Rapid three-step cleavage of RNA and DNA model systems promoted by a dinuclear Cu(II) complex in methanol. Energetic origins of the catalytic efficacy

Article abstract of DOI:10.1021/ja073780l

A dinuclear Cu(II) complex of 1,3-bis-N₁-(1,5,9- triazacyclododecyl)propane with an associated methoxide (2-Cu(II) ₂:(^-OCH₃)) was prepared, and its kinetics of reaction with an RNA model (2-hydroxypropyl-p-nitrophenyl phosphate (1, HPNPP)) and two DNA models (methyl p-nitrophenyl phosphate (3) and iso-butyl p-chlorophenyl phosphate (4)) were studied in methanol solution at _s^spH 7.2 ± 0.2. X-ray diffraction structures of 2-Cu(II)₂:(^-OH)(H₂O)(CF₃SO ₃^-)₃:0.5CH₃CH₂OCH ₂CH₃ and 2-Cu(II)₂:(^-OH)((C ₆H₅CH₂O)₂PO₂ ^-)(CF₃SO₃^-)₂ show the mode of coordination of the bridging ^-OH and H₂O between the two Cu(II) ions in the first complex and bridging ^-OH and phosphate groups in the second. The kinetic studies with 1 and 3 reveal some common preliminary steps prior to the chemical one of the catalyzed formation of p-nitrophenol. With 3, and also with the far less reactive substrate (4), two relatively fast events are cleanly observed via stopped-flow kinetics. The first of these is interpreted as a binding step which is linearly dependent on [catalyst] while the second is a unimolecular step independent of [catalyst] proposed to be a rearrangement that forms a doubly Cu(II)-coordinated phosphate. The catalysis of the cleavage of 1 and 3 is very strong, the first-order rate constants for formation of p-nitrophenol from the complex being ～0.7 s ^-1 and 2.4 × 10^-3 s^-1, respectively. With substrate 3, 2-Cu(II)₂:(^-OCH₃) exhibits Michaelis-Mentin kinetics with a k_cat/K_M value of 30 M^-1 s^-1 which is 3.8 × 10⁷-fold greater than the methoxide promoted reaction of 3 (7.9 × 10^-7 M ^-1 s^-1). A free energy calculation indicates that the binding of 2-Cu(II)₂:(^-OCH₃) to the transition states for 1 and 3 cleavage stabilizes them by -21 and -24 kcal/mol, respectively, relative to that of the methoxide promoted reactions. The results are compared with a literature example where the cleavage of 1 in water is promoted by a dinuclear Zn(II) catalyst, and the energetic origins of the exalted catalysis of the 2-Cu(II)₂ and 2-Zn(II)₂ methanol systems are discussed.

Full text of DOI:10.1021/ja073780l

Published on Web 08/23/2007
Rapid Three-Step Cleavage of RNA and DNA Model Systems
Promoted by a Dinuclear Cu(II) Complex in Methanol.
Energetic Origins of the Catalytic Efficacy
Zhong-Lin Lu,† C. Tony Liu, Alexei A. Neverov, and R. Stan Brown*
Contribution from the Department of Chemistry, Queen’s UniVersity,
Kingston, Ontario, Canada, K7L 3N6
Received June 4, 2007; E-mail: rsbrown@chem.queensu.ca
Abstract: A dinuclear Cu(II) complex of 1,3-bis-N1-(1,5,9-triazacyclododecyl)propane with an associated
methoxide (2-Cu(II)2:(-OCH3)) was prepared, and its kinetics of reaction with an RNA model (2-
hydroxypropyl-p-nitrophenyl phosphate (1, HPNPP)) and two DNA models (methyl p-nitrophenyl phosphate
(3) and iso-butyl p-chlorophenyl phosphate (4)) were studied in methanol solution at sspH 7.2 ( 0.2. X-ray
diffraction structures of 2-Cu(II)2:(-OH)(H2O)(CF3SO3-)3:0.5CH3CH2OCH2CH3 and 2-Cu(II)2:(-OH)((C6H5-
CH2O)2PO2-)(CF3SO3-)2 show the mode of coordination of the bridging -OH and H2O between the two
Cu(II) ions in the first complex and bridging -OH and phosphate groups in the second. The kinetic studies
with 1 and 3 reveal some common preliminary steps prior to the chemical one of the catalyzed formation
of p-nitrophenol. With 3, and also with the far less reactive substrate (4), two relatively fast events are
cleanly observed via stopped-flow kinetics. The first of these is interpreted as a binding step which is linearly
dependent on [catalyst] while the second is a unimolecular step independent of [catalyst] proposed to be
a rearrangement that forms a doubly Cu(II)-coordinated phosphate. The catalysis of the cleavage of 1 and
3 is very strong, the first-order rate constants for formation of p-nitrophenol from the complex being ∼0.7
s-1 and 2.4 × 10-3 s-1, respectively. With substrate 3, 2-Cu(II)2:(-OCH3) exhibits Michaelis-Mentin kinetics
with a kcat/KM value of 30 M-1 s-1 which is 3.8 × 107-fold greater than the methoxide promoted reaction of
3 (7.9 × 10-7 M-1 s-1). A free energy calculation indicates that the binding of 2-Cu(II)2:(-OCH3) to the
transition states for 1 and 3 cleavage stabilizes them by -21 and -24 kcal/mol, respectively, relative to
that of the methoxide promoted reactions. The results are compared with a literature example where the
cleavage of 1 in water is promoted by a dinuclear Zn(II) catalyst, and the energetic origins of the exalted
catalysis of the 2-Cu(II)2 and 2-Zn(II)2 methanol systems are discussed.
up to a factor of 1015-16 giving some of the most spectacular
rate enhancements known. Many of these contain active sites
with two or more metal ions (usually Zn2+ and in some cases
Mg2+, Ca2+, and Fe2+) as exemplified by ribonuclease H from
HIV reverse transcriptase,5 3′,5′-exonuclease from DNA poly-
merase I,6 the P1 nucleases,7 and phospholipase C.1-4 Not
surprisingly, intense research is directed at understanding the
origins of catalysis of phosphate diester cleavage provided by
metal ion containing systems.10-13 The earlier work10-13 and
more recent reports14-20 indicate that dinuclear complexes are
typically more reactive than their mononuclear counterparts,
although in some cases they are slower or only weakly
accelerating.18,21,22 Very recently we reported on the exalted
1. Introduction
Phosphodiesters are important biomolecules for the storage
of genetic information due to their great stability.1-7 The half-
times for hydrolysis of RNA8 and DNA9 at pH 7 and 25 °C are
reported to be 110 and up to 100 billion years, respectively.
Certain phosphodiesterase enzymes promote the cleavage by
† Presently at Beijing Normal University, College of Chemistry, Beijing,
China, 100875.
(1) Weston, J. Chem. ReV. 2005, 105, 2151.
(2) Cowan, J. A. Chem. ReV. 1998, 98, 1067.
(3) Wilcox, D. E. Chem. ReV. 1996, 96, 2435.
(4) Stra¨ter, N.; Lipscomb, W. N.; Klabunde, T.; Krebs, B. Angew. Chem., Int.
Ed. Engl. 1996, 35, 2024.
(5) Davies, J. F.; Hostomska, Z.; Hostomsky, Z.; Jordan, S. R.; Mathews, D.
A. Science 1991, 252, 88.
(6) Beese, L. S.; Steitz, T. A. EMBO J. 1991, 10, 25.
(7) Lahm, A.; Volbeda, S.; Suck, D. J. Mol. Biol. 1990, 215, 207.
(8) Schroeder, G. K.; Lad, C.; Wyman, P.; Williams, N. H.; Wolfenden, R.
Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 4052.
(9) Williams, N. H.; Wyman, P. Chem. Commun. 2001, 1268.
(10) Molenveld, P.; Engbertsen, J. F. J.; Reinhoudt, D. N. Chem. Soc. ReV. 2000,
29, 75.
(15) Feng, G.; Mareque-Rivas, J. C.; Williams, N. H. Chem. Commun. 2006,
1845.
(16) Mancin, F.; Rampazzo, E.; Tecilla, P.; Tonellato, U. Eur. J. Chem. 2004,
281.
(17) Yang, M.-Y.; Iranzo, O.; Richard, J. P.; Morrow, J. R. J. Am. Chem. Soc.
2005, 127, 1064.
(18) Iranzo, O.; Elmer, T.; Richard, J. P.; Morrow, J. R. Inorg. Chem. 2003,
42, 7737.
(19) Iranzo, O.; Richard, J. P.; Morrow, J. R. Inorg. Chem. 2004, 43, 1743.
(20) Iranzo, O.; Kovalevsky, A. Y.; Morrow, J. R.; Richard, J. P. J. Am. Chem.
Soc. 2003, 125, 1988.
(21) Bauer-Sienbenlist, B.; Meyer, F.; Farkas, E.; Vidovic, D.; Cuesta-Seijo, J.
A.; Herbst-Irmer, R.; Pritzkow, H. Inorg. Chem. 2004, 43, 4189.
(11) Williams, N. H.; Takasaki, B.; Wall, M.; Chin, J. Acc. Chem. Res. 1999,
32, 485.
(12) Mancin, F.; Scrimin, P.; Tecilla, P.; Tonellato, U. Chem. Commun. 2005,
2540.
(13) Morrow, J. R.; Iranzo, O. Curr. Opin. Chem. Biol. 2004, 8, 192.
(14) Yamada, K.; Takahashi, Y.-i.; Yamamura, H.; Araki, A.; Saito, K.; Kawai,
M. Chem. Commun. 2000, 1315.
9
11642
J. AM. CHEM. SOC. 2007, 129, 11642-11652
10.1021/ja073780l CCC: $37.00 © 2007 American Chemical Society

Cleavage of RNA/DNA System by Dinuclear Cu(II) Complex
A R T I C L E S
Scheme 1 a
Unlike Zn(II) complexes, those with Cu(II) are also highly
colored which can give information about changes in the
immediate coordination environment around the metal ion. As
will be shown, the 2-Cu(II)2 complex greatly catalyzes the
cleavages of both 1 and 3, and it also exhibits two clearly
defined steps confirmed with 3 and diester 4 which appear to
be associated with the binding of these substrates to 2-Cu(II)2.
a M ) Zn(II) or Cu(II), S ) phosphodiester substrate; charges omitted
for simplicity.
catalysis of the cleavage of an RNA model, 2-hydroxypropyl
p-nitrophenyl phosphate (HPNPP (1)), promoted by a dinuclear
Zn2+ complex (2-Zn(II)2) in methanol.23 The plot of kobsd for
the process vs [2-Zn(II)2] in the presence of 1 equiv of added
2. Experimental Section
2.1. Materials. Methanol (99.8% anhydrous), sodium methoxide (0.5
M) solution in methanol, dibenzyl phosphate, and Cu(CF3SO3)2 were
purchased and used without further purification. Methyl-p-nitrophenyl
phosphate (MNPP, 3) was prepared according to a general published
method30 while 2-methylpropyl p-chlorophenyl phosphate (MPClPP,
4) was prepared as described in the Supporting Information. 1,3-Bis-
N1-(1,5,9-triazacyclododecyl)propane (2) was synthesized according to
the published procedure.31 The sodium salt of 2-hydroxylpropyl
p-nitrophenyl phosphate (1) was prepared according to the literature
procedure32 with a slight modification.33 The dinuclear complex
(2-Cu(II)2:(-OCH3)) was prepared as a 2.5 mM solution in methanol
by sequential addition of aliquots of stock solutions of sodium
methoxide, 1,3-bis-N1-(1,5,9-triazacyclododecyl)propane,31 and Cu(CF3-
SO3)2 in relative amounts of 1:1:2. It has been found that this order of
addition is essential for the formation of the complex, and eVen then
its complete formation is achieVed only after 10-15 min (as monitored
by the increase in catalytic actiVity as a function of time).
methoxide (which attaches to the metal ions to form 2-Zn(II)2:
s
s
(-OCH3) and maintains the pH24 at 9.5-9.8) was linear with
a slope of k2obsd ) 275 000 M-1 s-1. This catalytic k2obsd value
is 108 larger than the methoxide promoted cyclization of 1 (2.56
× 10-3 M-1 s-1 23
) and far exceeds anything previously seen in
water. A similar plot for the methanolysis of a DNA model
(methyl p-nitrophenyl phosphate (MNPP (3)) exhibits Michae-
lis-Mentin behavior with KM and kmax values of 0.37 ( 0.07
mM and (4.1 ( 0.3) × 10-2 s-1. That two closely related
substrates exhibit such different kinetic behaviors seemed
unusual and was rationalized by invoking the mechanism given
in Scheme 1 where the rate-limiting step with 1 was its binding
(k1 or k2) while that with the slower reacting 3 was the chemical
step of methanolysis of the bound substrate (k3).
1
2.2. Methods. H NMR and 31P NMR spectra were determined at
+
400 and 162.04 MHz. The CH3OH2 concentration was determined
using a combination of glass electrode (Radiometer model no. XC100-
111-120-161) calibrated with standard aqueous buffers (pH ) 4.00 and
10.00) as described in previous papers.23,33 The sspH values in
methanol34 were determined by subtracting a correction constant of
-2.24 from the readings obtained from the electrode, while the
autoprotolysis constant was taken to be 10-16.77. The sspH values for
the kinetic experiments were simply measured from solutions of the
complexes which were made in situ by the addition of 1 equiv of ligand
2, 1 equiv of NaOCH3, and 2 equiv of Cu(OTf)2: the values determined
in this manner lie in the region 7.0-7.6, but mostly at 7.2-7.4 for the
concentrations where the catalysis was investigated. While the meth-
odology gives some variance in the measured sspH values particularly
at low concentrations of metal ion, we have found that the addition of
buffers to control the sspH retards the reaction probably due to
inhibition by the associated counterions binding to the catalyst. The
first and second spKavalues for 2-Cu(II)2:(HOCH3) (0.4 mM) were
s
determined from duplicate measurements to be 6.77 ( 0.01 and 7.82
( 0.03 by measuring the sspH at half neutralization whereby the
[2-Cu(II)2:(-OCH3)]/[2-Cu(II)2:(HOCH3)] or [2-Cu(II)2:(-OCH3)2]/[2-
Cu(II)2:(-OCH3)] ratios were 1.0. This involved treating 2-Cu(II)2:(--
OCH3), prepared as described above at 0.4 mM, with 0.5 equiv of 70%
HClO4 or NaOCH3 diluted to a 0.05 M stock solution in anhydrous
methanol and measuring the sspH.
Herein we present results with the Cu(II)2 complex of 2 that
supports the above mechanism. While it is true that Cu(II)
complexes prefer five- and six-coordinate environments which
are different from Zn(II) complexes, several of the former are
known to catalyze phosphoryl transfer reactions of phosphate
diesters and interesting information of relevance to the mech-
anism of action has been obtained from their study.16,22,25-29
2.3. Kinetics. The rates of cleavage of MNPP (3) (0.04 mM)
catalyzed by 2-Cu(II)2:(-OCH3) (5 × 10-5 to 6 × 10-4 M) were
followed by monitoring the appearance of p-nitrophenol at 320 nm at
25.0 ( 0.1 °C. The fast rates of binding steps of substrates 1, 3, and
4 (0.03 mM) to 2-Cu(II)2:(-OCH3) in anhydrous methanol were
followed by observing the changes in the Cu(II)2 band at 340 nm at
25 °C using a stopped-flow reaction analyzer with a 10 mm light path.
(22) Arca, M.; Bencini, A.; Berni, E.; Caltagiirone, C.; Devillanova, F. A.; Isaia,
F.; Garau, A.; Giorgi, C.; Lippolis, V.; Perra, A.; Tei, L.; Valtancoli, B.
Inorg. Chem. 2003, 42, 6929.
(23) 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.
(24) For the designation of pH in nonaqueous solvents we use the forms
recommended by the IUPAC, Compendium of Analytical Nomenclature.
DefinitiVe Rules 1997, 3rd ed.; Blackwell: Oxford, U.K., 1998. Since the
autoprotolysis constant of methanol is 10-16.77, neutral sspH is 8.4.
(25) Rossi, L. M.; Neves, A.; Ho¨rner, R.; Terenzi, H.; Szpoganicz, B.; Sugai, J.
Inorg. Chim. Acta 2002, 337, 366.
(29) Deal, K. A.; Park, G.; Shao, J.; Chasteen, N. D.; Brechbiel, M. W.; Planalp,
R. P. Inorg. Chem. 2001, 40, 4176.
(26) Deal, K. A.; Hengge, A. C.; Burstyn, J. N. J. Am. Chem. Soc. 1996, 118,
1713.
(27) Jagoda, M.; Warzeska, S.; Pritzkow, H.; Wadepohl, H.; Imhof, P.; Smith,
J. C.; Kra¨mer, R. J. Am. Chem. Soc. 2005, 127, 15061.
(28) Fry, F. H.; Fischmann, A. J.; Belousoff, M. J.; Spiccia, L.; Bru¨gger, J.
Inorg. Chem. 2005, 44, 941 and references therein.
(30) Kirby, A. J.; Younas, M. J. Chem. Soc. B 1970, 1165.
(31) Kim, J.; Lim, H. Bull. Korean Chem. Soc. 1999, 20, 491.
(32) Brown, D. M.; Usher, D. A. J. Chem. Soc. 1965, 6558.
(33) Tsang, J. S.; Neverov, A. A.; Brown, R. S. J. Am. Chem. Soc. 2003, 125,
1559.
(34) Gibson, G.; Neverov, A. A.; Brown, R. S. Can. J. Chem. 2003, 81, 495.
9
J. AM. CHEM. SOC. VOL. 129, NO. 37, 2007 11643

A R T I C L E S
Lu et al.
Figure 1. Absorbance vs time profile for the reaction of 0.5 mM 2-Cu(II)2:(-OCH3) and 0.03 mM 1 in methanol, sspH ) 7.4, followed at 340 nm.
-
In the case of 1, the second binding step is superimposed on that for
the production of p-nitrophenol which leads to a rise in absorbance as
shown in Figure 1. For the sets of experiments with 1 and 3 the [2-Cu-
(II)2:(-OCH3)] was varied from 0.25 to 1.0 mM. The two fast steps
(termed herein as the first and second binding events) for 4 (0.03 mM)
were followed at 340 and 380 nm also using stopped-flow spectro-
photometry at 25 °C with the [2-Cu(II)2:(-OCH3)] being varied from
0.4 to 1.0 mM. The first-order rate constants (kobsd) for first and second
events with all substrates were obtained by fitting the UV/vis absorbance
vs time traces to a standard biexponential model. The second-order
rate constants for the first binding event (k1) for each substrate were
determined from the slopes of kobsd vs [2-Cu(II)2:(-OCH3)] plots, while
the second binding event for each was found to be independent of the
[2-Cu(II)2:(-OCH3)] and is simply referred to as k2. Given in Table 2
are the rate constants with the original data being given in Tables 1S-
7S of the Supporting Information.
4 days. Crystals of 2-Cu(II)2:(-OH)((C6H5CH2O)2PO2-)(CF3SO3
)
2
were grown from a methanol solution containing 1 equiv each of 2,
NaOCH3 and the sodium salt of dibenzyl phosphate along with 2 equiv
of Cu(OTf)2. The methanol solution was allowed to evaporate slowly
over 4 days without protection from the atmosphere at ambient
temperature. Given in Table 1 are crystal data and structural refinement
details. The ORTEP diagrams at the 50% probability level for each of
the two complexes are given in Figure 7. Full structural details and
CIF files for each complex are given in the Supporting Information.
3. Results
Given in Figure 1 is the Abs vs time profile for the
methanolysis of 0.03 mM 1 promoted by 0.5 mM 2-Cu(II):
(-OCH3) spH ) 7.4 determined at 340 nm. The trace is
clearly biphasic indicating at least two events. The absorbance
vs time data were fit by NLLSQ to a standard biexponential
model for an A f B f C process under the assumption that
the absorbance change for the second step was constant and
controlled by the appearance of p-nitrophenol with a fixed ∆A2
of 0.05 abs units. The first-order rate constants for the first and
(
)
s
2.4. Mass Spectra. The methanolysis of 0.5 mM of MNPP by 0.5
mM of catalyst was also monitored by ESI mass spectroscopy with
the following conditions: ESI+ mode, declustering potential ) 80.0
V, N2 carrier gas, ion spray voltage ) 5500 V, resolution ) 10 000,
and direct syringe injection. The reaction progress was followed by
comparing the relative ratio of the sums of the integrated intensities of
all product species, or all the substrate species in solution vs the sums
of the integrated intensities of all product and substrate species in
second events are 10.4 s-1 and 0.72 s-1
.
Shown in Figure 2a and 2b are the plots of the first-order
rate constants for the first and second events observed for the
methanolysis of 0.03 M HPNPP obtained under pseudo-first-
solution (respectively, 2-Cu(II)2:(X-)(Y-)((CH3O)2PO2-) or 2-Cu(II)2:
-
(X-)(Y-)((NO2C6H4O)(CH3O)PO2-), where X- and Y- ) CF3SO3
,
order conditions of excess catalyst concentration, 0.25 mM <
3 or (CH3O)2PO2-. MS spectra were taken at time ) 0.7, 3.7, 13.7,
22.7, 42.7, and 116.7 min, and aliquots were removed from the reaction
mixture by syringe and manually injected. Representative examples of
the spectra are given in the Supporting Information.
s
s
[2-Cu(II):(-OCH3)] < 1.0 mM, pH ) 7.4 ( 0.2. The second-
order rate constant for the first event (k1), calculated as the linear
regression of the line, is 18 000 ( 700 M-1 s-1, and at the
95% confidence level, the rate constant (k2) for the second event
is not dependent on the [2-Cu(II)2:(-OCH3)], the average value
2.5. X-ray Diffraction. The crystal structures of 2-Cu(II)2:(-OH)-
-
(H2O)(CF3SO3-)3 and 2-Cu(II)2:(-OH)((C6H5CH2O)2PO2-)(CF3SO3
)
2
being 0.7 s-1
.
were determined using a Bruker SMART APEX II X-ray diffractometer
with graphite-monochromated Mo KR radiation (λ ) 0.710 73 Å),
operating at 50 kV and 30 mA over 2θ ranges of 3.86°-50.00° as
described in the Supporting Information. No significant decay was
observed during the data collection at -93 °C. Crystals of 2-Cu(II)2:
(-OH)(H2O)(CF3SO3-)3:0.5 CH3CH2OCH2CH3 suitable for study were
grown from a methanol solution containing 1 equiv each of 2 and
NaOCH3 along with 2 equiv of Cu(OTf)2 placed in the lower chamber
of a necked-down sealed tube, which was gently overlaid with an ether
layer which was allowed to diffuse into the methanolic solution over
To probe further the origins of the first and second events,
we switched to the DNA model, 3, which does not possess the
intramolecular 2-hydroxypropyl group and for which the release
of p-nitrophenol is slower than that from 1 by a factor of at
least 3200-fold for the methoxide catalyzed process.33,35 Shown
(35) The second-order rate constant for methoxide catalyzed methanolysis of 3
is kO2 Me- ) (7.9 ( 0.6) × 10-7 M-1 s-1 (see ref 23), while that for reaction
with 1 is 2.6 × 10-3 M-1 s-1
.
9
11644 J. AM. CHEM. SOC. VOL. 129, NO. 37, 2007

Cleavage of RNA/DNA System by Dinuclear Cu(II) Complex
A R T I C L E S
Table 1. Crystal Data and Structural Refinement Details for Crystals of 2-Cu(II)2:(-OH)(H2O)(CF3SO3-)3‚0.5Et2O and
2-Cu(II)2:(-OH)((C6H5CH2O)2PO2-)(CF3SO3
)
2
-
-
-
identification code
2-Cu(II)2:(-OH)(H2O)(CF3SO3
)
3
2-Cu(II)2:(-OH)((C6H5CH2O)2PO2-)(CF3SO3
)
2
empirical formula
C26H54Cu2F9N6O11.50S3;
C37H59Cu2F6N6O11 P S2
[Cu2(OH)(OH2)(C21H46N6)]
(CF3SO3)3‚0.5Et2O
1029.01
180(2) K
0.710 73 Å
formula weight
temperature
wavelength
crystal system
space group
1100.07
180(2) K
0.710 73 Å
triclinic
monoclinic
C2/m
unit cell dimensions
a ) 17.647(4) Å
a ) 11.8724(16) Å
R ) 77.490(2)°
b ) 14.0342(17) Å
â ) 70.893(3)°
c ) 15.446(2) Å
γ ) 73.312(3)°
2307.9(5) Å3
2
R ) 90°
b ) 14.230(3) Å
â ) 96.196(5)°
c ) 16.367(4) Å
γ ) 90°
volume
Z
4086.1(16) Å3
4
density (calculated)
absorption coefficient
F(000)
crystal size
theta range for data
collection
1.673 Mg/m3
1.296 mm-1
2124
1.583 Mg/m3
1.134 mm-1
1140
0.25 × 0.08 × 0.06 mm3
0.30 × 0.20 × 0.20 mm3
1.84° to 25.00°
1.93° to 25.00°
index ranges
-19 e h e 20,
-13 e h e 14,
-15 e k e 16,
-19 e l e 19
12 177
-16 e k e 16,
0 e l e 18
reflections collected
15 311
independent reflections
completeness to θ ) 25.00°
absorption correction
max and min transmission
refinement method
data/restraints/parameters
goodness-of-fit on F2
final R indices [I > 2σ(I)]
R indices (all data)
3760 [R(int) ) 0.1045]
15 333 [R(int) ) 0.0000]
99.7%
99.5%
empirical
TWINABS
0.3183 and 0.1805
full-matrix least-squares on F2
3760/3/324
0.8050 and 0.7273
full-matrix least-squares on F2
15 333/0/591
1.000
1.000
R1 ) 0.0830, wR2 ) 0.2244
R1 ) 0.1371, wR2 ) 0.2526
0.997 and -1.195 e‚Å-3
R1 ) 0.0519, wR2 ) 0.1385
R1 ) 0.0749, wR2 ) 0.1483
0.722 and -0.592 e‚Å-3
largest diff peak and hole
in Figure 3 is a plot of the kobsd vs varying [2-Cu(II)2:(-OCH3)]
where the rate of formation of the p-nitrophenol product was
followed at 320 nm by conventional UV/vis kinetics. The plot
shows Michaelis-Mentin behavior with strong binding. The
data for this can be fit to a universal binding equation, eq 1,36
that is applicable to both strong and weak binding situations:
promoted by 0.5 mM 2-Cu(II)2:(-OCH3) observed at λ ) 340
(for the first two steps) and 320 nm (for the final step) and
sspH ) 7.1 emphasizing the three temporally well-separated
events with rate constants of 2.4 s-1, 0.57 s-1, and 2.10 × 10-3
s-1
.
The absorbance vs time traces for the first two steps of the
reaction of 3 were monitored at 340 nm as a function of varying
kobsd ) kcat(1 + Kd*[Lim] + [Ex]*Kd - X)/(2Kd)/[Lim] (1)
s
[catalyst], 0.25 mM < [2-Cu(II):(-OCH3)] < 1.0 mM, pH )
s
7.2 ( 0.2. The first step is linear in [catalyst] (k1 ) 20 200 (
600 M-1 s-1), but the second step is independent of [2-Cu(II)2:
(-OCH3)], the average value for k2 being 0.53 ( 0.06 s-1 (see
Table 2 and Supporting Information).
The methanolysis reaction of 5 × 10-4 M each of 3 and 2-Cu-
(II)2:(-OCH3), in anhydrous methanol, was monitored by
electrospray mass spectrometry at various times after mixing
of the two components. The integrated intensities of all major
peaks containing the methanolysis product (dimethyl phosphate)
where [Lim] and [Ex] refer to total concentrations of limiting
and excess reagents and X is given in eq 2.
X ) (1 + 2Kd*[Lim] + 2*[Ex]*Kd + Kd *[Lim]2 -
2
2*Kd *[Ex][Lim] + [Ex]2*Kd ) (2)
2
2 0.5
NLLSQ fitting of the data to eqs 1, 2 gives a KM of 0.079 (
0.018 mM and a kcat for the fully bound substrate of (2.40 (
0.04) × 10-3 s-1
.
When the absorbance vs time profile for the methanolysis of
3 promoted by 2-Cu(II)2:(-OCH3) is monitored at 320-340 nm
on the stopped-flow time scale, there is evidence for three
events, with two rapid events coming before the one relating to
the release of p-nitrophenol from the bound substrate. Shown
in Figure 4 are traces for the methanolysis of 0.05 mM 3
or substrate (3) bound to 2-Cu(II)2 along with any counterions
present (e.g., respectively, 2-Cu(II)2:(X-)(Y-)((CH3O)2PO2
or 2-Cu(II)2:(X-)(Y-)((NO2C6H4O)(CH3O)PO2-, X-, Y-
)
-
)
triflate, 3, or product) were summed to obtain (Iprod + I3). The
reaction progress was monitored by observing the Iprod/(Iprod
+
I3) and I3/(Iprod + I3) ratios as a function of time (0.7, 3.7, 13.7,
22.7, 42.7, and 116.7 min). Figure 5 displays the results from
(36) Equation 1 was obtained from the equations for equilibrium binding and
for conservation of mass by using the commercially available MAPLE
software, Maple V, release 5; Waterloo Maple Inc.: Waterloo, Ontario,
Canada.
which one can calculate the first-order rate constants of kprod
0.031 ( 0.007 min-1 and k3 ) 0.022 ( 0.003 min-1
)
.
9
J. AM. CHEM. SOC. VOL. 129, NO. 37, 2007 11645

A R T I C L E S
Lu et al.
) 0.49 ( 0.03 s-1 at an average sspH of 7.1 ( 0.1 (see Table
2 and Supporting Information).
3.1. X-ray Diffraction Structures. Single-crystal X-ray
diffraction studies were done on crystals of 2-Cu(II)2 grown
under conditions where (a) the components 2, Cu(OTf)2, and
NaOCH3 were added to dry methanol in a 1:2:1 ratio, followed
by diffusion of an ether layer into the methanol solution over a
period of about 4 days and (b) the components 2, Cu(OTf)2,
NaOCH3, and the sodium salt of dibenzyl phosphate37 were
added to dry methanol in a 1:2:1:1 ratio, and the solution slowly
evaporated in the ambient atmosphere over a period of about 4
days. Shown in Figures 6 and 7 are the structures of 2-Cu(II)2-
(-OH)(H2O)(CF3SO3-)3:0.5CH3CH2OCH2CH3 and 2-Cu(II)2(--
-
OH)((C6H5CH2O)2PO2-)(CF3SO3
)
without the associated
2
triflate counterions (or CH3CH2OCH2CH3) while selected bond
distances and angles are given in Table 3; full details are given
in the Supporting Information.
4. Discussion
4.1. X-ray Diffraction Structures. The partial structures
shown in Figures 6 and 7, devoid of associated triflate
counterions, reveal that both metal ions are five-coordinate and
buried rather deeply in the ligand which resembles two
tricoordinating “ear-muffs”. These impede access to the metal
ions from all orientations except those perpendicular to the Cu-
Cu axis and distal to the C3 linking spacer. Apparently there is
some flexibility within the ligand that accommodates a variable
Cu-Cu distance which ranges from 3.26 to 3.68 Å in passing
from 2-Cu(II)2(-OH)(H2O)(CF3SO3-)3:0.5CH3CH2OCH2CH3 to
2-Cu(II)2(-OH)((C6H5CH2O)2PO2-)(CF3SO3-)2. In the former
the two Cu(II) ions and coordinated HO- and OH2 are nearly
planar, the intersection angle between the Cu1-(-OH)-Cu2
and Cu1(OH2)-Cu2 planes being only 1.1°. The doubly
coordinated phosphate diester in 2-Cu(II)2(-OH)((C6H5CH2O)2-
PO2-)(CF3SO3-)2 has its O3-P1-O4 plane twisted by 68.4°
relative to the Cu1-Cu2 axis. The bridging of the diester
between the two Cu(II) ions typifies the sort of motif seen in
dinuclear complexes where phosphate di- and monoesters are
coordinated to the complex.1,11,27,21,38 It is perhaps important
Figure 2. (Top) A plot of kobsd vs [2-Cu(II)2:(-OCH3)] for the first event
with NaHPNPP (3 × 10-5 M) at 340 nm in anhydrous methanol. The
gradient of the line is 18 000 ( 700 M-1 s-1 sspH ) 7.4 ( 0.2. (Bottom)
,
A plot of kobsd vs [2-Cu(II)2:(-OCH3)] for the second event with NaHPNPP
(3 × 10-5 M) at 340 nm in anhydrous methanol. The gradient is 0.022 (
0.013 M-1 s-1, intercept ) 0.690 ( 0.008 s-1 sspH ) 7.4 ( 0.2.
,
for the catalysis that the O3-P-O4 bond angle in 2-Cu(II)(--
OH)((C6H5CH2O)2PO2-)(CF3SO3
) is expanded to 118.8°
2
-
which is similar to that seen with other bridging phosphates in
dinuclear Cu(II) complexes11,38c,d and may be one reason for
the exalted reactivity of the coordinated phosphate diesters in
this system; the remaining four O-P-O bond angles are only
mildly distorted from tetrahedral, being 105°, 105°, 106°, and
110°. Of note is the fact that the coordinated lyoxide Cu1-
O5H-Cu2 is 3.189 Å away from the electrophilic P-center, and
when bis-coordinated, the O5H electron pairs are not oriented
correctly to assume a nucleophilic role. Thus, it seems likely
that if the metal coordinated lyoxide is the nucleophile, it must
shed one of the coordinating Cu(II) ions and rotate to allow it
to approach the phosphorus.
Figure 3. A plot of the first-order rate constants for methanolysis of 0.01
mM 3 vs [2-Cu(II)2:(-OCH3)]. The line through the data is a fit to the
expression in eqs 1, 2 giving a KM of 0.079 ( 0.018 mM and a kcat of (2.40
( 0.07) × 10-3 s-1
;
sspH ) 7.2 ( 0.2.
In order to investigate the behavior of a DNA derivative
which sterically more closely resembles 1 but is far less reactive,
we determined the Abs vs time profiles at 340 nm for the
reaction of 5 × 10-5 M 4 with varying [2-Cu(II)2:(-OCH3)]
(not shown). This substrate also exhibits the up/down behavior
in the UV/vis spectrum as a function of time with a linear
dependence on [catalyst] for the first step (k1 ) 8700 ( 300
M-1 s-1) and a second step that is independent of [catalyst], k2
Given the fact that both crystals were grown from anhydrous
methanol solutions (reported to contain 0.002% water or 1.0
(37) Since 1 and 3 react too quickly with methanol in the presence of the catalyst,
dibenzyl phosphate was chosen as a nonreactive phosphate for crystal
studies.
(38) (a) Feng, G.; Natale, D.; Prabaharan, R.; Mareque-Rivas, J. C.; Williams,
N. H. Angew. Chem., Int. Ed. 2006, 45, 7056. (b) Wall, M.; Hynes, R. C.;
Chin, J. Angew. Chem., Int. Ed. Engl. 1993, 32, 1633. (c) Young, M. J.;
Chin, J. J. Am. Chem. Soc. 1995, 117, 10577.
9
11646 J. AM. CHEM. SOC. VOL. 129, NO. 37, 2007

Cleavage of RNA/DNA System by Dinuclear Cu(II) Complex
A R T I C L E S
Figure 4. An absorbance vs time profile for the reaction of 0.05 mM 3 in the presence of 0.5 mM 2-Cu(II)2:(-OCH3) showing the three temporally
well-defined events; large graph, slow event of product formation followed at 320 nm; inset, the two fast preliminary events followed at 340 nm; T )
25 °C, sspH ) 7.1.
Table 2. Various Rate Constants for Binding and Chemical Steps
for Substrates 1, 3, and 4 Reacting with 2-Cu(II)2:(-OCH3), T )
25 °C
rate constant f/ k1 (M-1 s-1
substrate V
)
k2 (s-1
)
k3 (s-1
)
1a
3c
4e
18 000 ( 700 0.69 ( 0.01
7.0b
20 200 ( 600 0.53 ( 0.06 (2.40 ( 0.07) × 10-3 d
8700 ( 300 0.49 ( 0.03
N.O.f
s
a
spH ) 7.4 ( 0.2. b Assumed to be at least 10-fold faster than k2; see
s
text. spH ) 7.1 ( 0.2. d Based on saturation data in Figure 3 with KM
)
c
s
s
0.079 ( 0.018 mM, spH ) 7.2 ( 0.2. spH ) 7.1 ( 0.1. f Not observed.
e
Figure 6. Molecular structure of 2-Cu(II)2(-OH)(H2O)(CF3SO3-)3:0.5CH3-
CH2OCH2CH3 shown as an ORTEP diagram at the 50% probability level;
counterions omitted for clarity. Structural details given in the Supporting
Information.
solution where the triflate ions can be reasonably well solvated,
the bridging positions can be occupied by methoxide and
methanol so that, for the reaction of 3, the product will be a
dimethoxy ester as observed for the MS experiments.
Figure 5. A plot of the relative sums of the integrated intensities of the
product (Iprod) or substrate (I3) ESI peak ratios (Iprod/(Iprod + I3), O, and
I3/(Iprod + I3), 2) as a function of time for the methanolysis reaction of 0.5
mM each of 3 and 2-Cu(II)2:(-OCH3). Lines through the data are NLLSQ
fits to a standard exponential model giving kprod ) 0.031 ( 0.007 min-1
4.2. Kinetics. 4.2.1. HPNPP (1) and 2-Cu(II)2:(-OCH3).
The Abs340 nm vs time profile for the reaction of 1 with 2-Cu-
(II)2:(-OCH3) shown in Figure 1 is biphasic with at least two
discernible events. The data in Figure 2, where the kinetics of
these two steps are determined by stopped-flow spectropho-
tometry under pseudo-first-order conditions of excess [2-Cu-
(II)2:(-OCH3)] between 0.25 and 1.0 mM, indicate that the rate
of the first rapid rise in absorbance is linearly dependent on
[2-Cu(II)2:(-OCH3)], which is consistent with the formation of
a 1:(2-Cu(II)2:(-OCH3)) complex. At the concentrations of 2-Cu-
(II)2:(-OCH3) investigated which are at least 5-fold larger than
the [1], the absorbance change for the first event is constant
signifying that essentially all the substrate is bound within the
first event. This is consistent with the KM of 0.079 mM found
and k3 ) 0.022 ( 0.003 min-1
.
mM and used as supplied), it is somewhat surprising to see that
neither structure contains methoxide or methanol coordinated
to the metal ions, but rather contains only bridging hydroxide
or water. The preference for HO- or OH2 bridging between
the metal ions is not due to the difference in size, as molecular
modeling suggests that a methanol or methoxide can occupy
the bridging positions without any severe steric interactions.
Thus, it appears that the water and hydroxide are preferred due
to hydrogen bonded interactions with closely associated CF3SO3-
counterions in the crystal. We assume that when in methanol
9
J. AM. CHEM. SOC. VOL. 129, NO. 37, 2007 11647

A R T I C L E S
Lu et al.
rapid rise/fall behavior followed at 340 nm. When these two
events are followed at sspH ) 7.2 ( 0.2 by stopped-flow
spectrophotometry at 0.25 mM < [2-Cu(II):(-OCH3)] < 1.0
mM (all values being above the KM value so that the binding
should be essentially saturated), one determines that k1 ) 20 200
( 600 M-1 s-1 and k2 ) 0.53 ( 0.06 s-1
.
The observed kinetic behavior for substrate 4, which is far
less reactive than 1 or 3 but sterically similar to the structure of
1, closely parallels that of 3 for the first two binding steps where
the first event was determined to be first order in [catalyst] with
k1 ) 8700 ( 300 M-1 s-1 while the second step was
independent of [catalyst], k2 being 0.49 ( 0.03 s-1 at an average
spH of 7.1 ( 0.1. The k3 step that would pertain to the release
s
of p-chlorophenol is observed to be far slower than in the case
of 3 and was not followed for this substrate.
4.3. Electrospray MS Measurements. In order to probe the
nature of the products of the reaction, the ESI mass spectra of
aliquots removed from a methanol solution that was 0.5 mM in
each of 3 and 2-Cu(II)2:(-OCH3) were determined as a function
of time. These are conditions where all the phosphate material
should be bound 1:1 to the 2-Cu(II)2 complex. The ratios of
the sums of the total integrated intensity of all the major peaks
containing product (CH3O)2PO2-, (Iprod), and all those containing
the starting material 3, (I3) vs (Iprod + I3), were plotted as a
function of time as shown in Figure 5. At the end of the reaction,
all the 3 was consumed and the only peaks that were observed
were those attributable to 2-Cu(II)2:(OTf-)2((CH3O)2PO2-) and
2-Cu(II)2:(OTf-)((CH3O)2PO2-)2.
4.4. General Mechanistic and Energetic Considerations
of the Catalysis. The observed events for all three substrates
are consistent with the simplified process shown in Schemes 1
and 2. The major difference relates to the magnitude of the rate
constants for the k3 process which vary from .0.7 s-1 for
HPNPP to (2.40 ( 0.07) × 10-3 s-1 for 3 and is too slow to
measure within a reasonable time with 4. The clearest case for
three temporally well-separated events is that of the catalyzed
methanolysis of 3 where there is evidence consistent with sepa-
rate bimolecular catalyst:substrate binding and intramolecular
rearrangement events, followed by the slower chemical step for
the production of p-nitrophenol. The data in Table 2 indicate
that the two events that precede the chemical step have very
similar rate constants for all substrates which seems most consis-
tent with more-or-less common binding processes. The value
for the k1 step seems small for ligand exchange on Cu(II) com-
plexes, which in some cases can be very high ((2-20) × 108
M-1 s-1).39 However the rates of binding to M(II) complexes
in general are known40 to be highly sensitive to crowding effects
such as is the case here. The compactness of the 2-Cu(II)2-
(-OH)(H2O)(CF3SO3-)3 structure shown in Figure 7 suggests
that substrate coordination to the Cu(II) ion(s) cannot occur by
an associative process but rather requires that the complex
should open up, probably by an equilibrium dissociation of one
of the Cu-O(H2)-Cu bonds, to reveal a four-coordinate
Cu(II) with an open site to which the phosphate can then bind.
If the equilibrium concentration of the open form is small, as
expected, then the overall rate constant for the association of
Figure 7. Structure of 2-Cu(II)2(-OH)(C6H5CH2O)2PO2-)(CF3SO3-)2 at
50% probability level; counterions omitted for clarity. Details given in the
Supporting Information.
with 3 which indicates that these phosphate diester substrates
have a high affinity for the catalyst. The second event, which
also gives rise to the formation of p-nitrophenol product, is
independent of [2-Cu(II)2:(-OCH3)] within experimental un-
certainty and so must be a unimolecular process that we suggest
arises from a rearrangement of the first-formed 1:(2-Cu(II)2:
(-OCH3)) complex which places it into a configuration from
which an even faster production of p-nitrophenol product occurs.
Stopped-flow control experiments in the absence of the diesters
indicate that neither of these events is attributable to simple
dilution of the 2-Cu(II)2:(-OCH3) catalyst. The overall process
can be rationalized according to the simplified process presented
in Scheme 2 where the first step, having a k1 ) 18 000 M-1
s-1, leads to essentially complete formation of the 1:(2-Cu(II)2:
(-OCH3)) complex, while the second rearrangement step has
an average pseudo-first-order rate constant of k2 ) 0.69 s-1 at
spH ) 7.4 ( 0.2. The subsequent product forming step, k3 in
s
Scheme 1, must be faster than the second step (we have assumed
by at least 10-fold) which accounts for the apparent rise in
absorbance for the k2 step relative to the cases with the slower
reacting phosphates described below.
4.2.2. Methyl p-Nitrophenyl Phosphate (3) and 2-Cu(II)2:
(-OCH3). Since the second event with 1 and 2-Cu(II)2:(-OCH3),
which we attribute to an intramolecular rearrangement, appears
to be superimposed on a subsequent fast chemical process that
forms the observable p-nitrophenol product, we switched to the
DNA model 3 where the release of p-nitrophenol is far slower
than that with 1.23,35 When the kinetics of production of
p-nitrophenol are monitored by normal UV/vis spectrophotom-
etry at 320 nm under pseudo-first-order conditions as a function
of [2-Cu(II)2:(-OCH3)] at sspH ) 7.2 ( 0.2, one observes the
saturation kinetic plot shown in Figure 3 which can be analyzed
with eqs 1 and 2 to yield a KM of 0.079 ( 0.018 mM and a kcat
of (2.40 ( 0.07) × 10-3 s-1. The numerical value of KM
indicates that the binding of 3, and presumably closely related
phosphate diesters such as 1 and 4, is strong. Based on the above
analysis with 1 we expect three observed events for 3, with the
first two being attributed to binding and rearrangement and the
third to a slower chemical event that liberates the p-nitrophenol.
Indeed, these expectations for the first two fast events are borne
out by the data shown in Figure 4 in which the insert reveals a
(39) Tobe, M. L.; Burgess, J. Inorganic Reaction Mechanisms; Addison Wesley
Longman Ltd.: New York, 1999; pp 271-333,
(40) (a) Dukes, G. R.; Margerum, D. W. Inorg. Chem. 1972, 11, 2952. (b) Pitteri,
B.; Marangoni, G.; Viseutin, F. V.; Cattalini, L.; Bobbo, T. Polyhedron
1998, 17, 475.
9
11648 J. AM. CHEM. SOC. VOL. 129, NO. 37, 2007

Cleavage of RNA/DNA System by Dinuclear Cu(II) Complex
A R T I C L E S
Scheme 2 a
a Proposed reaction sequence for HPNPP (1) and 2-Cu(II)2:(-OCH3); charges omitted for clarity.
Table 3. Selected Bond Distances and Angles for 2-Cu(II)2(-OH)(H2O)(CF3SO3-)3:0.5CH3CH2OCH2CH3 and
-
2-Cu(II)2(-OH)(C6H5CH2O)2PO2-)(CF3SO3
)
2
as Determined from X-ray Diffractiona
-
parameter
2-Cu(II)(-OH)(H2O)
2-Cu(II)(-OH)((C6H5CH2O)2PO2
)
Cu-Cu (Å)
3.260
1.897
1.987
3.680
1.944
1.945
Cu1-OH (Å)
Cu2-OH (Å)
Cu1-O(H2); Cu2-O(H2) (Å)
Cu1-OP; Cu2-OP (Å)
O5-P (Å)
2.342, 2.342
2.022; 1.986
3.189
140.44
Cu1-OH-Cu2 (deg)
Cu1-OH2-Cu2 (deg)
OH-Cu1-OH2; OH-Cu2-OH2 (deg)
OH-Cu1-OP; OH-Cu2-OP (deg)
O3-P-O4 (deg)
118.46
88.22
76.66; 76.66
89.39; 89.00
118.83
a For a complete listing of band angles and distances as well as atomic coordinates, see the Supporting Information.
phosphate will be reduced accounting for the 9 000-20 000 M-1
s-1 values for k1 seen here. The second step is consistent with
an intramolecular rearrangement, probably to form a doubly
coordinated phosphate with a structure similar to what is shown
in Figure 7 with a coordinated dibenzyl phosphate. The slowness
of this step can also be attributed to the tight steric requirements
that may impede the required dissociation of the bound solvent
from the second Cu(II) prior to double coordination of the
phosphate.
binding.41 Using this number, the overall catalysis by the Zn-
(II)2 complex is 1.1 × 108-fold.
A second way to compare the catalyst efficacy is to measure
the rate acceleration relative to the methoxide reaction at the
sspH where the catalyst is operative (7.2 in the case of 2-Cu-
(II)2:(-OCH3), 9.5 in the case of 2-Zn(II)2:(-OCH3)).42 This
artificially enhances the computed catalysis for systems that
operate at lower “pH” values but is a generally accepted method
for enzymatic reactions where the efficacy of the catalyzed
process is analyzed at neutral pH’s where the enzymes operate.
In this case, at sspH 7.2 (or 9.5) the methoxide reactions of 1
and 3 would have first-order rate constants of 6.9 × 10-13 (1.4
× 10-10) s-1 and 2.1 × 10-16 (4.3 × 10-14) s-1, respectively.
For the 2-Cu(II)2 catalyst, where the rate-limiting step is the
intramolecular rearrangement with 1 or 3 having a rate constant
of ∼0.7 s-1, the respective accelerations are about 1012- and
The effectiveness of the catalysis of the chemical steps with
1 and 3 can be measured in several ways. First, one could
compare the effective second-order rate constant for the catalytic
reaction with 3 (given as kcat/KM ) 2.4 × 10-3 s-1/7.9 × 10-5
M ) 30 M-1 s-1) with that for the methoxide reaction (7.9 ×
10-7 M-1 s-1); by this measure, the acceleration imbued by
2-Cu(II)2:(-OCH3) is 3.8 × 107-fold. This is similar to the
catalysis afforded for methanolysis of 3 by 2-Zn(II)2:(-OCH3),23
where kcat/KM ) (4.1 ( 0.3) × 10-2 s-1/(0.37 ( 0.07) mM )
110 M-1 s-1, the acceleration relative to the methoxide reaction
being 1.4 × 108. In the case of the reactions with the RNA
model 1, the chemical step of production of p-nitrophenol is
far faster than the limiting rearrangement process, so the
calculation leads only to a lower limit for the acceleration. With
2-Cu(II)2:(-OCH3), assuming that the KM constant can be
approximated by that found for 3 and that the chemical step is
at least 10-fold faster than the rearrangement step (∼7 s-1), the
acceleration is 3.5 × 107-fold (calculated as 7 s-1/7.9 × 10-5
M/2.56 × 10-3 M-1 s-1). Even if one uses the rate constant
determined by the rate-limiting second rearrangement step (0.7
s-1), the acceleration is 3.5 × 106. Alternatively, if the first
binding step of the substrate to the complex is rate limiting at
18 000 M-1 s-1, the acceleration relative to the methoxide
reaction is computed as 7.0 × 106-fold. Comparison with the
2-Zn(II)2:(-OCH3) catalyst is also complicated by the fact that
the latter does not show Michaelis-Mentin kinetics, but rather
second-order kinetics with a rate constant of 275 000 M-1 s-1
where the rate-limiting step was proposed23 to be substrate
1013-fold. For a solution containing 1 mM of the 2-Zn(II)2
s
s
catalyst operating at pH 9.5, the acceleration of cleavage of 1
is 2 × 1012-fold, while the acceleration exhibited by 2-Zn(II)2:
(-OCH3)-bound 3 (which decomposes with a pseudo-first-order
rate constant of 4.1 × 10-2 s-1 23
)
is also about 1012.
A recent analysis of the reaction of a dinuclear Zn(II) complex
(5) reacting with HPNPP43 presents a third and thermodynami-
(41) A referee has suggested that there is good evidence from other studies
(see ref 17) with a dinuclear Zn(II) complex (5) in aqueous solution that
the binding involves the nonionized form of the catalyst (Zn(II)2:H2O) and
the 2-hydroxy deprotonated dianionic form of HPNPP rather than the Zn-
(II)2:-OH form of the catalyst and the protonated 2-OH form of HPNPP
which we favor on the basis of our studies. While it is true that these two
possibilities are kinetically equivalent and cannot be distinguished on the
basis of catalytic studies with 5 in water due to its relatively low activity,
the available results we have with 2-Cu(II)2:(-OCH3) or 2-Zn(II)2:(-OCH3)
reacting with HPNPP in methanol quickly rule out the former possibility.
The pKa for deprotonation of the 2-HO group of HPNPP in methanol should
be at least as large or greater than that of methanol (Kw/[MeOH] ) 10-16.77
/
30) or spKa ) 18.2. At a sspH of 7.2 or 9.5 where the 2-Cu(II) and 2-Zn-
(II) are soperative, the [HPNPP2-] would be one part in 1011 and one part
in 108.7 respectively. Since the second-order rate constants for binding of
the HPNPP substrate, in whatever form, by the 2-Cu(II) and 2-Zn(II)
catalysts are observed to be 18 000 and 275 000 M-1 s-1, respectively, the
reactions would have to exceed the diffusion limit of 1010 M-1 s-1 by at
least 104 if the dianionic form of the substrate was the active one.
(42) Since the autoprotolysis constant of methanol is 10-16.77, at sspH 7.2 or 9.5
the [CH3O-] is 2.69 × 10-10 or 5.4 × 10-8 M.
9
J. AM. CHEM. SOC. VOL. 129, NO. 37, 2007 11649

A R T I C L E S
Scheme 3
Lu et al.
(II)2:(-OCH3) we can estimate a lower limit for the kcat term as
0.7 s-1 from the rate-limiting intramolecular rearrangement and
estimate the KM term as 7.9 × 10-5 M based on the one found
for 3 (Vide supra). In reality the kcat term for the chemical step
must be much greater which will actually give a larger
stabilization of ∆G (an extra -1.4 kcal for each factor of 10 in
rate constant at 25 °C). For 2-Zn(II)2:(-OCH3) we see only
second-order kinetics for the reaction of catalyst and HPNPP
substrate to produce p-nitrophenol, the limiting rate constant
(k1obsd) being 275 000 M-1 s-1 which we have interpreted as
being due to the binding of HPNPP and catalyst. Using these
values, the lower limit for computed stabilization of the TS for
the intramolecular transesterification of HPNPP with CH3O-
bound by the Cu(II)2 and Zn(II)2 catalysts are -22.6 and -21.0
kcal/mol, while those for the reaction of 3 promoted by the same
two catalysts are -24.0 and -21.1 kcal/mol. These are very
large energy reductions for two dinuclear catalysts promoting
two different reactions where one involves a methoxide (either
metal-coordinated or free) acting as a base followed by an
intramolecular cylcization and subsequent formation of product,
while the other reaction involves methoxide acting as a
nucleophile on P.
cally explicit way of comparing the lyoxide and complex-
catalyzed reactions by analyzing the transition state energetics
and the stabilization of the lyoxide:substrate transition state
through its binding to the catalyst.44 Scheme 3 gives a
thermodynamic cycle describing both the methoxide reaction
and the 2-M(II)2:(-OCH3) complex promoted reaction. Given
in eq 3 is the expression for computing the free energy of
binding of the transition state for the catalyst promoted reaction
relative to the methoxide reaction (∆Gqstab.). The term “meth-
oxide reaction” which is given a short-hand designation of
(CH3O-:Sub.)q in Scheme 3 refers to both of the reactions with
1 and 3 where methoxide acts as a base or a nucleophile. In eq
4 is the free energy calculation specific for the reaction with
HPNPP and 2-Zn(II)2:(-OCH3) which does not show saturation
kinetics but does show an overall second-order reaction with a
first-order dependence on [catalyst]. The terms in eqs 3, 4 are
calculated from the following: (a) kcat/KM from the saturation
curve depicted in Figure 3 or that given in ref 23 for the reaction
of 3 with the Zn(II) complex; (b) Ka/Kw can be readily
interpreted as the binding constant of methoxide to the dinuclear
complex, where Ka refers to the first acid dissociation constant
for the formation of 2-Cu(II)2:(-OCH3) or 2-Zn(II)2:(-OCH3)
and Kw is the autoprotolysis constant for methanol (10-16.77);
The large TS binding energies of the 2-M(II)2 catalysts for 1
and 3 in methanol, while approximate given the assumptions,
invite comparison with the value of -9.6 kcal/mol afforded for
the cleavage of HPNPP by dinuclear Zn(II) complex 5 in
water.43 To our knowledge there are no such free energy com-
parisons possible from literature examples for the catalyzed
cleavage of DNA models, presumably probably because these
reactions are extremely slow in water unless catalyzed efficiently
as is the case here in methanol. The combination of these two
specific dinuclear complexes (whether Zn(II) or Cu(II) contain-
ing) and a medium effect engendered by the methanol, provides
rate enhancements exceeding anything reported in water so far.
Previously23 we attributed this medium effect in the light alcohol
solvents to a reduced dielectric constant that increases the poten-
tial energy of attraction between oppositely charged ions of the
sort involved in metal-catalyzed reactions of anionic substrates.
In addition, a lower polarity medium is well-known to promote
reactions where charge is dispersed in the TS. The application
of eqs 3, 4 allows one to assess the energetic consequences of
the medium effect on the various rate and equilibrium steps
shown in Scheme 3 for different catalytic systems as in eq 5
and (c) kOCH is the second-order rate constant for the methoxide
3
reactions in the absence of complex. Listed in Table 4 are the
various constants for the relevant terms in eq 3 for the reaction
with 3 and 1 with the 2-Cu(II)2 catalyst and 3 with the 2-Zn-
(II)2 catalyst and those in eq 4 for the reaction of HPNPP with
the 2-Zn(II)2 catalyst.
∆G‡stab ) (∆GBind + ∆GM + ∆Gcat) - ∆GN‡ on
)
(kcat/KM)(Ka/Kw)
- RT ln
(3)
[
kOCH
]
3
In Scheme 3, ∆Gbind, ∆Gcat, and ∆G‡ are the respective
Non
free energies for (1) binding of methoxide to the catalyst; (2)
the activation energy for unimolecular reaction of the Cat:-
OCH3:Sub. complex; and (3) the activation energy for the
reaction of methoxide alone with substrate. ∆GM is the free
energy for association of the substrate and Cat:-OCH3 complex.
Zn
cat
In eq 4, ∆G is the free energy for the second-order reaction
of the Cat:-OCH3 complex with substrate, the associated rate
(kcat/KM)MeOH [Ka/Kw]MeOH
∆∆G‡stab ) -RT ln
constant being defined as k1obsd
.
HOH
HOH
{(
)(
)
(kcat/KM)
[Ka/Kw]
∆G‡stab ) (∆GBind + ∆GZn) - ∆GN‡ on
)
cat
HOH
(kOH
)
(k1obsd)(Ka/Kw)
(5)
MeOH
(
)}
- RT ln
(4)
(kOMe
)
[
]
k
OCH3
(43) O’Donoghue, A. M.; Pyun, S. Y; Yang, M.-Y.; Morrow, J. R.; Richard, J.
P. J. Am. Chem. Soc. 2006, 128, 1615.
As described above, we do not see saturation kinetics for
HPNPP binding and reaction with either catalyst, but for 2-Cu-
(44) Wolfenden, R. Nature 1969, 223, 704.
9
11650 J. AM. CHEM. SOC. VOL. 129, NO. 37, 2007

Cleavage of RNA/DNA System by Dinuclear Cu(II) Complex
A R T I C L E S
Table 4. Kinetic Parameters for 2-M(II)2:(-OCH3) and Methoxide Ion Catalyzed Cleavage of 3 and HPNPP (1) at 25 °C in Methanol
parameter
3 (Cu(II))
3 (Zn(II))
HPNPP (Cu(II))
HPNPP (Zn(II))
kOCH (M-1 s-1
KM (M)
)
7.9 × 10-7 a
7.9 × 10-5 c
30
7.9 × 10-7 a
3.7 × 10-4 a
110a
2.56 × 10-3 b
7.9 × 10-5 d
9 × 103
2.56 × 10-3 b
-
3
k
cat/KM (M-1 s-1
)
275 000e
2.3 × 107 f
6.3 × 1012
e- 21.0
Ka/Kw (M-1
)
1010 f
2.3 × 107 f
2.5 × 109
-21.1
1010 f
(kcat/KM)(Ka/Kw) (M-2 s-1
∆G‡stab (kcal/mol)
)
30 × 1010
9 × 1013
-24.0
e- 22.6
a From ref 23. b From ref 33. c From data in Figure 3. d KM for HPNPP assumed to be the same as that determined for 3. e Given as second-order rate
constant observed in ref 23 for the reaction of Zn(II) catalyst with HPNPP (ko1bsd f Ka from half neutralization of 2-Cu(II)2:(-OCH3) and 2-Zn(II)2:(-OCH3)
)
by adding 0.5 equiv of acid to the 2-M(II)2:(-OCH3) complex, spKa ) 6.77 and 9.41, respectively.
s
Table 5. Computed ∆∆G‡stab Afforded from Each of the Ratios of
Constants Given in Eq 5 for the Reactions of Various Systems in
5. Conclusions
Promoting the Cyclization of HPNPP (1) at 25 °C
In the above we have provided structural data for two 2-Cu-
(II)2 complexes where the central coppers are bridged by HO-,
H2O, and HO-, (PhCH2O)2PO2- (Figure 7, respectively). These
are germane to the solution forms of the resting catalyst in
methanol where methoxide seems to be the active base or
nucleophile and to the state in which the reactive phosphate
diesters 1 and 3 studied here might be bound prior to their
cleavage. The kinetic studies of 2-Cu(II)2:-OCH3 with substrates
1, 3, and 4 demonstrate that all have two fast steps that are
interpreted as substrate binding to one of the Cu(II) centers,
followed by an intramolecular rearrangement to form a putative
double Cu(II) coordinated phosphate diester analogous to what
is shown in Figure 7. For two of these systems, 1 and 3, there
is a chemical event that leads to the production of p-nitrophenol
which is markedly accelerated relative to the background
reaction promoted by methoxide. In fact the acceleration of the
chemical step in the reaction of 1 is so effective that the
rate-limiting steps become those of substrate binding and
positioning.
total ∆∆
G‡stabmethanol
vs water
MeOH
HOH
[
−
RT ln
{
(kcat/Km
)
/
{−RT ln([Ka/Kw]MeOH
[Ka/Kw]HOH
/
[
−
RT ln
{
(kOH
)
/
HOH
MeOH
catalyst
(kcat/Km
(kcal/mol)a
)
}
]
)}
(kOMe
)
}]
comparison
V
(kcal/mol)
(kcal/mol)
(kcal/mol)
2-Zn(II)2 in
MeOH vs 5
in water
2-Cu(II)2 in
MeOH vs 5
in water
-7.6
-1.6
-2.2
-11.4
-13
-5.6
-5.2
-2.2
a The kcat/KM term is given as the second-order rate constant for 2-Zn(II)2
promoted cylcization of 1 (2.75 × 105 M-1 s-1), while that for 2-Cu(II)2 is
given as 0.7 s-1/7.9 × 10-5 M: the reported rate constant for the reaction
of 5 with HPNPP in water is 0.71 M-1 s-1 (ref 43).
where the kcat/KM term can be replaced by the apparent second-
order rate constant for the catalyst reacting with HPNPP or 3.
Ideally one would like to compare the same catalyst in water
and methanol, but this is not possible at present. Nevertheless,
the energy differences (∆∆G*stab) shown in Table 5 shed light
on which of the components contributes most to the observed
increase in catalysis in moving between the two systems. For
example, the Ka/Kw term has a value of 1.43 × 106 M-1 in
water with the Zn(II) catalyst 5,43 2.3 × 107 M-1 in methanol
with 2-Zn(II)2, and 1 × 1010 M-1 in methanol with 2-Cu(II)2.
The accelerations afforded to the cleavage of 1 and transes-
terification of 3 by 2-Cu(II)2:(-OCH3) catalyst are among the
fastest reported to date, the only faster ones being with our
previously reported23 2-Zn(II)2:(-OCH3) catalyst also in metha-
nol. An analysis of the free energy of binding of the 2-Cu(II)2
and 2-Zn(II)2 catalysts to the TS for the intramolecular cycliza-
tion of 1 and the phosphoryl transfer process of 3 indicates that
these two dinuclear systems exhibit similar values of -21 to
-24 kcal/mol which is significantly greater than what has been
heretofore reported for a dinuclear system catalyzing three
analogous phorphoryl cyclizations in water (-7.2 to -9.6 kcal/
mol).43 A detailed analysis partitions the origins of the differ-
ences in the free energies of stabilization of the methanol/2
system vs a water/5 system into three main terms that are
responsible for the ∆∆G‡stab where the main contributions arise
from the increased catalytic constants (kcat/KM).
Although the autoprotolysis constant for methanol (Kw
)
10-16.77) is lower than that of water, this only leads to an increase
in the Ka/Kw term if the sspKamethanol < (pKwa ater + 2.77). The
spKa for ionization of 2-Zn(II):(HOCH3) is 9.41, while the pKa
s
for 5 in water is 7.8-8.043 such that the respective and Ka/Kw
ratios for 5 and 2-Zn(II) differ by a factor of 16. This term
contributes -1.64 kcal/mol to the stabilization of the TS (∆∆
G‡stab) for the reaction of 2-Zn(II) in methanol vs 5 in water but
a more substantial -5.24 kcal/mol for the 2-Cu(II) complex in
methanol because its pKa of 6.77 is nearly 3 units lower than
s
s
that of the Zn(II) complex.
The results from this study reinforce our previous conten-
tions23 that the combination of the dinuclear catalysts and an
important medium effect contribute greatly to the acceleration
of the cleavage of phosphate diesters and that such a combina-
tion may fulfill a key role in related enzymatic processes.
The difference between the kOR values for reaction of 1 with
hydroxide in water (9.9 × 10-2 M-1 s-1 43
)
and methoxide in
methanol (2.56 × 10-3 M-1 s-1) contributes -2.2 kcal/mol to
the overall ∆∆G‡stab. However, the analysis shows that the
major part of the ∆∆G‡ stabilization in the case of the
2-Zn(II)2 complex results from the apparent second-order rate
constant (kcat/KM) for the reaction which is 3.9 × 105 times larger
than that seen in the case of 5 in water. The analogous value
for 2-Cu(II)2 is 1.3 × 104 times larger and, thus, contributes
roughly as much as its Ka/Kw term to the overall ∆∆G‡stab for
the reaction.
Acknowledgment. The authors gratefully acknowledge the
Natural Engineering Sciences and Research Council of Canada,
the Canada Foundation for Innovation, The Canada Council for
the Arts, and Queen’s University for financial support of this
work. They are also grateful to Dr. Ruiyao Wang for determin-
ing the X-ray diffraction structures of the 2-Cu(II)2 complexes.
9
J. AM. CHEM. SOC. VOL. 129, NO. 37, 2007 11651

A R T I C L E S
Lu et al.
-
In addition C.T.L. acknowledges NSERC Canada for a post-
graduate PGS-M scholarship.
Supporting Information Available: Tables of pseudo-first-
order rate constants for reactions of 1, 3, and 4 with 2-Cu(II)2:
(-OCH3); mass spectra at three different reaction times for 3
with equimolar 2-Cu(II)2:(-OCH3); structural reports and CIF
files for 2-Cu(II)2(-OH)(C6H5CH2O)2PO2-)(CF3SO3
) and
2
2-Cu(II)2(-OH)(H2O)(CF3SO3-)3:0.5CH3CH2OCH2CH3. This
material is available free of charge via the Internet at
http://pubs.acs.org.
JA073780L
9
11652 J. AM. CHEM. SOC. VOL. 129, NO. 37, 2007