Study on the transesterification of methyl aryl phosphorothioates in methanol promoted by Cd(II), Mn(II), and a synthetic Pd(II) complex

Article abstract of DOI:10.1021/ic102220m

Methanol solutions containing Cd(II), Mn(II), and a palladacycle, (dimethanol bis(N,N-dimethylbenzylamine-2C,N)palladium(II) (3), are shown to promote the methanolytic transesterification of O-methyl O-4-nitrophenyl phosphorothioate (2b) at 25 °C with impressive rate accelerations of 10 ⁶-10¹¹ over the background methoxide promoted reaction. A detailed mechanistic investigation of the methanolytic cleavage of 2a-d having various leaving group aryl substitutions, and particularly the 4-nitrophenyl derivative (2b), catalyzed by Pd-complex 3 is presented. Plots of k _obs versus palladacycle [3] demonstrate strong saturation binding to form 2b:3. Numerical fits of the kinetic data to a universal binding equation provide binding constants, K_b, and first order catalytic rate constants for the methanolysis reaction of the 2b:3 complex (k_cat) which, when corrected for buffer effects, give corrected (k_cat ^corr) rate constants. A sigmoidal shaped plot of log(k _cat^corr) versus _s^spH (in methanol) for the cleavage of 2b displays a broad _s^spH independent region from 5.6 ≥ _s^spH ≥ ～10 with a k ^minimum = (1.45 ± 0.24) × 10^-2 s^-1 and a [lyoxide] dependent wing plateauing above a kinetically determined _s^spK_a of 12.71 ± 0.17 to give a k ^maximum = 7.1 ± 1.7 s^-1. Bronsted plots were constructed for reaction of 2a-d at _s^spH 8.7 and 14.1, corresponding to reaction in the midpoints of the low and high _s ^spH plateaus. The Bronsted coefficients (β^LG) are computed as -0.01 ± 0.03 and -0.86 ± 0.004 at low and high _s^spH, respectively. In the low _s^spH plateau, and under conditions of saturating 3, a solvent deuterium kinetic isotope effect of k^H/k^D = 1.17 ± 0.08 is observed; activation parameters (ΔH_Pd^? = 14.0 ± 0.6 kcal/mol and ΔS_Pd^?= -20 ± 2 cal/mol?K) were obtained for the 3-catalyzed cleavage reaction of 2b. Possible mechanisms are discussed for the reactions catalyzed by 3 at low and high _s^spH. This catalytic system is shown to promote the methanolytic cleavage of O,O-dimethyl phosphorothioate in CD₃OD, producing (CD₃O)₂P=O(S^-) with a half time for reaction of 34 min.

Full text of DOI:10.1021/ic102220m

1
786 Inorg. Chem. 2011, 50, 1786–1797
DOI: 10.1021/ic102220m
Study on the Transesterification of Methyl Aryl Phosphorothioates in Methanol
Promoted by Cd(II), Mn(II), and a Synthetic Pd(II) Complex
David R. Edwards, Alexei A. Neverov, and R. Stan Brown*
Department of Chemistry, Queen’s University, Kingston, Ontario K7L 3N6, Canada
Received November 4, 2010
Methanol solutions containing Cd(II), Mn(II), and a palladacycle, (dimethanol bis(N,N-dimethylbenzylamine-2C,
N)palladium(II) (3), are shown to promote the methanolytic transesterification of O-methyl O-4-nitrophenyl
6
11
phosphorothioate (2b) at 25 °C with impressive rate accelerations of 10 -10 over the background methoxide
promoted reaction. A detailed mechanistic investigation of the methanolytic cleavage of 2a-d having various leaving
group aryl substitutions, and particularly the 4-nitrophenyl derivative (2b), catalyzed by Pd-complex 3 is presented.
Plots of kobs versus palladacycle [3] demonstrate strong saturation binding to form 2b:3. Numerical fits of the kinetic
data to a universal binding equation provide binding constants, K , and first order catalytic rate constants for the
b
corr
methanolysis reaction of the 2b:3 complex (k ) which, when corrected for buffer effects, give corrected (k ) rate
cat
cat
corr
cat
s
s
s
constants. A sigmoidal shaped plot of log(k ) versus pH (in methanol) for the cleavage of 2b displays a broad pH
= (1.45 ( 0.24) ꢀ 10
s
minimum
s
-2 -1
independent region from 5.6 e pH e ∼10 with a k
s
and a [lyoxide] dependent
= 7.1 ( 1.7 s . Brønsted plots
s
s
maximum
-1
wing plateauing above a kinetically determined pK of 12.71 ( 0.17 to give a k
s
a
s
s
were constructed for reaction of 2a-d at pH 8.7 and 14.1, corresponding to reaction in the midpoints of the low and
s
LG
high pH plateaus. The Brønsted coefficients (β ) are computed as -0.01 ( 0.03 and -0.86 ( 0.004 at low and
s
s
s
s
s
high pH, respectively. In the low pH plateau, and under conditions of saturating 3, a solvent deuterium kinetic isotope
H
D
q
q
Pd
effect of k /k = 1.17 ( 0.08 is observed; activation parameters (ΔH = 14.0 ( 0.6 kcal/mol and ΔS = -20 (
Pd
2
the reactions catalyzed by 3 at low and high pH. This catalytic system is shown to promote the methanolytic
cal/mol K) were obtained for the 3-catalyzed cleavage reaction of 2b. Possible mechanisms are discussed for
3
s
s
-
cleavage of O,O-dimethyl phosphorothioate in CD OD, producing (CD O) PdO(S ) with a half time for reaction
3
3
2
of 34 min.
2
2d,3
1
. Introduction
Phosphorothioate esters (1) are analogues of phosphate
structure and sites of metal ion coordination.
Phosphoro-
thioate modification of synthetic oligonucleotides confers
increased stability toward nuclease activity leading to a
number of useful applications in molecular biology. Anti-
sense oligonucleotide therapies incorporating phosphoro-
thioate internucleotide linkages have shown antiviral
activity toward HIV and Hepatitis B. The Dnd gene cluster
present in S. lividans and other bacterial genomes promotes
the sequence specific and stereoselective substitution of a
4
esters inwhichoneormore of thenon-bridging oxygen atoms
have been substituted with sulfur. Neutral phosphorothioate
triesters (1a) such as fenitrothion (O,O-dimethyl O-(3-methyl-
5
4-nitrophenyl) phosphorothioate and parathion (O,O-diethyl
6
O-4-nitrophenyl phosphorothioate) are important in agricul-
ture where they have seen widespread use as pesticides.
Phosphorothioate diesters (1b) and monoesters (1c) have
been employed as mechanistic probes for enzyme catalyzed
phosphoryl transfer reactions to deduce transition state
1
(
3) For selected examples see: (a) Forconi, M.; Lee, J.; Lee, J. K.; Piccirilli,
J. A.; Herschlag, D. Biochemistry 2008, 47, 6883. (b) Wrzesinski, J.; Wichlacz,
A.; Nijakowska, D.; Rebowska, B.; Nawrotb, B.; Ciesiolka, J. New J. Chem.
2010, 34, 1018. (c) Cernak, P.; Madix, R. A.; Kuo, L. Y.; Lehman, N. J. Inorg.
Biochem. 2008, 7, 1495.
(4) (a) Nakamaye, K.; Eckstein, F. Nucleic Acids Res. 1986, 14, 9679.
(b) Walker, G. T.; Nadeau, J. G.; Spears, P. A.; Schram, J. L.; Nycz, C. M.; Shank,
D. D. Nucleic Acids Res. 1994, 22, 2670.
(5) (a) Verma, S.; Eckstein, F. Annu. Rev. Biochem. 1998, 67, 99.
(b) Eckstein, F.; Gish, G. Trends Biochem. Sci. 1989, 14, 97.
(6) (a) Matsukura, M.; Koikeb, K.; Zen, G. Toxicol. Lett. 1995, 82/83,
435. (b) Wallace, T. L.; Bazemore, S. A.; Kornbrust, D. J.; Cossum, P. A. J. Exp.
Ther. 1996, 3, 1313. (c) Ushijima, K.; Shirakawa, M.; Kagoshima, K.; Park,
W.-S.; Miyano-Kurosaki, N.; Takaku, N. Biorg. Med. Chem. 2001, 9, 2165.
*
To whom correspondence should be addressed. E-mail: rsbrown@
chem.queensu.ca. Phone: 613-533-2400. Fax: 613-533-6669.
1) Quin, L. D. A Guide to Organophosphorus Chemistry; Wiley-Inter-
science: New York, 2000; p 369.
2) (a) Loverix, S.; Winqvist, A.; Stromberg, R.; Steyaert, J. Chem. Biol.
000, 7, 651. (b) Breslow, R.; Katz, I. J. Am. Chem. Soc. 1968, 90, 7376.
c) Holtz, K. M.; Catrina, I. E.; Hengge, A. C.; Kantrowitz, E. R. Biochemistry
000, 39, 9451. (d) Zhao, L.; Liu, Y.; Bruzik, K. S.; Tsai, M.-D. J. Am. Chem.
(
(
2
(
2
Soc. 2003, 125, 22. (e) Szewczak, A. A.; Kosek, A. B.; Piccirilli, J. A.; Strobel,
S. A. Biochemistry 2002, 41, 2516. (f) Lassila, J. K.; Herschlag, D. Biochemistry
008, 47, 12853.
2
pubs.acs.org/IC
Published on Web 02/01/2011
r 2011 American Chemical Society

Article
Inorganic Chemistry, Vol. 50, No. 5, 2011 1787
non-bridging phosphoryl oxygen with sulfur to form a
naturally occurring phosphorothioate DNA linkage.
Improved metal ion catalysis was achieved with Zn(II), Cd(II),
7
0
0
and Gd(III) for the transesterification of uridyl(3 ,5 )uridine
containing a phosphorothioate diester linkage where rate
accelerations for cyclization of up to 3600 relative to the
15
background reaction at pH 5.6 and 363 K were reported.
Several metallacycle Pd and Pt complexes have been devel-
oped for promoting the decomposition of phosphorothioate
1
6
triesters in water. Reports emanating from our lab have
described efficient palladacycle (3), Zn(II)-complex, and
Cu(II)-complex systems for the catalytic cleavage of neutral
phosphorothioate pesticides in alcohols such as methanol
9
where rate accelerations of >10 over the background reaction
17
were achieved. The methanolytic cleavage of a series of O,
O-dimethyl O-aryl phosphorothioates (1a, R = R = CH )
1
2
3
promoted by palladacycle 3 has been extensively investigated
and shown to proceed via a multistep pathway with rate
limiting ligand exchange on the Pd by substrates with good
leaving groups, switching to rate-limiting breakdown of a Pd-
-
bound 5-coordinate phosphorane (Pd: S(CH O) P(OAr))
3
3
Considerable effort has been devoted to studying solvoly-
tic reactions of phosphate esters, and a number of compre-
hensive reviews exist. By contrast, much less attention has
17d
with substrates having poor leaving groups.
8
Some efforts have been made to quantify the effects on
reactivity of oxygen for sulfur exchange for the cleavage of
phosphorothioates relative to phosphates. The thio effect is
defined as the ratio of rate constants for reaction of a phos-
phate ester relative to that of the corresponding phosphoro-
been directed toward therelatedchemistry ofphosphorothio-
ates. Transfer of the dimethoxy phosphorothioyl group
between oxyanion nucleophiles is best described as an A D
N
N
mechanism consistent with the linear free energy data for
O
S
9
thioate ester (k /k ). Hengge has determined the activation
parameters for hydrolysis of a contiguous series phosphoro-
thioate and phosphate mono-, di-, and triesters, all having a
attack of substituted phenoxides on fenitrothion in water.
Heavy atom kinetic isotope effects indicate a tighter, more
associative transition state for the reaction of parathion (1a,
12,18
1
2
common 4-nitrophenoxy leaving group.
Thio effects at
R = R =Et; X=4-NO ) relative to that of a corresponding
2
10
298 K of 12.6 can be calculated for hydrolysis of O,O-diethyl
O-4-nitrophenyl phosphate and O,O-diethyl O-4-nitrophe-
nyl phosphorothioate, 3.3 for O-ethyl O-4-nitrophenyl phos-
phate and O-ethyl O-4-nitrophenyl phosphorothioate and
0.13 for the dianions of O-4-nitrophenyl phosphate and O-4-
nitrophenyl phosphorothioate. By this analysis, the calcu-
lated thio effects for simple hydrolysis are small, being less
than an order of magnitude for all three classes of ester, but
thio effects determined for cleavage reactions of phosphate
and phosphorothioate esters catalyzed by enzymes and
ribozymes employing metalion cofactors are often verymuch
larger. For example the nucleotide phosphodiesterase from
Xanthomonas axonopodis (NPP) exhibits a thio effect of 48
for hydrolysis of O-methyl O-4-nitrophenyl phosphate/
phosphate triester. Dianionic phosphorothioate mono-
esters with aryloxy leaving groups are thought to react by a
highly dissociative DN þ AN mechanism. This proposal is
LG
consistent with the large negative β of -1.1 determined for
11
q
the hydrolysis of aryl phosphorothioate dianions, a ΔS of
þ29 cal/mol K for reaction of the 4-nitrophenyl phosphoro-
3
12
thioate dianion and the racemization observed in the hydro-
16
18
13
lysis of chiral 4-nitrophenyl [ O, O]phosphorothioate.
The transition state for hydrolysis of the diester O-ethyl
1
2
O-4-nitrophenyl phosphorothioate (1b, R = Et; R = H;
X = 4-NO ) is likely to be more associative than that of a
2
monoester but more dissociative than that of a triester.
0
0
Phosphorothioate analogues of uridyl(3 ,5 )uridine contain-
ing a non-bridging phosphoryl-sulfur undergo a complex
series of pH dependent reactions involving cyclization to
2f
O-methyl O-4-nitrophenyl phosphorothioate. The Ca(II)-
dependent cleavage of S -1,2-dipalmitoyl-sn-glycero-3-(1-
thiophospho-1-D-myo-inositol)phosphatidylinositol catalyzed
by a phosphatidylinositol phospholipase C from Streptomyces
0
0
,3 -cyclic phosphorothioates in competition with desulfur-
p
2
14
ization and isomerization.
We are aware of only one report describing attempts to
promote the solvolysis of a phosphorothioate monoester
8
2d
antibioticus displays a uniquely large thio effect of ∼10 .
2
þ
2þ
This particular thio effect is diminished by a factor of 400 by
substituting the Ca(II) cofactor with the soft thiophilic metal
with metal ions, where Mg and Cd were shown to inhibit
12
the rate of hydrolysis of O-4-nitrophenyl phosphorothioate.
(
15) Ora, M.; Peltomaki, M.; Oivanen, M.; L o€ nnberg, H. J. Org. Chem.
998, 63, 2939.
16) (a) Ryabov, A. D. In Palladacycles; Dupont, J., Pfeffer, M., Eds.;
Wiley-VCH: Weinheim, Germany, 2008; pp 307-339 and references therein;
b) Kazankov, G. M.; Sergeeva, V. S.; Efremenko, E. N.; Alexandrova, L.;
Varfolomeev, S. D.; Ryabov, A. D. Angew. Chem., Int. Ed. 2000, 39, 3117.
c) Kim, M.; Liu, Q.; Gabbaï, F. P. Organometallics 2004, 23, 5560. (d) Kim, M.;
(
7) (a) Wang, L.; Chen, S.; Xu, T.; Taghizzdeh, K.; Wishnok, J. S.; Zhou,
1
X.; You, D.; Deng, Z.; Dedon, P. C. Nat. Chem. Biol. 2007, 3, 709. (b) Yao, F.;
Xu, T.; Zhou, X.; Deng, Z.; You, D. FEBS Lett. 2009, 729.
(
(
8) (a) Thatcher, G. R. J.; Kluger, R. Adv. Phys. Org. Chem. 1989, 25, 99.
b) Hengge, A. C. Adv. Phys. Org. Chem. 2005, 40, 50. (c) Brown, R. S.;
Neverov, A. A. Adv. Phys. Org. Chem. 2007, 42, 271.
9) Omakor, J. E.; Onyido, I.; vanLoon, G. W.; Buncel, E. J. Chem. Soc.,
Perkin Trans. 2 2001, 324.
(
(
(
(
Gabbai, F. P. Dalton Trans. 2004, 3403. (e) Kim, M.; Picot, A.; Gabbai, F. P.
Inorg. Chem. 2006, 45, 5600.
(
(
(
(
10) Catrina, I. E.; Hengge, A. C. J. Am. Chem. Soc. 2003, 125, 7546.
11) Hollfelder, F.; Herschlag, D. Biochemistry 1995, 34, 12255.
12) Catrina, I.; Hengge, A. C. J. Am. Chem. Soc. 1999, 121, 2156.
13) Burgess, J.; Blundell, N.; Cullis, P. M.; Hubbard, C. D.; Misra, R.
(17) (a) Mohamed, M. F.; Neverov, A. A.; Brown, R. S. Inorg. Chem.
2009, 48, 1183. (b) Lu, Z.-L.; Neverov, A. A.; Brown, R. S. Org. Biomol. Chem.
2005, 3, 3379. (c) Neverov, A. A.; Brown, R. S. Org. Biomol. Chem. 2004, 2,
2245. (d) Liu, C. T.; Maxwell, C. I.; Edwards, D. R.; Neverov, A. A.; Mosey, N. J.;
Brown, R. S. J. Am. Chem. Soc. 2010, 132, 16599.
J. Am. Chem. Soc. 1988, 110, 7900.
14) Oivanen, M.; Ora, M.; Almer, H.; Stromberg, R.; L o€ nnberg, H.
J. Org. Chem. 1995, 60, 5620.
(
(18) Purcell, J.; Hengge, A. C. J. Org. Chem. 2005, 70, 8437.

1
788 Inorganic Chemistry, Vol. 50, No. 5, 2011
Edwards et al.
cat
ion Cd(II). The disparity in the magnitudes of observed thio
effectsdeterminedfor solution reactionsrelativeto enzymatic
transformations suggests that intimate interactions between
the metal ions and the non-bridging oxygen and sulfur atoms
play a pivotal role in catalysis. Despite this interesting apparent
Table 1. Second Order Rate Constants, k
Promoted by NBu OMe, Cd(ClO , Mn(OTf)
2
, for the Methanolysis of 2b
4
4
)
2
2
, and 3 at 25 °C and the Computed
Rate Accelerations Provided by the Metal Ions
s
a
cat -1 -1 b
c
metal system
NBu OMe
s
pH
k
2
(M
s
)
catalytic rate acceleration
-
7
19
4
(2.3 ( 0.2) ꢀ 10
consequence of Hard/Soft interactions in the enzymatic
reactions, it is surprising that so few studies of metal ion
promoted solvolytic reactions of phosphorothioate mono-
and diesters have been undertaken in an effort to establish a
mechanistic framework for the interpretation of thio effect
data in enzyme catalyzed phosphoryl- and phosphorothioyl
transfer. It is this subject that forms thebasis for the following
report.
6
Cd(ClO
4
)
Mn(OTf)2
2
10.0
9.6
11.2
0.56 ( 0.02
2 ꢀ 10
6
0.35 ( 0.1
1.5 ꢀ 10
4
11
3
(5.5 ( 1.7) ꢀ 10
2 ꢀ 10
a s
s
4
pH set by the addition of one-half equiv of NBu OMe relative to
b
M(II)]total and measured following reaction. The second order rate
[
constants, k , were determined from plots of kobs versus [M(II)]total as
2
c
described in the Supporting Information. Catalytic rate acceleration
determined as the ratio of second order rate constants for the metal
metal
OMe: (k
2
OMe
promoted reaction of 2b to that promoted by NBu
4
/k
2
).
19
Herein we report that the soft metal ions Cd(II), Mn(II),
and Pd(II) in palladacycle 3 are all effective promoters of the
methanolytic cleavage of O-methyl O-4-nitrophenyl phos-
phorothioates (2a-d). The reactions studied are models for
the transesterification of the phosphorothioate diester sub-
strates often employed in mechanistic studies of enzyme
catalyzed phosphororyl transfer including the NPP catalyzed
s
s
s
s
meaning thatneutral pHinmethanol is8.38. The pK values
a
of the substituted phenols in methanol used in this study can
22
befoundinapreviousreport. Kineticdatawereobtainedby
monitoring reaction progress using standard UV-vis spec-
trophotometry. In all cases, the reactions displayed excellent
first-orderkinetic traces with complete release of the expected
amount of product, and no evidence of significant product
2f,20
phosphorylation of an active site threonine (Thr90)
and
23
inhibition. Detailed descriptions of the kinetic methods
appear in the Supporting Information.
the Tetrahymena group I ribozyme catalyzed nucleotidyl
transfer to an external guanosine (CCCUCUA þ G
f
OH
3a
CCCUCU þ GA). A detailed mechanistic study is pre-
3. Results
sented for the cleavage reactions of 2 catalyzed by [Pd(II)(C,
N-dimethylbenzylamine)] (3). It is also shown that dimethyl
a. Methanolysis of 2b Promoted by Methoxide and
Metal Ion Systems, Cd(II), Mn(II), and 3. Phosphorothioate
3
-
phosphorothioate ((CH O) PdO(S )), which possesses a
3
2
s
s
LG
a
-5
more biologically relevant alkoxy leaving group ( pK
=
2b (9 ꢀ 10 M) was subjected to base-promoted metha-
1
8.2) is also a competent substrate for catalytic cleavage pro-
nolysis at 25 °C under pseudo-first order conditions of
moted by 3, giving a 34 min t1/2 for replacement of the CH O
excess NBu OMe (10-80 mM). Observed rate constants
4
3
group with a CD O group.
were determined by the method of initial rates using UV-
vis spectrophotometry monitoring the production of
4-nitrophenolate at 399 nm, and the second order rate
3
2
. Experimental Section
cat
constant, k2 , was computed as the slope of the linear
plot of kobs versus [NBu OMe], see Table 1. The effect of
A description of the materials used in this study is found in
the Supporting Information. The CH OH2 concentrations
þ
4
3
were determined potentiometrically using a combination
glass electrode (Radiometer model XC100-111-120-161)
calibrated with certified standard aqueous buffers (pH =
metal ions on the rate of 2b cleavage was determined in
methanol containing Cd(ClO ) , Mn(OTf) , and 3 at
4
2
2
s
25 °C. The solution pH values for this series of reactions
s
21
s
s
s
4
methanol were obtained by subtracting a correction constant
.00 and 10.00) as described previously. The
s
pH values in
were buffered at the pK of the first ionization of the
M(II)(HOCH ) by the addition of one-half equiv of NBu -
a
21
3
4
of -2.24 from the electrode readings, and the autoproto-
lysis constant for methanol was taken to be 10
-
16.77
2
M ,
OMe relative to [M(II)]total. Observed rate constants were
determined by fits of the absorbance versus time traces to
a standard first order exponential equation. Second order
cat
(
19) Smith, M. B.; March, J. Advanced Organic Chemistry, 5th ed.; Wiley
Interscience: New York, 2001; pp 338-342, and references therein.
20) Zalatan, J. G.; Fenn, T. D.; Brunger, A. T.; Herschlag, D. Biochem-
istry 2006, 45, 9788.
21) Gibson, G.; Neverov, A. A.; Brown, R. S. Can. J. Chem. 2003, 81,
95.
22) Neverov, A. A.; Liu, C. T.; Bunn, S. E.; Edwards, D. R.; White, C. J.;
Melnychuk, S. A.; Brown, R. S. J. Am. Chem. Soc. 2008, 130, 6639.
23) The observation that good first order kinetics are observed for the
rate constants (k2 ) were determined from the plots of
kobs versus [M(II)]total as described in the Supporting
Information. The computed second order constants and
catalytic rate accelerations appear in Table 1.
(
(
3
1
4
b. P NMR of 3 and Sodium O,O-Dimethyl Phosphoro-
thioate (2e) in Methanol. The P NMR spectrum (CH3-
(
31
(
OH, 202 MHz, referenced to 70% phosphoric acid)
obtained on an unlocked solution of neutral methanol
containing 3 (3.0 mM) and 2e (1.5 mM) at 298 K displays
signals at δ 54.0 and 28.2 ppm as shown in Figure 1.
Obtaining accurate integrals is difficult because of a
combination of a low signal-to-noise ratio and partial
broadening of the peaks; nonetheless an approximate
ratio of 1:1 is indicated.
full Abs versus time profile and that there is full release of product indicates
that there is no appreciable inhibition by product. This stems from the fact
that the product is a monoanionic phosphorothioate diester as is the starting
material, and both should have very similar binding constants to the catalyst.
The observation of saturation kinetics indicates that the starting material
and catalyst are in equilibrium, and so the catalyst must also be in
equilibrium binding with the product. If the establishment of equilibrium
is fast, then the total available pool of catalyst does not change during the
course of the reaction, so product inhibition will not be observed. On the
other hand, if the product binds far tighter than the starting material, which
would be the case if this were a hydrolysis process and the product was a
phosphorothioate mono ester, product inhibition in the form of burst
kinetics, followed by a subsequent step dependent on catalyst turnover
would be observed if the [catalyst] < [substrate]. If [catalyst] > [substrate],
then product inhibition would not be seen since the observed process is a
unimolecular decomposition of a fully formed substrate:catalyst complex.
c. UV-vis Titration of 3 with 2e in Methanol. Aliquots
-
5
of 2e (1.8 ꢀ 10 M) were added to a non-buffered
solution of methanol containing 3 and the UV-visible
spectrum recorded from 400 to 250 nm at 25 °C. The
change in absorbance at λ = 325 nm was recorded as a
function of [2e] as shown in Figure 2. The data were fit to

Article
Inorganic Chemistry, Vol. 50, No. 5, 2011 1789
-
5
Figure 3. Plot of kobs versus [3] for the methanolysis of 2b (2 ꢀ 10 M)
s
3 s
determined at [La(OTf) ] = [NaOMe] = 2.0 mM, pH 8.80 and 25 °C.
Fitting of the data to the expression giving in eq 2 gives kcat = (4.39 (
3
Figure 1. P NMR spectrum (CH
phosphoric acid) of a neutral methanol solution containing 3 (3.0 mM)
and 2e(1.5 mM) obtainedat298 K displayingtwo peaks atδ 54.0 and 28.2
ppm. The two peaks integrate in an approximate ratio of 1:1.
1
3
OH, 202 MHz, referenced to 70%
0
.21) ꢀ 10
-2 -1
s
and K
= (5.2 ( 2.4) ꢀ 10 M
5
-1
.
b
s
3þ
the range 7.06 e pH e 11.35. (La gives a small catalysis
of the reaction; this is a hard metal ion, and the reaction is
subject to profound catalysis only by soft metal ions, (vide
s
s
infra)). For the series of reactions run at pH 5.06, Yb-
(OTf) was substituted for La(OTf) owing to the lower
s
3 3
3þ
s
s
21
pK of the Yb :(HOCH ) complex. An excess of
a
3
s
NaOMe was simply added to the reactions run at pH
14.07 and 14.77 to maintain [H ].
s
þ
kobs ¼ kcatð1 þ K ½Sꢁ þ ½CatꢁK - XÞ=ð2K Þ=½Sꢁ ð2Þ
B
B
B
where
2
2
X ¼ ð1þ2K ½Sꢁþ2½CatꢁK þK ½Sꢁ
B
B
B
2
2
2 0:5
-
2KB ½Catꢁ½Sꢁþ½Catꢁ K
Þ
Plots of kobs versus [3] display saturation binding as
B
2
shown in Figure 3, and the data can be fit to eq 2 to
4
3
25 nm
Figure 2. Plot of ΔAbs
versus [2e] determined for the spectro-
=
photometric titration of 3 at 25 °C. Fit of the data to eq 1 provides K
b
provide a binding constant, K , and catalytic rate con-
b
5
-1
(
4.2 ( 1.3) ꢀ 10 M
.
stant, kcat. Under conditions of saturating [3] there is a
3
þ
2
4
5
linear dependence of the reaction rate on added [La ],
Figure 4. The second order rate constants for this buffer
catalysis, k2 , calculated as the gradients of the plots of
eq 1 to give a binding constant, K , of (4.2 ( 1.3) ꢀ 10
b
-
1
M
.
La
3
þ
ΔAbsobs ¼ ΔAbsmaxð1 þ Kb½3ꢁ þ ½2eꢁKb - XÞ=ð2KbÞ=½3ꢁ
kobs versus [La ] are given in Table 2. Also included in
Table 2 are the 2b:3 binding constants, K , and the
b
ð1Þ
corrected catalytic rate constants for the cleavage reac-
tion of this complex, kcat , determined by extrapolation of
where
corr
2
2
3
the plots of kobs versus [La ] to zero. Buffer catalysis was
þ
X ¼ ð1þ2K ½3ꢁþ2½2eꢁK þK ½3ꢁ
b
b
b
s
s
not observed for reactions run at pH 5.06 in the presence
2
2
2 0:5
3
of Yb . Control experiments performed in the absence
þ
-
2Kb ½2eꢁ½3ꢁþ½2eꢁ Kb Þ
d. pH/Rate Profile for the 3-Catalyzed Cleavage of 2b.
3
þ
s
of 3 indicate that the cleavage of 2b is promoted by La
alone, albeit slowly. For example the cleavage of 2b in a
methanol solution containing 1.7-6.7 mM La(OTf) at
5 °C and pH 8.4 provides an average observed rate
s
-
5
Substrate 2b (2 ꢀ 10 M) was subjected to 3-catalyzed
s
s
3
methanolysis at 25 °C, 5.06 e pH e 14.95. It was
s
s
2
determined early in the study that the use of bulky amines
(
to control reaction pH led to complex effects consistent
-
5 -1
i
NEt , Pr-morpholine, 2,2,6,6-tetramethylpiperidine, etc)
constant of (1.3 ( 0.2) ꢀ 10
s
that is independent of
3
s
s
metal ion concentration over the concentration range
corr
cat
s
investigated. Shown in Figure 5 is a plot of log(k
versus pH which, when fit to eq 3, provides a low pH
)
with buffer inhibition, and under certain conditions, buffer
catalysis. Accordingly, we turned to the use of a buffering
s
s
s
minimum
-2 -1
-
plateau of k
pH plateau of k
= (1.45 ( 0.24) ꢀ 10
s
and a high
= 7.1 ( 1.7 s connected via a
system comprising La ( OMe) ; where x = 1-5, to
2
x
s
s
maximum
-1
maintain a constant hydronium ion concentration over
s
kinetic pK of 12.71 ( 0.17.
s
a
(
24) Equations 1 and 2 were obtained from the equations for equilibrium
corr
maximum
þ
minimum
binding and for conservation of mass by using the commercially available
MAPLE software, Maple V Release 5, Waterloo Maple Inc., Waterloo,
Ontario, Canada.
logðkcat Þ ¼ logðk
ðK =ð½H ꢁ þ K ÞÞ þ k
a
a
Þ
ð3Þ

1
790 Inorganic Chemistry, Vol. 50, No. 5, 2011
Edwards et al.
Table 3. Kinetic Parameters for the Methanolysis of Phosphorothioates 2a-d
Catalyzed by 3 at T = 25 °C
s
s
s
s
LG
a
corr -1 a
cat (s
-1 a
substrate
pH
(
pK
)
k
)
b
K (M )
-
2
4
4
4
5
5
4
2
2
2
2
2
2
a
b
c
c
d
d
7.65
7.65
9.35
(2.4 ( 0.2) ꢀ 10
(1.85 ( 0.02) ꢀ 10
(9.8 ( 5.0) ꢀ 10
(4.4 ( 2.0) ꢀ 10
(8.0 ( 5.4) ꢀ 10
(5.9 ( 1.0) ꢀ 10
(4.2 ( 3.5) ꢀ 10
(1.8 ( 1.0) ꢀ 10
-
2
11.18
13.6
13.6
14.3
14.3
-
2
7.65
(2.2 ( 0.2) ꢀ 10
b
-2
14.1
(4.44 ( 0.03) ꢀ 10
(1.59 ( 0.07) ꢀ 10
-
2
7.65
b
-2
14.1
(1.0 ( 0.2) ꢀ 10
a
k
cat and K
b
corr
data determined from fits of the kobs versus [3] data to
eq 1, and the kcat data in the table are corrected for buffer catalysis unless
b
s
s
otherwise noted. Reactions run at pH 14.1 were performed in the
3þ
corr
absence of La , and so the kcat values listed in the table represent kcat
.
3
þ
-5
Figure 4. Plot of kobs versus [La ] for the methanolysis of 2b (2 ꢀ 10
-
5
s
s
M) determined at [3] = 5 ꢀ 10 M,
pH 8.80 and 25 °C. Fitting of the
La
2
-1 -1
data to a linear regression provides k
s
= 7.55 ( 0.64 M
s
and
corr
-2 -1
2
, r = 0.9929.
k
cat = (2.61 ( 0.20) ꢀ 10
corr
Table 2. Kinetic Constants kcat , K
La
b
, and k
2
for the Methanolysis of 2b
-1 b
s
Catalyzed by 3 (5.60 e
s
pH e 14.95, T = 25 °C)
corr -1 b
s
La -1 -1 a
s
pH
k
2
(M
s
)
kcat (s
)
b
K (M )
-
-
-
2
2
2
4
4
4
5
5
3
4
3
5
7
7
8
1
1
1
1
.60
.06
.65
.80
0.38
1.35
4.07
4.95
c
c
(1.23 ( 0.07) ꢀ 10
(1.02 ( 0.05) ꢀ 10
(1.41 ( 0.05) ꢀ 10
(4.7 ( 2.2) ꢀ 10
(8.0 ( 2.3) ꢀ 10
(9.8 ( 0.3) ꢀ 10
(5.2 ( 2.4) ꢀ 10
(1.8 ( 1.0) ꢀ 10
(9.2 ( 1.6) ꢀ 10
(6.6 ( 1.3) ꢀ 10
(5.6 ( 2.5) ꢀ 10
2.87 ( 0.12
7.6 ( 0.6
1.0 ( 0.2
14.8 ( 1.7
c
-2
(2.6 ( 0.2) ꢀ 10
-
2
(4.56 ( 0.09) ꢀ 10
-1
(3.3 ( 0.3) ꢀ 10
5.4 ( 0.2
c
8.9 ( 1.8
a
La
2
3þ
k determined from the slope of the plots of kobs versus [La ].
cat and K determined by fits of the kobs versus [3] data to eq 2, and the
b
k
b
corr
reported kc ac t is calculated by extrapolation to [buffer] = 0 as described
in the text. Where La -catalysis was not observed, the reported kcat
corr
Figure 6. (a) Brønsted plot of log(kcat ) versus
s
LG
pK
a
3þ
corr
s
(solid line, 2)
s
determined for the 3-catalyzed methanolysis of 2a-d at 25 °C,
s
pH
cat
corresponds to the computed k determined directly from eq 2.
corr
s
LG
a
7
0
.65. Linear regression gives log(kcat ) = (-0.01 ( 0.03)
s
pK
- (1.55 (
2
.40) (r = 0.094, 4 data). (b) Brønsted plot of log(kcat) versus
s LG
s
pK
a
(
2
0
dashed line, O) determined for the 3-catalyzed methanolysis of 2b,c,d at
s
5 °C,
s
pH 14.1 where a linear regression of gives log(kcat) = (-0.86 (
a
s
LG
2
.01)
s
pK
þ (10.39 ( 0.05), 3 data (r = 1.000, 3 data). (c) Catalytic rate
constant (b) for the 3-catalyzed cleavage of O,O-dimethyl phosphor-
othioate (2e) as described in the text, (Results section i below).
corr
values listed in Table 3
cat
constants kcat and K . The k
b
reflect correction of the kcat terms by extrapolation to
zero buffer concentration. Substrates 2b,c,d were also
subjected to 3-catalyzed methanolysis at the higher pH
s
s
of 14.1, 25 °C. The catalytic constants for 2c and 2d
appear in Table 3 whereas the data for 2b appear in
Table 2. In Figure 6 is a Brønsted plot of log(k ) versus
corr
cat
s
s
LG
s
s
pKa for reaction of substrates 2a-d at pH 7.65 which,
when fit to a standard linear regression, provides log-
( ) = (-0.01 ( 0.03) pK - (1.5 ( 0.4). Also included
in Figure 6 is the limited Brønsted plot determined for the
corr
cat
s
s
LG
a
k
corr
Figure 5. Plot of log(kcat ) versus
s
s
pH for the methanolysis of 2b (2 ꢀ
-
5
1
0
M) determined at 25 °C catalyzed by 3 (0) where a fit of the data to
s
s
maximum
s
-1 minimum
-2
3-catalyzed reaction of 2b,c,d at pH 14.1 which fits a
þ
eq 3 provides k
= 7.1 ( 1.7 s , k
= (1.45 ( 0.24) ꢀ 10
s
LG
a
-
1
corr
s
CD
, and a kinetic
pK
s a
of 12.7 ( 0.2. The kcat data (þ) obtained in
linear regression of log(kcat) = (-0.86 ( 0.01) pK
s
3þ
s
3
OD at [La ]/[NaOMe] = 2 and 1 appear in the Figure at
s
pH 7.65
(10.39 ( 0.05).
f. Activation Parameters for the 3-Catalyzed Cleavage
and 8.80, respectively.
of 2b. The catalyzed methanolysis of substrate 2b (5 ꢀ
-
5
s
e. Linear Free Energy Correlation for the 3-Catalyzed
Cleavage of Phosphorothioates 2a-d. Plots of k versus
10 M) was carried out at pH 8.8 in the presence of 3 =
s
-
4
3þ
1.2 ꢀ 10 M and [La ] = [NaOMe] = 1.68, 3.36, and
5.04 mM over a temperature range spanning 10-43 °C.
Under these reaction conditions it is assumed that 2b is
fully bound to 3 and therefore the observed rate constants
obs
[
3] were determined for the catalyzed methanolysis of
-
5
s
s
2
2
a-d (5 ꢀ 10 M) at 25 °C buffered at pH 7.65 with
mM La(OTf) and 1 mM NaOMe. The plots of k
3
obs
La
corr
versus [3] for these substrates showed strong saturation
binding and were fit to eq 2 to determine the catalytic
are interpreted as k . The k and kcat data were obtained
cat 2
as the gradients and y-intercepts, respectively, in the plots

Article
Inorganic Chemistry, Vol. 50, No. 5, 2011 1791
3
of kobs (or kcat) versus [La ]. Fitting the data to the
þ
q
Eyring equation provides ΔH = 14.0 ( 0.6 kcal/mol
Pd
q
Pd
and ΔS =-20 (2cal/mol K for the3-catalyzed cleavage
of 2b. The activation parameters for the corresponding
La(III)-catalyzed process are ΔH = 6.4 ( 0.4 kcal/mol,
ΔS = -33 ( 2 cal/mol K.
La
g. Solvent Deuterium Kinetic Isotope Effect. Substrate
3
q
La
q
3
-
5
2
b (4.3 ꢀ 10 M) was subjected to catalyzed methano-
- 3þ
4
lysis in the presence of 3 (0.2 to 1.2 ꢀ 10 M), 2 mM La
and 1 mM NaOMe in CD OD (99% deuterium content)
3
at 25 °C. A fit of the data to eq 2 provides K = (3.0 (
b
5
-1
-2 -1
2
correcting for buffer catalysis k
.2) ꢀ 10 M , k = (1.87 ( 0.11) ꢀ 10
s
is determined to be
and after
cat
corr
cat
1
-
2
-1
3þ
3
Figure 7. Partial H NMR (CD OD, 600 MHz) spectra obtained
5
(
[
1.19 ( 0.06) ꢀ 10 s . Under conditions where [La ] =
-
-4
corr
cat
following reaction of 2e (6.1 ꢀ 10 M) catalyzed by 3 (1.06 ꢀ 10 M)
NaOMe] = 2.0 mM, k
was determined as (2.4 (
. The kcat data appear in Figure 5 overlaid
3þ
in the presence of 2 mM La and 1 mM NaOCD
3
employing the
-
2
-1
corr
0
upon the data obtained in CH OH at pH 7.60 and
.3) ꢀ 10
s
described quench protocol at (a) 43.5 min; (b) 26.3 min; (c) 24.3 min;
(d) 9.0 min; and (e) 0 min.
s
s
3
8
.80 under otherwise identical conditions. The solvent
deuterium kinetic isotope effect is calculated to be k /k =
H
D
spectra illustrating the loss of the CH O-P signal intensity
3
1
.17 ( 0.08.
as the group undergoes conversion to CD O-P. An aver-
3
h. 4-Promoted Methanolysis of 2b. Substrate 2b (2 ꢀ
-4 -1
age observed rate constant of (3.4 ( 0.5) ꢀ 10
s
was
-
5
1
0
M) was subjected to methanolysis promoted by the
calculated from a standard first order exponential equation.
Under the conditions of the study it is determined that 2e
is >95% metal bound based on the binding constant
-
5
s
s
pincer palladacycle 4 (3 ꢀ 10 M) at 25 °C, pH 7.9
3
[La ] = 2.9 mM, [NaOMe] = 2.0 mM). The fit of the
þ
(
absorbance versus time data to a standard first order
exponential equation provided an observed rate constant
determined by spectrophotometric titration (K = 4.2 ꢀ
b
5
0 M ), and since the NMR experiment looks at the loss
-1
1
of both CH O groups, it therefore follows that for the loss
-
5 -1
of 1 ꢀ 10
s
that obtained in the absence of 4. The reaction of 2b was
which is not significantly different from
3
2e 26
of a single methoxy group kcat = 2kobs
.
The datum for
-
5
s
s
also carried out in the presence of 4 (3 ꢀ 10 M) at pH
reaction of 2e that appears in Figure 6 is seen to lie
significantly below the data corresponding to reaction
of 2a-d.
1
4.8 maintained with 10 mM NaOMe at 25 °C. These
conditions gave an observed rate constant of 3.7 ꢀ
-
6
-1
1
0
s
. By way of comparison the second order rate
constant for methoxide promoted cleavage of 2b is 2.3 ꢀ
4
. Discussion
-
7
-1 -1
1
0
M
s
providing a computed observed rate con-
-
9
-1
s
a. Methanolysis of 2b Promoted by Lyoxide and Metal
stant of 2.3 ꢀ 10
s
10 -fold rate acceleration over the background meth-
at pH 14.8. This represents a
s
3
Ions (Cd(II), Mn(II), Pd(II), La(III)). The second order
rate constant for methoxide attack on 2b (Table 1) is the
same as that determined for the base promoted hydrolysis
∼
2
oxide promoted reaction at that pH; however, this is
5
s
s
6
still some 10 -fold slower than the reaction catalyzed by 3
-
7
of O-ethyl O-4-nitrophenyl phosphorothioate (2.1 ꢀ 10
under similar reaction conditions.
i. Methanolysis of O,O-dimethyl Phosphorothioate (2e)
-
1
-1 10
s ) at the same temperature. It is interesting to
M
compare the rate of CH O-promoted cleavage of phos-
3
Promoted by 3 in d -Methanol. Five separate reaction
4
-
4
phorothioate 2b to that of O-methyl O-4-nitrophenyl
phosphate, which has a reported second order rate con-
solutions were prepared containing 1.1 ꢀ 10 M 3,
2.0 mM La(OTf) , and 1.0 mM NaOCD in 5 mL of
methanol (10% CH OH/90% CD OD). To each of these
3
3
-
7
-1 -1 22
stant of 7.9 ꢀ 10
M
s
.
Thus, the thio effect,
3
3
defined as the ratio of rate constants for reaction of the all
oxygen phosphate diester to that of the phosphorothioate
was added sodium O,O-dimethyl phosphorothioate 2e
-
5
(
6 ꢀ 10 M), and the reactions were allowed to progress
for a set time period (0-43.5 min) prior to quenching with
0 mM LiCl and 112 mM LiBr (these anions bind tightly
O
S
diester (k /k ), is 3.4 at 25 °C in methanol which com-
pares favorably to the thio effect of 3.3 for the hydrolysis
of O-ethyl O-4-nitrophenyl phosphate and O-ethyl O-4-
nitrophenyl phosphorothioate in water. The thio effects
reported here are for reaction at 25 °C, but it is empha-
sized that these values are necessarily dependent upon the
reaction temperature chosen for the comparison owing to
the small but significant differences in ΔH expected for
solvolytic cleavage of the two classes of substrate.
The solvolytic cleavage of phosphorothioates is efficiently
promoted by soft metal ions in methanol as demonstrated
5
to the metal ions in methanol and inhibit catalysis). The
quenched reactions were concentrated to dryness under
reduced pressure and then reconstituted in CD OD for
1
8
3
1
H NMR analysis (CD OD, 600 MHz). Reaction pro-
3
gress was determined by integrating the residual CH O-P
3
q
signal (δ 3.66 ppm, d, J = 12 Hz) relative to that of the
methylene protons of 3 (δ 3.98 ppm, singlet) which is
assumed to remain constant over the course of reaction.
2
7
1
Shown in Figure 7 is a stacked plot of the partial H NMR
-
6 -1
(
25) It must be noted that the value of 3.7 ꢀ 10
s
determined for the
(26) Since the observed process corresponds to the loss of both the CH O
groups, the pseudo-first order rate constant for the loss of a single group
would be twice as large, and this is the number we report.
(27) Examination of the activation parameters reported in ref 18 for
the base promoted hydrolysis of ethyl 4-nitrophenylphosphate and ethyl
4-nitrophenylphosphorothioate reveal an isokinetic temperature of ∼100 °C.
3
pincer complex is an upper limit, and may be at least partially due to trace
impurities (<1% of 3 or another catalytically active materials which are not
seen in the analysis of the pincer ligand, but contribute to catalysis. Never-
theless, the point of this comparison is to indicate that 4 does not have the
required two adjacent sites for catalysis.

1
792 Inorganic Chemistry, Vol. 50, No. 5, 2011
Edwards et al.
by the cleavage of 2b in the presence of Cd(II), Mn(II),
and palladacycle 3. The second order rate constants for
the cleavage of 2b at 25 °C in methanol containing Mn(II)
and Cd(II) and one-half equivalent of methoxide are
-
1
-1
similar at 0.35 and 0.56 M
at least 10 more efficient for the cleavage of 2b, having a
s , but palladacycle 3 is
5
4
-1 -1
second order rate constant of 5.5 ꢀ 10 M
s . A crude
estimate of the effectiveness of the metal ions at promot-
ing the methanolysis of 2b is arrived at by comparing the
rate of the methoxide promoted reaction to those ob6-
tained in the presence of metal ions: efficiencies of ∼10
are determined for Mn(II) and Cd(II) whereas 3 provides
Figure 8. Proposed structures for the complexes formed between 1 and
3
3
e in neutral methanol as determined by P NMR.
1
3
b. P NMR of a Methanol Solution Containing 3 and
1
1
1
31
a rate acceleration of ∼10 (Table 1). L o€ nnberg et al.
2e. The P NMR chemical shift in CD OD of sodium O,
3
have reported that the transesterification of uridylyl-
(
O-dimethyl phosphorothioate (2e) is 61.3 ppm whereas
the spectrum of a neutral methanol solution containing a
2:1 mixture of 3 and 2e displays peaks at 28 and 54 ppm
in the mM range (Figure 1). P NMR data are reported
for several palladacycle species bearing attached O,O-
dimethyl phosphorothioate ligands and serve as a guide in
the assignment of the peaks displayed in Figure 1. A mixture
of the dimeric complexes cis-[(μ-2e)(2-(2-pyridyl)phenyl-
0
0
0
0
(3 ,5 )uridine phosphoromonothioate [3 ,5 -Up(s)U] in
water containing 5 mM metal ion is accelerated over the
background reaction by 410 using Zn(II), 3600 with
Cd(II), and 2000 with Gd(III) at pH 5.6 and 363 K.
3
1
1
5
The exceptional catalytic efficiency for the metal pro-
moted cleavage of 2b in methanol relative to the metal
0
0
promoted cleavage of 3 ,5 -Up(s)U in water is intriguing
and suggests a pronounced medium effect provided by the
alcohol in stabilizing not only the bound form of the
M(II):2b complex, but also its ensuing transition state for
cleavage of the P-OAr bond.
C,N)Pd]
and trans-[(μ-2e)(2-(2-pyridyl)phenyl-C,N)Pd]
have been characterized by X-ray crystallography and solid-
2
2
3
1
1
16e
31
state MAS P{ H} NMR. The reported P NMR
spectrum consists of two peaks at δ 29.3 and 35.9 ppm
assigned to the cis-isomer and a single peak at 32.9 ppm
In the absence of any of the soft metal ion catalysts, a
3
þ
s
31
methanol solution containing 1.7-6.7 mM La at pH
assigned to the trans-isomer. A similar P NMR chemical
H C-
6 4
s
-
5 -1
8
.5 gives a k value of 1.3 ꢀ 10
s
for the methano-
lysis of O-methyl O-4-nitrophenyl phosphorothioate 2b,
shift at δ 29 ppm is reported for [Pd(C,N-(C
obs
(CH )dNOH)-2)(μ-2e)] in a mixed acetone-D O solvent
3
2
2
1
6d
this rate constant being experimentally invariant in re-
spect to [La ]. This behavior suggests a process where 2b
system. Assigning the peak at δ 28 ppm in Figure 1 to
symmetrical dimeric complex 5 is therefore consistent
with literature precedent although it is possible that the
actual structure consists of the phosphorothioate ligands
binding the two Pd centers with the non-bridging phos-
phoryl-oxygen and -sulfur to form an eight membered ring
chelate. Monomeric complexes of 2e bound to palladacycle
3
þ
3
undergoes saturation binding to a La species followed
þ
-
by unimolecular breakdown of a La(III) ( OCH ) (2b)
3 x
2
complex to give product. The closely related O-methyl
O-4-nitrophenyl phosphate undergoes methanolytic cleav-
-
6 -1
age with a rate constant of 7.5 ꢀ 10
s
under similar
3
þ
s
28
31
conditions of saturating La , 25 °C and pH 8.5,
demonstrating that this hard metal ion possesses no
special aptitude to promote the cleavage of the sulfur
containing 2b relative to its all oxygen analogue.
complexes have also been characterized by P NMR in
s
acetone-D
(2e)] having a peak in THF at 50.5 ppm and [Pd(C,
N-(C C-(CH )dNOH)-2)(2e)(py)] displaying a peak
at 53.6 ppm.
O including [Pd(2-(2-pyridyl)phenyl-C,N)(THF)-
2
1
6e
H
6
4
3
1
6d
Under conditions of saturating [3], where 2b is fully
These chemical shifts are similar to the
3
þ
bound to the palladacycle, the catalytic behavior of La
is somewhat different in that it provides an acceleration of
downfield peak seen in Figure 1 obtained in a methanol
solution of 3 and 2e, and accordingly we propose struc-
tures 6a or 6b as being consistent with the available NMR
data. Phosphorothioate 2e is suggested to exist as an
equilibrium mixture of dimeric complex 5 and monomeric
complexes 6 (See Figure 8) under the millimolar concen-
trations employed in the NMR experiment. However, the
concentration of any dimeric complex would be small
under the kinetic conditions employed in this study which
involve >10-fold dilution, and we see no kinetic evidence
for the intervention of dimers in the cleavage of these
phosphorothioates.
3
þ
]
the cleavage of Pd-bound 2b that is linear in [La
Figure 4). The second order rate constants for the
La -promoted process, determined as the gradients of
(
3
þ
3
þ
the plots of k versus [La ], vary by only a factor of ∼5
obs
-
1
-1
s
(
3 to 15 M
Under these reaction conditions the speciation of the
s ) over the range 7.65 e pH e 11.35.
s
3
La is dimeric with a pH dependent number of asso-
ciated methoxides ([La ( OMe) ]; where x = 1-5) which
are responsible for the observed buffer catalysis consis-
tent with previous studies emanating from this lab.
þ
s
s
-
2
x
8
c
On the basis of these results, one can conclude the
following: (1) in the absence of Pd the catalysis provided
by La -ions for the cleavage of 2b is modest and does not
interfere with the 3-catalyzed reactions studied here; and,
c. Binding Affinity of the Palladacycle 3 for Anionic
Phosphorothioates. The plots of kobs versus [3] for the
3
þ
catalytic cleavage of 2b at 25 °C display saturation
s
s
binding over the entire
pH range investigated. The bind-
ing constants were determined by fits of the kinetic data to
(
complex formed between 2b and 3 is linear with respect
2) the catalytic effect for the decomposition of the
s
eq 2, and for reactions run in the low pH domain (5.6 <
s
3
þ
s
5
to [La ] so correcting the catalytic rate constants to zero
buffer] is straightforward.
s
pH < 10.4) these fall in the broad range of (0.5 to 5 ꢀ 10 )
-
1
M . The binding constants determined for reaction of
[
s
s
2
a-d at pH 8.7 (Table 3) all fall within this general range
and show no obvious trend with respect to the pK of the
s a
s
(
28) Neverov, A. A.; Brown, R. S. Inorg. Chem. 2001, 40, 3588.

Article
Inorganic Chemistry, Vol. 50, No. 5, 2011 1793
aryloxy substituent. The binding constants determined
from the kinetic plots complement that determined for
rearrangement occurring prior to the intervention of a
chemical step involving cleavage of the P-OAr bond. An
alternative interpretation for the low dependence of the
2
e by UV-vis titration in neutral methanol where K is
b
5
-1
s
s
LG
computed to be 4.2 ꢀ 10 M . At higher pH there is an
reaction rate on pK might involve a rapid pre-equilib-
s a
s
apparent trend toward decreased binding affinity suggest-
ing that, at higher concentrations, methoxide is capable of
displacing 2b from 3 (Table 1, 11.35 < pH < 14.95). The
rium followed by a compensatory kinetic step so that the
respective Brønsted coefficients are of equal magnitude
s
s
1
2
but opposite sign (e.g., β þ β ≈ 0). A similar effect has
3
0
binding constants determined for catalyst 3 and 2 are
large for a mononuclear metal-containing species and
suggest that the soft Pd center has a special affinity for the
sulfur-containing anionic phosphorothioates. By way of
been forwarded for the acid catalyzed hydrolysis of esters
and also considered for the [Zn(II):(1,5,9-triazacyclodo-
decane)( OCH )] and [La ( OCH ) ] promoted cleav-
-
3þ
-
3
2
3 2
3
1
ages of carboxylate esters.
2
þ
comparison, it is reported that Zn forms a 1:1 complex
0
The datum for catalytic cleavage of O,O-dimethyl
phosphorothioate 2e is included on the plot of Figure 6
for comparison from which it can be seen that it reacts
0
with uridyl(3 ,5 )uridine phosphoromonothioate in water
-
1
s
15
with an estimated K of ∼30 M at pH 5.6 and 363 K.
b
s
-
1
Stronger binding of K = 293 M isobservedfor complex
some ∼44 times slower than the aryloxy phosphorothio-
b
s
formation between the monoester O-4-nitrophenyl phos-
phorothioate and Cd at pH 8 in water. It would
ates 2a-d at the same pH. Assuming that the methoxy
s
2
þ
s
s
12
derivative fits the same Brønsted relationship as do the
aryloxy derivatives, this observation is suggestive of a
change in rate limiting step on going from the activated
substrates 2a-d containing aryloxy leaving groups to the
less activated alkoxy leaving group of 2e. A line drawn
directly from the data point for reaction of 2d to 2e
appear that 3 has an unprecedented affinity for binding to
monoanionic phosphorothioates 2 in methanol, probably
because of soft metal/soft substrate interactions and in-
creased Coulombic attraction of the oppositely charged
species in the reduced dielectric constant medium. The
binding between catalyst and the ensuing transition state
for P-OAr cleavage must be greater still, otherwise cata-
3
2
LG
provides an upper limit of -0.4 for the β describing
3
3
the putative descending portion of the Brønsted plot. It
is stressed that this is only an upper limit as it assumes the
break point of the Brønsted plot occurs at the pK of
s a
lytic rate acceleration would not be achieved.
s
s
LG
d. pH Rate Profile for the 3-Catalyzed Cleavage of 2b.
s
corr
The kcat constants determined as described in the Results
section show an interesting pH dependence as shown in
the leaving group for 2d. Nonetheless, the significant
dependence of the reaction rate for 2e on leaving group
s
s
s
s
LG
Figure 5. There are two pH independent regions having
pKa is consistent with a chemical step involving con-
s
s
maximum
-1
computed first order rate constants of k
= 7.1 s
that differ by a factor of
00. The kinetic pK of 12.7 determined from Figure 5 is
siderable change in the P-OCH bonding being rate
3
minimum
-2 -1
and k
5
= 1.45 ꢀ 10
s
limiting, in contrast to the reaction of 2a-d.
Included in Figure 6 is a Brønsted plot for the 3-
s
s
a
s
ascribed to the ionization of a Pd-bound solvent molecule
in accordance with the process [Pd(C,N-(dimethylbenzy-
catalyzed cleavage of 2b-d determined at pH 14.1 and
s
s
25 °C corresponding to reaction along the high pH
s
LG
lamine)(2b)(CH OH)] rf [Pd(C,N-(dimethylbenzylamine)-
3
plateau. Linear regression of the data provides a β of
-
þ
s
(
is significantly higher than the pK of 10.8 determined
2b)(CH O )] þ H . The pK determined for this complex
-0.86 indicating that, in contrast to reaction at lower
3
s
a
s
s
s
pH, there is now a strong dependence of reaction rate on
a
s
s
LG
previously for [Pd(C,N-(dimethylbenzylamine)(pyridine)-
(
the pK
of the leaving group. The steepness of the
gradient for the Brønsted correlation determined at high
s
a
1
7b
CH OH)] which we attribute to the effect of replacing
3
s
s
s
a neutral pyridine ligand with a tightly bound and anionic
phosphorothioate 2b, converting the formally cationic Pd
to a neutral species prior to the acid dissociation of the
pH relative to that at low pH leads to an intersection
s
s
s
LG
point at a pK of 14.1. The β for the methanolysis of
a
O-methyl O-aryl phosphorothioates is not known, and so
no comparison can be made between the extent of P-OAr
bond cleavage for the background reaction and that
catalyzed by 3. A more complete analysis of the linear
Pd-bound CH OH.
3
e. Linear Free Energy Relationships for 3-Catalyzed
corr
Cleavage of Phosphorothioates 2a-d. The kcat values
s
for the 3-catalyzed cleavage of 2a-d at pH 7.65 and
free energy data is complicated by the absence of a known
β
s
s
s
Eq
2
erence in the pK of the aryloxy leaving groups. The
5 °C are relatively invariant despite the ∼5 pH unit diff-
for the reactions under question.
f. Proposed Reaction Mechanism for the 3-Catalyzed
s
LG
a
s
corr
s
s
LG
plot of log(k ) versus pK
a
s
provides a gradient of
Methanolysis of 2. (f.1). High pH Region above the
pK of the Palladacycle-Bound HOCH . In discussing
cat
s
LG
β
LG
s
s
= -0.01. Brønsted coefficients such as the β
a
3
determined in the present study are often used to gauge
the extent of bond cleavage involving the reporter group
and charge development at the transition state (TS) of
(30) (a) Taft, R. W. Jr. J. Am. Chem. Soc., 1952, 74, 2729, 3120; (b) Taft,
R. W., Jr. J. Am. Chem. Soc. 1952, 74, 3120. (c) Taft, R. W., Jr. J. Am. Chem.
Soc. 1953, 75, 4231.
2
9
LG
reactions. The near zero value of β for the reaction of
(31) Neverov, A. A.; Sunderland, N. E.; Brown, R. S. Org. Biomol. Chem.
s
s
2
a-d catalyzed by 3 at pH 7.65 strongly indicates that
2005, 3, 65.
LG
(
32) To clarify, the upper limit of β = -0.4 states that the actual value
charge changes on the leaving group oxygen have not
occurred to any appreciable extent at the TS and may argue
instead for a rate limiting ligand exchange or intramolecular
LG
might be significantly more negative (β < -0.4) but be of a larger absolute
magnitude overall.
(33) The rate constant for catalytic cleavage of 2e has not been corrected
for buffer effects. It should also be noted that this reaction was carried out in
methanol solvent containing 90% deuterium content. The combined effects
of buffer catalysis and any isotopic perturbation on the rate constant are not
known; however, it is probable that these are minimal considering that buffer
effects for 2a-d never account for more than 40% of the reaction flux and the
determined solvent deuterium kinetic isotope effect for 2b is 1.17.
(
29) (a) Leffler, J. E.; Grunwald, E. Rates and equilibria of organic
reactions; Wiley: New York, 1963. (b) Williams, A. Acc. Chem. Res. 1984,
7, 425. (c) Williams, A. Concerted Organic and Bioorganic Reaction Mecha-
nism; CRC Press: Boca Raton, FL, 2000.
1

1
794 Inorganic Chemistry, Vol. 50, No. 5, 2011
Edwards et al.
Scheme 1. Proposed Catalytic Cycle for Reaction of Phosphorothio-
ates 2 Promoted by 3 at High pH
Scheme 2. Possible Catalytic Mechanism for Reaction of Phosphor-
othioates Promoted by 3 at low pH
s
s
s
s
the mechanism for the 3-catalyzed methanolysis of series
2
s
we first address the reaction at high pH (Scheme 1).
Scheme 3. Alternative Possible Mechanisms Proceeding through For-
mally Neutral TS for the Reaction of Phosphorothioates Promoted by 3
at Low pH
s
Binding of O-methyl O-aryl phosphorothioates 2 to 3
generates the ground state complex 6 which undergoes
ionization of coordinated methanol to generate 6 with a
s
s
-
s
kinetically determined pK of 12.7. Intramolecular de-
a
s
livery of coordinated methoxide displaces the leaving
group in what might be either a concerted displacement
or alternatively a two-step process ultimately regenerat-
ing 6 containing an O,O-dimethyl phosphorothioate
ligand. Studies of the hydrolytic cleavage of phosphate
2
9b,c
10,18
diesters,
and some phosphorothioate diesters
suggest concerted processes proceeding by an associative
TS with slightly less departure of the aryloxy leaving
group in the TS involving the latter phosphorothioates.
It is also possible that the metal ion catalyzed methano-
lyses discussed herein are concerted, bearing in mind the
possibility that a (di)anionic phosphorane-like species
formed on the reaction pathway between starting material
and product might be sufficiently strongly bound by the
9
-1 -1
rate constant for methoxide attack would be 2 ꢀ 10 M s .
3
4
catalyst that it becomes a true intermediate. As shown in
Scheme 1, the nucleophile in the reaction is proposed to be
a Pd-bound species and not an external methoxide. This is
consistent with the observation that pincer complex 4 at
This value approaches the diffusion limit in methanol
M s ) and moreover does
not allow for the unfavorable equilibrium between
PdL (2b)(HOMe)] and [PdL (2b-H )(HOMe)] because
2
the pK of the latter would be on the order of -3 (vide
infra). Furthermore the reaction mechanism shown in
Scheme 2 fails to accommodate the poor reactivity seen
with pincer complex 4 at low pH.
Three alternative process, given in Scheme 3, posit that
a Pd-bound methoxide attacks a partially or fully proto-
nated Pd-bound 2b: (a) a general acid promoted cleavage
of 6 involving a leaving group assistance by lyonium ion;
or (b) protonation of the leaving group’s bridging oxygen
1
0
-1 -1 36
(estimated to be 5.2 ꢀ 10
þ
[
2
s
s
6
s
s
pH 14.8 catalyzes the reaction of 2b some 10 -fold slower
than does 3 at the same pH. The low level of activity
a
s
s
determined for 4 points to a requirement for the cataly-
tically competent 3 having two available cis-planar co-
ordination sites to accommodate both a substrate and the
s
s
methoxide nucleophile.
(
s
f.2). Low pH Region. The rate of reaction of substrate
s
s
s
-
2
b in the low pH plateau region from 5.5 e pH e 10, as
s s
shown in Figure 5, is only ∼500 times slower than that in
s
-
þ b
the high pH plateau. A small, normal solvent deuterium
s
(6 H ) , or the non-bridging oxygen of the PdO unit
H
kinetic isotope effect of k /k = 1.17 suggestive of the
D
-
þ n
6 H ) . These apparently satisfy the criterion of a pH
s
s
(
LG
absence of a proton in flight was determined at two points
neutral reaction, are consistent with a low β observed
in Figure 6, and do not suffer from a diffusion limited
s
LG
in the low pH independent plateau. The β in this region
s
is -0.01, indicating an insensitivity to the nature of the
leaving aryloxy group, and that little charge change has
occurred at that group in the TS. A plausible pH
restriction for an attack by external methoxide. Never-
theless, these are almost certainly ruled out by the pH/
s
s
s
s
rate profile in Figure 5 and considerations of the concen-
trations of reactive species. We deal first with the general
acid process depicted in Scheme 3a, specifically with sub-
independent reaction mechanism is shown in Scheme 2
involving attack of an external methoxide on a proto-
nated Pd-bound 2b. This reaction mechanism is similar to
that proposed by Kirby and Vargolis some time ago for
the decomposition of monoanionic aryl phosphate mono-
esters and is consistent with the small dependence of
s
þ
-9
strate 2b, noting that at pH 9 the [CH OH ] is 10 M,
2
s
3
corr
the k for the decomposition of the 3:2b complex is 1.58 ꢀ
cat
-1
-
2
s
s
-
10
s
and since pK = 12.7, [6 ] = 1/5000 [6]
a
.
total
Under these conditions, the computed second order rate
constant for methoxide attack with general acid catalysis
s
35
reaction rate on leaving group pK . However, consid-
a
s
-
2 -1
1
0
-1 -1
ering the rate constant of 1.23 ꢀ 10
s
Pd bound 2b at pH 5.6 and that [CH O ] = K
for cleavage of
auto
/
M, the second order
is 7.9 ꢀ 10
along with the solvent isotope effect of k /k = 1.17 (
.08, eliminates the general acid process with its proton in
flight.
For the processes in Scheme 3b, full protonation of
M
s , exceeding the diffusion limit, and
s
s
-
H
D
3
þ
-16.77
-5.6
-12
[
H ] = 10
/10
= 7 ꢀ 10
0
(
34) In a recent study of the methanolytic cleavage of a series of neutral
17c
phosphorothioate triesters promoted by 3, we have presented computa-
tional evidence that the catalyzed reaction is multistep, proceeding through a
tightly bound phosphorane intermediate, the breakdown of which is rate-
limiting for substrates with poor leaving groups.
either the bridging or non-bridging oxygens is required,
with a subsequent intramolecular attack on the phosphorus.
(
35) Kirby, A. J.; Vargolis, A. G. J. Am. Chem. Soc. 1967, 89, 415.
(36) Chen, R.; Freeman, G. R. J. Phys. Chem. 1995, 99, 4970.

Article
Inorganic Chemistry, Vol. 50, No. 5, 2011 1795
þ
Scheme 4. Hypothetical Acid Dissociations of the Dibasic Forms of
3:2 Complexes 6(2H ) where O-Protonation Can Occur on Either the
Bridging (b) or Non-Bridging (n) Oxygens
Finally, for ionization of the non-bridging O-H from
þ
-
þ n
6 H ) to form 6 , we assume the pK is close to that of
s a
-
s
3
(
s
s
2
a
pK but raised to ∼-2 because of a slight electrostatic
-
þ n
stabilization of the (6 H ) . For consistency in the
thermodynamic cycle small changes to any of the con-
stants can be accommodated using the relationship
1
3
2
4
a
s
s
3
a
K K = K K . The pK for ionization of the bridging
a
a
a
þ
-
þ b
O-H from the Pd-bound phenol of (6 H ) to form the
corresponding 6 is calculated from the thermodynamic
-
1
cycle where K K = K K . On the basis of these esti-
3
2
4
a
a
a
a
2
mates, one can calculate from K = K /K = [(6H ) ]/
1
þ m
þ n
e
a
a
-
þ n
14.8
(6 H ) ] = 10 . On this basis, the amount of (6 H )
-
[
material required to form the products is exceedingly
small and insufficient to account for the rate of the
reaction. To account for the observed rate constant of
-
þ b
We consider that the unimolecular process with (6 H )
of Scheme 3b involves an associative TS with concerted
Pd-bound methoxide attack and departure of the proton-
-
2
-1
-
þ n
1
.5 ꢀ 10
s
(6 H ) would have to decompose with a
-
2
14.8
unimolecular rate constant of (1.5 ꢀ 10 ) ꢀ 10
or
s , a value that approaches the lifetime of a
-
þ n
1
2 -1
ated leaving group. The analogous process with (6 H ) ,
if concerted, would involve displacement of the p-nitro-
phenoxide anion with or without general assistance by
solvent or perhaps by a synchronous translocation of the
proton from the PdO unit to the departing group with
possible solvent assistance. In the limit, this process could
become a two step one having a phosphorane intermediate
with subsequent Pd-ligand exchange and phosphorus pseu-
dorotation required to assist departure of the leaving group.
9
.5 ꢀ 10
-
13
transition state (10
s) which seems unlikely for such a
complicated process involving several bond-making and
bond breaking processes. A similar calculation for the
pathway involving (6H ) demonstrates that this too is a
þ m
4
0
non-viable species.
So what accounts for the rapid reaction in the low pH
s
s
region? In Scheme 5 is a proposed reaction mechanism
consistent with the available data. From pH 5.6 to 10.5,
s
s
þ m
-
þ b
Calculation of the concentration of the (6H ) , (6 H ) ,
and (6 H ) intermediates shown in Scheme 3b requires
palladacycle 3 binds substrates 2 with displacement of one
methanol to generate the key intermediate 6 (which from
the above discussion must exist as (6H ) as shown in
-
þ n
s
þ m
estimation of the microscopic pK values for dissociation
a
of the bridging or non-bridging H which is difficult.
s
þ
Scheme 4). This is probably an associative process con-
sistent with the generally accepted mechanism for ligand
1
2
Experimental determination of Ka and Ka values
Scheme 4) is not possible because of instability of the
Pd-complex at pH values below 4, but these are crudely
estimated as follows (for details of the analysis see
Supporting Information). The reported pK values for
the protonated phosphonium ions of trimethyl phosphate
and dimethyl phosphate in water are -3.6 and -2.6,
respectively, and a value of -3 is assumed at the outset
to be appropriate for the Ka constant for ionization of
the non-bridging proton of 6(2H ) to form the methanol
associated complex (6H ) . This ignores medium effect
differences on the solvation of the species on either side of
the equilibrium since these have similar charge types,
4
1
LG
(
exchange at Pd(II) centers. The lack of a significant β
s
s
s
for the ArO cleavage reaction in the low pH region
s
suggests that the rate limiting step cannot have substan-
tial changes in the P-OAr bonding, leading to the
simplest conclusion that a rate limiting intramolecular
rearrangement of 6 occurs, possibly generating complex 7
or a similarly disposed intermediate, with little perturba-
tion of the P-OAr bonding. A similar binding geometry
to that proposed for 7 has been observed for the square
planar bis-(O,O-diethyl dithiophosphato)platinum(II)
complex as characterized by X-ray crystallography, de-
a
3
7
2
þ
þ m
3
8
4
2
monstrating an internal S-P-S bond angle of 102.4°.
and any pK variations between phosphates and phos-
a
phorothioates, and also assumes that the Pd-complexa-
tion of the anionic S of 2b essentially converts the anionic
(
40) For the calculation predicated in Scheme 4, protonation on the
bridging oxygen of the aryloxy group should be far more difficult than on the
phosphoryl oxygen, making (6 H ) far less stable than (6 H ) . This rests
on two findings. The first is that in the study of the dissociation constant of
protonated trimethyl phosphate or dimethyl phosphate in water the
authors found no spectroscopic evidence for the presence of a protonated
methoxy phosphate, meaning that protonation adjacent to the P unit is less
favored than PdO protonation. Second, it is known from extensive studies of
the equilibrium transfer of the dialkylphosphoryl unit ((RO) PdO) between
oxyanionic nucleophiles (ref 29), that the O-Ar oxygen of phosphate tri- and
3
phosphorothioate into a neutral form. The pK for
9
s
1
-
þ b
-
þ n
s
a
þ
-
þ n
-
þ b
ionization of 6(2H ) to form (6 H ) or (6 H ) should
be lower than pK because the bound ligand is formally
neutral, and the starting complex is doubly protonated, so
s
s
4
37
a
s
1
a
pK is assumed to be ∼11.8, halfway between the 12.7
s
value determined experimentally for ionization of 3:2f
and the 10.8 value determined previously for ionization of
2
29b,c
17b
diesters has an effective charge of þ 0.83 and þ0.74 in water
which is
[
Pd(C,N-(dimethylbenzylamine)(pyridine)(CH OH)].
3
assumed to be the same as in methanol, and that for the bridging phenolic
1
7c
oxygen on phorphorothioate triesters is þ0.50.
This means that the
(
37) Az ꢀe ma, L.; Ladame, S.; Lapeyre, C.; Zwick; Lakhdar-Ghazal, A. F.
Spectrochim. Acta, A 2005, 62, 287.
38) Gibson, G. T. T.; Mohamed, M. F.; Neverov., A. A.; Brown, R. S.
Inorg. Chem. 2006, 45, 7891.
39) On the basis of the changes in the first and second acid dissociation
protonated form of the bridging oxygen of the Pd-bound substrates 2 should
s
be very acidic and have a
here.
s
pK
a
much lower that the values of ∼ -3 considered
(
(41) (a) Dies, V.; Cuevas, J. V.; Garcia-Herbosa, G.; Aullon, G.; Charmant,
J. P. H.; Carbayo, A.; Munoz, A. Inorg. Chem. 2007, 46, 568. (b) Soldatovic,
T.; Shoukry, M.; Puchta, R.; Bugarcic, Z. D.; van Eldik, R. Eur. J. Inorg. Chem.
2009, 2261. (c) Hohmann, H.; Suvachittanont, S.; van Eldik, R. Inorg. Chim.
Acta 1990, 177, 51.
(
-
2 4
constants of H PO bound between two Co(III) centers, it appears that two
metal ions have the effect of a single proton. By extension, a single metal ion
is predicted to have the effect of 1/2 a proton: (a) Edwards, J. D.; Foong,
S.-W.; Sykes, A. G. J. Chem. Soc., Dalton Trans. 1973, 829. (b) Williams,
N. H.; Cheung, W.; Chin, J. J. Am. Chem. Soc. 1998, 120, 8079.
(42) Gianni, M.; Caseri, W. R.; Gramlich, V.; Suter, U. W. Inorg. Chim.
Acta 2000, 299, 199.

1
796 Inorganic Chemistry, Vol. 50, No. 5, 2011
Edwards et al.
s
a
s
Scheme 5. Proposed Mechanism for the Reaction of Phosphorothioates at Low pH
a
Charges and the associated counterions have been omitted for clarity.
3
þ
new pathway for the La reaction at low pH (Scheme 2).
s
s
The angle strain imposed by the square planar binding
geometry of the Pd(II) center is expected to activate 7 for
nucleophilic attack by even weak nucleophiles such as solvent,
but the steric and geometric details of such a reaction are
3
þ
Under the reaction conditions the speciation of the La
is dimeric with a pH dependent number of associated
methoxides ([La ( OMe) ]; where x = 1-5) which are
responsible for the observed buffer catalysis. This is con-
sistent with the results obtained for our previous studies
involving La -promoted cleavage of anionic phosphate
s
s
-
2
x
opaque. The demonstrated lack of catalytic activity of 4
for the methanolysis of 2b in the low pH region provides
s
s
3
þ
evidence consistent with the proposed mechanism of
Scheme 5. For reaction of 2a-d it is proposed that rate
limiting formation of a doubly activated complex such as
27
30
43
diesters, neutral carboxylate, phosphonate, and phos-
4
4
45
phonothioate esters as well as phosphate triesters.
That cleavage of complex 6 is subject to significantly more
efficient La catalysis than is the cleavage of unbound phos-
phorothioate 2b suggests cooperativity between the pallada-
cycle and La systems in promoting methanolytic cleavage,
but this topic lies outside the scope of the current study.
7
is followed by fast chemical cleavage of the aryloxy
3þ
leaving group. Formation of 7 might be expected to display
a Brønsted dependence of ∼0 owing to a compensatory
3þ
effect of increased nucleophilicity for phosphorothioates
with high pKa leaving groups and a concomi-
s
s
tant decrease in the Lewis acidity of the corresponding
Pd(II) center. Activation of 2 for chemical cleavage by
bidentate coordination to Pd(II) as in 7 is an energetically
5
. Conclusions
The thiophilic metal ions Cd(II) and Mn(II) are effective
q
uphill process as indicated by the ΔH of 14.0 kcal/mol
promoters for the methanolysis of O-methyl O-4-nitrophenyl
phosphorothioate providing catalytic rate accelerations of
Pd
q
Pd
and ΔS of -20 cal/mol K. The negative entropy of
3
6
activation further suggests that 6 undergoes some restric-
tion of degrees of freedom in the transition state for
formation of 7, possibly resulting from the formation of
a strained four-membered ring. The details of the reaction
are not obvious, as it could follow a concerted or stepwise
process. In the latter case, breakdown of the 5-coordinate
phosphorane would be fast for aryloxy leaving groups,
but become slow, and possibly rate limiting from poorer
leaving groups such as methoxy. The significantly slower
symmetrical identity reaction of 2e relative to 2a-d is
consistent with such a pathway where chemical cleavage
from an intermediate has become slow relative to intra-
molecular rearrangement for displacement of the high
∼10 . These metal ions are often used in the study of enzyme
and ribozyme catalyzed phosphoryl transfer in conjunction
with thio effect data derived from the reaction of native
phosphate and sulfur modified phosphorothioate substrates.
It is often assumed in these studies that “metal ion rescue”
46
effects result directly from a synergistic effect of the en-
zyme/ribozyme acting in concert with the thiophilic metal
ions to bind and transform the phosphorothioate substrate.
The current study demonstrates that in instances where the
“metal ion rescue”experiments result inlow levels of returned
enzymatic activity, caution should be exercised to ensure that
the observable kinetic effects are not attributable to the metal
ions acting alone in solution to catalyze phosphothioyl transfer.
Palladacycle 3 is shown to be even more effective than
Cd(II) or Mn(II) in promoting the methanolysis of phos-
s
s
pK methoxy leaving group.
a
s
It should be noted that the pH independence of the
s
s
s
47
10
12
reaction determined for 2b at low pH (Figure 5) provides
phorothioates where rate accelerations of ∼10 and 10
no insight to the nature of the nucleophilic species since
the rate limiting step is proposed to occur prior to chemical
cleavage. However, the possibilities for the nucleophile
are narrowed by consideration of the following. Double
activation of the phosphorothioate as depicted in Scheme 2
completed the 4-coordination sphere for the Pd, thus
relative to the second order methoxide promoted reaction
s
s
were achieved for the cleavage of 2b in the low and high pH
domains, respectively. This represents an impressive catalytic
efficiency, particularly when one considers that the phospho-
diesterase NPP from X. axonopodis catalyzes the hydrolysis
min
(43) Lewis, R. E.; Neverov, A. A.; Brown, R. S. Org. Biomol. Chem. 2005,
suggesting an external nucleophilic mechanism. The k
-
1
3, 4082.
term of ∼0.02 s for reaction of 2b provides a lower limit
(44) Melnychuk, S. A.; Neverov, A. A.; Brown, R. S. Angew. Chem., Int.
for the rate of chemical cleavage and considering the
[
Ed. 2006, 45, 1767.
(45) Liu, T.; Neverov, A. A.; Tsang, J. S. W.; Brown, R. S. Org. Biomol.
Chem. 2005, 3, 1525.
-
s
s
CH O ] at pH 5.6, the external methoxide attack on
3
complex 7 at least approaches, and in all probability exceeds,
the diffusion limit in methanol as discussed above. For
these reasons we favor a process involving attack by
solvent methanol on a doubly activated complex such
as 7 leading to product formation.
(46) The use of metal ion rescue experiments is discussed in: Forconi, M.;
Herschlag, D. Methods Enzymol. 2009, 468, 91.
(47) Computed from the ratio of the average apparent second order rate
3
-1 -1
constant for the catalytic reaction (2.9 ꢀ 10
average of rate and K
data in the low pH 5.6 to 8.8 plateau, and 2.2 ꢀ 10
from the average of rate and K data in the high pH range in Table 2)
M
s
computed from the
5
b
-1 -1
M
s
b
The observation that the decomposition of 6 is subject
to buffer catalysis by La supports the intervention of a
-7
versus the second order rate constant for the methoxide reaction (2.3 ꢀ 10
3
þ
-1 -1
M
s
from Table 1).

Article
Inorganic Chemistry, Vol. 50, No. 5, 2011 1797
of R -O-methyl O-4-nitrophenyl phosphorothioate with a
Acknowledgment. The authors gratefully acknowledge
the financial assistance of the Natural Sciences and En-
gineering Research Council of Canada. This project also
received generous support (Grant HDTRA1-08-1-0046)
from the Defense Threat Reduction Agency - Joint
Science and Technology Office, Basic and Supporting
Sciences Division.
p
13 2f
reported acceleration of 10 . The efficacy of the cleavage of
b promoted by 3 relative to the corresponding methoxide
2
s
s
reaction inthe high pHplateaucan be compared with a value
6
17c
of 1.9 ꢀ 10 fold determined earlier for the cleavage of the
far more reactive triester p-nitrophenyl dimethyl phosphor-
othioate promoted by 3 in methanol. This points to an
interesting phenomenon whereby the same catalyst promotes
the reaction of a far less reactive substrate better than it does a
reactive substrate. It is also significant that 3 catalyzes the
Supporting Information Available: Procedures for the pre-
paration of 2a-e and corresponding analytical data, description
of the kinetic methods used, plots of observed rate constants
versus [metal] for the cleavage of 2b in the presence of Cd(II),
s
LG
cleavage of O,O-dimethyl phosphorothioate ( pK 18.2) in
s
a
-
CD OD to generate (CD O) PdO(S ) with a rate constant
-
3
3
2
Mn(II), La(III), plot of observed rate constant versus [ OMe]
-
4
-1
s
of 3.4 ꢀ 10
s
at what is essentially neutral pH. Examples
s
for the base promoted cleavage of 2b, tables of rate constants
s
s
of catalytic systems capable of cleaving alkoxy leaving groups
for the 3-catalyzed cleavage of 2b as a function of pH and
from phosphate diesters are rare making the 3-promoted
cleavage of 2e all the more noteworthy.
temperature, tables of rate constants for the 3-catalyzed
cleavage of 2a,c,d, and methodology for the speciation
calculations for the process in Scheme 4 (16 pages). This
material is available free of charge via the Internet at http://
pubs.acs.org.
22,48
(
48) Jagoda, M.; Warzeska, S.; Pritzkow, H.; Wadepohl, H.; Imhof, P.;
Smith, J. C.; Kr €a mer, R. J. Am. Chem. Soc. 2005, 127, 15061.