-O experiences formation of new bonds to solvent
hydrogen atoms, which could introduce a small normal or small
inverse contribution. Finally, new bending and torsional modes
in the transition state will result in an inverse contribution. In
the present case, the normal contribution from π-bond breaking
appears to be balanced by the other factors.
18
k
obs
=
0.9917. Since k3 is rapid, the observed KIE is due to formation
of the tetrahedral intermediate (k1). This can be explained
when the inverse contribution to the observed KIE from the
temperature-dependent factor (TDF) is only slightly larger
than the normal contribution from the temperature-independ-
ent factor (TIF).27,28
The carbonyl-C and carbonyl-O KIEs change somewhat on
going from acidic to neutral conditions (Table 1). It would be
tempting to assign the smaller carbonyl-C KIE in neutral
hydrolysis to a later transition state (more sp3 character), since
theoretical calculations predict that later transition states will
correlate with slightly smaller KIEs.17 However, to date, such
correlations have proved difficult to establish for most acyl
group transfer reactions.11 The carbonyl-O KIE goes from
essentially unity in acid-catalyzed hydrolysis to a normal KIE in
neutral hydrolysis (Table 1). The interpretation is again
complicated by transition state effects described in the acidic
hydrolysis case. However, the sizable normal KIE indicates that
breaking the carbonyl-O π bond plays the major role in
The theoretical maximum sulfur KIE is calculated to be near
1.015, while observed KIEs for SN1 and SN2 reactions range
from 1.009 to 1.018.20−22 In the present case, the leaving-S KIE
is 34kobs = 0.995. This measured value is statistically (>95% C.I.)
different from unity. Initially, this is surprising since leaving
heteroatom KIEs for oxoesters and amides are normal and in
the range of 18kobs = 1.0009 to 15kobs = 1.0050, respectively.23,24
In both amides and oxoesters, the breakdown of the tetrahedral
intermediate is faster than its formation. Leaving group KIEs on
formation of the tetrahedral intermediate are the result of
several factors. First, there is loss of the partial π bond on going
to the transition state, resulting in a normal contribution to the
overall KIE. Thioesters are not expected to show this normal
contribution due to the poor overlap between the 2p orbitals of
the carbonyl-C and the 3p orbital of sulfur.1 Second, another
small normal contribution to the observed effect is possible
when breakdown of the tetrahedral intermediate to products is
slightly rate-determining.23 PIX experiments indicate that this
contribution will be minimal for FTC. If all the normal
contributions to the leaving-heteroatom KIE are smaller for
thioesters than for amides and oxoesters, then what accounts
for the observed small inverse KIE? Formation of the transition
state will create new bending modes to sulfur. This may lead to
stiffer bonding to the sulfur and an inverse contribution to the
observed KIE. Catrina and Hengge reached a similar conclusion
in a study of phosphorothioate ester hydrolysis.25 In this case,
determining the overall observed KIE.
The leaving-S KIE for neutral hydrolysis is
34
k
obs
= 0.996,
very close to that measured in the acid-catalyzed case above and
is still statistically different than unity (>95%CI). The
interpretation is similar. The normal contributions to the
observed KIE are so small that what remains is a small inverse
secondary KIE due to creation of new bending modes in the
sp3-transition state.
Alkaline Hydrolysis. Kinetic investigations indicated that
alkaline hydrolysis is first-order in hydroxide, as is the case for
oxoesters.8,9,29 Our previous PIX results (kh/kex > 25) argue
that formation of the tetrahedral intermediate is rate-
determining.10 The formyl-H KIE for the alkaline hydrolysis
of FTC (Dkobs = 0.88) is considerably less inverse than that for
acidic and neutral hydrolysis, consistent with an earlier
transition state (less sp3 character).10 The formyl-H KIE for
alkaline hydrolysis of FTC can also be compared to that of its
oxoester counterpart, methyl formate.29 The formyl-H KIE for
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The Journal of Organic Chemistry
Note
FTC is more inverse (Dkobs = 0.88 versus Dkobs = 0.95), arguing
for a somewhat later transition state for FTC.10,30 Up to this
point, all of these results can be accommodated by the stepwise
mechanism given in eq 4.
The carbonyl-C KIE for alkaline hydrolysis is slightly less
inverse than that for neutral hydrolysis and slightly more
inverse than that for acidic hydrolysis. This trend cannot be
easily explained. On the basis of theoretical calculations, one
would expect the earliest transition state structure (alkaline
hydrolysis) to give the largest carbonyl-C KIE.17 The observed
carbonyl-O KIE is close to unity and similar to that for acidic
hydrolysis. It is clear from the small formyl-H KIE that the
transition state is somewhat early. As a result, the breaking of
the π bond to the carbonyl-O and the resulting normal
contribution to the KIE are minimized compared to acidic and
neutral conditions. The remaining aforementioned factors
appear to balance each other to create an overall observed
effect of unity.
Conclusions. KIE and PIX experiments indicate that acidic
and neutral hydrolysis of FTC follow a stepwise mechanism
with rate-determining formation of a tetrahedral intermediate.
The observed KIEs on alkaline hydrolysis of FTC can fit either
a stepwise mechanism with an early transition state for
formation of the tetrahedral intermediate or an asynchronous
concerted mechanism with both a small amount of C−O bond
formation and a small amount of C−S bond breaking. Leaving-
S KIE experiments on thioester hydrolysis may prove useful in
establishing the concerted mechanism in cases where one
observes both no 18O exchange (PIX) and a significant normal
sulfur KIE.
The solvent nucleophile-O KIE was measured in the same
manner as for the neutral hydrolysis above.10 In the alkaline
hydrolysis case, there are two possible nucleophiles present,
leading to two possible mechanisms. One mechanism involves
water as the nucleophile with general base assistance by
hydroxide; the other is hydroxide as the direct nucleophile.
Because water and hydroxide have different δ values, the
experiment gives two possible KIEs, depending on which
nucleophile one chooses. For the general base mechanism with
18
water as the nucleophile, the KIE is
k
obs
= 1.029, whereas, for
18
hydroxide as the actual nucleophile, the KIE becomes
k
=
obs
0.989. The choice between these two possibilities rests on the
magnitude of the formyl-H KIE. By theory, a very early
transition state is expected to give rise to an observed normal
solvent nucleophile-O KIE, whereas late transition states should
result in an inverse solvent nucleophile-O KIE.17 For the
oxoester, methyl formate, the choice of the general base
mechanism was clear because the formyl-H KIE was so small
(Dkobs = 0.95), leaving little doubt that the transition state for
formation of the tetrahedral intermediate was early.29 However,
in the alkaline hydrolysis of FTC, the formyl-H KIE is a slightly
larger inverse effect (Dkobs = 0.88), forcing one to consider the
alternative concerted mechanism (eq 1, middle pathway). Since
the leaving group and nucleophile are both good, the
conditions are right for this possible change in mechanism.10
The leaving-S KIE offers a possible way to distinguish
between the stepwise and concerted mechanisms. If the
stepwise mechanism is operating, the leaving-S KIE should be
near unity (or slightly inverse) because breaking the C−S bond
is fast in comparison to other mechanistic steps. If the
concerted mechanism is operating, a measurable normal
leaving-S KIE is possible when the transition state contains
significant C−S bond breaking. This expectation is similar to
empirical observations for p-nitrophenyl acetate hydrolysis,
where sizable leaving-O KIEs indicated the presence of the
concerted mechanism.12,13 The observed leaving-S KIE is 34kobs
= 1.000. This KIE is consistent with the stepwise mechanism
when the transition state for formation of the tetrahedral
intermediate is earlier for basic conditions than in the acidic and
neutral cases. The resulting smaller amount of C−O bond
formation will diminish the magnitude of the inverse
contributions to the KIE. Combined with the lack of C−S
bond breaking, the observed KIE for alkaline hydrolysis will
trend toward unity.
EXPERIMENTAL SECTION
■
Synthesis of Formylthiocholine. Formylthiocholine was synthe-
sized following our previously published synthetic scheme.10 Briefly, 2-
(dimethylamino)ethanethiol hydrochloride was added to the formyl−
acetic mixed anhydride. After completion, excess acetic anhydride,
acetic acid, and formic acid were removed under reduced pressure.
Crude formyl 2-(dimethylamino)ethanethiol hydrochloride (FDC)
was dissolved in acetone, deprotonated with PS-DIEA resin, and
methylated with methyl iodide. The precipitate was recrystallized in
absolute ethanol. Analytical data are as previously reported.10
Leaving-S KIE Procedures. These procedures were used to
determine the S-leaving atom KIE in acidic, neutral, and alkaline
conditions. (a) Alkaline conditions: FTC (14 mg, 51.1 μmol) was added
to 875 μL of 0.2 M NaCl. To this solution was added 125 μL of 0.2 M
NaOH. After 1 min, the reaction was quenched with 100 μL of 0.5 M
MES buffer at pH 6.2. The reaction mixture was immediately added to
a solution containing a slight excess of DTNB (22 mg, 55.5 μmol) in
10 mL of 50 mM MES buffer at pH 6.8.31 The solution was
immediately run through a 2 mL cation-exchange column with AG
50W resin. The column was washed with 20 mL of water and eluted
with 0.5 M TEAB at pH 8.5. The fractions (total volume 50 mL) were
assayed for free thiol using DTNB. The free thiol containing fractions
were collected, and the buffer was removed by rotary evaporation. The
samples were further dried by lyophilization and analyzed by IRMS.
(b) Neutral conditions: FTC (14 mg, 51.1 μmol) was added to 1000 μL
of 50 mM MES buffer at pH 6.8 containing 150 mM NaCl. The
mixture was allowed to react until the reaction reached 20−80%
hydrolysis, as determined by DTNB assays (∼5 h).31 The mixture was
added to a solution of excess DTNB and subjected to cation-exchange
chromatography as previously described. (c) Acidic conditions: FTC
(14 mg, 51.1 μmol) was added to 875 μL of water. To this solution
was added 125 μL of 1.6 M HCl. The mixture was allowed to react
until the reaction reached 20−80% hydrolysis, as determined by
DTNB assays (∼2 h). The mixture was added to a solution of excess
DTNB and subjected to cation-exchange chromatography as
previously described. Controls: Recrystallized FTC was applied to
However, the observed leaving-S KIE can also fit an
asynchronous concerted mechanism. If the sulfur KIE for
formation of the tetracoordinate transition state is small and
inverse (as in acidic and neutral hydrolysis) and the C−S bond
is only slightly broken (a small normal effect), the two KIEs
may result in the observed KIE of unity. The transition states
for these two possible mechanisms are given in structures I and
II, respectively.
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The Journal of Organic Chemistry
Note
the cation exchange column in the presence of DTNB. Elution with
water was followed by elution with TEAB. FTC was present in TEAB
eluted fractions. These fractions were concentrated and submitted for
IRMS analysis, which showed the 34δ to be unchanged from original
FTC within the precision of analysis. Next, completely hydrolyzed
FTC plus DTNB were applied to the cation exchange column and
eluted first with water, followed by TEAB. The fraction where FTC
normally eluted was concentrated and submitted for IRMS analysis.
After combustion, no SO2 was detected.
AUTHOR INFORMATION
Corresponding Author
*E-mail: lrobins@uw.edu (L.I.R.).
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a grant from the National Science
Foundation (CHE-1049689). We posthumously thank Profes-
sor W. W. Cleland of the University of Wisconsin for use of his
isotope ratio mass spectrometer and for his long-term interest
in our research. We thank Professor Alvan Hengge of Utah
State University for useful comments.
Carbonyl-O and Carbonyl-C KIE Procedures. The carbonyl-C
and carbonyl-O KIEs were measured in a single experiment. (a) Basic
conditions: A solution containing 875 μL of 0.20 M NaCl and 16 mg of
FTC (57 μmol) was prepared. To this solution was added 125 μL of a
solution containing 40 μL of 1.0 M NaOH in 160 μL of water. The
reaction was monitored by the DTNB assay described above, and the
fraction of reaction at quenching was determined. At the appropriate
time, the reaction was quenched by addition of 50 μL of MES buffer at
pH 6.2. The final pH was always greater than 5.0. The reaction mixture
was added to an anion exchange column (acetate form), washed with
30 mL of water. The product, formate was eluted with 0.1 M NaCl in
6 mL fractions. A 1.5 mL aliquot of 1.5 M MES buffer at pH 6.3 was
added to the combined fractions containing formate. This solution was
evaporated to a small volume (∼1 mL) and transferred to a round-
bottom flask that was equipped with two stopcocks. One stopcock was
on a side arm that was capped with a septum. The second stopcock
was for attachment to the high vacuum line. The solution was dried
under high vacuum at 70 °C overnight. While under vacuum, 2 mL of
anhydrous DMSO containing 250 mg of I2 was added through the side
arm to the dried formate, and the resulting CO2 was collected into a
liquid nitrogen trap as previously described.10 Isotopic analysis gave
the δ for both the oxygen and the carbon atoms. The above δ values
and those for formate isolated after complete alkaline hydrolysis were
used in the KIE calculation. (b) Neutral conditions: The reaction
mixture contained 16 mg of FTC (57 μmol) in 1000 μL of 0.050 M
MES buffer at pH 6.8. The final pH was not allowed to drop below pH
5.0. Assay, isolation, and isotopic analysis were performed in the same
as for the alkaline hydrolysis. (c) Acidic conditions: The reaction
solution contained 16 mg of FTC (57 μmol) in 1000 μL of 0.20 M
HCl. Fraction of reaction was determined by the DTNB assay. The
product of the reaction under acidic conditions is formic acid, which
undergoes rapid 18O exchange. To avoid this problem, the substrate,
FTC, must be analyzed by an indirect method. To accomplish this, the
reaction mixture after partial hydrolysis was first applied to an anion
exchange column (acetate form) and the 30 mL water wash containing
unreacted FTC was collected. The FTC (from partial hydrolysis) was
then quantitatively hydrolyzed with NaOH and applied to a second
anion exchange column identical to the first. This column was washed
with water, followed by typical elution of formate with 0.10 M NaCl as
described above. Oxidation of formate and isotopic analysis were as
described above. Next, a sample of FTC was completely hydrolyzed
with aqueous NaOH. For both the partial and the quantitatively
hydrolyzed samples, formate contains one oxygen from the carbonyl-O
of FTC and one oxygen from the nucleophile acquired during alkaline
hydrolysis. The 18δ from the nucleophile is identical for both the
partial and the quantitatively hydrolyzed samples because the solvent
nucleophile is in great excess. This allows the carbonyl-O KIE to be
calculated from the measured 18δ from both samples. Controls:
Formate of known isotopic composition was eluted from the anion
exchange column in the absence of FTC. The known 18δ of formate
did not change. FTC was also passed through the anion exchange
column. FTC was detected in the water wash. However, elution with
0.10 M NaCl did not produce formate, as expected.
REFERENCES
■
(1) Yang, W.; Drueckhammer, D. G. J. Am. Chem. Soc. 2001, 123,
11004.
(2) Castro, E. J. Sulfur Chem. 2007, 28, 401.
(3) Hupe, D. J.; Jencks, W. P. J. Am. Chem. Soc. 1977, 99, 451.
(4) Bruice, T.; Bruno, J. J.; Chou, W.-S. J. Am. Chem. Soc. 1963, 85,
1659.
(5) Castro, E. A. Chem. Rev. 1999, 99, 3505.
(6) Bruice, T. C.; Fedor, L. R. J. Am. Chem. Soc. 1964, 86, 4886.
(7) Fedor, L. R.; Bruice, T. C. J. Am. Chem. Soc. 1965, 87, 4138.
(8) Hershfield, R.; Schmir, G. L. J. Am. Chem. Soc. 1972, 94, 1263.
(9) Hershfield, R.; Schmir, G. L. J. Am. Chem. Soc. 1973, 95, 3994.
(10) Robins, L. I.; Meisenheimer, K. M.; Fogle, E. J.; Chaplan, C. A.;
Redman, R. L.; Vacca, J. T.; Tellier, M. R.; Collins, B. R.; Duong, D.
H.; Schulz, K.; Marlier, J. F. J. Org. Chem. 2013, 78, 12029.
(11) Marlier, J. F. Acc. Chem. Res. 2001, 34, 283.
(12) Hess, R. A.; Hengge, A. C.; Cleland, W. W. J. Am. Chem. Soc.
1998, 120, 2703.
(13) Hengge, A. C.; Hess, R. A. J. Am. Chem. Soc. 1994, 116, 11256.
(14) Bentley, T. W.; Ebdon, D. N.; Kim, E. J.; Koo, I. S. J. Org. Chem.
2005, 70, 1647.
(15) Brown, R. S.; Bennet, A. J.; Slebocka-Tilk, H.; Jodhan, A. J. Am.
Chem. Soc. 1992, 114, 3092.
(16) Bender, M. L.; Heck, H. d. A. J. Am. Chem. Soc. 1967, 89, 1211.
(17) Hogg, J. L.; Rodgers, J.; Kovach, I.; Schowen, R. L. J. Am. Chem.
Soc. 1980, 102, 79.
(18) Marlier, J. F.; Frey, T. G.; Mallory, J. A.; Cleland, W. W. J. Org.
Chem. 2005, 70, 1737.
(19) Marlier, J. F.; O’Leary, M. H. J. Am. Chem. Soc. 1990, 112, 5996.
(20) Hargreaves, R. T.; Katz, A. M.; Saunders, W. H. J. Am. Chem.
Soc. 1976, 98, 2614.
(21) Friedberger, M. P.; Thornton, E. R. J. Am. Chem. Soc. 1976, 98,
2861.
(22) Huskey, W. P. In Enzyme Mechanisms from Isotope Effects; Cook,
P. F., Ed.; CRC Press: Boca Raton, FL, 1991; p 38.
(23) Sawyer, C. B.; Kirsch, J. F. J. Am. Chem. Soc. 1973, 95, 7375.
(24) Marlier, J. F.; Campbell, E.; Lai, C.; Weber, M.; Reinhardt, L. A.;
Cleland, W. W. J. Org. Chem. 2006, 71, 3829.
(25) Catrina, I. E.; Hengge, A. C. J. Am. Chem. Soc. 2003, 125, 7546.
(26) Venkatasubban, K. S.; Davis, K. R.; Hogg, J. L. J. Am. Chem. Soc.
1978, 100, 6125.
(27) Westaway, K. C. J. Labelled Compd. Radiopharm. 2007, 50, 989.
(28) Hengge, A. C. In Secondary Isotope Effects; Kohen, A., Limbach,
H.-H., Eds.; Taylor & Francis: Boca Raton, FL, 2006.
(29) Marlier, J. F. J. Am. Chem. Soc. 1993, 115, 5953.
(30) Bilkadi, Z.; De Lorimier, R.; Kirsch, J. F. J. Am. Chem. Soc. 1975,
97, 4317.
ASSOCIATED CONTENT
■
(31) Ellman, G. L.; Courtney, K. D.; Andres, V., Jr.; Feather-Stone, R.
M. Biochem. Pharmacol. 1961, 7, 88.
S
* Supporting Information
The equations for calculating the KIEs from substrate analysis
and product analysis and the relationship between δ notation
and isotope ratios are included. This material is available free of
charge via the Internet at http://pubs.acs.org.
1908
DOI: 10.1021/jo502472m
J. Org. Chem. 2015, 80, 1905−1908