Mechanistic studies of la3+- And Zn2+-catalyzed methanolysis of aryl phosphate and phosphorothioate triesters. Development of artificial phosphotriesterase systems

Article abstract of DOI:10.1039/b502569a

The methanolyses of a series of O,O-diethyl O-aryl phosphates (2,5) and O,O-diethyl S-aryl phosphorothioates (6) promoted by methoxide and two metal ion systems, (La³⁺)₂(^-OCH₃)₂ and 4:Zn²⁺:^-OCH₃ (4 = 1,5,9- triazacyclododecane) has been studied in methanol at 25°C. Bronsted plots of the log k₂ values vs. _s^spK_a for the phenol leaving groups give β_lg values of -0.70. -1.43 and -1.12 for the methanolysis of the phosphates and -0.63, -0.87 and -0.74 for the methanolysis of the phosphorothioates promoted by the methoxide, La ³⁺ and Zn²⁺ systems respectively. The kinetic data for the metal-catalyzed reactions are analyzed in terms of a common mechanism where there is extensive cleavage of the P-XAr bond in the rate-limiting transition state. The relevance of these findings to the mechanism of action of the phosphotriesterase enzyme is discussed. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b502569a

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A R T I C L E
3+
2+
Mechanistic studies of La - and Zn -catalyzed methanolysis of
aryl phosphate and phosphorothioate triesters. Development of
artificial phosphotriesterase systems
Tony Liu, Alexei A. Neverov, Josephine S. W. Tsang and R. Stan Brown*
Department of Chemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6.
E-mail: rsbrown@chem.queensu.ca; Fax: 613-533-6669; Tel: 613-533-2400
Received 21st February 2005, Accepted 2nd March 2005
First published as an Advance Article on the web 18th March 2005
The methanolyses of a series of O,O-diethyl O-aryl phosphates (2,5) and O,O-diethyl S-aryl phosphorothioates (6)
3
+
−
2+
−
promoted by methoxide and two metal ion systems, (La )
2
( OCH
3
)
2
and 4:Zn : OCH
3
(4 = 1,5,9-
◦
s
s
triazacyclododecane) has been studied in methanol at 25 C. Brønsted plots of the logk
2
values vs. pK
a
for the
phenol leaving groups give blg values of −0.70, −1.43 and −1.12 for the methanolysis of the phosphates and −0.63,
3
+
2+
−
0.87 and −0.74 for the methanolysis of the phosphorothioates promoted by the methoxide, La and Zn systems
respectively. The kinetic data for the metal-catalyzed reactions are analyzed in terms of a common mechanism where
there is extensive cleavage of the P–XAr bond in the rate-limiting transition state. The relevance of these findings to
the mechanism of action of the phosphotriesterase enzyme is discussed.
3
+
Introduction
that the La -catalyzed methanolysis of the phosphorus esters
is very sensitive to the nature of the leaving group, contrasting
Phosphorus triesters and phosphonate esters of general struc-
3+
the relative insensitivity for La -catalyzed methanolysis of the
corresponding aryl acetate esters.
the second-order rate constants for La -catalyzed methanolysis
of p-nitrophenyl acetate and phenyl acetate are very similar at
8 and 29 dm mol s ,
and 5a vary by 1000-fold at ca. 2 dm mol s and ca. 1.3 ×
dm mol s respectively.
ture 1 (LG = leaving group, Z = alkyl, aryl, alkoxide or
14,15
s
s
At the pH optimum of 9
1,2
aryloxide) are widely used as insecticides, acaricides and
3+
3
OP-based chemical warfare (CW) agents. Owing to their
toxicity and lingering effects on the environment, consider-
able effort has been directed toward methods of facilitating
the controlled decomposition of these materials, particularly
through hydrolysis and oxidation. Transition metal ions and
lanthanides and certain mono- and di-nuclear complexes thereof
are known to promote the hydrolysis of neutral phosphate
3
−1 −1 14,15
3
while those for methanolysis of 2
3
−1 −1
−3
3
−1 −1
13
10
3,4
5,6
and/or phosphonate esters, and Pt and Pd metallocycles are
remarkably efficacious for thiophosphate pesticide hydrolysis.
7
By contrast, metal-catalyzed alcoholysis reactions of
organophosphates have received scant attention, although our
recent work indicates that this is an effective strategy for the
8
destruction of neutral organophosphate esters. For example,
−
3
3+
a methanolic solution of 2 mmol dm in each of La and
NaOCH catalyzed the methanolysis of paraoxon (2, also a
3
+
3
The large leaving group effect on the rate of the La -catalyzed
methanolysis of these phosphate triesters prompted the present
simulant for the phosphonofluoridate chemical weapon G-
agents) by 10 -fold relative to the background reaction at
9
3+
3+
investigation of the La - and Zn -catalyzed methanolysis of
a series of phosphate (2,5) and phosphorothioate (6) esters. As
will be shown, the mechanism of this reaction proceeds through
a rate-limiting transition state having a very large degree of
cleavage of the P-leaving group bond and is thus very different
from that determined for the metal-catalyzed methanolysis reac-
tions of the corresponding phenoxy acetate esters. These findings
have important implications for the mechanism of action of
s
s
9,10
3+
near-neutral pH
and ambient temperature. La , and other
11
lanthanides, also promote the ethanolysis of paraoxon and the
2
+
2+
1
,5,9-triazacyclododecane complexes of Zn or Cu , as their
2+
−
monomethoxy forms (4:M : OCH
the methanolysis of 2 and of the P=S derivative, fenitrothion
3) for which La is ineffective. A preliminary analysis of the
3
), give excellent catalysis of
3
+
12
(
13
effects of structural variation in the phosphate on the rates of
catalyzed methanolysis of 2, its phenyl counterpart 5a, and the
two corresponding O,O-diethyl S-phenyl- and O,O-diethyl S-(p-
nitrophenyl)-phosphorothioate derivatives, 6a and 6b, indicated
2
+
phosphotriesterase, a dinuclear Zn -containing enzyme isolated
from a soil bacterium, that effectively catalyzes the hydrolysis of
16
paraoxon.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 5 2 5 – 1 5 3 3
1 5 2 5

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Experimental
(b) General UV/vis kinetics
Materials used to prepare solutions for kinetic determinations:
(
a) Materials
−
3
sodium methoxide (0.5 mol dm ), 1,5,9-triazacyclododecane
3
-Nitrophenol (99%), pentafluorophenol (99+%), 4-chloro-
−3
(
97%), tetrabutylammonium hydroxide (1.0 mol dm ),
phenol (99+%), 4-chloro-2-nitrophenol (98%), 4-methoxy-
La(OTf)
3
(99.999%), anhydrous methanol (99.8%) and N-
phenol (99%), 4-methoxythiophenol (97%), 4-fluorothiophenol,
ethylmorpholine (99%) were all commercially available
NaHSO , diazabicycloundecane (98%), and diethyl chlorophos-
4
(
Aldrich), while Zn(OTf)
2
(98%) was obtained from Strem
phate (97%) were purchased from Aldrich and used as sup-
plied. 4-Chlorothiophenol (98%, Acros Organics) and 3,5-
dichlorothiophenol (98%, Matrix Scientific) were used as sup-
plied.
−3
Chemical. Stock solutions (5 mmol dm ) of each of the phos-
phate and phosphorothioate substrates were prepared in 99.8%
anhydrous acetonitrile (Aldrich). La(OTf) , sodium methoxide,
3
and tetrabutylammonium hydroxide stock solutions were made
Phosphates 2 and 5a as well as phosphorothioates 6a and
b were available from a previous study. All the other esters
−3
in anhydrous methanol to 50 mmol dm . Zn(OTf)
2
stock
1
3
6
solutions in anhydrous methanol were formulated between 50
were prepared according to the following general protocol.
CAUTION: phosphorothioates 6, and the phosphate triesters
−3
and 70 mmol dm while the 1,5,9-triazacyclododecane stock
−3
solution in methanol was made between 30 and 50 mmol dm .
2
and 5 are all acetylcholinesterase inhibitors, and should be
2+
2+
−
For each kinetic run using Zn , the 4:Zn : OCH
was made in situ by adding known amounts of the Zn(OTf)
,5,9-triazacyclododecane and tetrabutylammonium hydroxide
3
catalyst
synthesized and used with due attention to safety protocols.
Phosphates: 10 mmol of the appropriate phenol was added
slowly to a solution of 1.6 mL diazabicycloundacene (DBU)
in 20 mL of dry THF under argon and the resulting mixture
was cooled in an ice bath while slowly adding 10 mmol
diethylphosphoryl chloride. The mixture was then allowed to
stir at room temperature for 3 h. The products were extracted
three times with 20 mL of diethyl ether and washed twice
3
,
1
stock solutions to anhydrous methanol such that the final
2+
volume was 2.5 ml with the final ratios of ligand, Zn and
−
s
s
OCH
3
being 1 : 1 : 0.5 to self-buffer the solution at pH 9.1. The
kinetics were followed by monitoring the change in absorbance
−4
−3
of (0.5 to 2) × 10 mol dm of 5d, 5b, 5e, 5c and 6a, 6c, 6e,
6
b, 6f and 6d at 344, 276, 290, 428, 260, 266, 290, 277, 265,
with 20 mL of saturated NaHSO , twice with 20 mL of 10%
4
and 270 nm respectively. The absorbance vs. time data were
fit to a standard first-order exponential equation to obtain the
pseudo-first-order rate constants, kobs. The rates of reaction were
monitored in duplicate at 4–7 different catalyst concentrations
phosphate buffer and five times with 20 mL of water. The ether
layer was dried with sodium sulfate and, following filtration, the
volatiles were removed by rotary evaporation. Purification was
achieved by flash chromatography over silica gel eluting with
ethyl acetate–hexane (1 : 5 ratio) to produce the products in
yields of around 60%.
The general method for the synthesis and purification of the
phosphorothioate esters was the same as for the phosphotriesters
except that the thiophenols, diethyl chlorophosphate and dry
THF were mixed together before introducing DBU. The yields
for the syntheses of phosphorothioate esters were around 55%.
−3
obs
from 0.4 to 2.0 mmol dm . Second-order rate constants, k
2
,
for the catalyzed reaction were obtained from plotting kobs vs.
2+
−
[
4:Zn : OCH
3
] and are presented in Tables 1 and 2.
The La -catalyzed reactions were monitored by UV/vis
spectrophotometry under buffered (N-ethylmorpholine,
3+
−3
s
1
7 mmol dm ) conditions at three pH values between 8.34
s
−3
−3
and 9.14 in the presence of (0.2 to 2.0) × 10 mol dm
La(OTf) . This was done to ensure that the optimum rate of
the reaction for all substrates plateaued by pH 9.0. The rates
of methanolysis of (0.5 to 2) × 10 mol dm solutions of 5d,
3
1
31
All of the above esters had H NMR, P NMR, and mass
spectra consistent with the structure.
s
s
−3
−4
3+
2+ −
Table 1 Second-order rate constants (k
solvent, T = 25 C
2
3
) for the methanolysis of phosphates 2 and 5 promoted by methoxide, La and 4:Zn : OCH in methanol
◦
a
s
s
b
(OMe)
3
−1 −1
La c
3
−1 −1
4:Zn:OMe
3
−1 −1
Aryloxy-phosphate
pK
a
pK
a
k
2
/dm mol
s
k
2
/dm mol
s
k
2
/dm mol
s
Pentafluoro (5b)
5.53
6.32
7.14
8.39
9.38
10.0
10.20
8.84
0.201 ± 0.002
1070 ± 40
185 ± 7
d
23.0 ± 0.6
11.4 ± 0.4
1.3
−
2
4
-Chloro-2-nitro (5c)
10.64
11.30
12.41
13.59
14.33
14.7
(6.4 ± 0.1) × 10
−
3
2
p-Nitro (2)
(1.02 ± 0.03) × 10
23.2 ± 0.9
−
−
m-Nitro (5d)
p-Chloro (5e)
p-H (5a)
(6.0 ± 0.1) × 10
2.42 ± 0.07
0.58 ± 0.01
4
−2
−3
(6.3 ± 0.1) × 10
(1.98 ± 0.07) × 10
(8.04 ± 0.04) × 10
−
4 d
−3 d
1.4 × 10
1.97 × 10
(6.50 ± 0.01) × 10−
5 e
(2.2 ± 0.1) × 10
−4 e
(2.25 ± 0.05) × 10−4 e
p-Methoxy (5f)
a
b s
c
La
−3
pK
a
values in water from ref. 18. pK
a
values in methanol from refs. 10, 15, and 17. k
2
determined in a 17 mmol dm N-ethylmorpholine buffer
s
s
d
e
1
at pH 9.1.
section.
2 3
k values from ref. 13. From duplicate initial rate measurements monitored by H NMR in CD OD as described in the Experimental
s
3+
2+ −
Table 2 Second-order rate constants (k
solvent, T = 25 C
2
3
) for the methanolysis of phosphorothioates 6 promoted by methoxide, La and 4:Zn : OCH in methanol
◦
a
s
s
b
OMe
3
−1 −1
La
3
−1 −1
4:Zn:OMe
3
−1 −1
ArylS-phosphorothioate
pK
a
pK
a
k
2
/dm mol
s
k
2
/dm mol
s
k
2
/dm mol
s
c
d
d
p-Nitro (6b)
4.61
5.07
5.97
6.54
6.68
6.95
8.4
8.9
10.1
0.12
12.4
0.84 ± 0.01
0.97 ± 0.01
3
,5-Dichloro (6d)
0.152 ± 0.001
14.0 ± 0.5
1.23 ± 0.05
0.46 ± 0.01
−
−
2
−2
p-Chloro (6c)
p-Fluoro (6e)
p-H (6a)
(1.88 ± 0.03) × 10
(11.6 ± 0.01) × 10
(5.34 ± 0.06) × 10
2
−2
−2
−2
10.7c
10.9
11.2
(1.11 ± 0.02) × 10
−3 d
d
4.8 × 10
0.48
(4.2 ± 0.1) × 10
(2.22 ± 0.03) × 10−
3
(9.3 ± 0.2) × 10−2
(1.46 ± 0.04) × 10
p-Methoxy (6f)
a
c
b s
(MeOH)
(water)
Aqueous pK
a
values from ref. 16e. pK
a
a
values in methanol computed from two-point linear regression pK
a
= 1.2(pK
a
) + 2.83.
s
s
s
d
La
Experimental pK
values from ref. 19. Kinetic k
2
values from ref. 13.
1
5 2 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 5 2 5 – 1 5 3 3

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5
b, 5e, 5c and 6c, 6e, 6f, 6d were monitored at 348.6, 275, 286,
In Figs. 1, 2 and 3, as aids to visualizing the data, are presented
Brønsted plots of the second-order rate constants for metho-
3
86.7, 235, 288, 239.6, and 281 nm respectively to obtain the
3
+
3+
2+
−
pseudo-first-order rate constants (kobs) at each [La ], and the
gradients for the kobs vs. [La ] plots were calculated to give
xide-, La - and 4:Zn : OCH
3
-catalyzed methanolysis of the
3
+
s
phosphate and phosphorothioate esters vs. the pK of the conju-
a
s
obs
3+
−
−
the overall k
2
values for the La -catalyzed methanolysis at
gate acids of the ArO or ArS leaving groups. Linear regressions
for the methoxide reactions of the phosphates (except 5b which
falls well under the Brønsted plot in Fig. 1) and phosphoroth-
ioates (except 6b) are given in eqns. (1) and (2) respectively.
s
s
pH 9.1 which are given in Tables 1 and 2. Kinetic data for 2,
5
a, 6a and 6b were obtained from ref. 13. Initial rate kinetic
3
+
1
data for La -catalyzed methanolysis of 5f were obtained by H
NMR spectrometry by observing the rate of disappearance of
starting material and the rate of formation of product for the
first 10% of the reaction in CD OD. Into two separate NMR
3
−
3
tubes were placed 10 mmol dm of substrate 5f along with
−
3
3+
−
1
.5 mmol dm of catalyst (1 : 1 ratio of La : OCH
3
) in 1 mL
of CD
3
OD. The tubes were immersed in a thermostated bath at
◦
1
25 C and the H NMR spectra were monitored periodically
over 155 h. The integrations of the signals corresponding to
the aromatic protons in the starting material and product were
used to determine the initial rates which were converted into
the second-order rate constants given in Table 1.
2
+
Kinetic data for Zn -catalyzed methanolysis of 5f were deter-
−
3
mined similarly in duplicate using 10 mmol dm of substrate,
−
3
5
mmol dm each of Zn(OTf)
2
and 1,5,9-triazacyclododecane
−
3
and 2.5 mmol dm of Bu
periodically monitored by H NMR over 45 h. The initial rate for
4
NOCH in 1 mL of CD OD,
3
3
1
Fig. 1 Brønsted plots of the log second-order rate constant for methox-
the methoxide reaction with 5f was determined from duplicate
−
3
−3
ide attack on phosphates 2 and 5 as well as on phosphorothioates 6
reactions of 10 mmol dm of substrate and 25 mmol dm of
sodium methoxide followed for 45 h to 23% completion.
The pseudo-first-order rate constants for methanolysis of
the other phosphorus esters promoted by methoxide were
determined by UV/vis (monitored at the same wavelength as
s
s
(
except 6b) vs. pK
a
values for the corresponding phenols or thiophenols;
linear regressions for the phosphate (except 5b) and phosphorothioate
except 6b) data give slopes of −0.70 ± 0.05 (solid line, ꢀ) and −0.76 ±
0.08 (dashed line, ᭺) respectively.
(
3
+
for La -catalyzed methanolysis) in duplicate at four different
−
−3
[
OCH
3
] ranging from 0.01 to 0.04 mol dm . The second-order
OMe
rate constants for these (k
2
) were computed as the gradients
of the kobs vs. [methoxide] plots and are given in Tables 1 and 2.
Results
Given in Tables 1 and 2 are the second-order rate constants
for the methanolysis of the phosphates and phosphorothioates
−
3+
2+
−
promoted by OCH
3
, La and 4:Zn : OCH . In all cases,
3
these were determined from the gradients of the plots of kobs vs.
x+
2+
[
methoxide] or [metal ]total. In the case of 4:Zn , the solutions
s
were self-buffered at pH 9.1 through half neutralization of the
catalyst ([4:Zn : OCH
with 4:Zn showed that the catalytic activity was entirely
due to the methoxide form, 4:Zn : OCH
s
2
+
−
2+
3+
Fig. 2 Brønsted plot of the log second-order rate constant for La -
3
]/[4:Zn :HOCH
3
] = 1). Earlier work
2
+
and 4:Zn2+
:
−
OCH -catalyzed methanolysis of phosphates 2 and 5 vs.
3
s
2
+
−
12b
4:Zn:OMe
pK values for the corresponding phenols; linear regressions through
a
s
3
,
and the k
2
3+ 2+
−
the La and 4:Zn : OCH
3
data (except 5b) give gradients of −1.43 ±
values reported in Tables 1 and 2 were calculated based on the
0
.08 (solid line, ꢀ) and −1.12 ± 0.13 (dashed line, ᭺) respectively. Note
3
+
concentration of the catalytically active species. La catalysis for
that the points on lower right for the p-methoxy derivative (5f) are
3
+
all substrates was determined by observing the kobs vs. [La ]total
coincident.
s
s
−3
at the pH value of 9.14 in a 17 mmol dm N-ethylmorpholine
3
+
buffer system. Earlier work on the La -catalyzed methanolysis
of paraoxon had shown that the catalytic rate constant plateaus
between pH ca. 8.3 and ca. 9.2, so it was important to see
s
8
s
that the same trend exists for all the other phosphate and
phosphorothioate esters, which is confirmed in this work. Given
in Tables 1 and 2 are the observed second-order rate constants
s
s
8
determined at pK
a
9.1, where earlier analysis indicated that
>
90% of the catalysis occurs through a single form of the
3+
−
catalyst, namely La
2
( OCH ) , vide infra.
3
2
s
s
Also given in Tables 1 and 2 are the pK
a
values for the phenols
and thiophenols in methanol. While experimental methanol
s
s
17,18
pK
a
values for most of the phenols are available
or can
be calculated from their water values by the linear regressions
MeOH
H O
2
given in refs. 10 and 17 (pK
a
= 1.12pK
a
+ 3.56), to our
s
s
knowledge the only two available thiol pK
are thiophenol (10.9) and p-nitrothiophenol (8.4). Using
these two values, a computed linear regression of pK
a
values in methanol
3+
19
Fig. 3 Brønsted plot of the log second-order rate constant for La - and
2
+
−
s
s
4
:Zn : OCH
3
-catalyzed methanolysis of phosphorothioates 6 vs. pK
a
s
s
(MeOH)
a
=
values for the corresponding thiophenols; linear regressions through the
3+ 2+
(water)
1.2(pK
a
) + 2.83 is proposed to relate the aqueous and
values for the unknown aryl thiols.
−
La and 4:Zn : OCH
3
data give gradients of −0.87 ± 0.10 (solid line,
methanolic pK
a
ꢀ) and −0.74 ± 0.06 (dashed line, ᭺) respectively.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 5 2 5 – 1 5 3 3
1 5 2 7

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OMe
OMe
s
s
HOAr
La
OMe
logk
2
2
(phosphate) = (6.20 ± 0.70) − (0.70 ± 0.05) pK
a
(1)
(2)
logk
logk
logk
logk
2
2
2
2
(phosphate) = (1.94 ± 0.10)logk
2
(phosphate)
OMe
+
(4.65 ± 0.26)
(7)
(8)
logk
(phosphorothioate) = (6.00 ± 0.86)
s
s
HSAr
4:Zn:OMe
−
(0.76 ± 0.08) pK
a
(phosphate) = (1.49 ± 0.11)logk
2
(phosphate)
+
(2.78 ± 0.27)
For the metal-catalyzed reactions, the Brønsted plots of which
are given in Figs. 2 and 3, the appropriate linear regressions
for the phosphates (excluding 5b) are given in eqns. (3)and (4)
whereas those for the phosphorothioates (except 6b) are given
in eqns. (5) and (6).
La
OMe
(phosphorothioate) = (1.15 ± 0.11)logk
2
(phosphorothioate)
OMe
+
(2.10 ± 0.18)
(9)
4:Zn:OMe
(phosphorothioate) = (0.99 ± 0.06)logk
2
(phosphorothioate)
La
s
s
HOAr
logk
2
(phosphate) = (17.60 ± 1.07) − (1.43 ± 0.05) pK
a
(3)
+ (0.79 ± 0.11)
(10)
4
:Zn:OMe
logk
2
(phosphate) = (13.05 ± 1.59)
s
s
HOAr
Discussion
−
(1.12 ± 0.13) pK
a
(4)
(5)
The various second-order rate constants for the reactions of the
phosphates and phosphorothioates are presented in Tables 1 and
La
s
s
HSAr
logk
logk
2
2
(phosphorothioate) = (8.93 ± 1.01) − (0.87 ± 0.10) pK
a
s
s
2
along with the leaving group phenol pK
a
values in methanol
4
:Zn:OMe
s
(phosphorothioate) = (6.64 ± 0.69)
and water. For the aryloxy esters the HOAr pK
all known or can be computed from known linear regressions
relating the methanol and aqueous pK
we are aware, the HSAr pK
are only known for the thiophenol and for p-nitrothiophenol,
and if we assume that there is also a linear dependence of
these methanol and aqueous pK
two-point linear regression of pK
a
values are
s
s
s
HSAr
−
(0.74 ± 0.06) pK
a
(6)
10,15,17,18
a
values.
As far as
Shown in Figs. 4 and 5 are alternative presentations of the
data where the methoxide rate constants are plotted vs. the
La or 4:Zn : OCH rate constants for all the phosphate and
phosphorothioate substrates. The linear regressions for these are
given in eqns. (7) and (8), and (9) and (10) respectively.
s
s
a
values in methanol for the arylthiols
3
+
2+
−
3
a
values one can compute a
s
s
(MeOH)
(water)
a
= 1.2(pK
a
) + 2.83
s
s
from which the pK
a
values for the other thiols may be estimated.
Although the gradient of this linear regression is quite similar
17
to that reported for the phenols (1.12), these computed thiol
s
s
pK
a
values are viewed as estimates only for the construction of
various Brønsted plots for the phosphorothioate esters.
3
+
In constructing the various Brønsted plots for the La -
catalyzed processes, we used the second-order rate constants ob-
3
+
tained as the gradients of plots of Dkobs/D[La ] under buffered
s
s
La
3+
conditions at pK
a
9.1, so the k
2
values are presented per La
ion even though we have determined that the actual catalytic
3
+
−
2
species are dimers (La )
( OCH )2,3,4 the relative importance
3
s
s
8,13,20 3+
of which depend on the pK
a
and the [dimer].
For the La
catalytic study, the aryloxy phosphate esters encompass a range
6
7
La
of 10 in leaving group acidity and about 10 in k
2
but only
. The second-order
-catalyzed reactions were
OMe
about three orders of magnitude in k
rate constants for the 4:Zn : OCH
determined under self-buffered conditions at pK
2
2
+
−
3
s
s
a
9.1 through
4
:Zn:OMe
half neutralization of the catalyst, and the k
2
values
reported in Tables 1 and 2 are computed on the basis of the
active form.
OMe
La
4:Zn:OMe
Fig. 4 Plots of logk
2
vs. logk
2
(ꢀ) or logk
2
(᭺) for the
◦
3+
methanolysis of phosphate esters at 25 C: slope for La plot is 1.94 ±
0
.10; slope for 4:Zn:OMe plot is 1.49 ± 0.11 all data included. Note that
the data points for the p-methoxy derivative (5f) in lower left corner are
(a) Uncatalyzed reaction of methoxide
coincident.
OMe
The Brønsted plots of logk
2
data for methoxide reactions
s
of the phosphate and phosphorothioate esters vs. the pK
a
s
values shown in Fig. 1 provide reasonable linear correlations,
the respective blg values being −0.70 ± 0.05 and −0.76 ± 0.08.
s
s
In both series the derivatives having the lowest pK
a
values of
the leaving phenol/thiophenol (5b and 6b) are less reactive
s
s
than predicted on the basis of the equilibrium pK data, and
a
cursory inspection reveals that the plots have some curvature,
with the latter points lying below the indicated lines. Non-
linear Hammett and Brønsted behaviours have been observed
2
1
for the hydroxide reactions of substituted aryl benzoates
22
23
and acetates and Schowen reported that logk
2
values for
methoxide reactions of aryl acetates and carbonates in methanol
s
s
are not linearly related to the pK values for ionization of the
a
corresponding phenols. Our previous study of the methanolysis
of aryl acetates also showed some discontinuity in the Brønsted
OMe
s
s
plot of logk
vs. phenol pK
a
for the most acidic phenols
15
such as pentafluoro- and 2,4-dinitro-phenol. Such curvature,
if observed, does not result from a change in mechanism or
rate-limiting step in the reaction but is related to a greater
importance of the resonance and inductive interactions in the
equilibrium acid dissociation constants (on which the Hammett
OMe
La
4:Zn:OMe
Fig. 5 Plots of logk
2
vs. logk
2
(ꢀ) or logk
2
(᭺) for the
3+
◦
methanolysis of phosphorothioate esters at 25 C: slope for La plot is
.15 ± 0.10; slope for 4:Zn:OMe plot is 0.99 ± 0.06.
1
1
5 2 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 5 2 5 – 1 5 3 3

View Article Online
s
s
and pK
a
values are based) than in the kinetic processes
measures the change in the Brønsted blg for the TS relative to
the beq for equilibrium transfers of acyl or phosphoryl groups
between oxyanion nucleophiles. In the case of the transfer of
where far less charge development generally occurs in the rate-
limiting TS.
21–23
In the present study we have chosen to omit
26
the data for the pentafluorophosphate and phosphorothioate
derivatives, 5b and 6b, from calculation of the gradients of the
Brønsted plots. The blg values of −0.70 and −0.76 respectively
for the methoxide reaction of esters 2 and 5 (except 5b)
and 6 (except 6b) can be compared with the corresponding
hydroxide blg values of −0.41 to −0.35 for 2-aryloxy-2-oxo-1,3-
the (EtO)
2
P=O group, the beq value is −1.87 with the O–Ar
oxygen in the starting material having a net effective charge of
+0.87. When methoxide is the nucleophile, the Leffler parameter
of blg/beq = 0.37 suggests that the P–OAr cleavage is 37% of the
way from starting material to product.
For phosphorothioates 6 reacting with methoxide, a similar
sort of analysis for the reaction proceeding through a two- or
one-step reaction can be invoked. In this case one has no exact
measure of the effective charge on the S-atom in the ArS–P unit,
but based on comparison of the known effective charges on the
24,25
dioxaphosphorinanes
on diethyl aryl phosphate triesters,
attack on diethyl S-aryl phosphorothiolates.
−0.43 and −0.44 for hydroxide attack
16e,26
and −0.42 for hydroxide
16e
24
25
Khan and Kirby, and later Rowel and Gorenstein, dis-
cussed the reaction of oxyanions with some 2-aryloxy-2-oxo-
dioxaphosphorinanes concluding that, for strong nucleophiles
such as hydroxide and methoxide, the mechanism was consistent
with, although not uniquely so, a two-step process that proceeds
via a five-coordinate intermediate as in eqn. (11).
S and O atoms of ArS–C(=O)CH
and ArO–C(=O)CH
of 0.4
3
3
30a
and 0.7 respectively, one might expect that S is less positive
than O in the case of the ArX–P unit. Assuming the effective
charge on S is 0.5–0.6, the Leffler a for a concerted P–SAr
cleavage can be computed as ca. 0.45–0.50.
3
+
2+ −
(
b) La - and 4:Zn : OCH -catalyzed methanolysis
3
(
11)
Given in Figs. 2 and 3 are the respective Brønsted plots
for the metal-catalyzed methanolysis of the phosphate and
phosphorothioates. For phosphates 2 and 5, the respective blg
The relatively low blg values of ca. 0.4 obtained with these
nucleophiles is consistent with little cleavage of the P–OAr bond
in the TS, and supports a process having a rate-limiting k step
with preferential breakdown of the intermediate to the product.
However, as the nucleophiles became weaker, the blg values
increased, supporting either a two-step process where the break-
3+
2+
−
values for La and 4:Zn : OCH are large and negative, being
3
1
−
1.43 ± 0.08 and −1.12 ± 0.13 respectively if we exclude
the pentafluoro derivative 5b which deviates downward from
both plots, probably due to the known difficulties in plotting
23
3+
kinetic data against ionization data of phenols. The La and
2
4
down became more rate limiting or a change in mechanism to
2+
−
4
:Zn : OCH Brønsted plots for the phosphorothioates given
3
2
5
a concerted, S 2 displacement at P.
N
in Fig. 3 have blg values of −0.87 ± 0.10 and −0.74 ± 0.06
More recently, Williams and co-workers provided convincing
evidence for a single transition state for the transfer of the
diphenylphosphoryl group between phenoxide anions in water.
In that case, a Brønsted plot of the second-order rate constants
for reaction of the phenoxides with p-nitrophenyl diphenyl
respectively.
The large negative blg values for the metal-ion-catalyzed
methanolysis of the phosphate esters suggests a process where
there is considerable cleavage of the leaving group in the
transition state, far more than is the case for the methoxide
27
phosphate against the pK
a
of the nucleophile’s conjugate acids
3+
2+
−
reaction. In our previous study of the La and 4:Zn : OCH -
3
gave a straight line with bnuc = 0.66 with no evidence of a break
promoted methanolysis of acetate esters having aryloxy and
which would have been required if there was an intermediate
15
some alkoxy leaving groups, we determined that both metal
−
produced. In the case of the HO nucleophile reacting with
diethyl aryloxy phosphates, they judged, by analogy with the
above reactions, that HO displacement of aryloxy leaving
groups was probably concerted, although with little cleavage
s
s
systems exhibited Brønsted plots with a break at ca. pK
a
1
4.7, consistent with a catalyzed process comprising a pre-
−
equilibrium binding of the metal ion followed by kinetic steps
proceeding through a reversibly-formed intermediate as shown
in Scheme 2. With good leaving groups the internal attack of
metal-coordinated methoxide on the transiently bound ester
was rate limiting (blg ≈ 0), but with poor leaving groups
the breakdown of the metal-coordinated anionic tetrahedral
intermediate was rate limiting (blg = −0.71). In that case, it
appeared that metal ion coordination actually stabilized the
tetrahedral intermediate, but the situation with the present
phosphate/phosphorothioate esters is clearly different because
26
of the ArO–P bond. This conclusion is supported by the
1
8
O-phenoxy kinetic isotope effect of 1.006 for hydroxide-
28
promoted cleavage of paraoxon 2 which was interpreted as
being consistent with a bond order of 0.75 for the P–OAr bond
in the “S 2-like transition state of an associative mechanism
with concerted, asynchronous departure of the leaving group”.
Our data for methanolysis of the aryl phosphates 2 and 5
and diethyl S-aryl phosphorothioates 6 indicate that the blg
values are more negative by ca. 0.3 units than is the case for
N
there is a large dependence of the rate on the leaving group in the
1
6e,24,25
the hydroxide reactions.
treatment” described by Jencks and Williams, the Brønsted
lg value for nucleophilic attack of methoxide on the ary-
Following the “effective charge
s
s
pK region where blg is zero for the reaction of the carboxylate
a
29
30
esters. Accordingly we suggest that, for the metal-catalyzed
b
loxyphosphates suggests a process where the rate-limiting tran-
sition state has appreciable changes in the P–OAr bond. This
could be viewed as resulting from either a two-step process with
the attack step largely rate limiting due to the fact that the
methoxide nucleophile is a far poorer leaving group than any of
31
the aryloxy anions, or more likely with a concerted process as
shown in Scheme 1. The extent of breaking of the P–OAr bond
in the TS can be measured by the Leffler parameter, a, which
X+
−
3+
−
2+ −
Scheme 1
Scheme 2
M
– OCH
3
refers to (La )
2
( OCH
3
)
2
3
or 4:Zn : OCH .
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 5 2 5 – 1 5 3 3
1 5 2 9

View Article Online
Scheme 3 When X = O, a = 0.76.
III
III
III
methanolysis of these phosphates/phosphorothioates, there is
no evidence for a change in rate-limiting step for aryloxy or
arylthio leaving groups and, by inference, no evidence for an
intermediate.
toluene of Sc , Yt and the trivalent Ln ions from aqueous
nitrate solutions by coordination to neutral organophosphorus
(P=O) compounds correlates with the Taft r* values of the
3
2d,e
3+
substituents.
These considerations indicate that for the La -
-promoted methanolysis of the phosphates, the
2
+
−
Although we have never observed evidence of saturation
kinetics, it is difficult to envision any mechanism for the metal-
promoted reactions where the ion does not bind to the phosphate
to provide Lewis activation toward attack. Indeed, phosphate
or 4:Zn : OCH
observed blg may be a lower limit because it will be reduced by
the positive b
3
b
.
Presented in Scheme 3 is a proposed mechanism of reaction
for the La -catalyzed reaction of the phosphate which earlier
studies have shown is largely (>90%) attributable to the in-
volvement of a bis-methoxy-bridged dimer, (La )
32
3+
complexation of lanthanides and actinides is well known and
2
+
33
structures are known for Zn complexes of phosphine oxides
and tritoluoyl phosphate.
34
3+
−
2
( OCH ) ,
3
2
8
We analyze the present Brønsted plots as being consistent
with the mechanism in eqns. (12) and (13)
7 in Scheme 3. In Scheme 3, for the sake of visual clarity,
3
+
we have omitted the methanols of solvation on each La as
well as any possible associated triflates about which we have
no information. It seems unlikely that a methoxy group bound
3
+
between two La ions will be sufficiently nucleophilic to attack
(
(
12)
13)
3
7
the phosphate, so we propose that the pre-equilibrium binding
of phosphate to form 8 induces opening of one of the methoxy
bridges to reveal a kinetically active form (9) with one of the
3
+
La ions acting as a Lewis base, the other serving to deliver the
methoxide intramolecularly. Since the Leffler parameter, a, for
where there is a rapid pre-equilibrium binding followed by an
intramolecular concerted displacement of the leaving group.
Given in eqns. (14) and (15)
3
+
the La -catalyzed reaction is blg/beq = −1.43/−1.87 = 0.76, the
transition state for this reaction (10) has extensive cleavage of
the P–OAr bond, and this large value is most consistent with
a concerted reaction within the complex. Catalytic turnover
requires a final dissociation of the diethyl methyl phosphate with
the reformation of 7. In the case of the 4:Zn : OCH -catalyzed
reaction a similar mechanism is envisioned but this time the
transition structure (shown below as structure 12)
obs
(b + b1)pKa
k
2
= K
b
k
1
= C
b
C
1
10
b
(14)
(15)
obs
logk
2
= {logC
b
+ logC
1
} + (b
b
+ b
1
)pK
a
2
+
−
3
are two forms of the kinetic expression for this process where
and b refer to the Brønsted blg values for the binding and
kinetic steps, and pK refers to the acid dissociation constant
b
b
1
a
for the conjugate acid of the leaving groups. (Although these
expressions are derived for the concerted pathway, which we
favour on the basis of the large and negative blg values for
the metal-catalyzed reaction, they have the identical form for a
two-step process proceeding through an intermediate where the
formation of the intermediate is rate limiting.) The experimental
Brønsted blg values are thus (b
the influence of the leaving group on the pre-equilibrium binding
step and the kinetic step. It is difficult to predict an exact value for
b
+ b
1
), a composite measure of
35
b
b
and what few data there are available in the literature suggest
will be four-coordinate with a Leffler a of blg/beq
=
that this should be (+), but probably not large. For example,
−1.12/−1.87 = 0.60. This also signifies extensive dissociation
3
+
Rackham reported that the europium shift reagent Eu (2,2,6,6-
of the P–OAr bond in the transition state, but less so than in the
−
3+
tetramethylheptane-3,5-dione )
3
binds trimethyl phosphate and
case of La catalysis.
−
1
3
triphenyl phosphate with constants of 348 and 23.2 mol dm
While one might expect that the positively charged metal ion
might stabilize an anionic penta-coordinate intermediate, the far
more negative blg values observed for the metal-ion-catalyzed
reactions, compared to those for the methoxide reactions, sug-
gest that the metal ions do not stabilize sufficiently any putative
respectively, and suggested that the fall in the binding constant
can be attributed to steric bulk and the inductive withdrawal
of the phenoxy group relative to the methoxy group. Du
Preez and Preston have also reported that the extraction into
36
1
5 3 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 5 2 5 – 1 5 3 3

View Article Online
OMe
4:Zn(OMe)
La
intermediate to direct the reaction through a stepwise pathway.
(c) Comparision of k
2
and k
2
or k
2
3
+
The reason why the La reaction exhibits a significantly more
3
+
2+
−
Comparison of the La and 4:Zn : OCH
3
kinetic data in
2
+
negative blg than does the Zn reaction is not immediately
OMe
Tables 1 and 2 with the corresponding k
that the metal ion systems are more effective than methoxide for
promoting the methanolysis of this series of phosphates and
phosphorothioates. For the phosphates 2 and 5, the k
ratio varies from 5000 for the pentafluorophenoxy derivative 5b
to ca. 3 for the phenoxy derivative 5a. Over the same series, the
2
kinetic data indicates
obvious, but could result from better attack/departure angles
3
+
2+
in the 6-membered La TS relative to the 4-membered Zn
TS or from a greater electrostatic stabilization of the transition
La
OMe
2
/k
2
3
+
structure in the case of the more highly charged La system.
Given the fact that the catalysis becomes better with the aryloxy
s
s
anion leaving groups with lower pK
a
values for their conjugate
4:Zn:OMe
OMe
k
2
/k
2
ratio varies only by 40-fold (115 to ca. 3). On
3
+
2+
acids, it does not seem likely in either the La or Zn cases that
direct coordination of the leaving group to the metal ion is a
significant factor in the catalysis.
the other hand, the phosphorothioates show very little variation
La
OMe
4:Zn:OMe
OMe
of the k
2
/k
2
or k
2
/k
2
ratios with leaving groups,
the values being 40–100 throughout the arylthio series for the
former and 5–8 for the latter.
Shown in Figs. 4 and 5 are the plots of logk
3
+
2+
In the case of La - and Zn -catalyzed methanolysis of
the phosphorothioate esters the observed blg values of −0.87
and −0.74 also signify an associative mechanism with some
departure of the leaving group, but it is difficult to assign
the extent of the bond cleavage since the beq value is not
known for the phosphoryl transfer between thiol and oxygen
nucleophiles. It is highly instructive to consider the graph shown
OMe
2
vs. log
La
4:Zn:OMe
k
2
or logk
2
for the phosphates and phosphorothioates.
As was the case in our earlier study of the metal-catalyzed
15
methanolysis of esters, we assume here that the mechanism
of methoxide-promoted methanolysis is similar enough for all
the phosphate esters with the nucleophilic addition being either
3
+
in Fig. 6 which is a plot of the logk
2
constants for the La -
rate limiting or concerted with leaving group departure that
2
+−
and 4:Zn OCH
3
-catalyzed reactions for the entire series of
OMe
the rate constant, k
2
, can be used as an empirical measure
phosphate and phosphorothioate esters. The fact that the slopes
for each series of substrate are very similar and without breaks
of the composite effects of structural changes that incorporate
both electronic and steric effects. This approach has advantages
over the Brønsted or Hammett plots since the latter rely on
+3
over some seven orders of magnitude for the La reaction
2
+
and five orders of magnitude for the Zn reaction indicates
that there is no change of mechanism throughout either series.
Furthermore, the slopes of 1.30 ± 0.05 and 1.17 ± 0.04 for
the phosphate and the phosphorothioate reactions are some
substituent-induced changes to equilibrium pK processes which
a
may not be appropriate for how a substituent influences a
particular reaction where the rate-limiting step(s) have less
charge development on the leaving group. An additional benefit
is that this treatment does not rely on experimental or estimated
pK values for a given substituent which, if in error, may
influence the conclusions based on the slopes of Brønsted plots.
The slopes of the lines in Fig. 4 for methanolysis of the
phosphates are 1.94 and 1.49 for La and the Zn com-
plex respectively, indicating that catalytic enhancement of the
methanolysis reaction by the metal ion gets better as the leaving
3
+
measure of the greater efficacy of the La reaction which may
relate to the higher net positive charge or better geometry for
s
s
3
+
a
the La -catalyzed processes. Finally, the fact that both the
phosphate and phosphorothioate esters lie essentially on the
same line indicates that there cannot be any special catalytic
assistance to the departure of the leaving group since one would
expect a different effect for assistance of departure of the softer
3
+
2+
2
+
38
3+
SAr group by Zn relative to the ‘hard’ La . This is drastically
different to a situation we have previously reported where, for
group gets better. The data also suggest that for leaving groups
s
s
having pK
a
values larger than 15, the second-order rate constant
3
+
2+
the methanolysis of P=S and P=O phosphates, La and Zn
for the methoxide reaction will be larger than those for either
metal-catalyzed reaction. The slopes of the lines in Fig. 5 for
the phosphorothioates are, within experimental uncertainty,
essentially unity for both the La - and Zn -catalyzed reactions.
Importantly, the fact that all the plots in Figs. 4 and 5 are linear
with no break over the range of substrates studied indicates that
there is no change in mechanism or rate-limiting step for one of
the reactions which is not manifested in the other.
2
+
or Cu show very different catalytic effects implying that in
these materials coordination to the phosphoryl O and S are
12
required components of catalysis. Thus on the basis of the
current evidence, it is reasonable that the phosphorothioate
methanolysis promoted by both metal-containing systems is
concerted and proceeds analogously to the process depicted in
Scheme 3.
3
+
2+
(d) Relevance to the phosphotriesterase enzyme
The above study has a direct bearing on the mechanism of
2
+
action of phosphotriesterase (PTE), a Zn -contaning enzyme
found in the soil-dwelling bacterium Pseudomonas diminuta
which has been shown to degrade pesticides such as paraoxon
15
and parathion. X-Ray diffraction shows that the active site
2
+
˚
comprises two Zn ions separated by 3.4 A, one of which
is coordinated to the protein by two His imidazoles and an
aspartate, and the second by two histidines: both metal ions are
−
bridged by the oxygens of N-carboxy lysine and an OH or water
16c,39
having a kinetic pK
a
of 5.8–5.9
which is thought to be the
X-Ray diffraction studies also showed
that inhibitors such as triethyl phosphate and di-iso-propyl
16a,39,40
active nucleophile.
2
+
methylphosphonate bind to the more solvent-exposed Zn ion
41
with P=O coordination. The metal ions can be removed from
2
+
2+
2+
2+
the wild-type enzyme and replaced by Cd , Ni , Co and Mn ,
and all reconstituted enzymes show activity. The native enzyme
1
8
has been shown to react with paraoxon in O-labelled water to
La
4:Zn:OMe
Fig. 6 Plots of logk
2
vs. logk
2
for the methanolysis of phos-
18
produce the (EtO)
chiral O-ethyl phenylphosphonothioic acid to give a product that
2
P(=O) OH product, and also to react with
◦
phate (ꢁ) and phosphorothioate (ꢀ) esters at 25 C: the linear regression
La
4:Zn:(OMe)
for the phosphate ester plot is logk
2
= (1.30 ± 0.05)logk
2
+
42
2
is inverted. Detailed kinetic studies of the hydrolysis of a series
of diethyl aryl phosphates and phosphorthioates conducted on
(
1.03 ± 0.10), r = 0.9930; the linear regression for the phosphorothioate
La
4:Zn:(OMe)
2
esters is logk
2
= (1.17 ± 0.04)logk
2
+ (1.18 ± 0.05), r =
2
+
0
.9952.
the (Zn )
2
wild-type enzyme by Hong and Raushel indicated
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 5 2 5 – 1 5 3 3
1 5 3 1

View Article Online
that the respective Brønsted blg values of Vmax vs. pK
a
of aryl
of the Army, Army Research Office, Grant No. W911NF-04-1-
43
phosphates and phosphorothioates in water were −2.2 and −1.0
respectively, the high value for the phosphates being interpreted
as arising from a significantly dissociative mechanism with a
0057 and the Defense Threat Reduction Agency, Joint Science
and Technology Office (06012384BP). T. L. thanks the Summer
Student Work Experience Program at Queen’s University.
16e
quite product-like transition state.
There is much in common between the reactivity of the
enzyme for both phosphates and phosphorothioates and that
References and notes
1 A. Toy and E. N. Walsh, Phosphorus Chemistry in Everyday Living,
American Chemical Society, Washington, DC, 2nd edn., 1987, ch.
3
+
−
2
which is operative for our very simple dinuclear (La )
( OCH
3
)
2
2
+
−
and mono-nuclear 4:Zn : OCH
3
systems. The most obvious
1
8–20; L. D. Quin, A Guide to Organophosphorus Chemistry, Wiley,
similarity comes from comparison of the large Brønsted blg
we see for both sets of substrates and those exhibited by the
wild-type enzyme. However, very much higher blg values are
New York, 2000; M. A. Gallo and N. J. Lawryk, Organic Phosphorus
Pesticides. The Handbook of Pesticide Toxicology, Academic Press,
San Diego, CA, 1991; P. J. Chernier, Survey of Industrial Chemistry,
VCH, New York, 2nd edn., 1992, pp. 389–417.
2
+
2+
reported for the Cd - and Mn -enzymes (−3.0 and −4.3
respectively) which clearly are far greater than one expects for
2 K. A. Hassall, The Biochemistry and Uses of Pesticides, VSH,
16e
Weiheim, 2nd edn., 1990, pp. 269–275.
Y.-C. Yang, J. A. Baker and J. R. Ward, Chem. Rev., 1992, 92, 1729;
Y.-C. Yang, Acc. Chem. Res., 1999, 32, 109; Y.-C. Yang, Chem. Ind.
simple P–OAr bond cleavage in water. The latter blg values
3
4
are obtained by plotting the rate data against the aqueous
pK
and electrostatic stabilization of the leaving anion in the active
site. In fact, if the pK values in the lower dielectric constant
solvents MeOH or DMSO were used, which tends to expand
a
values which likely do not reflect faithfully the solvation
(
London), 1995, 32, 334.
H. Morales-Rojas and R. S. Moss, Chem Rev., 2002, 102, 2497 and
a
refs. cited therein.
5 S. H. Gellman, R. Petter and R. Breslow, J. Am. Chem. Soc., 1986,
19
1
1
08, 2388; R. S. Brown and M. Zamkanei, Inorg. Chim. Acta, 1985,
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the pK
a
scales of phenols, this would lessen considerably the
b
lg values. Nevertheless, the kinetic data do indicate a very large
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liming step of the enzymatic phosphoryl transfer, similar to
what we have observed in our simple catalytic system which
we have interpreted in terms of a concerted mechanism. All the
other data from the enzyme studies, for example the inversion
of configuration in hydrolysis of an optically active starting
1
8
material and O-incorporation of solvent water to the hydrolytic
1
2, 6235; C. A. Bunton, P. Scrimmin and P. Tecilla, J. Chem. Soc.,
42
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Conclusions
The above study indicates that metal-catalyzed methanolyses
of these series of phosphate and phosphorothioate triesters
proceed by a mechanism that involves considerable cleavage of
the leaving group in the rate-limiting step, in fact more cleavage
than in the analogous methoxide attack on the same esters.
3
+
−
The data indicate that while the dinuclear (La )
2
( OCH
3
)
2
system has a large and negative blg for phosphate methanolysis
¯
210; M. Yamami, H. Furutachi, T. Yokoyama and H. Okawa, Inorg.
2
2
+
−
(
−1.43), that of the mononuclear complex 4:Zn : OCH
3
is
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slightly less at −1.15. These large negative values are best
interpreted in terms of a concerted displacement mechanism
with considerable departure of the leaving group bond in the
rate-limiting step. The reason for the difference in blg of the
3
+
2+
La and Zn complexes may be a result of differences in total
(
+) in the vicinity of the transition structure, or geometric
3+
differences where the La -containing transition structure is
more optimally aligned for internal methoxide displacement of
the leaving group. It is interesting to note that, despite the fact
that metal-catalyzed hydrolysis of phosphate triesters has been
extensively investigated for many years, in no case of which
we are aware has such a significant catalytic effect over such
a wide series of substrates been observed. A significant aspect
5,6
7
G. M. Kazankov, V. S. Sergeeva, L. A. Efremenko, S. D. Varfolomeev
and A. D. Ryabov, Angew. Chem., Int. Ed., 2000, 39, 3117; G. M.
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3
+
2+
of the efficacy of the La and Zn catalysts in methanol must
arise from a medium or solvent effect since in general the metal
hydroxo counterparts in water are significantly less reactive than
our metal methoxide catalysts in methanol and even less reactive
than hydroxide itself. Given that the phosphotriesterase enzyme
8
9
J. S. W. Tsang, A. A. Neverov and R. S. Brown, J. Am. Chem. Soc.,
2
003, 125, 7602.
For the designation of pH in non-aqueous solvents we use the forms
2
+
contains a dinuclear Zn active site, it would be interesting to
compare the efficacy for alcoholysis promoted by an appropriate
10
described by Bosch and co-workers based on the recommendations
of the IUPAC Compendium of Analytical Nomenclature. Definitive
2
+
2+
−
dinuclear Zn -containing catalyst with that of 4:Zn : OCH ,
3
Rules 1997, Blackwell, Oxford, UK, 3rd edn., 1998. If one calibrates
a study which is currently underway in these laboratories.
the measuring electrode with aqueous buffers and then measures
s
s
the pH of an aqueous buffer solution, the term pK
electrode is calibrated in water and the ‘pH’ of the neat buffered
methanol solution then measured, the term pK is used; and if a
a
is used; if the
s
s
Acknowledgements
a
correction factor of −2.24 (in the case of methanol) is subtracted
The authors gratefully acknowledge the financial assistance
of the Natural Sciences and Engineering Research Council of
Canada, Queen’s University and the United States Department
s
s
from the latter reading, then the term pK
a
is used.
10 Given that the autoprotolysis constant of methanol is 10−16.77
−
3
2
s
s
a
(mol dm ) , the neutral pK in methanol is 8.4; see E. Bosch, F.
1
5 3 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 5 2 5 – 1 5 3 3

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1, 495; We have determined that the maximal activity for the La -
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s
s
triesters lies in the pK
the activity is attributed to La
a
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3+
−
2
( OCH
3 2
) . Thus, the conditional
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43 The content of the information does not necessarily reflect the
position or the policy of the federal government, and no official
endorsement should be inferred.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 5 2 5 – 1 5 3 3
1 5 3 3