A simple DNase model system comprising a dinuclear Zn(II) complex in methanol accelerates the cleavage of a series of methyl aryl phosphate diesters by 1011-1013

Article abstract of DOI:10.1021/ja8006963

The di-Zn(II) complex of 1,3-bis[N₁,N′₁-(1,5,9- triazacyclododecyl)]propane with an associated methoxide (3:Zn(II) ₂:^-OCH₃) was prepared and its catalysis of the methanolysis of a series of fourteen methyl aryl phosphate diesters (6) was studied at _s^spH 9.8 in methanol at 25.0 ± 0.1°C. Plots of k_obs vs [3:Zn(II)₂: ^-OCH₃]_free for all members of 6 show saturation behavior from which K_M and k_cat^max were determined. The second order rate constants for the catalyzed reactions (k _cat^max/K_M) for each substrate are larger than the corresponding methoxide catalyzed reaction (k₂^-OMe) by 1.4 × 10⁸ to 3 × 10⁹-fold. The values of k_cat^max for all members of 6 are between 4 × 10 ¹¹ and 3 × 10¹³ times larger than the solution reaction at _s^spH 9.8, with the largest accelerations being given for substrates where the departing aryloxy unit contains ortho-NO ₂ or C(=O)OCH₃ groups. Based on the linear Bronsted plots of k_cat^max vs _s^spK _aof the phenol, β_Ig values of -0.57 and -0.34 are determined respectively for the catalyzed methanolysis of regular substrates that do not contain the ortho-NO₂ or C(=O)OCH₃ groups, and those substrates that do. The data are consistent with a two step mechanism for the catalyzed reaction with rate limiting formation of a catalyst-coordinated phosphorane intermediate, followed by fast loss of the aryloxy leaving group. A detailed energetics calculation indicates that the catalyst binds the transition state comprising [CH₃O ^-:6]^?, giving a hypothetical [3:Zn(II) ₂:CH₃O^-:6]^? complex, by -21.4 to -24.5 kcal/mol, with the strongest binding being for those substrates having the ortho-NO₂ or C(=O)OCH₃ groups.

Full text of DOI:10.1021/ja8006963

Published on Web 04/29/2008
A Simple DNase Model System Comprising a Dinuclear Zn(II)
Complex in Methanol Accelerates the Cleavage of a Series of
Methyl Aryl Phosphate Diesters by 1011-1013
Alexei A. Neverov, C. Tony Liu, Shannon E. Bunn, David Edwards,
Christopher J. White, Stephanie A. Melnychuk, and R. Stan Brown*
Department of Chemistry, Queen’s UniVersity, Kingston, Ontario, Canada, K7L 3N6
Received January 28, 2008; E-mail: rsbrown@chem.queensu.ca
Abstract: The di-Zn(II) complex of 1,3-bis[N1,N1′-(1,5,9-triazacyclododecyl)]propane with an associated
methoxide (3:Zn(II)2:-OCH3) was prepared and its catalysis of the methanolysis of a series of fourteen
s
methyl aryl phosphate diesters (6) was studied at pH 9.8 in methanol at 25.0 ( 0.1 °C. Plots of kobs vs
s
max
[3:Zn(II)2:-OCH3]free for all members of 6 show saturation behavior from which KM and kcat
were
determined. The second order rate constants for the catalyzed reactions (kcatmax/KM) for each substrate
are larger than the corresponding methoxide catalyzed reaction (k2–OMe) by 1.4 × 108 to 3 × 109-fold. The
max
values of kcat
for all members of 6 are between 4 × 1011 and 3 × 1013 times larger than the solution
s
reaction at pH 9.8, with the largest accelerations being given for substrates where the departing aryloxy
s
unit contains ortho-NO2 or C(dO)OCH3 groups. Based on the linear Brønsted plots of kcatmax vs sspKaof the
phenol, ꢀlg values of -0.57 and -0.34 are determined respectively for the catalyzed methanolysis of “regular”
substrates that do not contain the ortho-NO2 or C(dO)OCH3 groups, and those substrates that do. The
data are consistent with a two step mechanism for the catalyzed reaction with rate limiting formation of a
catalyst-coordinated phosphorane intermediate, followed by fast loss of the aryloxy leaving group. A detailed
energetics calculation indicates that the catalyst binds the transition state comprising [CH3O-:6]q, giving a
hypothetical [3:Zn(II)2:CH3O-:6]q complex, by -21.4 to -24.5 kcal/mol, with the strongest binding being
for those substrates having the ortho-NO2 or C(dO)OCH3 groups.
Introduction
of cleavage of p-nitrophenoxide generally occurs with intramo-
lecular participation of the 2-hydroxypropyl group.
Phosphate diesters are highly resistant toward solvolytic
cleavage1 making them ideal linking groups to be entrusted with
the preservation of genetic information in RNA and DNA.
Numerous enzymes are known to cleave biologically important
phosphate diesters with several of these containing metal ions
such as Zn2+, Mg2+, Ca2+, and Fe2+.2 Because of the remark-
able rate enhancements of up to 1017 for P-O cleavage achieved
by the enzymes,3 much research has centered on the ability of
small, metal containing complexes to promote the cleavage of
phosphate diesters.4 Many of the recently reported studies have
provided invaluable mechanistic information on the role by
which the metal ions, in specially tailored dinuclear Zn(II)
complexes,4a promote the cleavage of simple RNA models such
as 2-hydroxypropyl p-nitrophenyl phosphate (1),5 where the rate
In an ongoing program to investigate metal ion promoted
alcoholysis of carboxylate esters and phosphate esters,6 we have
shown that the light alcohols such as methanol and ethanol
induce a remarkable enhancement of the catalysis of cleavage
of 1 and related compounds by uncomplexed metal ions such
Zn2+ and La3+7 as well as complexes 2:Zn(II):-OCH3 and
3:Zn(II)2:-OCH3.8 The most recent study of the cyclization of
a series of 2-hydroxypropyl aryl phosphates mediated by
(5) (a) Feng, G.; Mareque-Rivas, J. C.; Williams, N. H. Chem. Commun.
2006, 1845. (b) Mancin, F.; Rampazzo, E.; Tecilla, P.; Tonellato, U.
Eur. J. Chem. 2004, 281. (c) Yang, M.-Y.; Iranzo, O.; Richard, J. P.;
Morrow, J. R. J. Am. Chem. Soc. 2006, 127, 1064. (d) O’Donoghue,
A. M.; Pyun, S. Y.; Yang, M.-Y.; Morrow, J. R.; Richard, J. P. J. Am.
Chem. Soc. 2006, 128, 1615. (e) Iranzo, O.; Elmer, T.; Richard, J. P.;
Morrow, J. R. Inorg. Chem. 2003, 42, 7737. (f) Iranzo, O.; Richard,
J. P.; Morrow, J. R. Inorg. Chem. 2004, 43, 1743. (g) Iranzo, O.;
Kovalevsky, A. Y.; Morrow, J. R.; Richard, J. P. J. Am. Chem. Soc.
2003, 125, 1988.
(1) Schroeder, G. K.; Lad, C.; Wyman, P.; Williams, N. H.; Wolfenden,
R. Proc. Nat. Acad. Sci. U.S.A. 2006, 103, 4052.
(2) (a) Cowan, J. A. Chem. ReV. 1998, 98, 1067. (b) Wilcox, D. E. Chem.
ReV. 1996, 96, 2435. (c) Sträter, N.; Lipscomb, W. N.; Klabunde, T.;
Krebs, B. Angew. Chem., Int. Ed. Engl. 1996, 35, 2024.
(6) (a) Brown, R. S.; Neverov, A. J. Chem. Soc. Perkin 2002, 2, 1039.
(b) Brown, R. S.; Neverov, A. A.; Tsang, J. S. W.; Gibson, G. T. T.;
Montoya-Peláez, P. J. Can. J. Chem. 2004, 82, 1791. (c) Brown, R. S.;
Neverov, A. A. Metal catalyzed alcoholysis reactions of carboxylate
and organophosphorus esters. In AdVances in Physical Organic
Chemistry; Richard, J. P., Ed.; Elsevier: Oxford, 2007; Vol. 42, pp
271–331.
(3) Wolfenden, R.; Snider, M. J. Acc. Chem. Res. 2001, 34, 938.
(4) For compendia of references on various metal containing complexes,
see: (a) Mancin, F.; Tecillia, P. New J. Chem. 2007, 31, 800. (b)
Weston, J. Chem. ReV. 2006, 105, 2151. (c) Molenveld, P.; Engbertsen,
J. F. J.; Reinhoudt, D. N. Chem. Soc. ReV. 2000, 29, 75. (d) Williams,
N. H.; Takasaki, B.; Wall, M.; Chin, J. Acc. Chem. Res. 1999, 32,
485. (e) Mancin, F.; Scrimin, P.; Tecilla, P.; Tonellato, U. Chem.
Commun. 2006, 2540. (f) Morrow, J. R.; Iranzo, O. Curr. Opin. Chem.
Biol. 2004, 8, 192.
(7) (a) Tsang, J. S.; Neverov, A. A.; Brown, R. S. J. Am. Chem. Soc.
2003, 125, 1559. (b) Neverov, A. A.; Brown, R. S. Inorg. Chem. 2001,
40, 3588. (c) Liu, C. T.; Neverov, A. A.; Brown, R. S. Inorg. Chem.
2007, 46, 1778.
9
10.1021/ja8006963 CCC: $40.75
2008 American Chemical Society
J. AM. CHEM. SOC. 2008, 130, 6639–6649 6639

A R T I C L E S
Neverov et al.
s
3:Zn(II)2:-OCH3 in methanol indicated that the transition state
of the complex catalyzed reaction is stabilized by -21 to -23
kcal/mol relative to the methoxide catalyzed reaction. This
stabilization energy approaches the -20 to -23 kcal/mol or so
transition state stabilization energy that is seen for efficient
enzymes that promote the cleavage of phosphodiesters,1,9
bearing in mind that the solvents and substrates are different. It
is apparent that the synergism between an appropriately
configured dinuclear Zn(II) catalyst and the medium effect
imbued by methanol accelerates these phosphoryl transfer
reactions to rates greatly exceeding anything so far reported
for RNA models in water.
reactions for series 6 at pH 9.815 in methanol. In addition we
s
report on an unusual effect where ortho-nitro and carbomethoxy
functionalized derivatives of 6 (6a,c,d,g,h,l) react faster than
their counterparts not having this substitution pattern, and we
give a detailed energetics analysis of the catalyzed and meth-
oxide reactions for these substrates.
Experimental Section
Materials. Methanol (99.8% anhydrous), sodium methoxide
(0.50 M solution in methanol, titrated against N/50 certified standard
aqueous HCl solution and found to be 0.49 M), tetrabutylammonium
hydroxide in methanol (1 M, titrated against N/50 certified standard
aqueous HCl solution and found to be 1.087 M), Zn(CF3SO3)2, 2,4-
dinitrophenol (97%), 2-chloro-4-nitrophenol (97%), 4-chloro-2-
nitrophenol (98%), 2,4,5-trichlorophenol (99%), 4-nitrophenol
(98%), 2-nitrophenol (98%), 4-methoxyl-2-nitrophenol (tech.),
4-chlorophenol (99+%), 3-nitrophenol (99%), methyl salicylate
(ReagentPlus, g99% GC), phenol (99%), 4-methoxyphenol (99%),
3-methoxyphenol (96%), sodium carbonate, imidazole (99%),
Amberlite IR-120H ion-exchange resin (functionalized as sulfonic
acid), sodium (solid in kerosene, 99%), and dimethyl phosphate
(96%) were purchased from Aldrich and used without further
purification. HClO4 (70% aqueous solution, titrated to be 11.40 M)
was purchased from Acros Organics and used as supplied. Methanol-
d4 was obtained from Cambridge Isotope Laboratories, Inc.
The metal ion catalyzed cleavage of phosphate diesters
serving as DNA models has also seen extensive study4 but, due
to their lower reactivity than 1, the great bulk of the substrates
studied are those with good leaving groups such as nitro- and
dinitrophenoxy.10 There are some important exceptions where
the reactions are reported to proceed in water with poor leaving
groups,11,12 and there are two fine studies of the hydrolysis of
a series of methyl aryl phosphates mediated by so-called ligand
exchange inert complexes, namely Co(III)-tris(3-aminopropyl)-
amine (4)13 and the bis[1,4,7-triazacyclononane:Co(III)(-OH)]2
complex (5).14
(10) (a) Blasko, A.; Bruice, T. C. Acc. Chem. Res. 1999, 32, 475. and
references therein. (b) Moss, R. A.; Park, B. D.; Scrimin, P.; Ghirlanda,
G. J. Chem. Soc., Chem. Comm. 1996, 1627. (c) Moss, R. A.; Zhang,
J.; Bracken, K. J. Chem. Soc., Chem. Comm. 1997, 1639. (d) Sumaoka,
J.; Miyama, S.; Komiyama, M. J. Chem. Soc., Chem. Comm. 1994,
1755. (e) Morrow, J. R.; Buttrey; L. A.; Shelton, V. M.; Berback,
K. A. J. Am. Chem. Soc. 1992, 114, 1903. (f) Breslow, R.; Zhang, B.
J. Am. Chem. Soc. 1994, 116, 7893. (g) Takeda, N.; Irisawa, M.;
Komiyama, M. J. Chem. Soc. Chem. Comm. 1994, 2773. (h) Hay,
R. W.; Govan, N. J. Chem. Soc., Chem. Commun. 1990, 714. (i)
Schneider, H.-J.; Rammo, J.; Hettich, R. Angew. Chem., Int. Ed. Engl.
1993, 32, 1716. (j) Ragunathan, K. G.; Schneider, H.-J. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 1219. (k) Gómez-Tagle, P.; Yatsimirsky, A. K.
J. Chem. Soc., Dalton Trans. 1998, 2957. (l) Roigk, A.; Hettich, R.;
Schneider, H.- J. Inorg. Chem. 1998, 37, 751. and references therein.
(m) Gómez-Tagle, P.; Yatsimirski, A. K. J. Chem. Soc., Dalton Trans.
2001, 2663. (n) Jurek, P. E.; Jurek, A. M.; Martell, A. E. Inorg. Chem.
2000, 39, 1016.
As a continuation of our investigation of the catalytic ability
of 3:Zn(II)2:-OCH3 we have studied its mediation of the
methanolyses of fourteen methyl aryl phosphates (6a-n). The
results reported herein show that all the substrates react via a
mechanism involving a pre-equilibrium binding, followed by a
rate limiting cleavage, where the half-times for the release of
the substituted phenol (phenoxide) from the {3:Zn(II)2:-OCH3:
6} Michaelis complex range from 0.14 s to 33 min. These
correspond to accelerations for the series ranging from 4 × 1011
to 3 × 1013 relative to the background methoxide promoted
(11) Jagoda, M.; Warzeska, S.; Pritzkow, H.; Wadepohl, H.; Imhof, P.;
Smith, J. C.; Krämer, R. J. Am. Chem. Soc. 2006, 127, 15061.
(12) (a) Tsubouchi, A.; Bruice, T. C. J. Am. Chem.Soc. 1994, 116, 11614.
(b) Tsubouchi, A.; Bruice, T. C. J. Am. Chem. Soc. 1996, 117, 7399.
(13) Padovani, M.; Williams, N. H.; Wyman, P. J. Phys. Org. Chem. 2004,
17, 472.
(14) Williams, N. H.; Cheung, W.; Chin, J. J. Am. Chem. Soc. 1998, 120,
8079.
(15) For the designation of pH in non-aqueous solvents, we use the forms
recommended by the IUPAC, Compendium of Analytical Nomencla-
ture. Definitive Rules 1997 3rd ed., Blackwell, Oxford, U. K. 1998.
If one calibrates the measuring electrode with aqueous buffers and
then measures the pH of an aqueous buffer solution, the term wwpH is
used; if the electrode is calibrated in water and the “pH” of the neat
buffered methanol solution then measured, the term swpH is used; and
if the electrode is calibrated in the same solvent and the “pH”’ reading
(8) (a) Neverov, A. A.; Lu, Z.-L.; Maxwell, C. I.; Mohamed, M. F.; White,
C. J.; Tsang, J. S. W.; Brown, R. S. J. Am. Chem. Soc. 2006, 128,
16398. (b) Lu, Z.-L.; Liu, C. T.; Neverov, A. A.; Brown, R. S. J. Am.
Chem. Soc. 2007, 129, 11642. (c) Bunn, S. E.; Liu, C. T.; Lu, Z.-L.;
Neverov, A. A.; Brown, R. S. J. Am. Chem. Soc. 2007, 129, 16238.
(9) Raines, R. T. Chem. ReV. 1998, 98, 1045.
is made, then the term sspH is used. Since the autoprotolysis constant
s
of methanol is 10-16.77, neutral pH is 8.4.
s
9
6640 J. AM. CHEM. SOC. VOL. 130, NO. 20, 2008

Simple DNase Model System
A R T I C L E S
Table 1. sspKa Values in Methanol of the Phenols Corresponding to
Phosphates 6, the λcat Wavelengths Used to Monitor the
3:Zn(II)2:-OCH3 Catalyzed Methanolysis Reaction, and the λbase
Wavelengths Used to Follow the –OCH3 Promoted Methanolysis
Reaction
NaOCD3 was prepared by adding sodium metal to CD3OD; the
resultant solution was titrated against certified standard aqueous
HCl solution and found to be 0.502 ( 0.006 M. Lithium chloride
was purchased from Anachemia Chemical LTD whereas methyl
2-hydroxy-5-nitrobenzoate (98%) was obtained from Alfa Aesar
and was used as supplied. The lithium salts of all phosphate diesters
(6a-n) were synthesized according to a general method.13 The
lithium salts were converted into the corresponding acids by passing
them through a column containing approximately 30 g of Amberlite
IR-120H ion-exchange resin. Each of 6a-n had 1H NMR, 31P
NMR and exact MS spectra consistent with the structure (see
Supporting Information). 1,3-Bis-N1-(1,5,9-triazacyclododecyl)pro-
pane (3) was prepared as described.16 The dinuclear 3:Zn(II)2:
-OCH3 complex was prepared as a 2.5 mM stock solution in
anhydrous methanol at ambient temperature by sequential addition
of aliquots of stock solutions of sodium methoxide, 3, and
Zn(CF3SO3)2 such that the relative ratios were 1:1:2. This order of
addition is essential for the formation of the catalyst complex which
takes ∼40 min in methanol (as monitored by the change in catalytic
activity over time).
phosphate
sspKa phenol
λcat (nm)
λbase (nm)
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
7.88
9.35
359
323
418
345
300
320
424
291
340
284
282
345
280
292
270a
395
426
386
315
400
409
10.29
10.64
10.73
11.18
11.28
11.37
12.41
13.59
13.93
14.21
14.33
14.77
387
a Methoxide reaction of 6a was monitored by following the
disappearance of starting material at 270 nm. Since this process involves
both aryl and P attack, the amount of each was determined from NMR
analysis of the product mixture, see text.
1
Methods. H NMR and 31P NMR spectra were determined at
+
400 and 162.04 MHz. The CH3OH2 concentrations were deter-
mined potentiometrically using a combination glass electrode
(Radiometer model # XC100–111–120–161) calibrated with certi-
fied standard aqueous buffers (pH ) 4.00 and 10.00) as described
in a previous paper.17 The sspH values in methanol were determined
by subtracting the correction constant of -2.2417 from the electrode
readings and the autoprotolysis constant for methanol was taken
UV–Visible Kinetics in Methanol. The rates of the 3:Zn(II)2:
-OCH3 catalyzed reactions of 6e,f, and h-n were monitored by
UV–visible spectrophotometry at 25.0 ( 0.1 °C. A 2.5 mM stock
solution of 3:Zn(II)2:-OCH3 in methanol was prepared in an oven-
dried vial as above. Portions of this solution were diluted to
concentrations ranging from 0.4 mM e [3:Zn(II)2:-OCH3] e 2.2
mM in anhydrous methanol inside a UV cell. The methanolysis
reaction was initiated by the addition of the substrates (final
concentration 4 × 10-5 M).
to be 10-16.77 M2. We have found that the addition of buffers to
s
s
control the pH inhibits the catalytic reaction, probably due to the
associated counterions that bind to the catalyst, so all the kinetic
s
s
studies are done under buffer free conditions although the pH is
always found to be in the range of 9.8 ( 0.2 at the concentrations
of catalyst employed.
The methoxide catalyzed reactions of phosphates 6a-g and i
were also followed by UV–visible spectrophotometry at 25.0 (
0.1 °C. Tetrabutylammonium hydroxide (TBA--OH; 1.087 M in
methanol) was used as the base and it was assumed to be completely
converted to TBA--OCH3 in methanol. For each substrate, the basic
reaction media were prepared in duplicate at three to five concentra-
tions ranging from 0.02 to 0.40 M TBA--OCH3 along with 0.05
or 0.1 mM of substrate in methanol. The initial rates of the reactions
were obtained by fitting the first 5–10% of the abs. vs time traces
The sspKa values in methanol of the corresponding phenol leaving
group for 6c, 6f, 6i, 6j, 6m, and 6n were obtained from previous
s
work18 and the pKa values for 6a, 6b, and 6d in methanol were
s
determined by measuring the sspH at half-neutralization with
s
NaOMe. These values were plotted against the known pKavalues
s
s
s
in water and fit by linear regression to the relationship, pKaMeOH
) (1.08 ( 0.03)pKaHOH + (3.50 ( 0.20). This relationship was
s
s
used to interpolate the pKavalues in methanol for the 6e, 6h, 6k,
and 6l phenols. The sspKa values for the corresponding phenol
leaving groups are listed in Table 1 along with the wavelengths at
which the appearance of each product was monitored in the kinetic
studies described below.
by linear regression and converted to first order rate constants (kobs
)
by dividing them by the expected absorbance change (∆Abs
expressed as δꢀ/[concentration]) if the reaction were to reach 100%
completion. The concentration of water at 0.02 M of TBA--OH in
methanol was titrated (Karl Fisher) to be 46 ( 2 mM and at 0.08
M it was found to be 160 ( 10 mM. Anhydrous methanol as
received was found to contain 16 ( 1 mM of water. It was assumed
that the base promoted reaction was associated with methoxide and
was not significantly affected by the presence of water at these
concentrations.
NMR determination of reaction products. Three samples, each
containing 2.5 mM of 6f (premixed with one equivalent of NaOMe
immediately before the introduction of the catalyst) and 2.5 mM
of 3:Zn(II)2:-OCH3 in 1.6 mL of anhydrous methanol, were
prepared and allowed to sit at room temperature for 12 h. The
reaction was then quenched by addition of two equivalents of HClO4
(from a stock solution in methanol) to each sample. Methanol was
carefully removed under vacuum and the resulting yellow oil/solid
was redissolved in 800 µl of CD3OD. 1H NMR (400 MHz) spectra
were taken for all three samples to determine the products of the
reaction.
Stopped-Flow Kinetics in Methanol. The rates of the metal
complex catalyzed reactions for phosphate diesters 6a-d, g, and
h were followed using an Applied Photophysics SX-17MV stopped-
flow reaction analyzer with a 10 mm light path thermostatted at
25.0 ( 0.1 °C. A 2.5 mM stock solution of 3:Zn(II)2:-OCH3 in
methanol was prepared in an oven-dried vial, stoppered with a
needle-permitting lid and then sealed with Parafilm under nitrogen
gas. After approximately 45 to 60 min (to allow the complex to
form fully), portions of this were used to prepare stock catalyst
solutions of concentrations ranging from 0.4 mM < [3:Zn(II)2:
-OCH3] < 2.2 mM, which were loaded into one syringe of the
stopped-flow reaction analyzer. Substrate solutions at concentrations
of 8 × 10-5 M were loaded into the second syringe. After mixing,
the final substrate concentration was 4 × 10-5 M. At each
concentration of catalyst, five kinetic runs were recorded and the
average values were used for the kobs for appearance of phenol
product vs [catalyst] plots.
Results
(16) Kim, J.; Lim, H. Bull. Korean Chem. Soc. 1999, 20, 491.
(17) Gibson, G.; Neverov, A. A.; Brown, R. S. Can. J. Chem. 2003, 81,
495.
(18) Liu, T.; Neverov, A. A.; Tsang, J. S. W.; Brown, R. S. Org. Biomol.
Chem. 2006, 3, 1525.
Identification of Reaction Products. An 1H NMR study was
conducted with 6f and 3:Zn(II)2:-OCH3, 2.5 mM each in
anhydrous methanol. After 12 h the reaction was quenched, the
9
J. AM. CHEM. SOC. VOL. 130, NO. 20, 2008 6641

A R T I C L E S
Neverov et al.
Table 2. Second Order Rate Constants (k2–OMe) for the Methoxide
Promoted Methanolysis of Substrates (6a-g, i) at T ) 25.0 ( 0.1
°C
phosphate
k2–OMe (M-1s-1
)
(7.7 ( 0.4) × 10-5
(2.4 ( 0.5) × 10-6
(2.0 ( 0.1) × 10-6
(1.9 ( 0.1) × 10-6
(1.0 ( 0.1) × 10-6
a
b
6a
6b
6c
6d
6e
6f
(7.9 ( 0.6) × 10-7c
(3.6 ( 0.2) × 10-7
(1.4 ( 0.1) × 10-7
6g
6i
a The k2
value of (7.7 ( 0.4) × 10-5 M-1 s-1 is for attack on P
–OMe
Figure 2. Plot of kobs vs [3:Zn(II)2:-OCH3]free for the catalyzed metha-
to give dimethyl phosphate as the product as well as the corresponding
phenoxide. Attack on the ring to give the anisole product along with
nolysis of 6c (4 × 10-5 M) determined from the rate of appearance of
s
s
product phenol at 418 nm, pH 9.8 ( 0.2, and 25.0 ( 0.1 °C. Fitting the
methyl phosphate proceeds with a rate constant of (6.9 ( 0.4) × 10-4
data to the expression given in eq 1 gives KM ) (3.7 ( 0.9) × 10-4 M,
M-1 s-1
.
b The k2
value of (2.4 ( 0.5) × 10-6 M-1 s-1 is for
–OMe
max
kcat
) 0.89 ( 0.09 s-1, and an intercept (A)) 0.174 ( 0.002 mM.
attack on P to give dimethyl phosphate as the product as well as the
corresponding phenoxide. Attack on the ring to give the anisole product
along with methyl phosphate proceeds with a rate constant having an
[CH3O-] (0.02–0.08 M in methanol). Due to the slowness of
the reactions, the rate constants were determined from initial
rates as described above. Substrates 6j-n reacted too slowly
for reliable rate constants to be determined in a reasonable time.
Because the 1H NMR results described above indicate that
∼90% of the methoxide reaction with 6a proceeds via nucleo-
philic attack on the ring resulting in Ar-OP cleavage, with 10%
resulting from attack on P to produce P-OAr cleavage, respective
second order rate constants of (6.9 ( 0.4) × 10-4 M-1 s-1 and
(7.7 ( 0.4) × 10-5 M-1 s-1 for each process were determined
from the overall rate of disappearance of starting material at
270 nm. In the case of the second fastest substrate reacting with
methoxide (6b), the product distribution between Ar-OP cleav-
age and P-OAr cleavage was determined by following both the
initial rate of appearance of the phenoxide product, and the
disappearance of the starting material. From the expected
absorbance of phenoxide (based on the extinction coefficient
at 395 nm) ∼90% of the reaction generates phenoxide product,
so we can estimate an upper limit for the rate constant for ring
upper limit of 2.4 × 10-7 M-1 s-1
.
c From ref 7b.
Figure 1. Brønsted plot for the methoxide promoted methanolysis of
phosphates (6a-g, i). The eight data points (9) for the base attack on the
–OMe
P were fit to a standard linear regression of log k2
) (-0.57 ( 0.06)
sspKa + (0.14 ( 0.68), r2 ) 0.9279. The two data points (O) for the formation
of 2,4-dinitroanisole and 2-Cl-4-nitroanisole were fit to a separate standard
attack as 2.4 × 10-7 M-1 -1
s
. A two-point Brønsted plot is
–OMe
s
s
linear regression of log k2
) (-2.4) pKa + 15.4.
shown in Figure 1 for the methoxide attack on the ring of the
–OMe
s
s
starting ester (6a and 6b), log k2
The Brønsted plot for the methoxide attack on P which is shown
) (-2.4) pKa + 15.4.
solvent removed and the residue dissolved in CD3OD. The
p-nitrophenol product exhibited peaks at 8.13 ppm (2H, d, ArH,
J ) 9.09 Hz) and 6.89 ppm (2H, d, ArH, J ) 9.09 Hz). The
ratio of the intensities of the p-nitrophenol hydrogens and those
of the dimethyl phosphate product at 3.69 ppm (6H, d, OCH3,
J ) 10.86 Hz) was 4:6 (see Supporting Information).
–OMe
in Figure 1 fits a standard linear regression of log k2
)
s
s
(-0.57 ( 0.06) pKa + (0.14 ( 0.68). The fit incorporates all
substrates since those with an ortho-NO2, Cl or carbomethoxy
group fit on the same line defined by substrates without ortho
groups.
Methanolysis of 6a-n Promoted by 3:Zn(II)2:-OCH3 in
Methanol. The plots of kobs for catalyzed methanolysis of 6a-n
vs [3:Zn(II)2:-OCH3]free all exhibit saturation kinetics within
the concentration range of the catalyst used for the study (see
Supporting Information). Previously we established that triflate
ion inhibits the catalysis afforded by 3:Zn(II)2:-OCH3 with an
inhibition constant of Ki ) 14.9 mM8a from which one can
To determine whether the methoxide reaction with 6a
proceeds via attack on P or on the aryl group with formation of
methyl phosphate and 2,4-dinitroanisole, a solution comprising
5 mM of 6a and 0.2 M of NaOMe in 2 mL of anhydrous
methanol was allowed to react at room temperature overnight
and then, after neutralization with aqueous HCl, methanol was
removed under vacuum and the residue dissolved in CD3OD.
The 1H NMR spectrum showed a 9:1 ratio of the 2,4-
dinitroanisole to 2,4-dinitrophenoxide products (see Supporting
Information). The same sample was again dried under vacuum
and redissolved back into anhydrous methanol for MS analysis.
The TOF-EI+ MS indicated the presence of 2,4-dinitroanisole
determine the free catalyst concentration ([3:Zn(II)2:-OCH3]free
)
at any [triflate]. The representative plots of kobs vs [3:Zn(II)2:
-OCH3]free shown in Figures 2 and 3 show that saturation
kinetics were found for phosphates with both good aryloxy
leaving groups (6c) and with poor ones (6k). The results for
the 3:Zn(II)2:-OCH3-catalyzed methanolysis of 6f have been
reported in an earlier study.8a
at m/z ) 198.0312 amu [M+], matching the calculated m/z of
+
198.0277 amu for C7H6N2O5
.
Kinetics of Methoxide Promoted Reactions. In Table 2 are
presented the second order rate constants (k2–OMe) for the
methoxide promoted cleavage of phosphates 6a-g, i determined
at 25.0 ( 0.1 °C under pseudofirst order conditions of excess
Inspection of all the plots (Figures 2 and 3 and those in
Supporting Information) reveals that there is a significant
x-intercept which we assume results from one Zn2+ dissociating
from the catalyst at low concentrations, and the following
9
6642 J. AM. CHEM. SOC. VOL. 130, NO. 20, 2008

Simple DNase Model System
A R T I C L E S
phosphate triesters with aryloxy leaving groups20 but closer to
the ꢀlg values for methoxide attack on diethyl aryl phosphates
(-0.70 ( 0.0521), as well as the methoxide (-0.728c) and
hydroxide (-0.69,14 -0.62, or -0.5422) promoted cyclization
of 2-hydroxypropyl aryl phosphates (1). Kirby and Younas23
demonstrated that the attack of oxyanion nucleophiles on methyl
aryl phosphate anions can proceed via three competitive
pathways involving CH3-OP, Ar-OP, and ArO-P cleavage,
but the initial rate method used here for monitoring the
production of phenoxide or phenol products for all of the
substrates except 6a and 6b probes the desired reaction
proceeding through nucleophilic attack on P.
With 6a and 6b product studies show that about 90 and 10%
of the respective reactions involve attack on the aryloxy group,
from which we can determine the rate constants given in Table
2 for the methoxide attack on P for these substrates. The two
open circles on the Brønsted plot of Figure 1 represent the rate
Figure 3. Plot of kobs vs [3:Zn(II)2:-OCH3]free for the catalyzed metha-
nolysis of 6k (4 × 10-5 M) determined from the rate of appearance of
s
s
product phenol at 282 nm, pH 9.8 ( 0.2, and 25.0 ( 0.1 °C. Fitting the
data to the expression given in eq 1 gives KM ) (1.4 ( 0.4) × 10-4 M,
kcatmax ) (7.4 ( 0.5) × 10-4 s-1 and an x-intercept A ) 0.13 ( 0.01 mM.
constants observed for attack on the aryloxy rings of 6a,b and
simplified data treatment method was devised to account for
the loss of catalyst integrity at lower concentrations. For all
substrates, the kinetics data in the kobs vs [3:Zn(II)2:-OCH3]free
plots were fitted to a nonlinear least-squares (NLLSQ) model
given in eq 1, which is an equation that is applicable to both
strong and weak binding situations:8a
–OMe
the slope of the two point linear regression, log k2
)
(-2.4)sspKa + 15.4, indicates such a large drop in rate with
s
s
increasing phenol pKa that none of the other substrates is
expected to encounter competitive methoxide promoted Ar-OP
cleavage.
A. Williams suggested some time ago that there is little
evidence that acyclic phosphate diesters react with oxyanion
nucleophiles by mechanisms other than concerted ones24 and
the concerted or synchronous mechanism is supported by heavy
atom kinetic isotope effect studies.25 However, there is now
good evidence that diesters having ꢀ-hydroxy groups capable
of acting as intramolecular nucleophiles, such as uridine 3′-
phosphate diesters and probably 2-hydroxypropyl phosphates,26
do react via the formation of stable pentacoordinate phosphorane
intermediates.27 The ꢀlg value we see here for the methoxide
reaction with 6 is consistent with either an associative concerted
reaction with little cleavage of the ArO-P bond in the transition
state or a two step mechanism with rate limiting formation of
a phosphorane intermediate. Williams’ charge mapping24,28 for
the hydrolysis of aryloxy phosphate diesters considers the
charges on the starting material and phenolate oxygens are 0.74
and -1.0 in aqueous media. If we assume that these same values
obtain for reactions in methanol, as in Scheme 1 we suggest
that the methoxide promoted cleavage of 6, if concerted, is
highly associative and has proceeded only 33% of the way along
the reaction path leaving a residual charge of +0.17 (0.74–0.57)
kobs ) kcat(1 + KB × [S] + [Cat] × KB - X) ⁄ (2KB) ⁄ [S]
(1)
where:
X ) (1 + 2KB × [S] + 2 × [Cat] × KB + KB2 × [S]2 -
2 × KB2 × [Cat][S] + [Cat]2 × KB )
2 0.5
and KB refers to the Cat + 6 binding constant (units of M-1),
the reciprocal of which is defined here as the Cat:6 dissociation
constant (KM) in units of M. The [Cat] term in eq (1) used for
the fitting corresponds to the actual concentration of Viable
catalyst, free from triflate inhibition, which is derived according
to the expression [Cat] ) ([3:Zn(II)2:-OCH3]free - A), where
A is an independently fitted parameter generally having the value
e0.2 mM, corresponding to the observable intercept.
Given in Table 3 are all kinetic data determined for the
3:Zn(II)2:-OCH3 -catalyzed methanolysis of 6a-n, as well as
the acceleration for methanolysis of the catalyst-bound substrate
(3:Zn(II)2:-OCH3:6) relative to the background (base-promoted)
reactions at sspH 9.8. In Figure 4 is a Brønsted plot of log (kcat
)
max
vs sspKa for all the substrates from which it is apparent that there
are two lines, one for substrates having o-NO2 or o-CO2CH3
groups lying above a second for all the other substrates. Linear
regressions of these have respective gradients of ꢀlg ) -0.34
( 0.01 and ꢀlg ) -0.59 ( 0.03. Also shown on the plots are
(20) Khan, S. A.; Kirby, A. J. J. Chem. Soc. B 1970, 1172.
(21) Liu, T.; Neverov, A. A.; Tsang, J. S. W.; Brown, R. S. Org. Biomol.
Chem. 2005, 3, 1525.
(22) Brown, D. M.; Usher, D. A. J. Chem. Soc. 1966, 6558.
(23) (a) Kirby, A. J.; Younas, M. J. Chem. Soc. B 1970, 1154. (b) Kirby,
A. J.; Younas, M. J. Chem. Soc. B 1970, 1165.
two points for methyl 2-tert-butyl-4-nitrophenyl phosphate (∆,
s
kcat ) 8.5 × 10-4 -1
s
; pKa ) 12.03) and dimethyl phosphate
s
(0, kcat ) 2.2 × 10-6 -1
s ; pKa ) 18.16) which we will deal
s
(24) (a) Williams, A. Concerted Organic and Bio-Organic Mechanisms;
CRC Press: Boca Raton, FL, 2000; pp 161–181. (b) Bourne, N.;
Williams, A. J. Am. Chem. Soc. 1984, 106, 7591. (c) Bourne, N.;
Chrystiuk, E.; Davis, A. M.; Williams, A. J. Am. Chem. Soc. 1988,
110, 1890. (d) Ba-Saif, S. A.; Davis, A. M.; Williams, A. J. Org.
Chem. 1989, 54, 5483.
s
with later.
Discussion
Methoxide Reaction. The methoxide reaction for the eight
most reactive substrates (6a-g, i) adheres to the Brønsted
relationship log k2–OMe ) (-0.57 ( 0.06) sspKa + (0.14 ( 0.68).
The ꢀlg of -0.57 is at the low end of a range of -0.64 to -0.94
reported recently for the hydroxide promoted hydrolysis of
methyl aryl phosphates.14,19 It is somewhat larger than the value
of -0.35 for attack of the good nucleophile, hydroxide, on
(25) (a) Cassano, A. G.; Anderson, V. E.; Harris, M. E. J. Am. Chem. Soc.
2002, 124, 10964. (b) Hengge, A. C.; Tobin, A. E.; Cleland, W. W.
J. Am. Chem. Soc. 1996, 117, 5919.
(26) Brown, D. M.; Usher, D. A. J. Chem. Soc. 1966, 6558.
(27) Lönnberg, H.; Strömberg, R.; Williams, A. Org. Biomol. Chem. 2004,
2, 2165.
(28) (a) Williams, A. Acc. Chem. Res. 1984, 17, 425. (b) Williams, A.
Chem. Soc. ReV. 1986, 15, 125.
(29) (a) Humphry, T.; Forconi, M.; Williams, N. H.; Hengge, A. C. J. Am.
Chem. Soc. 2002, 124, 14860. (b) Humphry, T.; Forconi, M.; Williams,
N. H.; Hengge, A, C. J. Am. Chem. Soc. 2004, 126, 11864.
(19) Zalatan, J. G.; Herschlag, D. J. Am. Chem. Soc. 2006, 128, 1293.
9
J. AM. CHEM. SOC. VOL. 130, NO. 20, 2008 6643

A R T I C L E S
Neverov et al.
obs
Table 3. Kinetic Constants (Maximum Rate Constant (kcatmax), Dissociation Constant (KM), and the Second Order Rate Constant (k2
kcatmax/KM)) for the Methanolysis of 6a-n Catalyzed by 3:Zn(II)2:-OCH3 at sspH 9.8 and 25.0 ( 0.1 °C
)
max
kcat
phosphate diester
(s-1 a
)
KM (M)a
k2obs (M-1 s-1 b
)
catalytic acceleration at sspH 9.8
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
5.1 ( 0.3
0.44 ( 0.01
0.89 ( 0.09
0.69 ( 0.03
(3.3 ( 0.5) × 10-4
(3.2 ( 0.2) × 10-4
(3.7 ( 0.9) × 10-4
(1.7 ( 0.4) × 10-4
(2.6 ( 1.0) × 10-4
(3.7 ( 0.8) × 10-4
(3.6 ( 0.2) × 10-5
(4.5 ( 0.5) × 10-5
(1.8 ( 0.6) × 10-4
(1.3 ( 0.2) × 10-4
(1.4 ( 0.4) × 10-4
(6.9 ( 0.2) × 10-5
(2.3 ( 0.5) × 10-4
(1.5 ( 0.3) × 10-4
15000 ( 3000
1400 ( 200
2400 ( 600
4100 ( 200
140 ( 60
6.2 × 1011
1.7 × 1012
5.2 × 1012
4.3 × 1012
4.2 × 1011
6.1 × 1011
1.1 × 1013
5.7 × 1012
1.0 × 1012
(3.6 ( 0.4) × 10-2
(4.1 ( 0.3) × 10-2
0.415 ( 0.008
110 ( 27
12000 ( 1000
6200 ( 100
83 ( 33
c
0.278 ( 0.006
(1.5 ( 0.1) × 10-2
(2.9 ( 0.1) × 10-3
(7.4 ( 0.5) × 10-4
(3.9 ( 0.1) × 10-2
(5.3 ( 0.3) × 10-4
(3.5 ( 0.1) × 10-4
22 ( 4
1.1 × 1012c
4.4 × 1011c
3.3 × 1013c
5.3 × 1011c
6.2 × 1011c
5.3 ( 1.9
570 ( 20
2.3 ( 0.5
2.3 ( 0.4
max
a kcat
and KM determined by fits of the kobs vs [Cat] data to eq 1 as described in text. b Computed as kcatmax/KM. c Acceleration values for substrates
–OMe
6h, 6j-n were predicted by estimating the k2
equation for the Brønsted plot for the base-promoted reaction in Figure 1.
values for the based-catalyzed reaction for these less active substrates using the linear regression
max
behavior with kcat
values spanning 4 orders of magnitude
while the corresponding acidity of the leaving group phenols
spans 7 orders of magnitude. However, there are two different
Brønsted plots in Figure 4 of slopes -0.34 and -0.59
intersecting at the point for methyl 2,4-dinitrophenyl phosphate.
The shallower gradient plot incorporates substrates with ortho-
nitro and carbomethoxy derivatives (6a,c,d,g,h,l) and lies above
the steeper gradient plot comprising all other substrates (termed
here “regular” substrates). We will deal with these in turn.
Recent studies of the heavy atom kinetic isotope effect29a on
the cleavage of labeled methyl p-nitrophenyl phosphates bound
to a di-Co(III) complex (5), coupled with the ꢀlg of -1.38
reported14 for the cleavage of four aryl methyl phosphates from
various derivatives of 5, were interpreted29a,b in terms of a two-
step mechanism with fast formation of a phosphorane interme-
diate followed by rate limiting cleavage of the aryloxy leaving
group. The ꢀlg of -0.59 for catalyzed transesterification of
“regular” substrates 6 suggests a different process is utilized
by the 3:Zn(II)2:-OCH3 catalyst relative to the di-Co(III) catalyst
5. Since the general catalytic reaction pathway involving
substrate binding and chemical transformation should have
common characteristics for related phosphodiester substrates,
it is useful to view the results obtained here within the context
Figure 4. Brønsted plots of log (kcatmax) vs the sspKa values for the 3:Zn(II)2:
-OCH3-catalyzed methanolysis of phosphates 6a, c, d, g, h, and l (O) which
fits a linear regression of log kcatmax ) (-0.34 ( 0.01) sspKa + (3.4 ( 0.2);
r2 ) 0.9933 and phosphates 6b, e, f, i-k, m, and n (9) which fits a linear
max
s
regression of kcat
) (-0.59 ( 0.03) pKa + (5.2 ( 0.4); r2 ) 0.9816.
s
The datum for methyl 2-tert-butyl-4-nitrophenyl phosphate (7) (∆, kcat
)
s
8.5 × 10-4 -1
s ; pKa ) 12.03) sits below both Brønsted lines and that for
s
dimethyl phosphate (0, kcat ) 2.2 × 10-6 -1
s
; sspKa ) 18.16) rests near the
of three pieces of our earlier work. These are: 1) for 6f the plot
Brønsted line for phosphates that do not include ortho - NO2 or car-
bomethoxy groups: neither of the latter two points is used to define the
linear regression (see text).
max
of kcat
vs the [-OCH3]/[3:Zn(II)2] ratio is bell-shaped,
maximizing at unity,8a suggesting the TS stoichiometry com-
prises a 1:2:1:1 ratio of ligand, Zn(II), methoxide and substrate;
2) the X-ray diffraction structure8c of the closely related
3:Zn(II)2:-OH complex shows a hydroxide bridged between the
two Zn(II) ions, and this motif is undoubtedly maintained for
the methoxide complex in methanol solution, at least prior to
substrate binding. The X-ray diffraction structure8b of a related
3:Cu(II):(-OH):((C6H5CH2O)2PO2-) complex shows that both
the dibenzyl phosphate and the hydroxide are bridged between
two 5-coordinate metal ions; and 3) the 3:Zn(II)2:-OCH3-
promoted intramolecular cyclization of a series of 2-hydrox-
ypropyl aryl phosphate diesters (1) with aryloxy leaving groups
is proposed8b,c to occur by a multistep pathway involving two
binding steps and a chemical cleavage step (kcat) which is rate
Scheme 1. Charge Mapping for a Concerted Displacement of
Substrates 6
still on the aryloxy group in the TS. The difference between
the solvation characteristics of methanol and aqueous media
could modify the charges on the starting materials somewhat,
but the differences are not expected to be large enough to alter
the analysis greatly.
limiting when the corresponding phenol of the aryloxy leaving
s
group has a high pKa, and very fast (nonrate limiting) when
s
s
s
the phenol has a low pKa.
3:Zn(II)2:-OCH3 -Catalyzed Reaction. The complex-cata-
lyzed reactions of all the substrates exhibit consistent saturation
Substrates 6 react slower than their hydroxypropyl counter-
parts (∼5000-fold for the 4-nitro derivatives) but should follow
9
6644 J. AM. CHEM. SOC. VOL. 130, NO. 20, 2008

Simple DNase Model System
A R T I C L E S
Scheme 2. Metal ion charges omitted for simplicity
Scheme 3. Leffler index28 R is Given as ꢀlg/ꢀeq ) -0.59/-1.83 ) 32% and Represents the Extent of the Departure of the OAr Group from
the P at the Transition State
the same general process for placement of the substrate into a
doubly activated complex as shown in simplified Scheme 2,
from which a slower alkoxide mediated cleavage step (kcat)
occurs. The products of the reaction are dimethyl phosphate
and the corresponding phenol/phenoxide, depending on the sspKa
of the phenol.
strong that the reaction is deflected from an associative concerted
reaction into a two-step process with rate limiting formation of
a phosphorane intermediate (int. in Scheme 3) followed by fast
aryloxy anion departure.
ii. Methanolysis of Phosphates 6a,c,d,g,h and l. Also shown
in Figure 3 is a second Brønsted plot with ꢀlg ) -0.34 for
substrates containing ortho-NO2 or C(dO)OCH3 groups (but
not other ortho groups like Cl or tert-butyl) which lies above
the regression line comprising the “regular” substrates. The rate
i. Methanolysis of Phosphates 6b,e,f,i-k,m and n. The lower
Brønsted plot shown in Figure 4 has a ꢀlg of -0.59 ( 0.03
which is essentially the same as that observed for the methoxide
max
acceleration due to this ortho effect is not small when we
reaction, and less than -0.97 ( 0.05 we observed for the kcat
s
s
vs pKa plot for 3:Zn(II)2:-OCH3-catalyzed cyclization of the
s
compare substrates having similar phenol pKa values. For
s
example, the kcatmax for substrate 6l (2-(methoxycarbonyl)phenol,
sspKa ) 14.21) is 53 and 73 times larger than the respective
hydroxypropyl aryl phosphates 1.8c In Scheme 3 are two possible
pathways for cleavage of the bound diester within the 3:Zn(II)2:
-OCH3:6 complex. Coordination of the phosphate anion to two
Zn(II) ions gives the aryloxy oxygen a net positive charge of
∼0.83,14,30 similar to that of a phosphate triester. Because it is
difficult to envision that a methoxide coordinated between two
Zn(II) ions is nucleophilic enough to attack the coordinated
phosphate, we propose, as shown in Scheme 3, that one of the
bridging methoxide-Zn(II) bonds is broken to allow an intramo-
lecular delivery of the methoxide from a monocoordinated
Zn(II)-methoxide, although strictly speaking, the data do not
rule out the possibility of attack of an external methoxide31 on
the 3:Zn(II)2-bound phosphate. If the reaction is concerted, the
ꢀlg ) -0.59 suggests the cleavage of the P-aryloxy bond would
have progressed only 32% of the way with the oxygen bearing
+0.24 charge in an associative, but early, TS. An associative
mechanism would be greatly enhanced via electrophilic activa-
kcatmax terms for substrates 6k (3-methoxyphenol, sspKa ) 13.93)
s
s
and 6m (phenol, pKa ) 14.33).
The ortho groups in question must bring on some energeti-
cally favorable interaction for the catalyzed process and while
it is not immediately clear how this is achieved, there are limited
possibilities. One involves a different reaction specific to these
catalyst-bound substrates involving ipso-attack on the ring by
an external or internal methoxide to give Ar-OP cleavage and
the corresponding anisole product. However, no anisole products
could be found (at the limits of detection e5%) for the catalyzed
reaction of 6a, 6g, and 6m, the only identifiable products being
the phenols resulting from P-OAr cleavage. A second involves
a possible general acid catalysis of the leaving group departure
by a closely situated Zn(II)-O(H)CH3 as has been discussed for
Zn2+(H2O)x ion catalysis of RNA model cleavage,33 although
the implication is that any general acid assistance must be unique
to the presence of the highly nonbasic ortho NO2 and
C(dO)OCH3 entities. The effect cannot be a simple steric one,
since the o-chloro derivative shows no deviation from the regular
tion by the catalyst through charge removal from the coordinated
- 32
(CH3O)(OAr)PO2
.
Possibly the stabilization is sufficiently
(30) We assume that binding of a phosphate between two Zn(II) centers
has the same net effect as a single protonation based on the
observations for the first and second acid dissociation constants of
(32) Patrick, J.; O’Brien, P. J.; Herschlag, D. Biochemistry 2002, 41, 3207.
They present a fine discussion of the ramifications of electrophilic
assistance of catalysis via binding of the leaving group to a metal ion
in the context of the alkaline phosphatase catalyzed reaction of aryl
and alkyl phosphate monoesters. .
-
H2PO4 bound between two Co(III) centers: Edwards, J. D.; Foong,
S.-W.; Sykes, A. G. J. Chem. Soc., Dalton Trans. 1973, 829.
(31) For the fastest substrate (6a), the kcatmax value is 5 s-1. Attack of free
s
methoxide cannot be ruled out because at pH 9.8, the [-OCH3] is
s
10–7 M and if it reacts at the diffusion limit of 1010 M-1 s-1, the upper
limit for this reaction would be 1000 s-1.
(33) Mikkola, S.; Stenman, E.; Nurmi, K.; Yousefi-Salakdeh, E.; Strömberg,
R.; Lönnberg, H. J. Chem. Soc., Perkin Trans. 1999, 2, 1619.
9
J. AM. CHEM. SOC. VOL. 130, NO. 20, 2008 6645

A R T I C L E S
Neverov et al.
Figure 6. Reaction coordinate diagram for the formation of a phosphorane/
di-Zn(II) stabilized complex (Int.1) without the extra binding stabilization
of the ortho-group X; and (Int.2) with the extra binding stabilization of the
ortho-group × showing the shift of the TS to the left.
values given in Table 3 that the o-substituted substrates (e.g.,
6g, 6h, 6l) do bind somewhat tighter to the catalyst in the ground
state, but of course in order to produce an increase in reaction
rate the rate-limiting transition state would have to bind tighter
yet37 due to the proposed electrophilic assistance or second-
sphere coordination. According to the reaction coordinate
diagram shown in Figure 6, such additional stabilization of the
phosphorane complex shifts the TS to the left with less
weakening of the P----OAr bond and less charge neutralization
on the aryloxy group, leading to a less negative Brønsted ꢀlg as
observed.
Our proposed mechanism of a catalyzed two-step reaction
involving rate-limiting formation of a 3:Zn(II)2 complexed
phosphorane intermediate invites comparison with two earlier
model studies of the hydrolysis of aryl methyl phosphates
mediated by Co(III) complexes 4 and 5. For the dinuclear Co(III)
complexed phosphate diesters (5),14 the data are consistent with
a two-step reaction with fast formation a phosphorane inter-
mediate followed by slow expulsion of the aryloxy leaving group
to form a highly strained di-Co(III) complexed phosphate having
two conjoined four-membered rings. Formation of a highly
strained product requires an increased cleavage of the P-OAr
bond in the TS accounting for the large negative ꢀlg value of
-1.38.14 An important consequence of the large Brønsted ꢀlg
relative to that for the hydroxide promoted reaction (-0.6414),
is that catalyzed cleavage of substrates with good leaving groups
is much more effective than for substrates with poor leaving
groups.
In the second case of the hydrolysis of a series of 6 promoted
by 4, N. Williams and co-workers13 surmised that the most
probable mechanism involved two steps with an intramolecular
attack of a Co(III)--OH on a transiently coordinated phosphate.
That the observed Brønsted ꢀlg changed from -0.17 to -1.05
at a pKa(lg) of ∼6 is consistent with a change in mechanism
from rate limiting formation of a phosphorane intermediate with
good leaving groups to breakdown with poorer ones. This
interpretation would be consistent with what we observe, with
the important modification that there is no break in the Brønsted
plots that would signify a change in the rate limiting step to
breakdown over the sspKa range encompassed by the substrates
used here. We suggest that the smaller ꢀlg of -0.59 (or -0.34
in the case of the ortho-substrates) observed for the 3:Zn(II)2:
-OCH3 catalyzed reaction is most consistent with a two step
Figure 5. UV spectra in methanol for 0.34 mM 2-nitrophenol (solid line),
the corresponding phenoxide (dotted line), and that for the complex of 0.34
mM in each of 3:Zn(II)2:-OCH3 and 2-nitrophenol (dashed line).
substrate plot, and the datum point for the o-tert-butyl derivative
(2-tert-butyl-4-nitrophenyl methyl phosphate (7)) lies consider-
ably below both lines in Figure 4. Rather, it is more likely that
the o-NO2 or C(dO)OCH3 groups enhance the interaction with
the catalyst in a way that decreases the development of negative
charge on the departing oxygen, leading to a lower magnitude
of ꢀlg due to what might be termed electrophilic assistance or
possibly second-sphere coordination34 that enhances increased
ion-dipole interaction, hydrogen bonding or hydrophobic inter-
action between catalyst and transforming substrate in the TS.
Electrophilic assistance of the departure of aryloxy leaving
groups via general acid assistance has been proposed to explain
the low ꢀlg observed for the cleavage of uridine-3′-phosphate
aryl esters promoted by bovine pancreatic RNase A35 and more
recently to explain the rapid cleavage of methyl 8-dimethy-
lamino-1-naphthyl phosphate with a variety of nucleophiles
under acidic conditions where intramolecular general acid
assistance plays a key role.36
The UV/vis spectra of the corresponding o-substituted phenols
show evidence of an enhanced binding to the 3:Zn(II)2:-OCH3
complex which is not seen for phenols having similar sspKavalues
but no o-substituent. In Figure 5 are displayed spectra of
2-nitrophenol, its phenoxide and that of 2-nitrophenol in the
presence of 3:Zn(II)2:-OCH3. The latter shows a new band from
the phenol or phenoxide which can only arise from a higher
order phenoxide: 3:Zn(II)2:-OCH3 interaction. Similar sets of
spectra are generated with 2,4-dinitrophenol, and 2-carbomethox-
yphenol, suggesting that these two o-substituted phenols are also
deprotonated and form higher order complexes with 3:Zn(II)2:
-OCH3. This is not the case with 4-nitrophenol as its UV/vis
spectrum does not change in the presence of 3:Zn(II)2:-OCH3
(see Supporting Information).
Although the above refers specifically to the phenol product
with 3:Zn(II)2:-OCH3, there is weak indication from the KM
(34) Raymo, F. M.; Stoddart, J. F. Chem. Ber. 1996, 129, 981.
(35) Davis, A. M.; Regan, A. C.; Williams, A. Biochemistry 1988, 27, 9042.
(36) Kirby, A. J.; Lima, M. F.; Da Silva, D.; Roussev, C. D.; Nome, F.
J. Am. Chem. Soc. 2006, 128, 16944.
(37) (a) Jencks, W. P. Catalysis in Chemistry and Enzymology; Dover
Publications: New York, 1987. (b) Wolfenden, R. Acc. Chem. Res.
1972, 5, 10. (c) Lienhard, G. E. Science 1973, 180, 149.
9
6646 J. AM. CHEM. SOC. VOL. 130, NO. 20, 2008

Simple DNase Model System
A R T I C L E S
–OMe
the Free Energies of Activation for kcat (∆Gqcat), and the Free Energies of Stabilization of the Methoxide Transition State through Binding to
Table 4. (kcat/KM)(Ka/Kw) and (kcat/KM)/k2
Constants and Computed Free Energies of Formation of Michaelis Complex (∆GBind - ∆GM),
3:Zn(II)2 (∆∆Gqstab) for the Reaction of 3:Zn(II)2:-OCH3 with Substrates 6a-n at 25.0 ( 0.1 °C in Methanola,b
substrate
(kcat/KM)/k2–OMe
(kcat/KM)(Ka/Kw) (M-2 s-1 c,d
)
∆GBind - ∆GM (kcal/mol)e
∆Gq (kcal/mol)f
∆Gq (kcal/mol)f
∆∆Gq (kcal/mol)
cat
Non
stab
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
1.94 × 108
5.8 × 108
1.2 × 109
2.2 × 109
1.4 × 108
1.4 × 108
3.3 × 1010
1.3 × 1010
5.9 × 108
9.2 × 108
3.4 × 108
5.2 × 1010
2.4 × 107
4.3 × 107
3.43 × 1011
3.2 × 1010
5.5 × 1010
9.4 × 1010
3.2 × 109
2.5 × 109
2.5 × 1011
1.4 × 1011
1.9 × 109
5.3 × 108
1.3 × 108
1.3 × 1010
5.3 × 107
5.3 × 107
-14.8
-14.8
-14.7
-15.1
-14.9
-14.7
-16.1
-16.0
-15.1
-15.3
-15.3
-15.7
-15.0
-15.2
16.4
17.9
17.5
17.6
19.4
19.3
17.9
18.1
19.9
20.9
21.7
19.5
21.9
22.1
23.0
25.1
25.2
25.2
25.6
25.7
26.2
26.1
26.8
27.8
28.0
28.3
28.4
28.7
-21.4
-22.0
-22.4
-22.7
-21.1
-21.1
-24.4
-24.0
-22.0
-22.2
-21.6
-24.5
-21.5
-21.8
a Italicized numbers are for o-NO2 and C(dO)OCH3 derivatives. b ∆Gqstab computed from application of kinetic and equilibrium constants to eq (2);
–OMe
c
k2
constants from Table 2. kcat/KM from Table 3. d Ka determined from half-neutralization8b to be 10-9.41; Kw ) 10-16.77; Ka/Kw ) 2.3 × 107.
e Computed as (∆GBind - ∆GM) ) -RT ln((Ka/Kw)/KM). f Computed from ∆Gqcat ) -RT ln(kcat/(kT/h)) or ∆GqNon ) -RT ln(k2–OMe/(kT/h)) from the
Eyring equation where (kT/h) ) 6 × 1012 s-1 at 298 K.
process with rate limiting formation of the phosphorane
intermediate. The fast departure of the leaving group in our case,
but not with 4 or 5, perhaps results from a conformational
flexibility of 3:Zn(II)2, not seen with the Co(III) complexes,
that accommodates the transforming substrate while it passes
along the reaction coordinate, electrostatically stabilizing the
phosphorane intermediate, its formation, pseudorotation and its
breakdown, and also complementing the substrate’s geometric
changes in passing between four- and five-coordinate phosphorus.
For 3:Zn(II):-OCH3 reacting with the “regular” substrates
6, the Brønsted ꢀlg of -0.59 (Figure 4) is the same as observed
for the methoxide reaction for the eight most rapidly reacting
substrates (ꢀlg ) -0.57, Figure 1). This indicates that substrates
with both good and poor leaving groups will be catalyzed to
the same extent, at least for the “regular” substrates. More
importantly, the smaller ꢀlg of -0.34 observed for the catalyzed
reaction of the group of ortho-nitro- and carbomethoxy-
substituted substrates indicates that this catalyst promotes the
cleavage of substrates with poorer leaving groups more than
ones with better leaving groups, bearing in mind our categoriza-
tion of the leaving group ability is based solely on phenol sspKa.
iii. Catalyzed Methanolysis of Dimethyl Phosphate. On the
methyl aryl phosphates even though the leaving groups are
substantially different. There are at least four possible processes
consistent with the observations, namely whether the attacking
methoxide is external, or metal-coordinated, and whether the
reaction is concerted or two-step, although for the reasons
reiterated previously, we favor (although not rigidly so) the
metal-coordinated methoxide proceeding through a two step
process with rate limiting formation of the phosphorane. For
such a symmetrical reaction occurring via a metal-delivered
methoxide, microscopic reversibility requires that methoxide
departure must also be metal-promoted. Thus, the five-coordinate
intermediate must be geometrically mobile enough that pseu-
dorotation and methoxide interchange on the Zn(II) cannot limit
the rate. For that process, and a mechanism involving an external
nucleophile proceeds via two steps, one anticipates a gradual
change in the rate limiting step from formation to breakdown
of the 5-coordinate intermediate. That the datum point for
dimethyl phosphate lies slightly below the extrapolated line may
signify that there is the onset of the anticipated break attributable
to the expected change in rate limiting step, but more study
with several additional derivatives having poor leaving groups
is required before this can be confirmed.
-
lower Brønsted plot in Figure 4 is a point (0) for (CH3O)2PO2
iv. Energetics Associated with the Catalyzed Reaction. The
acceleration afforded by 3:Zn(II)2:-OCH3 (or a chemical
equivalent such as 3:Zn(II)2 + (-OCH3)) in cleaving 6 can be
determined for 0.9 mM dimethyl phosphate reacting with 1.0
s
mM 3:Zn(II)2:-OCD3 in CD3OD at ambient temperature, pH
s
judged in comparison with the CH3O--promoted reaction.
9.8. This was determined from quantitative mass analysis of
the recovered dimethyl phosphate for the intensities of the M+
and (M++3) peaks checking for the replacement of OCH3 with
OCD3.38 The observed rate constant (kcat) for formation of
(CH3O)(CD3O)PO2-, corrected for an incomplete binding of
substrate and statistically for the expulsion of CH3O vs CD3O
from a putative phosphorane intermediate, is (2.27 ( 0.03) ×
10-6 s-1 or t1/2 ) 85 h which compares favorably with a kcat of
(9 ( 3) × 10-6 s-1 reported for the transesterification of
dimethyl phosphate catalyzed by a di–Cu(II) complex in CD3OD
at 25 °C.11,39
–OMe
Relative to the k2
values given in Table 2 for substrates
obs
6a-g,i, the catalytic second order rate constants (given as k2
) kcatmax/KM in Table 4) are larger by 1.4 × 108 to 3 × 109-
fold, the substrates with ortho-NO2 or C(dO)OCH3 groups
(6b,c,g) exhibiting the largest enhancements. These realistically
have errors of ( 20% due to the relatively low accuracy placed
–OMe
on the KM dissociation constants as well as the k2
values
determined for the slow methoxide reactions, but such errors
do not detract in any major way from the significant enhance-
The fact that dimethyl phosphate fits very near the Brønsted
(39) The dinuclear Cu(II) catalyst reported in ref 11 is indeed unusual
because it is reported to transesterify the CH3O group dimethyl
phosphate at 50 °C about 6 times faster than it does the CH3O group
of methyl-p-nitrophenyl phosphate (6f) and about 5.5 times faster than
it cleaves the p-nitrophenoxy group of the latter. We do not see this
phenomenon with 3:Zn(II)2:-OCH3.
s
line of ꢀlg -0.59 derived from the log kcat vs pKa data for
s
“regular” phosphates40 provides some evidence that the mech-
anism for its catalyzed cleavage is the same as that for the
(38) Melnychuk, S. A.; Brown, R. S. unpublished results. For experimental
details pertaining to this experiment, see Supporting Information.
(40) A ꢀlg of-0.60 is computed when all data points for “regular” methyl
aryl phosphates and that for dimethyl phosphate are considered.
9
J. AM. CHEM. SOC. VOL. 130, NO. 20, 2008 6647

A R T I C L E S
Neverov et al.
Scheme 4
ment provided by 3:Zn(II)2:-OCH3. An alternative comparison
max
of the kcat
observed for the bound substrates 6a-g,i with
the pseudofirst order rate constant calculated from the methoxide
–OMe
s
s
k2
at the pH where the catalyst is active (9.8) gives
accelerations ranging from 4 × 1011 to 1 × 1013. The latter
accelerations provided for the DNA model substrates compare
favorably with the 1 × 1012 to 4 × 1012 accelerations previously
obtained8c for the intramolecular transesterification of a series
of RNA models (2-hydroxypropyl aryl phosphates) in methanol,
although in that study none of the special ortho-substrates was
studied.
A revealing way of assessing catalytic efficacy quantifies the
free energy difference between the transition state for the
methoxide reaction of 6 (CH3O-:6)q and a catalytic transition
state comprising 3:Zn(II)2:-OCH3 plus 6 (or any of its kinetic
equivalents such as 3:Zn(II)2 + -OCH3 + 6). This comparison,
widely used for comparing enzyme and man-made catalyst-
promoted reactions relative to background reactions,41,42 proved
very useful in a recent analysis of the origin of the acceleration
afforded by 3:Zn(II)2:-OCH3 to transesterification of 2-hydrox-
ypropyl aryl phosphates 1 and 6f.8c,b Shown in Scheme 4 is a
thermodynamic cycle involving both the methoxide reaction and
the 3:Zn(II)2:-OCH3 promoted (or kinetically equivalent) reactions
of 6a-m for which eq 2 provides the stabilization free energy
(∆∆Gqstab.) difference between [3:Zn(II)2:-OCH3:6]q and [CH3O-:
6]q. The analysis and definition of terms is identical with the
treatments given previously8b,c and need not be repeated here.
Figure 7. Activation energy diagram for the reaction of CH3O– and
3:Zn(II)2:-OCH3 with substrate 6e at standard state of 1 M and T ) 25.0
( 0.1 °C showing the calculated energies of binding the methoxide by
3:Zn(II)2, of binding of the substrate to 3:Zn(II)2:-OCH3, and the calculated
-OMe
activation energies associated with kcat and k2
.
which fit into a narrow range of -21 to -22 kcal/mol for the
‘regular’ substrates without the ortho-NO2 or C(dO)OCH3
substituents and a more expanded range of -22.4 to -24.5 kcal/
mol for the ortho-substituted substrates. This is as expected from
the Brønsted plots shown in Figures 1 and 4 which have
essentially identical slopes of ∼-0.57 and -0.59 for the
methoxide and catalyzed reactions of the substrates with no
‡
ortho substituents, so it is expected that the overall ∆∆G
stab
s
for these substrates will not change appreciably with pKa.
s
However the shallower Brønsted plot in Figure 4 for the
catalyzed reactions of the o-NO2 and C(dO)OCH3 substituted
substrates means that the ∆∆Gqstab should become increasingly
negative with increasing substrate sspKa. Further dissection
indicates that the Ka/Kw term contributes -10.0 kcal/mol to the
∆∆G*stab ) (∆GBind - ∆GM + ∆Gc*at) - ∆G*Non ) -RT ln ×
(kcat ⁄ KM)(Ka ⁄ Kw)
(2)
k-2 OMe
[
]
‡
–OMe
∆∆G
for all substrates, while the (kcat/KM)/k2
terms,
stab
The k2–OMe values and kcat/KM values in eq 2 are from Tables
2 and 3 respectively and for slow reactions where the experi-
pertaining to the apparent second order rate constant for the
catalytic reaction relative to the methoxide reaction, contribute
a somewhat larger and variable amount (-10.0 to -14.6 kcal
–OMe
mental k2
values are not available, these are estimated
–OMe
/mol), with the largest negative numbers being for the substrates
(without error limits) from the Brønsted relationship k2
)
s
s
s
s
–OMe
with high phenol pKavalues that also contain the o-NO2 or
-0.57 pKa + 0.14. Listed in Table 4 are the (kcat/KM)/k2
and (kcat/KM)(Ka/Kw) constants and computed ∆∆Gqstab values
C(dO)OCH3 substituents. A small contribution comes from the
KM values which fall into a narrow range of -4.7 to -6.0 kcal/
mol, with the larger negative numbers generally belonging to
substrates with o-NO2 or C(dO)OCH3 substituents.
(41) Wolfenden, R. Nature 1969, 223, 704.
(42) For analyses of this treatment, see: Yatsimirsky, A. K. Coord. Chem.
ReV. 2005, 249, 1997. and references therein.
The ∆∆Gqstab for the catalyzed reaction of dimethyl phosphate
can be estimated from the experimental data only if we
extrapolate a value for its methoxide reaction43 of 3.2 × 10-11
(43) The rate constant for methoxide attack on the P of dimethyl phosphate
(DMP) is not known but might be estimated from the extrapolation
of the Brønsted plot as described in the text, or from literature work
as follows: Guthrie, J. P., J. Am. Chem. Soc. 1977, 99, 3991, has
computed the bimolecular rate constant for hydroxide attack on DMP
at 25 °C in water of 6.8 × 10–12 M-1 s-1. On the other hand, from
the extrapolation of high temperature data, Williams and Wyman
(Williams, N. H.; Wyman, P. Chem. Commun. 2001, 1268) have
determined that a di-neopentyl type phosphate diester (bis(2,2-
dimethyl-3-(p-carboxyphenyl)propyl) phosphate) reacts with HO- with
M-1 -1
from the Brønsted relationship in Figure 1, and assume
s
a KM value of 2 × 10-4 M from the average of the experimental
values for the “regular” phosphate (Table 3). With these values,
the ∆∆Gqstab is -21.7 kcal/mol.
The energies associated with the various components of the
thermodynamic cycle shown in Scheme 3 are easily appreciated
via visualization of the activation energy diagram exemplified
for the reaction of 6e in Figure 7. At a standard state of 1 M,
methoxide attack has a ∆Gqnon of 25.6 kcal/mol. For the
a rate constant of 1 × 10-15 M-1 s-1 1
. Where comparisons can be
made,6–8 the attack of methoxide on diesters in methanol is only
slightly less (a factor of 2-4) than those reported for attack of HO-
on phosphate diesters in water, so the above two rate constants might
be considered as limits for the reaction of CH3O- on P
9
6648 J. AM. CHEM. SOC. VOL. 130, NO. 20, 2008

Simple DNase Model System
A R T I C L E S
catalyzed process, preliminary binding of both methoxide and
substrate to the catalyst is accompanied by a reduction in free
energy of -14.9 kcal/mol. The ∆Gqcat of 19.4 kcal/mol
computed for kcat brings the activated complex of [3:Zn(II)2:
-OCH3:6e]‡ to a level that is -21.1 kcal/mol lower than the
transition state for the methoxide reaction.
3-Zn(II)2 is a poor catalyst for the cleavage of phosphate diesters
in water,46 but is an exceptionally good one in methanol, it
seems that the net positively charged catalyst is an important,
but not exclusive, component probably acting electrostatically
to assemble and orient the reaction partners and subsequently
escort the transforming anionic substrate through a low energy
path along the reaction coordinate. To exert its maximum
catalytic effect, this system requires an intimately associated
low dielectric constant/polarity environment that appears to be
satisfied by light alcohols such as methanol.
Conclusions
The catalysis of the cleavage of the 6a-n and dimethyl
phosphate brought about by a simple system comprising
3:Zn(II)2:-OCH3 and a medium effect provided by the methanol
solvent stands out among all known catalytic systems for the
cleavage of a well-defined series DNA models. A recent report19
of alkaline phosphatase (AP) catalyzed hydrolysis of a series
of methyl aryl phosphates that included several of the substrates
studied here (6f,i,j,m), gave kcat/KM values 230–880-fold smaller
than what we find for the methanolysis promoted by the
3:Zn(II)2:-OCH3. Of course the natural substrates for AP are
phosphate monoesters for which the catalyzed reactions are ∼1017-
fold faster than the solution reaction, but the 1011-fold overall rate
enhancement observed for AP promoted diester hydrolysis relative
to the solution reaction19 is substantial. In the present case, 3:Zn(II)2:
-OCH3 accelerates the methanolytic cleavage of the methyl aryl
phosphate diesters by factors of between 4 × 1011 and 3 × 1013
relative to the solution reaction at sspH 9.8.
It has been pointed out5g that enzymes that promote the
cleavage of phosphodiesters1,9 provide ∼20 to 23 kcal/mol of
stabilization energy to the transition state of the catalyzed
reaction relative to that of the background reaction. In the present
case, the ∆∆Gqstab of -21 to -24 kcal/mol for 3:Zn(II)2:-OCH3
promoted cyclization of substrates 6 in methanol approaches
that exhibited by such efficient enzymes, bearing in mind the
solvent differences for the enzymatic and small molecule catalyst
studied here. The present model system also exhibits an
interesting enhanced catalysis of o-NO2 and C(dO)OCH3
substrates that suggests that an improvement in efficiency might
be realized in a situation where there is an energetically
favorable interaction between the catalyst and the leaving group
of the phosphodiester. A most important part of these observa-
tion is the reduced gradient for the Brønsted plot for these ortho-
The available data are consistent with a mechanism for the
chemical cleavage involving a two step process with rate-
limiting formation of a phosphorane intermediate or, less likely
in our opinion, an associative concerted process with little
cleavage of the aryloxy leaving group in the transition state. It
is interesting that the same catalyst also promotes the cyclization
of a series of 2-hydroxypropyl aryl phosphates with rate
accelerations very similar to what we see here for a quite
different reaction involving an nucleophilic attack of methoxide
on a phosphate doubly activated by Zn(II)---O-P-O---Zn(II)
coordination. Whether the reaction involves an external meth-
oxide or intramolecular delivery of metal-coordinated methoxide
cannot be ascertained from the kinetics, but intuitively we favor
the latter for its chemical efficiency wherein all the catalytically
required components (3:Zn(II)2, substrate and -OCH3) are
recruited into one higher order complex prior to initiating the
chemical cleavage step.44
substituted derivatives which predicts that those substrates with
s
poorer leaving groups (as judged by the pKa value for the
s
corresponding phenol) are more effectively cleaved by the
catalyst relative to the methoxide background reaction. These
observations suggest that one should be able to design transition
metal ion complexes that enhance the cleavage of substrates
having leaving groups with a built-in additional affinity for the
catalyst.
Acknowledgment. We gratefully acknowledge the financial
assistance of the Natural Sciences and Engineering Research Council
of Canada (NSERC), The Canada Council of the Arts (CCA), and the
Canada Foundation for Innovation (CFI). We are indebted to Prof.
Jik Chin for helpful discussions. In addition, S.E.B. thanks NSERC
for an Undergraduate Summer Research Award, C.T.L. thanks NSERC
for a PGS-2 postgraduate scholarship, and R.S.B. thanks the CCA for
a Killam Research Fellowship, 2007-2009. Finally, we are indebted
to Dr. Y.-M. She of the Mass Spectrometry Facility at Queen’s
University for his expertise in determining the mass spectra of the
product mixtures resulting from the catalyzed methanolysis of dimethyl
phosphate.
An essential component of the very high catalytic rates we
see in methanol must arise from the medium effect. It is likely
that enzyme active sites comprise a domain of effectively
reduced dielectric constant decorated with oriented functional
groups to create a specific medium, or what we can term a
“molecular bottle”, tailored for a given reaction.45 Because
Supporting Information Available: Tables of pseudofirst order
rate constants for reactions of 6a-n with 3:Zn(II)2:-OCH3;
NMR and mass spectroscopic data for the identity of substrates
6a-n; UV/vis spectra for various ortho-substituted phenols in
the presence of 3:Zn(II)2:-OCH3; mass spectrometric data for
the catalyzed methanolysis of dimethylphosphate in CD3OD
promoted by 3:Zn(II)2:-OCD3: (25 pages). This material is
available free of charge via the Internet at http://pubs.acs.org.
(44) When a solution containing 1 mM of 3:Zn(II)2:-OCH3 (measured sspH
) 9.8) is treated with 0.9 mM of dimethyl phosphate, the measured
sspH is 9.5, signifying that the methoxide originally attached to the
catalyst is retained when it is bound to the phosphate: Edwards, D.;
Brown, R. S. , Unpublished observations.
(45) Richard, J. P.; Ames, T. L. Bioorg. Chem. 2004, 32, 354.
(46) Kim and Lim (ref 16) have reported that the cleavage of bis(2,4-
dinitrophenyl) phosphate promoted by 3:Zn(II)2 in water as a function
of pH is not markedly different from the catalysis afforded by the
mono Zn(II) complex of 1,5,9-triazacyclododecane, the rate constant
for the former being 7 × 10–3 M-1 s-1 at pH 7.
JA8006963
9
J. AM. CHEM. SOC. VOL. 130, NO. 20, 2008 6649